Advances in Pharmacology Volume 50 Roles of Vasopressin and Oxytocin in Memory Processing

THE ROLES OF VASOPRESSIN AND OXYTOCIN IN MEMORY PROCESSING SERIAL EDITORS J. Thomas August Daryl Granner Baltimore,...

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THE ROLES OF VASOPRESSIN AND OXYTOCIN IN MEMORY PROCESSING

SERIAL EDITORS J. Thomas August

Daryl Granner

Baltimore, Maryland

Nashville, Tennessee

Ferid Murad Houston, Texas

______________________________________________________________________________________________________ ADVISORY BOARD

R. Wayne Alexander

Floyd E. Bloom

Boston, Massachusetts

Leroy Liu

Thomas F. Burke

Leroy Liu

Houston, Texas

Piscataway, New Jersey

Anthony R. Means

G. Alan Robison

Durham, North Carolina

Houston, Texas

John A. Thomas

Thomas C. Westfall

San Antonio, Texas

St. Louis, Missouri

THE ROLES OF VASOPRESSIN AND OXYTOCIN IN MEMORY PROCESSING By

Barbara B. McEwen Professor Emeritus Department of Psychology Southern Connecticut State University New Haven, Connecticut

ADVANCES IN

PHARMACOLOGY VOLUME 50

Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK

This book is printed on acid-free paper. Copyright ß 2004, Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (www.copyright.com), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2004 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 1054-3589/2004 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, E-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’ For all information on all Academic Press publications visit our Web site at www.academicpress.com ISBN: 0-12-032951-4 PRINTED IN THE UNITED STATES OF AMERICA 04 05 06 07 08 9 8 7 6 5 4 3 2 1

In Memory of David De Wied

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Acknowledgments

Special homage is due to the many scientists whose cited publications form the very essence of this ﬁeld of research, and without which this book would not have been possible. A special note of appreciation is due to the researchers listed below who read selected chapters in which I discussed their speciﬁc research contributions. William A Banks, GRECC, Veterans Affairs Medical Center and Division of Geriatrics, Department of Internal Medicine, Saint Louis University School of Medicine, St Louis, Missouri, USA. Bill, thank you for your patience, and for the invaluable guidance you provided this psychology-trained author in meeting the challenges she faced in discussing the interaction of vasopressin and oxytocin with the bloodbrain barrier. The late Michael Bunsey, formerly Assistant Professor of Psychology at Kent State University in Kent, Ohio, USA. Michael’s particular contributions during the preparation of this text are discussed in a subsequent memorial tribute paid in honor of his memory. Robert Dantzer, director of research, Neurobiologie Integrative, rue Camille Saint-Saens, 33077 Bordeaux Cedex, France. Robert, I am especially grateful to you for clarifying for me your distinction between vasopressin’s arousal effects on general memory processing, and its unique and distinctive role in social memory. The late David De Wied, founder and former director of the Rudolf Magnus Institute for Neurosciences, Department of Medical Pharmacology, Utrecht University, Universteitsweg 100, 3584 GG Utrecht, the Netherlands.

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Acknowledgments

My humble tribute to this great man, whose constant support and friendship were of key importance to the publication of this work, is given in a subsequent memorial tribute. George F. Koob, Professor and Director, Division of Psychopharmacology, Department of Neuropharmacology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California, USA. I thank you, George Koob, for a ﬁrst rate critique of my discussion of the research by you and your colleagues, which highlighted the role of arousal mechanisms in this research ﬁeld. The two researchers listed below are also deserving of my special gratitude for the assistance each provided, enabling some discussion of their signiﬁcant research contributions. Richard D. Broadwell, Bethesda, Maryland, USA. Y-C Du, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai, People’s Republic of China. This book’s existence also depended on the special skills and emotional support provided by friends and colleagues during one or more stages of its preparation. Mona Gustafson Afﬁnito, Duane Harmon, and Jim Hurlbut, my former colleagues from the Department of Psychology at Southern Connecticut State University (SCSU) in New Haven, Connecticut, read and commented on the initial versions of this book. Their advice to proceed with my vision of its organization provided the emotional thrust I needed to begin the book that was ﬁnally written. Stuart Solomon, my long-time friend and colleague from SCSU, read and commented on the numerous versions of this text throughout the many years of its writing. His excellent editing greatly improved its readability, while faithfully preserving the content of its message. His penetrating questions and incisive comments sent me back to ‘‘the drawing board’’ on many occasions forcing me to reevaluate, reorganize, and revise passages throughout the text. In short, Stu has collaborated on many fronts in the writing of this book and deserves far greater recognition than is provided by this acknowledgement. My very dear friend, Amy Huie-Li, spent many hours tracking down resource materials and obtaining journal articles from the Medical Library at the Yale School of Medicine in New Haven, Connecticut. Additionally, the constancy of her devoted friendship has meant a great deal to me throughout the course of this writing. I would also like to thank her husband, Heung Ming Li, whose valuable computer skills enabled the reproduction of the ﬁgures used in this text. I very much appreciate the efﬁciency with which my requests for additional source materials were processed by Beth Paris, Library Technical

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Assistant at Hilton C. Buley Library at SCSU, and Elaine Edwards, Head of Interlibrary Services at the Cheshire Public Library in Cheshire, Connecticut. A special thank you to Joke Jaarsma, Senior Publisher, Department of Pharmacology, Pharmaceutical Science, and Toxicology at Elsevier Publishers in Amsterdam, the Netherlands. My decision to ‘‘go for the gold’’ and submit the manuscript to Elsevier Science Publishing Company bode well for the book’s fate. Its fortune was sealed when Joke accepted it for publication and orchestrated the behind-the-scenes work needed for its editing, marketing, and production for ﬁnal publication. I am especially appreciative of Joke’s understanding, patience, and steadfast support during the latter period of its preparation.

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Tribute

Tribute to Michael Bunsey I was very sad to learn of the untimely death of Michael Bunsey. In 1997 Michael donated valuable time and effort to read and critique selected chapters in this text. I will never forget, and always be grateful for his kind words and valuable commentary, especially those comments relating to my discussion of vasopressin’s role in attentional processing. All of his excellent suggestions were used in subsequent revisions of the chapters he reviewed.

Tribute to David De Wied The recent death of David De Wied is a tragic loss to the scientiﬁc community and a deep personal loss to family, friends and colleagues alike. His research in the areas of neuropharmacology and behavioral pharmacology has earned worldwide recognition. This is particularly true of his pioneering work on the effects of vasopressin and oxytocin on memory processing, the subject of this book. When I ﬁrst wrote to David in 1997, he was delighted to learn about the book and readily agreed to read and comment on the chapters that discussed the research carried out by him and his colleagues. Throughout the course of our subsequent correspondence, he unreservedly supported my attempts to maintain an objective position regarding the theoretical controversy, This, together with his warmth and understanding, openness to my questions, and practical advice regarding publication matters were of inestimable help to me during the course of preparing the book for publication. It is my sincere hope that the book will prove to be a worthy tribute to his invaluable contributions to this important area of research. xi

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Contents

Preface

PART

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I

General Introduction to Vasopressin and Oxytocin: Structure/Metabolism, Evolutionary Aspects, Neural Pathway/Receptor Distribution, and Functional Aspects Relevant to Memory Processing Barbara B. McEwen

I. Metabolic Aspects 1 A. Biosynthesis of VP and OT 2 B. Metabolic Degradation of VP and OT 6 II. Evolutionary Considerations and Comparative Study 6 III. VP and OT Cell Systems, Pathways, and Receptors: Characteristics and Distribution 9 A. VP-ergic and OT-ergic Cell Groups and Their Pathways in the CNS 9 1. Methods Used to Localize VP and OT Systems in the CNS 9 2. Distinction between Magnocellular and Parvocellular VP-ergic and OT-ergic Cellular Systems 11 3. Hypothalamic VP-ergic and/or OT-ergic Cells 13 a. Hypothalamic SON 13

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b. Hypothalamic PVN 13 c. Afferent Input into the PVN 16 d. PVN as a Visceral Effector Integrative Center 17 e. Suprachiasmatic Nucleus 17 4. Extrahypothalamic VP-ergic and/or OT-ergic Cell Systems 17 a. Overview 17 b. Sexually Dimorphic and Gonad-Dependent Circuitry 21 5. VP-ergic and OT-ergic Fibers and Terminals of Unknown Origin 22 B. Vasopressin and Oxytocin Receptors 22 1. Distinction between Ionotropic and Metabotropic Receptor Transmission 22 a. Ionotropic Receptor Transmission 22 b. Metabotropic Receptor Transmission 26 2. Characterizing and Localizing V1 and V2 types of VP Receptors in Peripheral and Neural Tissues 28 a. Vasopressin V1a Receptor 28 i. Localization 28 ii. Receptor-Mediated Signaling Pathway 31 b. Vasopressin V2 Receptor 34 3. Classification and Localization of OT Receptors 35 C. Vasopressin and Oxytocin as Neurotransmitters and Neuromodulators in the CNS 37 IV. Functional Aspects Relevant to Memory Processing: Actions at Nonneural Receptor Sites 38 A. Hormonal VP Actions on V1 Receptors in the Peripheral Vasculature and Memory Processing 38 B. VP and OT Interactions with Blood Vessels in the Brain: Role in Memory Processing? 38 1. VP–OT Interactions with Extracerebral and Intracerebral Vasculature 40 a. Roles of Extracerebral and Intracerebral Vasculature in Brain Perfusion and Circulation 40 b. Interaction with Extracerebral Vasculature: Homeostatic Regulation of Brain Perfusion 41 c. Interaction with Intracerebral Vasculature 42 i. Regulation of Local Blood Flow 42 ii. Nutrient Exchange Across the Blood–Brain Barrier 43 C. Actions of VP and OT in Nonneural Tissue Sites and Glucose Regulation: Role in Memory Processing 44 1. Functional Role for VP and OT in Glucose Storage and Utilization 44

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2. Stress-Associated Actions of VP and OT at Nonneural Tissue Sites and Enhanced Glucose Availability to the Brain: Role in Memory Processing? 44 a. VP and OT Action in the Pancreas and Liver: Direct Influence on Glucose Metabolism 44 b. VP and OT Influences on Stress Hormones: Indirect Influence on Glucose Metabolism 45 c. Relation between Glucose-Induced Enhancement of Memory Processing, Stress, and VP- and/or OT-Induced Release of Stress Hormones that Enhance Glucose Availability to the Brain 45 D. Stress-Induced VP/OT Effects on Receptors in the Adrenal Medulla and Anterior Pituitary Gland: Relevance for Memory Processing 46 1. VP/OT Actions on Receptors in the Adrenal Medulla and Memory Processing 46 2. Support for Stress-Associated VP and OT Influence on the Pituitary–Adrenocortical Axis 47 a. Hypothalamic VP and OT Activation of the Pituitary– Adrenocortical Axis during a Stress Response 47 b. Types of Stressors that Activate VP and OT Cell Groups Involved in the Regulation of Hormonal Release from the Anterior Pituitary 47 c. Stress Hormones Released from the Anterior Pituitary and the Adrenal Cortex: Effects on Learning and Memory 48 i. ACTH 48 ii. -Endorphin 48 iii. Corticosterone 49

PART

II

De Wied and Colleagues I: Evidence for a VP and an OT Inﬂuence on MP: Launching the ‘‘VP/OT Central Memory Theory’’ Barbara B. McEwen

I. Chapter Overview 51 II. Major Task Paradigms Used by De Wied and Associates in Their VP/OT Memory Research 52 A. Multitrial Two-Way Active Avoidance Task: The Shuttlebox Task 52

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VI. VII.

Contents

B. Multitrial One-Way Active Avoidance Task: The Pole Jump Task 52 C. Single-Trial Step-Through Passive (Inhibitory) Avoidance Task 55 Role for Vasopressin in Facilitating Memory Consolidation and Retrieval 55 A. Avoidance Conditioning Paradigms: Selected Studies 56 1. Selected Studies 56 a. De Wied (1965) 56 b. De Wied and Bohus (1966) 58 c. De Wied (1971) 59 d. Ader and De Wied (1972) 61 e. Bohus and Colleagues (1972) 62 f. King and De Wied (1974) 65 g. Hagan and Colleagues (1982) 69 B. Appetitive Learning Paradigms 72 1. Selected Studies 72 a. Garrud and Colleagues (1974) 72 b. Bohus (1977) 73 c. Vawter and Van Ree (1995) 75 d. Vawter and Colleagues (1997) 77 Oxytocin: A Natural Amnestic in Aversive Learning Situations 78 A. Introduction 78 1. Selected Studies 79 a. Walter and Colleagues (1975) 79 b. Kovacs and Colleagues (1978) 80 c. Bohus and Colleagues (1978a) 80 d. Bohus and Colleagues (1978b) 81 e. Gaffori and De Wied (1988) 86 Effects of VP on Retrograde Amnesia: Effect on Memory Retrieval? 90 A. Introductory Comments: Retrograde Amnesia and Memory Retrieval 90 1. Selected Studies 90 a. Rigter and Colleagues (1974) 90 b. Bohus and Colleagues (1982) 93 VP and OT Appear to Have No Important Effects on the Learning Phase of Memory Processing 94 Theoretical Propositions of the ‘‘VP/OT Central Memory Theory’’ and Relevant Evidence 97 A. Proposition 1: VP Facilitates Memory Consolidation and Retrieval 97 B. Proposition 2: OT Attenuates Memory Consolidation and Retrieval 99

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C. Proposition 3: VP and OT Do Not Have a Major Role in the Learning Phase of Memory Processing 100

De Wied and Colleagues II: Further Clariﬁcation of the Roles of Vasopressin and Oxytocin in Memory Processing Barbara B. McEwen

I. Chapter Overview 103 II. Establishing the Roles of Endogenous VP in Memory Processing: The Brattleboro Rat Model 104 A. Introductory Comments 104 B. Early Research Studies by De Wied and Colleagues with the Brattleboro Rat 105 1. Selected Studies 105 a. De Wied et al. (1975) 105 b. Bohus et al. (1975) 106 C. Inconsistencies in the Research Literature Regarding the Putative Brattleboro Diabetes Insipidus Retention Deficit 109 D. Colony-Specific Heritable Traits and Inconsistent Findings Concerning a Retention Deficit in the Brattleboro DI Rat 110 1. Selected Studies 111 a. Herman et al. (1986a) 111 b. Herman et al. (1986b) 112 E. Brattleboro Rat Retention Deficit I: A Primary or Secondary Effect of VP Deficiency? 113 F. Brattleboro Rat Retention Deficit II: An Arousal-Mediated Phenomenon? 113 G. Brattleboro Rat Model Revisited: Recent Findings by De Wied and Colleagues 114 1. Selected Study: De Wied et al. (1988) 114 H. Section Summary and Concluding Remarks 115 III. Further Study of the Role of Endogenous VP and OT in Memory Processing: Peripheral and/or Central Mechanisms? 116 A. Introductory Comments 116 B. Neutralizing Peripheral or Centrally Circulating VP or OT by Antiserum Treatment: Effect on Memory Processing 117 1. Selected Study: Van Wimersma Greidanus et al. (1975a) 117 C. Correlational Studies 1: Avoidance Retention and AVP Levels in the Blood 118 1. Selected Studies 118 a. Thompson and De Wied (1973) 118 b. Van Wimersma Greidanus et al. (1979a) 119

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c. Mens et al. (1982a) 120 d. Laczi et al. (1983c) 121 D. Correlational Studies 2: Avoidance Retention and AVP Levels in the CSF 124 1. Selected Studies 124 a. Van Wimersma Greidanus et al. (1979a) 124 b. Mens et al. (1982a) 124 c. Laczi et al. (1984) 125 E. Correlational Studies 3: Avoidance Retention and AVP Levels in Selected Brain Structures 126 1. Selected Studies 126 a. Laczi et al. (1983a) 126 b. Laczi et al. (1983b) 127 F. Section Summary 129 IV. Vasopressin-Induced Increase in Behavioral Arousal Is Not Essential for Its Effect on Memory Processing 129 A. Introductory Comments 129 1. Strategy 1: Dissociation of the Behavioral and Endocrine Effects of Peripherally Administered Vasopressin—Use of DG-AVP and Other C-Terminal VP Metabolites 130 a. Selected Study: Gaffori and De Wied (1985) 130 2. Strategy 2: Evidence That the Arousal Effect of Peripherally Injected VP Is Not Essential for Its Influence on Memory Storage 132 a. Selected Study: Skopkova et al. (1991) 132 V. Peripherally Administered Neurohypophysial Peptides and Central Memory Processing 133 A. Does Peripherally Injected VP or OT Reach Central Memory-Processing Sites? 133 1. Selected Study: De Wied et al. (1984a) 134 VI. Theoretical Propositions of the ‘‘VP/OT Central Memory Theory’’: Continued 134 A. Proposition 1: VP Facilitates Memory Consolidation and Retrieval 135 B. Proposition 3: VP and OT Have No Major Role in the Learning Phase of Memory Processing 136 C. Proposition 4: Central VP-ergic and OT-ergic Circuitry and Not Peripherally Circulating Hormones Are the Primary Means by Which Neurohypophysial Peptides Influence Memory Processing 136 D. Proposition 5: VP and OT Modulate Memory Processing Directly and Not by an Indirect Influence on Behavioral Arousal 138

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E. Proposition 6: The Effect of Exogenously Administered VP and OT on Memory Processing Is Due to Action Exerted at Central and Not Peripheral Receptor Sites 139

De Wied and Colleagues III: Brain Sites and Transmitter Systems Involved in theVasopressin and Oxytocin Inﬂuence on Memory Processing Barbara B. McEwen

I. Introductory Remarks 141 II. Localizing Central Sites for the Memory-Modulating Effects of VP and OT by Means of Lesioning and Microinjection Techniques 141 A. Thalamic-Limbic Lesions: Influence on the Memory-Modulating Effects of Adrenocorticotropic Hormone-Like Peptides and of VP and/or OT 142 1. Selected Studies 143 a. Van Wimersma Greidanus et al. (1974) 143 b. Van Wimersma Greidanus et al. (1975b) 144 c. Van Wimersma Greidanus and De Wied (1976b) 144 d. Van Wimersma Greidanus et al. (1979b) 145 e. Van Wimersma Greidanus et al. (1979c) 146 2. Summary: Lesion Studies 148 B. Microinjection of VP, OT, or Their Antisera into Discrete Brain Sites 149 1. Microinjections of VP or OT into Selected Brain Sites 149 a. Van Wimersma Greidanus et al. (1973) 149 b. Kovacs et al. (1979a) 149 c. Kovacs et al. (1979b) 150 d. Bohus et al. (1982) 150 2. Microinjections of VP or OT Antiserum into Selected Brain Sites 151 a. Kovacs et al. (1980a) 151 b. Kovacs et al. (1982a) 151 c. Veldhuis et al. (1987) 152 d. Van Wimersma Greidanus and Baars (1988) 153 3. Summary: Microinjection Studies 153 III. Interaction between VP/OT Peptides and Brain Catecholamines in Memory Processing 155 A. The Behavioral/Biochemical Protocol: Influence of VP, OT, or Their Antisera on PA Behavior and Catecholaminergic Neurotransmission 156

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1. Selected Studies 156 a. Kovacs et al. (1977) 156 b. Kovacs et al. (1979a) 157 c. Veldhuis et al. (1987) 158 2. Summary 158 B. The Behavioral Protocol: Effect of Selective Lesions in a Catecholaminergic Projection System on VP-Induced Avoidance Retention 159 1. Selected Studies 159 a. Kovacs et al. (1979b) 159 b. Kovacs et al. (1980a) 162 C. The Biochemical Protocol: The Effect of VP, OT, or Their Antisera on Catecholaminergic Transmission in Selected Brain Sites 162 1. Effect of Centrally Injected AVP or OT on Catecholamine Neurotransmission 162 a. Selected Studies 162 i. Tanaka et al. (1977a) 162 ii. Van Heuven-Nolsen et al. (1984a) 163 iii. Van Heuven-Nolsen et al. (1984b) 163 iv. Van Heuven-Nolsen and Versteeg (1985) 164 b. Summary 166 2. Intraventricularly Injected VP or OT Antiserum: Effect on Catecholamine Neurotransmission in Selected Brain Sites 166 a. Selected Studies 166 i. Versteeg et al. (1979) 166 ii. Kovacs and Telegdy (1983) 167 b. Summary 168 IV. Interaction between VP and Catecholamines of Peripheral Origin during Memory Processing 169 A. Selected Studies: Borrell et al. (1983a,b) 169 V. Theoretical Propositions of the ‘‘VP/OT Central Memory Theory’’: Continued 173 A. Proposition 7: The Central Anatomical Substrate for the Memory-Modulating Effects of VP and OT Includes Brainstem and Forebrain Limbic System Structures That Are Implicated in Memory Processing 173 B. Proposition 8: Neurohypophysial Peptides Interact with Central Catecholaminergic Neurotransmitters in Mediating Their Influence on Memory Processing, and the VP/NA-ergic Interactional Effect Appears to Be Dependent on an Intact Hormonal Epinephrine System for Its Expression 174

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De Wied and Colleagues IV: Research into Mechanisms of Action by Which Vasopressin and Oxytocin Inﬂuence Memory Processing Barbara B. McEwen

I. Chapter Overview 177 II. VP and OT: Precursors of Metabolic Fragments That Exert Memory-Modulating Effects in the Brain? 178 A. The Neuropeptide Concept 178 B. Early Behavioral Evidence of a Role for VP and OT Metabolic Fragments in Memory Processing 178 C. Biochemical Support for the Formation of Biologically Active VP and OT Neuropeptide Fragments in the Rat Brain 180 1. In Vitro Research 180 a. Selected Studies 180 i. Burbach and Lebouille (1983) 180 ii. Burbach et al. (1983b) 182 2. In Vivo Research 185 a. Selected Studies 185 i. Burbach et al. (1984) 185 ii. Stark et al. (1989) 187 D. Demonstration of the Behavioral Activities of VP and OT C-Terminal Peptides 188 1. Selected Studies 188 a. Burbach et al. (1983a) 188 b. Burbach et al. (1983b) 189 c. Kovacs et al. (1986) 189 d. Gaffori and De Wied (1986) 190 e. De Wied et al. (1987) 191 E. Localizing Brain Receptor Sites for the Behaviorally Active VP and OT C-Terminal Fragments 192 III. Characterizing the Brain Receptors That Mediate the Effects of VP and OT on Memory Processing 193 A. Selected Study: De Wied et al. (1991) 193 IV. VP, OT, and Hippocampal Theta Rhythm during Paradoxical Sleep 195 A. Hippocampal Theta Activity: Characteristics, Genesis, and Behavioral Correlates 195 B. VP, OT, Hippocampal Theta Activity, and Memory Consolidation during Paradoxical Sleep 196 1. Hippocampal Theta Activity during Paradoxical Sleep: A Neural Correlate of Memory Consolidation? 196

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2. Influence of VP and OT on Hippocampal Theta Activity during Paradoxical Sleep: Relevance for Memory Processing 197 a. Selected Studies 197 i. Urban and De Wied (1975) 197 ii. Urban and De Wied (1978) 200 iii. Bohus et al. (1978b) 202 iv. Urban (1981) 203 V. Effects of VP and OT on Neuronal Activities in the Septal–Hippocampal System, and Memory Processing 204 A. Introductory Comments: Electrophysiological Study of the Neurotransmitter and Neuromodulatory Activities of VP and OT 204 1. Selected Studies 205 a. Joels and Urban (1982) 205 b. Joels and Urban (1984a) 208 c. Joels and Urban (1985) 210 d. Urban and De Wied (1986) 212 e. Van den Hooff et al. (1989) 214 f. Urban and Killian (1990) 216 g. Chepkova et al. (1995) 218 VI. Theoretical Propositions of the ‘‘VP/OT Central Memory Theory’’: Concluded 220 A. Proposition 9: VP(1–9) and OT(1–9) Are Precursors of Metabolic Fragments That Modulate Memory Processing in the Brain 221 B. Proposition 10: The Influence of Vasopressin on Hippocampal Theta Activity during Paradoxical Sleep Is Related to Its Role in Memory Consolidation 223 C. Proposition 11: Vasopressin Exerts Both a Neurotransmitter and a Neuromodulator Action on Neurons in the Septal–Hippocampal System; Its Neuromodulator Action at Glutamatergic Synaptic Sites Is Especially Important for Memory Consolidation 223

PART

III

Research Studies of Koob and Colleagues: The ‘‘Vasopressin Dual Action Theory’’ Barbara B. McEwen

I. Overview 227 II. Memory Tasks Used in VP/Memory Research 229 A. Pole-Jump Footshock Avoidance Task 229

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IV.

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VI. VII.

VIII.

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B. Single-Trial Inhibitory (Passive) Avoidance Task 230 C. Single-Trial Water (Food)-Finding Task 230 D. Radial Maze Task 231 Studies with Pole-Jump Active Avoidance, and Single-Trial Passive Avoidance Paradigms 231 A. Selected Studies 231 1. Le Moal et al. (1981) 231 2. Koob et al. (1981) 232 3. Lebrun et al. (1984) 234 4. Koob et al. (1985a) 235 5. Lebrun et al. (1985) 237 6. Koob et al. (1986) 238 Aversive Effects of Behaviorally Active Doses of Peripherally Administered VP 240 A. Selected Studies 240 1. Dantzer et al. (1982) 240 2. Bluthe et al. (1985a) 242 3. Bluthe et al. (1985b) 244 Research Studies with the Single-Trial Water (Food)-Finding Task 245 A. Selected Studies 245 1. Ettenberg et al. (1983a) 245 2. Ettenberg et al. (1983b) 248 3. Ettenberg (1984) 249 Research Study with the Radial Maze Task 250 A. Selected Study: Packard and Ettenberg (1985) 250 Physiological Research 253 A. Evidence Relevant to the Issue of Whether VP Crosses the Blood–Brain Barrier 253 1. Selected Study: Deyo et al. (1986) 253 B. Electroencephalographic Activational Effect Induced by Behaviorally Active Doses of Peripherally or Centrally Administered AVP 254 1. Selected Study: Ehlers et al. (1985) 254 The ‘‘VP Dual Action Theory’’ 256 A. Opposing Views Concerning the Means by Which Exogenous Vasopressin Influences Retention Behavior 256 B. Propositions of the ‘‘VP Dual Action Theory’’ 257 1. Proposition 1: The Posttraining Retention Effect Induced by Treatments That Increase Peripherally Circulating AVP Is Unlikely to Be Due to a ‘‘Nonmemorial’’ Performance Effect 257

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2. Proposition 2: Increased Blood Pressure Is an Intervening Causal Factor in the Learning/Memory Effects Induced by Treatments That Lead to High Levels of Peripherally Circulating VP 258 3. Proposition 3: An Experimental Treatment, Which Increases Plasma VP to a Sufficient Degree to Influence Learning and Memory, Results in Aversive Stimulus Properties Causally Linked to the VP-Induced Pressor Effect 258 4. Proposition 4: The Aversive/Pressor Accompaniments of High Levels of Peripherally Circulating VP Produce Arousal Effects That, in Turn, Mediate the Effects of the Peptide on Learning/ Memory in Avoidance and Appetitive Learning Tasks 259 5. Proposition 5: The Learning/Memory Effects Observed after Experimental Treatments That Increase Brain Levels of VP Are Due to a Direct Action on the Neurophysiological Substrates of the Arousal System 259 6. Proposition 6: The Learning/Memory Effects That Occur after Experimental Treatments That Increase Peripheral Levels of VP Do Not Result from a Direct Action on Central VP Receptors 260 IX. The Theoretical Controversy between the De Wied et al. and Koob et al. Research Groups 261 A. Two lines of Evidence Presented by De Wied and Associates 261 B. Two Lines of Evidence Offered by Koob and Colleagues 262 X. Commentary Relevant to the Koob et al. Position 263

Contributions of Sahgal and Colleagues: The ‘‘Vasopression Central Arousal Theory’’ Barbara B. McEwen

I. Chapter Overview 265 II. Behavioral Arousal and Central Arousal System Constructs, and Their Relevance for Cognitive Behavior 266 A. Introductory Remarks 266 B. Views on Behavioral Arousal and the Arousal System: The Hebbian Influence 266 C. The Yerkes–Dodson Postulated Arousal/Performance Curve Revisited: Qualifying Comments by Hebb and Eysenck 267 D. Broadbent’s Two-Component Model of Behavioral Arousal and Its Relevance for the ‘‘VP Central Arousal Theory’’ 269 III. Relationship Between the ‘‘VP Central Arousal Theory’’ and the ‘‘VP Dual Action Theory’’ 271 IV. Sahgal’s Critique of the Research Practices of De Wied and Colleagues 272

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V. Research Practices and Objectives of Sahgal and Colleagues 273 VI. Task Paradigms and Research Findings 274 A. Passive Avoidance Task 274 1. Task Description 274 2. Studies Using the Passive Avoidance Task 274 a. Sahgal et al. (1982) 274 b. Sahgal and Wright (1983) 275 B. Autoshaped Lever Touch Task 276 1. Task Description 276 2. Studies Using the Autoshaped Lever Touch Task 277 a. Sahgal (1983) 277 b. Andrews et al. (1983) 278 C. Combined Active/Passive Avoidance Task 280 1. Task Description 280 2. Studies Using the Combined Active/Passive Avoidance Task 281 a. Sahgal and Wright (1984) 281 b. Sahgal (1986) 283 D. Delayed Matching and Nonmatching to Position Tasks 285 1. Task Description 285 2. Studies Using Delayed Matching and/or Nonmatching to Position Tasks 286 a. Sahgal (1987a) 286 b. Sahgal et al. (1990) 288 VII. Chapter Summary and Commentary on the ‘‘VP Central Arousal Theory’’ 290 A. Propositions Derived from the ‘‘VP Central Arousal Theory’’ and Relevant Evidence 290 1. Proposition 1: Increments in Peripheral or Central Levels of Vasopressin Raise the Baseline Behavioral Arousal Level, and by Implication Activity in Its Underlying Neurophysiological Substrate 290 2. Proposition 2: The Behavioral Effects of VP on Learning and Memory Tasks Are Attributable to Increasing Baseline Arousal Level (Reflected in the Postulated U-Shaped Arousal–Performance Efficiency Curve) and Not to a Direct Facilitation of the Neural Processes Mediating Information Acquisition, Storage, and Retrieval 291

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IV

Role of Attentional Processing in Mediating the Inﬂuence of Vasopressin on Memory Processing Barbara B. McEwen

I. Introductory Remarks 295 II. Beckwith and Colleagues 296 A. Research with Human Subjects 296 1. Overview 296 2. The Studies 297 a. Beckwith et al. (1982) 297 b. Beckwith et al. (1983) 298 c. Beckwith et al. (1984) 301 d. Till and Beckwith (1985) 302 e. Beckwith et al. (1987a) 306 B. Research with Animal Subjects 307 1. Overview 307 2. Description of the White/Black Reversal Discrimination Task 308 3. Research Studies 309 a. Couk and Beckwith (1982) 309 b. Beckwith and Tinius (1985) 310 c. Beckwith et al. (1987b) 311 d. Tinius et al. (1989) 314 III. Vasopressin and Attentional Processing: Bunsey, Strupp, and Colleagues 315 A. Introductory Remarks 315 B. Research Demonstrating a Role for Vasopressin Fragment AVP(4–9) in Selective Attention in Rats 316 1. Selected Study: Bunsey et al. (1990) 316 IV. Other Lines of Evidence Supporting a Role for Vasopressin in Attentional Processing 326 A. Animal Research: Divided Attention in Laboratory Rats 327 1. Meck (1987) 327 B. Human Research: Electrophysiological Measures of Attentional Processing 327 1. Fehm-Wolfsdorf and Colleagues 327 2. Timsit-Berthier et al. (1982) 331 V. Research Summaries: Beckwith and Colleagues, and Bunsey, Strupp, and Colleagues 332 A. Beckwith and Colleagues 332

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B. Bunsey, Strupp, and Colleagues 334 VI. Chapter Commentary: Vasopressin, Attention, and Memory Processing 335

PART

V

Expansion of Vasopressin/Oxytocin Memory Research I: Peripheral Administration Barbara B. McEwen

I. Introductory Remarks 339 II. Animal Research Literature 340 A Aversive Paradigms 340 1. Conditioned Taste Aversion 340 a. Selected Study: Vasopressin 340 b. Selected Study: Oxytocin 341 2. Conditioned Response Suppression 342 a. Selected Study: Vasopressin 342 3. Shuttlebox Footshock Avoidance Conditioning 343 a. Selected Studies: Vasopressin 343 i. Hagan (1982) 343 ii. Hamburger-Bar et al. (1985) 345 iii. Hamburger et al. (1985) 347 b. Selected Study: Oxytocin 347 4. Single-Trial Inhibitory (Passive) Avoidance Conditioning 349 a. Selected Studies: Vasopressin 349 i. Hostetter et al. (1980) 349 ii. Rigter (1982) 349 iii. Alescio-Lautier and Soumireu-Mourat (1990) 350 iv. Faiman et al. (1987, 1988) 351 v. Baratti et al. (1989) 351 vi. Faiman et al. (1991) 351 b. Selected Study: Oxytocin 352 5. Ethologically Relevant Avoidance Behavior in Mice 354 a. Selected Studies: Vasopressin 354 i. Leshner and Roche (1977) 354 ii. Roche and Leshner (1979) 356 B. Appetitive Paradigms 357 1. Autoshaped Lever Touch Response Conditioning 357 a. Selected Studies: Vasopressin 357 i. Messing and Sparber (1983) 357

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ii. Messing and Sparber (1985) 359 iii. Mundy and Iwamoto (1987) 361 2. Visual Discrimination Learning 364 a. Selected Studies: Vasopressin 364 i. Hostetter et al. (1977) 364 ii. Sara et al. (1982) 366 iii. Mulvey et al. (1988) 368 iv. Alescio-Lautier and Soumireu-Mourat (1990) 369 3. The Radial Maze Task 371 a. Task Description 371 b. Selected Studies: Vasopressin 372 i. Buresova and Skopkova (1980) 372 ii. Buresova and Skopkova (1982) 373 iii. Van Haaren et al. (1986) 374 iv. Strupp (1989) 374 4. Socially Transmitted Information Regarding Food Preferences 377 a. Task Description 377 b. Selected Studies: Vasopressin 378 i. Strupp et al. (1990) 378 ii. Bunsey and Strupp (1990) 379 III. Human Research Literature 380 A. Influence of VP and/or OT on Human Cognitive Processing 380 1. Effect on Memory Processing of OT Treatment Given for the Therapeutic Induction of Second-Trimester Abortion 380 a. Selected Studies 380 i. Ferrier et al. (1980) 380 ii. Kennett et al. (1982) 382 2. Intranasal Administration of VP and OT in Young Healthy Male and/or Female Human Subjects 383 a. Selected Studies 383 i. Fehm-Wolfsdorf et al. (1984) 383 ii. Bruins et al. (1992) 386

Expansion of Vasopressin/Oxytocin Memory Research II: Brain Structures and Transmitter Systems Involved in the Inﬂuence of Vasopressin and Oxytocin on Memory Processing Barbara B. McEwen

I. Chapter Overview 389 II. Central Neural Structures Involved in VP and/or OT Effects on Memory Processing 390

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A. The Hippocampus 390 1. Effects of Lysine Vasopressin and OT and Metabolites on Memory Processing in an Aversive Paradigm 390 a. Selected Study: Ibragimov (1990) 390 2. Effect of VP on Memory Processing in an Appetitive Paradigm 391 a. Selected Studies 391 i. Alescio-Lautier et al. (1989) 391 ii. Metzger et al. (1989) 393 iii. Metzger et al. (1993) 394 B. The Septal Area 397 1. Effects of AVP and AVP Receptor Antagonists on Memory Processing in an Active Avoidance Paradigm 397 a. Selected Study: Engelmann et al. (1992a) 397 2. Effects of AVP and/or AVP Receptor Antagonists on Memory Processing in a Spatial Learning Task 399 a. Selected Studies 399 i. Engelmann et al. (1992b) 399 ii. Everts and Koolhaas (1999) 401 C. The Parvocellular Hypothalamic VP-ergic System 402 1. Introductory Comments 402 2. Selected Study: Herman et al. (1991) 402 III. VP and/or OT Interaction with Central Neurotransmitter Systems and Memory Processing 405 A. The Nigrostriatal DA System 405 1. Selected Study: Hamburger-Bar et al. (1984) 405 B. The Cholinergic System 407 1. VP–ACh Interactional Effects and Memory Processing in a Passive Avoidance Paradigm 407 a. Introductory Comments 407 b. Selected Studies 408 i. Faiman et al. (1987) 408 ii. Faiman et al. (1988) 408 iii. Baratti et al. (1989) 409 iv. Faiman et al. (1991) 410 2. OT–ACh Interactional Effects and Memory Processing in a Passive Avoidance Paradigm 412 a. Introductory Comments 412 b. Selected Study: Boccia and Baratti (2000) 412 IV. Endogenous AVP and/or OT and Memory Processing 415 A. Endogenous AVP and Avoidance Learning 415 1. Selected Study: Engelmann et al. (1992a) 415

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B. Endogenous AVP and OT and Memory Processing in an Aversive Paradigm 417 1. Selected Study: Ermisch et al. (1986) 417 C. Endogenous OT and Spatial Memory 418 1. Selected Study: Ferguson et al. (2000) 418

Expansion of Vasopressin/Oxytocin Memory Research III: Research Summary and Commentary on Theoretical and Methodological Issues Barbara B. McEwen

I. Introductory Remarks 421 II. Research Summary 1: Peripherally Administered VP and/or OT and Memory Processing—Studies Reviewed in Chapter 9 422 A. Animal Research 422 1. Vasopressin and/or Oxytocin and Learning 422 2. Vasopressin and/or Oxytocin and Memory Consolidation and Retrieval 425 a. Aversive Paradigms 425 b. Appetitive Paradigms 427 3. Vasopressin and Short-Term Memory 430 B. Human Research 430 C. Peripherally Administered VP and/or OT, and Memory Processing: General Conclusions 432 III. Research Summary 2: Central Aspects of VP-ergic and OT-ergic Involvement in Memory Processing—Studies Reviewed in Chapter 10 434 A. Central Structures That Mediate VP and/or OT Influences on Memory Processing 434 1. The Hippocampal Area 434 2. The Septal Area 436 3. The Hypothalamic Parvocellular VP-ergic System 437 B. AVP and Perhaps OT Interact with Catecholamine and Acetylcholine Neurotransmitters in Memory Processing 438 1. The Nigrostriatal DA System 438 2. The Cholinergic System 439 C. Endogenous VP and OT, and Memory Processing 440 IV. Controversial Issues Concerning Interpretation of the Inﬂuence of Peripherally Administered VP on Learning/Memory Tasks 441 A. Pressor/Aversive Properties of Peripherally Administered AVP: Essential for VP-Enhanced Memory Processing? 441

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B. Peripherally Administered VP and the Postulated Arousal– Performance Efficiency Relationship: Relevant Findings 442 V. Methodological Issues: Subject, Treatment, and/or Task Variables May Affect the Outcome or Interpretation of Experimental Studies on VP/ Memory Research 444 A. Subject Variables 445 1. Pharmacogenetic Factors 445 2. Age Differences 445 3. Individual Differences in Baseline Task Proficiency and in Memory Accessibility 446 B. Treatment Variables 446 1. Dose-Dependent and Time-Dependent Effects 446 2. Acute versus Chronic Treatment Regimens and Long-Term Memory 447 C. Task Variables 448 1. Amount of Pretraining and Training Experience in the Task 448 2. Level of Task Difficulty 448 3. Motivational Factors 449 a. Need for Sufficient Motivational Incentives to Promote Efficient Learning 449 b. Arousal Effects and the Interpretation of VP/Memory Research 449 D. Measures of Retention 449 E. Methodological Issues: Conclusions 450

PART

VI

Research Contributions of Dantzer, Bluthe, and Colleagues to the Study of the Role of Vasopressin in Olfactory-Based Social Recognition Memory Barbara B. McEwen

I. Overview 453 II. The Olfactory-Based Social Recognition Memory Task 454 III. Research Findings 455 A. Effects of Peripherally and Centrally Circulating VP and OT on Conspecific SRM 455 1. Selected Studies 455 a. Dantzer et al. (1987) 455 b. Le Moal et al. (1987) 457

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B. Brain Sites Involved in VP-Mediated Olfactory-Based SRM 458 1. Selected Study: Dantzer et al. (1988) 458 C. Central Circuitry Mediating the Effect of VP on Olfactory-Based SRM Is Androgen Dependent 459 1. Selected Studies 459 a. Bluthe et al. (1990) 459 b. Bluthe and Dantzer (1990) 462 c. Bluthe and Dantzer (1992) 464 D. The Vomeronasal System and VP-ergically Modulated Olfactory-Based SRM 465 1. Introductory Remarks 465 2. Selected Study: Bluthe and Dantzer (1993) 465 E. AVP Neurotransmission Is Involved in Olfactory-Based SRM in Mice as well as in Rats 467 1. Selected Study: Bluthe et al. (1993) 467 IV The ‘‘VP Dual Action Theory’’ and Olfactory-Based SRM 468 A. Interpretation of SRM Effects Produced by Pharmacological Doses of Peripherally and Centrally Administered Vasopressin 468 B. A Role for Endogenous VP in Olfactory-Based SRM Independent of VP-Induced Arousal Effects 469 C. Two Functionally Distinct VP-ergic Systems in the Rodent Brain 470 V Commentary I: Roles of VP in Mediating Olfactory-Based SRM 470 A. The ‘‘VP Dual Action Theory’’ and Olfactory-Based SRM 470 B. Sexual Dimorphy and Olfactory-Based SRM 471 C. The Vomeronasal Organ and the Contribution of AVP to Olfactory-Based SRM 472 VI Commentary II: Contribution of the California/Bordeaux Research Teams in VP Memory Research 473

Expansion of Olfactory-Based Social Recognition Memory Research: The Roles of Vasopressin and Oxytocin in Social Recognition Memory Barbara B. McEwen

I. Introductory Remarks 475 II. Test Paradigms for Assessing SRM 475 A Social Recognition Test 475 B. The Social Discrimination Test 476 C. The Multitrial Social Recognition Test

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III. Effects of Peripherally and/or Centrally Administered VP, OT, or Their Metabolic Fragments on SRM in Laboratory Rats and Mice 477 A. Vasopressin and Related Peptides 477 1. General Comments 477 2. Peripheral Administration 477 a. Selected Studies 477 i. Popik et al. (1991) 477 ii. Sekiguchi et al. (1991a) 480 iii. Popik and Van Ree (1992) 481 B. Oxytocin and Related Peptides 483 1. Section Overview 483 2. Peripheral Administration 484 a. Selected Studies 484 i. Popik and Vetulani (1991) 484 ii. Popik et al. (1992) 485 iii. Popik et al. (1996) 487 iv. Arletti et al. (1995) 489 3. Central Administration 489 a. Selected Study: Benelli et al. (1995) 489 IV. Sex Differences and the VP/OT Inﬂuence on SRM 490 A. General Comments 490 1. Selected Studies 491 a. Axelson et al. (1999) 491 b. Engelmann et al. (1998) 493 V. Inﬂuence of Septal–Hippocampal VP and OT on SRM 495 A. General Comments 495 1. Selected Studies 496 a. Van Wimersma Greidanus and Maigret (1996) 496 b. Engelmann et al. (1994) 498 c. Landgraf et al. (1995) 504 d. Everts and Koolhaas (1997) 506 e. Everts and Koolhaas (1999) 508 VI. VP/OT and the Olfactory System 509 A. General Comments 509 1. Selected Studies 509 a. Dluzen et al. (1998a) 509 b. Dluzen et al. (1998b) 511 c. Dluzen et al. (2000) 513 VII. VP and OT in the Medial Preoptic Area and SRM 516 A. General Comments 516 1. Selected Study: Popik and Van Ree (1991) 516 VIII. VP and OT Genetic Knockout Models and SRM 518 A. General Comments 518 1. Selected Studies 519

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a. Engelmann and Landgraf (1994) 519 b. Ferguson et al. (2000) 521 IX. Chapter Summary and Commentary 524 A. General Comments 524 B. Peripherally Administered AVP and SRM 524 C. OT and SRM 526 D. Brain Structures and Pathways Mediating the VP and OT Influence on SRM 527 E. VP/OT Knockout Models and SRM 528

PART

VII

Brain–Fluid Barriers: Relevance for Theoretical Controversies Regarding Vasopressin and Oxytocin Memory Research Barbara B. McEwen

I. Chapter Overview 531 II. Brain–Fluid Barriers: Blood-Brain Barrier and Blood–Cerebrospinal Fluid Barrier 532 A. Historical Development of the Concept of Brain–Fluid Barriers 532 B. Blood–Brain Barrier 533 C. CSF Formation and Circulation, Blood–CSF Barriers, and the Circumventricular Organs 535 1. Choroid Plexus and the Formation of CSF 535 2. CSF Circulation Within and Around the CNS 539 3. Other Brain–Fluid Barrier Structures: Arachnoid Membrane, Phagocytic Cells, and CVOs 539 D. Functional Operations of Brain–Fluid Barriers and Their Regulation 542 III. Origin and Fate of VP and OT within the CSF 543 A. CSF VP and OT Are of Central Origin 543 B. Transport of VP and OT from Sites of Central Origin to Ventricular CSF 545 C. Does CSF Serve a Conduit as well as a Sink Function in VP and OT Transport? 547 IV. Means by Which Peripherally Injected VP and OT Might Induce Behavioral Effects 549 A. Means by Which Peripherally Injected Hormones Could Enter the Brain 549

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B. Means by Which VP and OT Could Induce Behavioral Effects Other than by Entering the Brain 552 V. Research Relevant to the View That Peripherally Circulating AVP Can Penetrate Brain-Fluid Barriers 554 A. Penetration of Blood–Fluid Barriers: Blood-to-Brain Transport 554 1. Type I Research Approach: Methods and Findings 554 a. Relevant Studies 554 i. Heller et al. (1968) 554 ii. Ang and Jenkins (1982) 556 iii. Mens et al. (1983) 557 iv. Deyo et al. (1986) 558 b. Evaluation and Commentary: Type I Approach 559 2. Type II Research Approach 560 a. Oldendorf Single-Pass Technique: Methodology and Results 560 i. Landgraf et al. (1979) 560 ii. Ermisch et al. (1982) 562 b. Vascular Brain Perfusion Technique: Methodology and Results 562 i. Zlokovic et al. (1990) 564 ii. Zlokovic et al. (1992) 565 c. Evaluation and Commentary: Type II Approach 566 B. Penetration of Brain–Fluid Barriers: Brain-to-Blood Transport of AVP and OT 567 1. Introductory Remarks 567 2. Relevant Studies 568 a. Brain-to-Blood Transport of AVP: Banks et al. (1987a) 568 b. Brain-to-Blood Transport of OT: Durham et al. (1991) 570 3. Efflux Systems in the Brain for VP and OT: Relevance for Transport of VP and OT Across Brain–Fluid Barriers 573 VI. AVP Inﬂuences Permeability of Brain–Fluid Barrier to Nutrient Transport 575 A. Introduction 575 B. Relevant Research 576 1. AVP and Brain–Fluid Barrier Permeability to the RNA Precursor, Orotic Acid 576 a. Selected Study: Landgraf et al. (1978) 576 2. AVP and Brain–Fluid Barrier Permeability to Large Neutral Amino Acids 577 a. Selected Studies 577 i. Brust (1986) 577 ii. Reith et al. (1987) 579 iii. Brust and Diemer (1990) 580

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iv. Ermisch et al. (1992) 582 v. Reichel et al. (1996) 583 vi. Reichel et al. (1995) 585 C. Vasopressin Influence on Nutrient Transport Across the BBB: A Role in Memory Processing? 587 D. Ermisch–Landgraf Proposal on the Functional Significance of Interaction of VP with the BBB 588 VII Chapter Summary and Commentary 590

PART

VIII

Closing Remarks: Review and Commentary on Selected Aspects of the Roles of Vasopressin and Oxytocin in Memory Processing Barbara B. McEwen

I. Chapter Overview 593 II. In Retrospect: Historical Highlights in the Study of the Roles of VP and OT in Memory Processing 594 III. VP and OT and Memory Processing: Avoidance and Appetitive Learning Paradigms 595 A. VP and Memory Processing 595 1. Central versus Peripheral Locus of Action for the MemoryProcessing Effects of Peripherally Administered VP 595 a. Contrasting Views of De Wied et al. and Koob et al. 595 b. Commentary 597 2. Position Statements on the Role of Peripherally Circulating VP in Memory Processing 598 a. De Wied and Colleagues 598 b. Koob and Colleagues 599 c. Commentary 599 3. Importance of the Arousal System in Mediating the Influence of VP on Memory Processing 600 a. Viewpoints of De Wied and Colleagues and of the Arousal-Centered Theorists 600 i. De Wied and Colleagues 600 ii. Arousal-Centered Theorists 600 b. Relevant Research 601 i. De Wied and Colleagues 601 ii. Arousal-Centered Theorists 602

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c. Commentary 603 4. Influence of VP on Attentional Processing and Its Relationship to Memory Processing 604 a. Views of Beckwith and Colleagues and of Strupp, Bunsey, and Colleagues 604 b. Supportive Evidence of a VP Role in Attentional Processing 605 c. Commentary: Relationship between the Effects of VP on Activation/Arousal, Attention, and Memory Processing 606 i. Relationship between the Role of VP in Attention Processing and its Effects on Memory Processing 606 ii. Activation/Arousal Effect of VP and its Putative Role in Attention-Dependent Memory Processing 607 5. Interactions between VP and Catecholaminergic and Cholinergic Systems: Relevance for Memory Processing 608 a. VP–Catecholamine Interactions in Memory Processing: Relevant Evidence 608 i. De Wied and Colleagues 608 ii. Hamburger-Bar and Associates 609 b. VP–Cholinergic Interactional Effects in Memory Processing: Studies of Faiman and Associates 610 c. Commentary: Mechanisms by Which These VP–Transmitter Interactions Might Influence Memory Processing 611 6. Influence of VP on Hippocampal Theta Rhythm During REM Sleep and Memory Processing 612 7. Neuromodulatory Action of VP in the Septum and Hippocampus: Relevance for Memory Processing 613 a. Neurotransmitter and Neuromodulator Actions of VP in the Septum and Hippocampus: In Vivo Studies with Exogenous AVP and Related Peptides 613 b. Neuromodulatory Action of VP in the LS after Stimulation of VP-ergic cells in the Diagonal Band of Broca and the Bed Nucleus of the Stria Terminalis: An In Vivo Study 615 c. Significance of the Neuromodulatory Action of VP for Memory Processing 615 d. Commentary: Neuromodulatory Action of VP and Long-Term Memory Formation in the LS and VH 616 B. OT and Memory Processing 617 1. OT as an Amnestic Neuropeptide in Stress-Associated Learning Paradigms 618

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2. Effects on Memory Processing of Treatments That Modulate Peripheral and Central Levels of OT 618 a. Evidence Obtained with Treatments That Increase Peripheral and/or Central Levels of OT 618 i. Peripherally Administered OT and OT Fragments 618 ii. Centrally Administered OT and OT Fragments 619 iii. Interpreting Some of the Inconsistent Results 620 b. Evidence Obtained with Treatments That Reduce Endogenous OT or Interfere with Its Receptor Transmission 622 i. Behavioral Studies 622 ii. Hippocampal Theta Activity during Paradoxical Sleep, and Memory Processing 622 3. Means by Which Peripherally and/or Centrally Circulating OT Might Influence Memory Processing 623 a. Roles of OT in Cerebral Circulation, Glucose Metabolism, and Release of Stress Hormones: Relevance for Memory Processing 623 b. Role of the Arousal System in Mediating OT Effects on Memory Processing 623 c. OT–Catecholaminergic Interactions in Memory Processing 624 d. OT–Cholinergic Interactions in Memory Processing 624 4. Commentary 625 C. The Neuropeptide Concept 626 1. Relation of the Neuropeptide Concept to the ‘‘VP/OT Central Memory Theory’’ 626 2. Relevant Observations and Experimental Findings 627 a. Biochemical/Anatomical Evidence 627 i. Formation of Bioactive C-Terminal VP and OT Metabolites 627 ii. Specific Binding Sites for C-Terminal VP and OT Fragments? 627 b. Behavioral and Electrophysiological Experimental Evidence 628 i. Behavioral Studies 628 ii. Electrophysiological Studies 630 3. Commentary: Research Update 630 a. Research Support for the Neuropeptide Concept: VP(4–8) as a New Memory-Enhancing Molecule 630 b. VP(4–8) Receptor Localization and Characterization of Its Ligand–Receptor Interactions in the Rat Brain 631

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c. The VP(4–8) Receptor Intracellular Signaling System That Mediates the Effects of the Peptide on Memory Processing 632 IV. VP, OT, and Rodent Olfactory-Based Social Recognition Memory 634 A. VP and SRM 634 1. Views and Research of Dantzer, Bluthe, and Colleagues 634 a. Role for the Androgen-Independent VP-ergic System in SRM 634 b. Role for the Androgen-Dependent VP-ergic System in SRM 634 i. Defining Characteristics and Linkage to the Accessory Olfactory System 634 ii. Evidence in Support of a Role for Androgen-Dependent VP in SRM 635 2. Contributions to This Line of Inquiry from Other Research Groups 635 a. Introductory Remarks 635 b. Androgen-Dependent VP and SRM 636 c. Androgen-Independent VP and SRM 636 i. Olfactory System 636 ii. Hippocampus 637 d. Postulated Short-Term Memory and Long-Term-Memory Components in SRM, and the Relevance of VP for These Components 637 3. Commentary 639 B. OT and SRM 640 1. Introductory Remarks 640 2. Relevant Evidence 640 a. Treatments That Increase Peripheral or Central Levels of OT: Dose-Related Attenuation and Facilitation of SRM 640 b. Treatments That Reduce Peripheral or Central Levels of OT 641 c. OT-ergic Representation in the Main Olfactory Pathway: Relevance for Conspecific Recognition 643 3. Commentary Concerning the Role of OT in Memory Processing in Avoidance Learning Paradigms, and in Rodent Olfactory-Based Conspecific Recognition 645 a. The Case for Two Functionally Distinct OT-ergic Systems Involved in Memory Processing 645 b. Adaptive Value of an OT-ergic Amnestic Action in Stressful Contexts and an OT-ergic Memory-Enhancing Action in Prosocial Encounters 646

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V. Closing Remarks: Future Considerations 647 A. Questions and Issues of Long-Standing Interest 647 1. How Do Peripherally Administered VP and OT Exert Centrally Mediated Behavioral Effects? 647 2. Arousal and Attentional Mechanisms as Mediators of the Effects of VP and OT on Memory Processing 648 B. Methodological Issues: Protocols and Paradigms, and the Use of VP and OT Knockout Models in VP/OT Memory Research 648 1. Protocols 648 2. Paradigms 649 3. Use of VP and OT Gene Knockout Models in VP/OT Memory Research 650 C. The Role of OT in Memory Processing 650 1. Appetitive Learning Tasks 650 2. OT–Classic Transmitter Interactions in Aversive and Appetitive Learning Encounters 651 3. Neuromodulatory Actions of OT in the Septum and Hippocampus? 651 D. VP, OT, and Olfactory-Based SRM 652 1. VP and Behaviorally Active VP Metabolites: Differential Effects on Short-Term and Long-Term Components of SRM? 652 2. Dose-Dependent and Site-Dependent Effects of OT on Olfactory-Based SRM 652 E. Molecular Aspects of the Roles of VP and OT in Memory Processing 652 F. A Step Toward Integration in VP and OT Memory Research 653 1. VP and/or OT Roles in Memory Processing: From Stress Stimulation to Memory Modulation 653

References 655 Index 709 Contents of Previous Volumes

727

Preface

Vasopressin (VP) and oxytocin (OT) are peptides that are synthesized within cells of the hypothalamus, in certain limbic system structures of the brain and also in some peripheral tissues (e.g., adrenal gland, and reproductive organs). When circulating in the blood they function as hormones, and when secreted from nerve terminals in the brain they function as neurotransmitters and neuromodulators. These and many other peptides have functional roles in peripheral tissues and because they are also present as neurotransmitters in the brain, they are commonly referred to as ‘‘neuropeptides’’. VP and OT are linked by virtue of their evolutionary history, structural similarity and the frequency with which their functions appear to be similar, opposing, or integrative in nature. Both as peripheral hormones and central neurotransmitters, VP and OT participate in a diverse array of actions involved in self-preservation (e.g., maintenance of salt/water balance, blood pressure control, antifebrile activity), species-preservation (e.g., reproductive physiology and behavior) and adaptive stress responding (including learning and memory processes). The pioneering studies of David De Wied in the mid 1960s on the putative roles of VP and OT in memory processing attracted worldwide and continuing research interest. This text focuses on the contributions of many of these investigators, and the issues, theoretical controversies, and problems that have arisen in the development of this research area. Part I (Chapter 1) provides an introduction to, and background on, information on these two neuropeptides, important for the subsequent discussion of their roles in memory processing. More speciﬁcally it: (1) describes their molecular structures, biosynthesis and metabolism; (2) speciﬁes what is known or theorized about their evolutionary histories within the vertebrate subphylum; (3) identiﬁes localizes the brain structures which

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synthesize these peptides and the pathways that release them into the: (a) portal circulation as pituitary releaser factors, (b) systemic circulation as hormones and (c) central brain sites as neurotransmitters and neuromodulators; (4) describes what is known about the nature and localization of VP and OT receptors, both in the nervous system and in peripheral tissues; (5) offers several explanations of how VP and OT receptors in nonneural structures may mediate a VP and/or OT inﬂuence on memory processing. Parts II through VIII provide a detailed description of the last thirty-ﬁve plus years of research on the inﬂuence VP and OT on memory processing, and the theoretical views proposed to specify the mechanisms by which this inﬂuence is achieved. With the exception of a few studies using human subjects, the research presented in this text has been carried out with laboratory rats and mice. Part II (Chapters 2 through 5) describes the research and theoretical views of De Wied and colleagues, whose work in the mid-1960s ﬁrst drew scientiﬁc attention to a possible contribution of these neuropeptides to memory consolidation and retrieval. The studies selected for review have been organized according to the major issues and questions that guided their investigative efforts. Their theoretical viewpoint, herein referred to as the ‘‘VP/OT Central Memory Theory,’’ is presented as a series of 11 major propositions, described in the summary sections of Chapters 2 through 5. This view proposes that VP and OT participate in opponent fashion in memory consolidation and its retrieval, with VP serving to enhance, and OT to inhibit these processes. Another major tenet of their theory is that the central but not the peripheral receptors of these neuropeptides mediate their memory processing functions. Thus, the behavioral effect induced by peripherally administered VP- or OT-related neuropeptides was attributed to their crossing the blood-brain barrier and inﬂuencing memory processing via actions at central receptor sites. However, the presence of this barrier, which prevents the passage of many nonlipid soluble substances from entering the brain from the blood, has posed a problem for this theoretical position. This problem was responsible, in part, for a major theoretical controversy initiated in the 1980s in which De Wied and colleagues defended the ‘‘VP/OT Central Memory Theory’’ against the objections posed by Koob and colleagues and by Sahgal and colleagues. Part III (Chapters 6–7) discusses the contributions of research investigators who focused on VP’s inﬂuence in MP and postulated a prominent place for the arousal system in mediating this inﬂuence. These VP-arousal centered viewpoints directly challenged the major premise of De Wied and colleagues that these neuropeptides inﬂuence memory storage and retrieval by their mnemonic actions within memory processing brain sites. Chapter 6 discusses the ‘‘VP Dual Action Theory’’ of memory processing and relevant research of Koob and colleagues. According to these researchers, VP’s peripheral hormonal and central neuronal actions both interact with

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the arousal system to inﬂuence memory processing, but do so by different mechanisms. The former increase the arousal level by means of its pressor/ aversive effects, the latter by directly interacting with the central arousal system. Thus, it is the peptide’s arousal effect (both direct and indirect) that secondarily affects learning/memory performance, rather than an effect on brain mechanisms responsible for the structural changes involved in memory processing. Chapter 7 presents Sahgal et al.’s viewpoint, herein referred to as the ‘‘VP Central Arousal Theory’’. This viewpoint was formulated independently of the ‘‘VP Dual Action Theory’’ but like the former, postulated a VP-induced arousal effect as mediating the neuropeptide’s inﬂuence in MP. Sahgal and associates focused on a neuronal VPergic interaction with the central arousal system, and initially accepted the possibility that peripherally administered AVP might enter the brain via the blood-brain barrier. It was theorized that once in the brain VP directly interacted with the central arousal system and increased the subject’s arousal level. Moreover, in accordance with the proposed arousal level/performance efﬁciency curve, the VP-increased arousal level is expected to enhance or impair learning / memory performance depending on the individual’s initial level of arousal in the test situation, and the relation of this to ‘‘optimal’’ level. Thus, Koob et al. and Sahgal et al agreed that a VP-induced arousal effect, instead of a direct VPergic mnemonic action, explained the neuropeptide’s inﬂuence on MP. Nevertheless, their initial differences led to research inquiry along distinctive lines speciﬁc to each of their theoretical viewpoints. Sahgal et al subsequently withdrew their original skepticism that a VP-induced pressor/aversive action was the means by which peripherally administered VP inﬂuenced the arousal system. As a result, the two theoretical viewpoints became virtually identical to one another. Part IV (Chapter 8) discusses the views and experimental ﬁndings of two sets of investigators that studied VP’s effect on attention. These investigators have linked their study of VP’s inﬂuence on attention to its putative role in MP in two major ways. Beckwith and colleagues, who have used human as well as laboratory animal subjects in their studies, theorize that VP’s facilitation on learning and memory paradigms is due to a primary effect on attention. Their viewpoint appears to be consistent with the suggestion that attentional mechanisms operate during both memory formation (selective attending sensory information placed into storage), and retrieval (removal of selectively attended memories from storage). In contrast to Beckwith and colleagues primary focus on VP’s role in attentional processing, Strupp, Bunsey and colleagues have studied VP s inﬂuence in a number of cognitive processes (attention, learning, memory consolidation and retrieval) and link VP’s inﬂuence on these cognitive processes with its interaction with the central arousal system in a manner similar to, but not identical with the theoretical views of Sahgal and colleagues (Chapter 7).

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Preface

Part V (Chapters 9 through 11) discusses VP/OT-memory processing research by a number of other investigators. Although these studies were not speciﬁcally designed to support or refute the major theoretical viewpoints previously discussed, many of the ﬁndings are relevant to them. In addition to the valuable data their studies have provided, the contributions of these researchers include: 1) an extension of the paradigms used in VP/OT memory research from primarily active and passive avoidance tasks to a wide variety of appetitive tasks; 2) investigation of factors potentially responsible for the numerous discrepancies in laboratory ﬁndings concerning VP’s role in appetitive learning tasks; 3) the recognition of a variety of VP interactions with subject, treatment and task variables that further clarify VP’s role in memory processes. Part VI (Chapters 12 and 13) examines the evidence for VP’s and OT’s participation in the mediation of olfactory-based social recognition memory. Chapter 12 describes the pioneering contributions to this ﬁeld of inquiry by Dantzer and Bluthe, two members of the Koob et al. group whose earlier work in connection with the ‘‘VP Dual Action Theory’’ was described in Chapter 6. The olfactory-based social recognition memory (SRM) paradigm was adopted to limit problems of interpretation of results due to the intrusion of performance factors such as arousal and emotionality with the variety of appetitive and avoidance paradigms previously used (Dantzer, 1998; p 409). This olfactory-based SRM task was used to investigate conspeciﬁc recognition resulting from a species-typical type of memory processing. On the basis of their research, these investigators postulated the existence of two types of VPergic systems: one system inﬂuences social recognition memory via its interaction with the central arousal system, as occurs in the tasks described in Chapter 6; the other operates independently of the arousal system. Chapter 13 describes more recent studies by investigators who have expanded the social recognition memory research area. The relevance of the ﬁndings to the theoretical views and research of Dantzer and Bluthe, as well as to memory as assessed in other learning paradigms, is discussed. The researchers whose contributions are discussed in this chapter have included OT in their studies, promising greater clariﬁcation of the speciﬁc roles played by both neuropeptides in social recognition memory and, by extension, in memory processing tested in other learning paradigms. Part VII (Chapter 14) explores the signiﬁcance of the blood-brain barrier (BBB) for VP/memory research. The blood/brain barrier and the blood/cerebrospinal ﬂuid barrier separate the central and peripheral ﬂuid compartments in which VP and OT circulate. Therefore, it is important to discuss what is known and theorized about the way the peptides interact with these barriers, especially for designing and evaluating experimental protocols that administer the neuropeptides peripherally. Moreover, the question of whether VP and OT, and/or their metabolites, can penetrate

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the BBB is an important issue in the theoretical debate between the ‘‘VP/OT Central Memory Theory’’ and the ‘‘VP Dual Action Theory’’. Part VIII (Chapter 15) reviews and comments on theoretical positions and research evidence concerning the mechanism by which VP and OT might inﬂuence memory processing tested in avoidance and appetitive learning paradigms and in the olfactory-based social recognition memory task. The chapter opens with a summary of the highlights of progress in VP/OT memory processing research from its inception in the mid-1960s to the present. Subsequent discussion details some issues arising from the controversial theories and some of the research bearing directly or indirectly on them. The chapter closes with a number of general suggestions concerning the course of future study in this area of research. The book should be of particular interest to students or research investigators concerned with the neuropeptidergic-underpinnings of memory processing. The detailed description of the research studies provide sufﬁcient information to assist in evaluative assessment of the major theoretical views presented in this book. The studies, theories, controversies, and issues discussed in this text are useful for stimulating and guiding future studies in this research ﬁeld, and are relevant to the clinical treatment of memory disorders.

Barbara B. McEwen June 9, 2004

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Barbara B. McEwen

Part I

General Introduction to Vasopressin and Oxytocin: Structure/Metabolism, Evolutionary Aspects, Neural Pathway/Receptor Distribution, and Functional Aspects Relevant to Memory Processing

I. Metabolic Aspects

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Vasopressin (VP) and oxytocin (OT) are nonapeptides (single linear chains of nine amino acids), derived from large precursor proteins (propeptides), which in turn are derived from even larger molecules known as ‘‘prepropeptides.’’ The prepropeptide differs from the propeptide by an initial sequence of amino acids, called the signal sequence, which occurs in the N-terminal portion of the prepropeptide molecule. In peptide synthesis, the amino group of one amino acid is bonded with the carboxyl group of the adjoining amino acid [by convention the peptide is written so that the amino group (N terminus) defines the beginning of the peptide, and the carboxyl group (C terminus) defines its end]. The side chains of the amino acids identify the various peptides and are responsible for their physical and functional attributes. The amino acid components of a peptide or protein are called ‘‘residues.’’ The biological synthesis and Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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subsequent processing of the VP and OT prepropeptides are briefly outlined below.

A. Biosynthesis of VP and OT VP and OT are synthesized within cells located in the brain and in certain peripheral organs of the body. In the brain, VP and OT are synthesized in cell groups within the hypothalamus; several of these cell groups release hormones into the systemic circulation or into the portal circulation of the anterior pituitary gland and others release neurotransmitters at synaptic targets within the brain. VP is also synthesized in certain extrahypothalamic brain sites, such as limbic system structures in the forebrain. In peripheral tissues, there is evidence that VP is synthesized in the anterior pituitary, adrenal, and thymus glands, and in male and female reproductive structures (ovaries, uterus, and testes) (Clements and Funder, 1986), and that OT is produced at a low level in male and female reproductive structures in a number of species (e.g., Ivell et al., 1998). Whatever the location of their synthesis, VP and OT are derived from separate precursor molecules (Fig. 1) (Clements and Funder, 1986; Norman and Litwack, 1987). The VP precursor embodies three functional peptides: VP, a VP-associated neurophysin (VP-Np), and a glycopeptide; the OT precursor contains two functional peptides: OT and an OT-associated neurophysin (OT-Np). The Nps have been proposed as carrier proteins that

FIGURE 1 Preprovasopressin and preprooxytocin. Proteolytic maturation proceeds from top to bottom for each precursor. The organization of the gene translation products is similar in either case except that a glycoprotein is included in the proprotein of VP in the C-terminal region. Dark stippled bars of the NPs represent conserved amino acid regions; lightly stippled bars represent variable C and N termini. Source: Norman and Litwack, 1987 (Figs. 4–7, p. 141). Copyright ß 1987 by Academic Press.

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assist in neurosecretory granule packaging and axonal transport of VP and OT, protecting each from proteolytic degradation (Breslow, 1979, as cited in Clements and Funder, 1986). The major steps involved in the formation, storage, and release of these functional peptides are depicted in Figs. 2 and 3. This description of VP and OT biosynthesis, processing, and release is based on discussions by Clements and Funder (1986), Roberts (1984), Majzoub (1985), Norman and Litwack (1987), and Richter (1985). The VP and OT genes encoding each of the prepropeptides exhibit the same three-exon organization. Exons are coding regions of the gene separated by intervening noncoding segments called introns. The first exon encodes the tripeptide signal, the VP (or OT) nonapeptide, and the highly variable initial (N-terminal) portion of the VP- or OT-associated neurophysin (VP-Np or OT-Np, respectively). The second exon encodes

FIGURE 2 Hypothetical model of biosynthesis, translocation, processing, and release of peptides in a peptidergic neuron. Translocation of mRNA occurs on the rough endoplasmic reticulum (R.E.R.) in the soma yielding a propeptide or precursor protein molecule (P1) in the cisternal space of the R.E.R. The packaging of the P1 into secretory granules occurs in the Golgi body. The secretory granule represents the site of post-translational processing to smaller peptide products (P1–Pn), which can occur either in the soma or in the axon during axonal transport. The peptide products (P1–Pn) are released from the neuron terminals by depolarization in the presence of extracellular Calcium. Source: Gainer et al., 1977 (Fig. 9, p. 378). Copyright ß 1977 by Rockefeller University Press. Reprinted with permission.

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FIGURE 3 Vasopressin synthesis, transport, and secretion in the magnocellular neuron. A stimulus results in propagation of an action potential to axonal terminals in the posterior pituitary, leading to secretion. Vasopressin mRNA is transcribed from the vasopressin gene in the nucleus and transported to the cytoplasm, where it is translated on rough endoplasmic reticulum into preproVPNp. PreproVPNp is transported and processed intracellularly. Source: Majzoub, 1985 (Fig. 2, p. 468). Copyright ß 1985 by Raven Press. Reproduced by courtesy of Lippincott, Williams and Wilkins, new owners of this copyrighted material.

the central part of the Np (10 to 76 amino acid residues in length). The third exon encodes the final section of NP (77 to 95 amino acid residues) and, in the case of VP, the glycoprotein extension (39 residues). Figure 4 depicts the gene structure and prepropeptide structure for the vasopressin precursor. Briefly, the VP (or OT) gene, [i.e., the stretch of deoxynucleotide (DNA) that encodes the amino acid sequence for the peptide precursor] is transcribed onto a precursor messenger ribonucleic acid (mRNA) within the cell nucleus. The mRNA is then translocated to cytoplasmic ribosomes that, induced by the signal sequence in the prepropeptide, are attached to the rough endoplasmic reticulum (RER). Here the genetic code, embodied in the mRNA template, is translated into the VP (or OT) prepropeptide molecule by the ribosomes, transfer RNAs, and a host of protein and nonprotein cofactors. Subsequent processing within the RER involves removal of the signal sequence, formation of disulfide bonds (–S–S–) between the cysteine residues (one in the nonapeptide and seven in the associated neurophysin), and formation of the glycopeptide. The VP (or OT) propeptide molecules then enter the Golgi body, where they are sequestered, along with a number of processing enzymes, into membrane-bound vesicles (secretory granules). Additional posttranslational processing occurs within these secretory

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FIGURE 4 Proposed structure of the VP gene, its translation product, and the matured peptides. The gene encodes arginine VP, NP, and glycoprotein and contains two introns. The gene product, provasopressin, is indicated in the middle. Negative numbers indicate amino acid sequence of the signal sequence and positive numbers indicate the prohormone sequence. The attached open circle on the matured glycoprotein product at the top right stands for carbohydrate. Source: Norman and Litwack, 1987 (Figs. 4–6, p. 141). Copyright ß 1987 by Academic Press.

granules either while they are in the soma or during axonal transport to the neuronal terminals, where they are stored. After electrical or chemical stimulation of the cell, the peptide and its neurophysin are released into the blood stream (by a neuroendocrine cell) as a hormone or into a synaptic junction (by a VP- or OT-ergic neuron) as a neurotransmitter. This release requires simultaneous depolarization at receptor sites and entry of extracellular calcium ions at the storage sites in the axonal terminals. The neurophysin remains bound to the peptide during storage, where it may prevent the small peptide from diffusing away from the storage granule before release (Norman and Litwack, 1987; Roberts, 1984). In addition, the hormone may remain attached to its neurophysin on immediate release from the axon terminal, but once the peptide–neurophysin complex is dissociated the free hormone attaches to its receptor membrane. Attachment to the carrier protein may prolong the life of the peptide (VP or OT) because the half-life of the hormone is increased dramatically from about 3 min to about 10–20 min when complexed with Np in the blood (Norman and Litwack, 1987). The molecular structures of vasopressin and oxytocin are depicted in Fig. 5. As Fig. 5 shows, each molecule consists of a covalent ring structure closed by a disulfide bridge, and a linear tripeptide tail. The NH2 group represents the C terminus of each peptide and indicates that the glycine residue has been amidated (i.e., the free COOH group has been converted to CONH2). The two peptides differ only in the amino acids at positions 3 and 8. The covalent ring of VP is pressinamide (pressinoic acid) and the tripeptide tail is Pro-Arg-Gly-NH2 (PAG). The covalent ring structure of OT is tocinamide (tocinoic acid) and the linear tripeptide tail is Pro-Leu-GlyNH2 (PLG).

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FIGURE 5 Molecular structures of arginine vasopressin and oxytocin. Source: Feldman et al., 1997 (Chapter 11, p. 463). Copyright ß 1997 by Sinauer Associates, Inc. Figure reprinted with permission. Legend adapted with permission.

B. Metabolic Degradation of VP and OT Claybaugh and Uyehara (1993) have discussed the metabolic degradation of the two peptides. First, in connection with whole body clearance, the peptides are removed from the circulation and metabolized primarily in the kidney and liver and, probably to some degree, in the small intestine. The chemical degradation of VP and OT in the kidney is thought to occur in the lumen of the proximal tubule after filtration from the plasma. The proteolytic enzymes metabolically degrade the peptides by hydrolyzing peptide bonds. Depending on the site of activity the enzymes are classified as either exopeptidases (hydrolyze peptides from either the C- or N-terminal region) or endopeptidases (cleave internal bonds of the peptide) (Brownstein, 1989). Peptidases are specific to the peptide bond they hydrolyze [e.g., trypsin hydrolyzes bonds involving only the basic amino acids (lysine and arginine)] (Brownstein, 1989). In addition to whole body clearance of these peptides, there is evidence that VP and OT released at synaptic sites in the brain are also subject to degradation by peptidase enzymes. This metabolism may be the means by which these peptides are removed from synaptic sites once neurotransmission has been completed (Claybaugh and Uyehara, 1993) and it may produce some OT and VP metabolites having significant functions, for example, a role in memory processing (see discussion in Chapter 5).

II. Evolutionary Considerations and Comparative Study

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Archer and Chauvet (1995) cite evidence for the presence of a neurohypophysial hormone-like molecule in various invertebrates, including gastropod mollusks (freshwater and seawater snails), cephalopod mollusks (octopus), annelids (earthworm), and arthropods (locust insects). Although the function of the peptide in these invertebrates is not yet known, its

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presence indicates that the gene encoding the peptide existed at least 600 million years ago, that is, before invertebrate–vertebrate divergence (Archer and Chauvet, 1995). VP- and OT-like hormones are both present in all vertebrates except cyclostomes, the most primitive vertebrate group (e.g., Pacific hagfish), which possess only a single neurohypophysial hormone, vasotocin (AVT). Thirteen OT- and VP-like peptides have been found among the vertebrates studied thus far (Table I). Archer (1980) developed the following model to accommodate these observations. In early vertebrate evolution a single ancestral gene that encoded AVT underwent a gene duplication that resulted in the formation

TABLE I

Peptide

Structures of Vertebrate Neurohypophysial Peptidesa Vertebrates among which peptide is found

Sequence Oxytocin-Like Peptides

Oxytocin

1 2 3 4 5 6 7 8 9 Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (NH2)

Mesotocin

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Ile-Gly (NH2)

Seritocin Isotocin Glumitocin Valitocin Aspargtocin Asvatocin Phasvatocin

Cys-Tyr-Ile-Gln-Ser-Cys-Pro-Ile-Gly (NH2) Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Ile-Gly (NH2) Cys-Tyr-Ile-Ser-Asn-Cys-Pro-Gln-Gly (NH2) Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Val-Gly (NH2) Cys-Tyr-Ile-Asn-Asn-Cys-Pro-Leu-Gly (NH2) Cys-Tyr-Ile-Asn-Asn-Cys-Pro-Val-Gly (NH2) Cys-Phe-Ile-Asn-Asn-Cys-Pro-Val-Gly (NH2)

Placentals Some marsupials Lungfishes Nonmammalian tetrapods Marsupials Lungfishes Bufo regularis Bony fishes Rays Spiny dogfish Spiny dogfish Spotted dogfish Spotted dogfish

Vasopressin-Like Peptides Vasopressin Lysipressin

1 2 3 4 5 6 7 8 9 Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly (NH2) Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly (NH2)

Phenypressin Vasotocin

Cys-Phe-Phe-Gln-Asn-Cys-Pro-Arg-Gly (NH2) Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly (NH2)

a

Mammals Pig Macropodids Didelphids Peramelids Macropodids Nonmammalian vertebrates

Residues different from those of oxytocin or vasopressin are indicated by boldface type. Key: Asn, asparagine; Cys, cysteine; Gln, glutamine; Gly, glycine; Ile, isoleucine; Phe; phenylalanine; Pro, proline; Leu, leucine; Ser, serine; Tyr, tyrosine; Val, valine. Adapted from Archer and Chauvet (1995).

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of two lineages in the VP/OT superfamily. The original gene continued to encode the VP-like peptide, vasotocin. The duplicated gene encoded the OT-like peptide. In the bony fish, the OT-like gene underwent two point mutations that produced isotocin from vasotocin with amino acid substitutions at position 4 (isoleucine to serine) and position 8 (arginine to isoleucine). Thus, vasotocin and isotocin are present in bony fish. In early tetrapod evolution, a point mutation in the gene encoding isotocin produced mesotocin, in which the amino acid at position 4 was changed from serine to glutamine. Vasotocin and mesotocin are the two neurohypophysial peptides found in amphibians, reptiles, and birds. In placental mammals, oxytocin was formed as a result of a point mutation in the gene encoding mesotocin (leucine replaced isoleucine at position 8 of the molecule), and vasopressin was formed after the occurrence of a point mutation in the gene encoding vasotocin (phenylalanine replaced isoleucine at position 3 of the molecule). A subsequent point mutation in the gene encoding arginine vasopressin (AVP), in which lysine replaced arginine at position 8 of the molecule, occurred only in the pig family, resulting in the formation of lysine vasopressin (LVP). Although most species have the two types of neurohypophysial hormones, secondary duplications of the VP-like and/or OT-like peptides have occurred in some groups of marsupial mammals, and of OT-like peptides in certain cartilaginous fish (e.g., sharks) (Archer and Chauvet, 1995) (see Table I). Some of these genetic changes were without functional significance, indicating neutral evolution, but others produced biological alterations, indicating selective or Darwinian evolution. The genetic changes that led to the formation of oxytocin appear to illustrate neutral evolution because mesotocin and oxytocin have roughly equal uterotonic or milk-ejecting actions in rat and rabbit, and mesotocin, which exhibits the same function as oxytocin in placental mammals, has been conserved in marsupial mammals. An example of Darwinian evolution occurred in the formation of vasopressin because, unlike vasotocin, this hormonal peptide no longer acts on uterine and mammary gland receptors. The two types of neurohypophysial hormones in the various vertebrate classes are paralleled by two types of neurophysins. The VP-like and OT-like neurophysins are distinguished by the amino acids present at positions 2, 3, 6, and 7, and these same residues are present in all vertebrate classes on the evolutionary path from bony fish to placental mammals. The evolutionary stability of both neurophysins is evident from the fact that each type possesses seven disulfide bridges within the same molecule location; this stability may have played a role in the preservation of the three-dimensional structure of the molecule. The 39-residue C-terminal glycopeptide, which is observed in the VP, but not the OT, precursor molecule in mammals, appears to have been lost from the OT-like precursor in early tetrapod evolution. It is

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present in the proisotocin precursor in bony fish but is absent from the promesotocin precursor in amphibians, reptiles, and birds as well as in mammals. In general, the variability that occurs in the exon segments of the genes encoding the VP/OT superfamily appears to be primarily in the first and third exon segments because the second exon segment, which encodes the large middle section of the neurophysin, appears to have been highly conserved. In summary, after the duplication in the ancestral gene, subsequent evolutionary changes in the VP and OT lineages were fairly modest, as evidenced by the following observations: (1) conformational structures between the VP- and OT-like nonapeptides and between the two neurophysins are similar; (2) following the duplication of the ancestral gene only two peptides, a VP-like peptide and an OT-like peptide, are usually present in each vertebrate species; and (3) with the exception of cartilaginous fishes, species in the same vertebrate class almost always possess the same VP- and OT-like nonapeptides and neurophysins (Archer and Chauvet, 1995).

III. VP and OT Cell Systems, Pathways, and Receptors: Characteristics and Distribution

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A. VP-ergic and OT-ergic Cell Groups and Their Pathways in the CNS An overview of the distribution of known and putative VP-ergic and OT-ergic cell groups and their fiber pathways is provided in Fig. 6. Subsequent discussion in this section briefly summarizes the methods used to localize these systems in the brain, as well as information concerning the cell groups and their efferent and afferent inputs that characterize the circuitry of the hypothalamic and extrahypothalamic systems of these peptides. 1. Methods Used to Localize VP and OT Systems in the CNS Present knowledge concerning the anatomical localization of VP and OT cell bodies, axon pathways, and terminals has relied mainly on immunocytochemical [ICC; also known as immunohistochemical (IHC)] staining techniques in preparation for light microscopic, electron microscopic, or autoradiographic viewing conditions. The description of the techniques given below rests in large part on discussions in Feldman et al. (1997, pp. 28–31, 35–36, 43–45, and 49–50). Briefly, the ICC staining technique consists of three essential procedural steps: development of antibodies against the antigen (e.g., peptide), labeling of the antibody molecule to enable visibility under microscopic viewing, and antibody attachment to its antigen. Antibody production can be

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FIGURE 6 Major vasopressinergic and oxytocinergic pathways in the rat brain and their probable sites of origin. (a) Vasopressin. (b) Oxytocin. Source: Feldman et al., 1997 (Fig. 11.11, p. 466). Copyright ß 1997 by Sinauer Associates, Inc. Figure reprinted with permission. Legend adapted with permission.

accomplished by injecting the antigen into a host species (e.g., rabbit) and collecting the antiserum after development of the immune reaction. Alternatively, a cell culture may be used that is composed of hybrid cells derived by fusing antigen-sensitized tissue cells (e.g., spleen cells from a mouse) and immortal, rapidly replicating tumor cells. The combination of antibody production and cell replication provides a continuous supply of antibodies specific to the designated antigen. Depending on the method of viewing, the label attached to the antibody molecule may be a highly fluorescent compound such as fluorescein (light microscopy), a heavy metal such as colloidal gold (electron microscopy), or a radioactive isotope (autoradiography). The label may be applied to the antibody before or after its linkage to the antigen. Antibody–antigen linkage occurs when the sections containing the peptide (antigen) are incubated with the antiserum containing the antibodies. When viewed under the microscope, the labeled antibody–antigen complex is visible and localized.

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Autoradiography, a technique used to map radioactively labeled cellular components, has been used effectively with ICC staining (described in Carlson, 1991; Feldman et al., 1997). The radioactively labeled substance (neurotransmitter, hormone, enzyme that synthesizes the transmitter, etc.) is incorporated into the brain tissue either by in vitro incubation or in vivo injection into the experimental animal. The brain tissue selected for study is sectioned, placed on microscopic slides, and prepared for autoradiogram development in a dark room. Here, the slides are either dipped into a photographic emulsion or placed against photographic film, and left for several weeks. During this time the autoradiogram is formed (i.e., silver halide grains in the emulsion or in the film are darkened by the charged particles emitted from the radioactive compound during its constant disintegration). When seen under standard microscopic viewing conditions the radioactively labeled peptide is localized as darkened areas in the autoradiogram, and its concentration is indicated by the degree of darkening. Under dark-field illumination, the areas containing the peptide show up as light areas. More recently, readability of the autoradiograms has been enhanced by digitally applied ‘‘false colors’’—warmer colors (reds, yellows) depicting brain areas that contain greater concentrations of the labeled substance. De Kloet et al. (1985a) used in vitro incubation with tritium-labeled ligands and autoradiography to map binding sites for AVP and OT in the rat brain. Brain sections were incubated with either tritium-labeled arginine-vasopressin ([3H ]AVP) or tritium-labeled oxytocin ([3H]OT) before preparation of the autoradiograms. Receptor concentration at the affected brain sites was quantified by computerized densitometry of the film images. Combining ICC staining with lesioning techniques has especially facilitated mapping of VP- and OT-ergic pathways. For example, De Vries and Buijs (1983) used ICC staining to map the disappearance of VP-ergic and OT-ergic brain pathways 2.5 to 3 weeks after either bilateral lesioning of the hypothalamic paraventricular nucleus or the bed nucleus of the stria terminalis, or horizontal knife cuts that separated the septum from underlying structures. 2. Distinction between Magnocellular and Parvocellular VP-ergic and OT-ergic Cellular Systems The large-sized [magnocellular (mgc)] cells that synthesize either VP or OT and their respective neurophysins are neuroendocrine cells, which project to the capillaries in the posterior pituitary lobe, where they secrete their contents as hormones into the systemic circulation. The small-sized [parvocellular (pvc)] cells that synthesize these neuropeptides serve one of a number of specialized roles in the brain: some of these cells secrete releasing hormones into the portal circulation of the anterior pituitary gland; others activate, inhibit, or modulate activity in other neurons in the brain; and still

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others synapse on blood vessels in the brain, where they may influence vascular dynamics. The mgc cells are found in certain nuclei of the hypothalamus: supraoptic nucleus (SON), paraventricular nucleus (PVN), and so-called hypothalamic ‘‘accessory nuclei’’ (more or less compact structures or loose collections of individual neurons in various parts of the hypothalamus), and in the bed nucleus of the stria terminalis (BNST) (Sofroniew, 1985a) (see Fig. 7). These cells project primarily to the posterior pituitary gland (neural lobe) and secrete their contents as hormones into the systemic circulation; about 50% of these projections originate in the PVN and SON and 50% in the less studied accessory nuclei (Hatton, 1990). Earlier study suggested that mgc output was confined solely to the neural lobe (Sofroniew, 1985a). However, there have been occasional observations that mgc VP and OT projections may reach some of the brain regions innervated by pvc

FIGURE 7 Sagittal view of the rat brain, depicting the approximate topography of the more prominent groups of vasopressin and oxytocin neurons. The numbers 1–5 refer to large groups of so-called accessory magnocellular neurons, located outside of the supraoptic (son) and paraventricular (pvn) nuclei, for which there is no generally accepted nomenclature. The number 2 corresponds to the anterior commissural nucleus (acn). In addition, parvocellular vasopressin neurons are present in the regions depicted by dashed lines: bn, bed nucleus of the stria terminalis; lc, locus coeruleus; ls, lateral septum; ma, medial amygdala; ms, medial septum; ph, posterior hypothalamus; scn, suprachiasmatic nucleus; td, nucleus of the diagonal tract of Broca. Source: Sofroniew, 1985a (Fig. 1, p. 96). Copyright ß 1985 by Elsevier Science Publishers B, V.

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neurons, and it has been suggested that axon collaterals may be responsible for at least some of this output (see Hatton, 1990). These mgc neuroendocrine cells function as neurons—they respond to stimuli by generating electrical impulses that traverse the axon, and they exhibit calcium-dependent release of the hormone by exocytosis at the axonal terminals (Swanson and Sawchenko, 1983). The pvc VP and OT cells form distinct groupings within the PVN and SON, and VP-ergic pvc neurons are also found in several other hypothalamic sites, as well as in brain structures lying outside the hypothalamus (extrahypothalamic brain sites). IHC studies have shown that with rare exceptions separate neuronal populations within the brain synthesize and release either OT or VP and their respective neurophysins (Vandesande and Dierickx, 1975; Van Leeuwen and Swaab, 1977). 3. Hypothalamic VP-ergic and/or OT-ergic Cells The most studied hypothalamic structures that synthesize VP and/or OT are the supraoptic nucleus (SON), paraventricular nucleus [PVN, or paraventricular nucleus of the hypothalamus (PVH)] and suprachiasmatic nucleus (SCN). a. Hypothalamic SON There are no pvc subdivisions within the SON and this nucleus consists almost entirely of mgc neurons (Hatton, 1990). The ratio of VP-ergic to OT-ergic cells in the SON, at least in the rat, is about 1:1 (Swaab et al., 1975; Vandesande and Dierickx, 1975). Although the two cell populations are not totally segregated from one another, the VP-ergic cells are concentrated mainly in the posteroventral portion, and the OT-ergic cells in the anteroventral portion, of the nucleus (Swaab et al., 1975; Vandesande and Dierickx, 1975). Hatton (1990) cites evidence for direct or indirect afferent input to the SON from a number of brainstem (e.g., noradrenergic A1 and A2 cell groups in medulla) and forebrain [other hypothalamic sites, subfornical organ (SFO), amygdala, lateral septum (LS), diagonal band of Broca (DBB), and main and accessory olfactory bulb) structures. Some of this input is related to cardiovascular (brainstem input) and fluid (SFO) control, but the functions of much of the input have not as yet been clarified. b. Hypothalamic PVN The PVN is differentiated into eight well-defined subdivisions, three containing mgc neurons and five containing pvc neurons (Swanson and Kuypers, 1980) (see Fig. 8). VP and OT cells are concentrated in different portions of the pvc and mgc subdivisions. For example, the anterior and medial mgc subdivisions are composed almost exclusively of OT cells, whereas more or less equal numbers of OT and VP cells are concentrated in different parts of the posterior mgc subdivision of the nucleus (Sawchenko and Swanson, 1982). The mgc neurons project to

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FIGURE 8 Summary diagrams to show the relationships between various cell groups in the parvocellular (pvc) and magnocellular (mgc) (stippled) divisions of the PVH. The reconstructions were made from a brain cut in the frontal plane. Top: PVN as viewed from the side (sagittal). Bottom: PVN as viewed from above (horizontal). Because of its vertical orientation adjacent to the wall of the third ventricle, the periventricular part of the pvc division is not illustrated in the upper figure. Key: am (anterior mgc part); mm (medial mge part); pm (posterior mgc part); ap (anterior pvc part); lp (lateral pvc part); mp (medial pvc part); dp (dorsal pvc part); pv (periventricular part). Source: Swanson & Kuypers, 1980 (Fig. 3, p. 560). Copyright ß 1980 by Alan R. Liss, Inc. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

the posterior pituitary lobe via the internal zone of the median eminence (conduit to the posterior pituitary gland), and the pvc neurons project to neural or vascular sites either within the hypothalamus or outside of it (exohypothalamically). One major intrahypothalamic projection site for the pvc neurons is the external zone of the median eminence (EZME, conduit to anterior pituitary gland). There, VP and OT secreted into the portal circulation promote the release of certain anterior pituitary hormones (e.g., adrenocorticotropic hormone (ACTH), gonadotropic

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hormones [follicle-stimulating hormone (FSH) and luteinizing hormone (LH)], and prolactin) (Swanson et al., 1980; Vandesande et al., 1977). The posterior pituitary and the median eminence are examples of circumventricular organs (CVOs). These are specialized brain sites that border the brain ventricles and collectively act as an interface between the blood and the brain. They include the subcommissural organ (SCO), the subfornical organ (SFO), the vascular organ of the lamina terminalis (OVLT), the pineal body, and the area postrema (AP) (see Fig. 9). With one exception (the SCO), these brain areas lack a blood–brain barrier. As a result, receptors in these structures (e.g., the SFO and the OVLT) can directly monitor the internal milieu and, after transduction of blood-borne chemical stimuli into neural impulses, relay these impulses to brain processing sites. Similarly, effectors in these structures [e.g., mgc or pvc VP and OT neurosecretory terminals in the portal zone of the median eminence (ME) or posterior

FIGURE 9 Circumventricular organs of the human brain (AP, area postrema; ME, median eminence; NH, neural lobe of hypophysis; OVLT, organum vasculosum of lamina terminalis; PI, pineal body; SCO, subcommissural organ; SFO, subfornical organ; CP, choroid plexus). With the exception of the SCO, all circumventricular organs are located outside the blood– brain barrier and are permeable to peptides. Source: Weindl and Sofroniew, 1985 (Fig. 2, p. 141). Copyright ß by Springer-Verlag. Reprinted with permission.

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Barbara B. McEwen

pituitary gland] can release their contents directly into the portal circulation, influencing release of anterior pituitary hormones, or into the posterior pituitary, reaching peripheral targets via the systemic circulation (see discussion in Weindl and Sofroniew, 1981). Parvocellular neurons appear to project to the SFO and OVLT in addition to the ME (Sofroniew, 1985a). Parvocellular VP and OT neurons in the PVN also project to autonomic nervous system (ANS) centers in the brainstem and spinal cord; ventricular sites, where they may secrete directly into the cerebrospinal fluid (CSF) (e.g., Buijs et al., 1978; see discussion in Chapter 14); and blood vessels, where they may influence vascular dynamics (Jojart et al., 1984; see discussion in Chapter 14). VP-ergic projections from the pvc divisions of the PVN include the dorsal parabrachial nucleus (dorsal PBN), the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (DMV), the nucleus ambiguus (NA), the lateral reticular nucleus, and A1 and A2 (noradrenergic areas in the medulla) and the intermediolateral cell column (IML) in the spinal cord (Sawchenko and Swanson, 1982; Sofroniew and Schrell, 1981). The DMV and NA are centers for parasympathetic output to visceral organs. VPcontaining cells of PVN origin also project to the spinal cord, where VP axons terminate in the IML, which contains sympathetic preganglionic cells. Thus VP-ergic projections to parasympathetic and sympathetic autonomic centers in the brainstem and spinal cord may play a role in the modulation of various visceral activities such as cardiovascular regulation. VP-ergic axons also end in the substantia gelatinosa of the spinal cord (a site of interaction between thin fibers mediating pain and temperature sensations and the large fibers mediating cutaneous sensory information to the CNS) (Nieuwenhuys et al., 1988). OT-ergic pvc fibers from the PVN descend to the brainstem and spinal cord, where they innervate the locus coeruleus of the pons (Swanson, 1977), the spinal nucleus of the trigeminal nerve in the medulla (Swanson and Kuypers, 1980), several ANS centers in the brainstem (parabrachial nucleus [Swanson, 1977], dorsal vagal complex [Nilaver et al., 1980], and spinal cord [intermediolateral cell column (Buijs, 1978; Nilaver et al., 1980; Swanson, 1977)]). c. Afferent Input into the PVN Afferents to VP-ergic and OT-ergic cells in the PVN include fiber projections from visceral relay structures in the brainstem (e.g., NTS, PBN, and the A2 cell group in ventrolateral medulla), from CVOs (e.g., the SFO and OVLT), which monitor the blood for the status of the internal milieu, and from the brainstem (raphe nuclei and locus coeruleus) and forebrain (e.g., medial amygdala and BNST) limbic system processing sites. The ANS projections from the brainstem carry visceral messages from several brainstem centers (e.g., NTS [A1 cell group], PBN, and the

General Introduction to VP and OT

17

ventrolateral medulla [A2 cell group]). Moreover, this visceral input appears to differentially innervate the three mgc and pvc output divisions of the PVN (i.e., output to ANS centers, anterior and posterior lobes of pituitary gland) (Cunningham and Sawchenko, 1988; Sawchenko and Swanson, 1981) (see Fig. 10). The humorally generated messages received by the SFO and OVLT monitor in part, plasma osmolality, and neurons in these CVOs convert these blood-borne messages into neural impulses before relay to mgc and/ or pvc neurons in the SON or PVN. Direct or indirect neural input to PVN mgc and pvc neurons also originates in numerous hypothalamic nuclei [lateral hypothalamic area (LHA), ventromedial nucleus (VMN), DMN, preoptic region], and in certain forebrain limbic system structures (BNST, olfactory bulb, amygdala, and septal area). d. PVN as a Visceral Effector Integrative Center Swanson and Sawchenko (1983) view the PVN as ‘‘a visceral effector integrative center’’ because its neurons mediate homeostatic regulation by means of three output systems: hormonal output via the posterior pituitary lobe, a releasing action on certain anterior pituitary hormones, and neural output via the ANS. Evidence reviewed by Swanson and Sawchenko (1983) indicates that essentially separate, topographically distinct cell populations of VP and OT neurons project to the posterior pituitary (mgc divisions), EZME (medial pvc group), and brainstem and spinal cord ANS centers (e.g., lateral pvc group) (see Fig. 11). Afferent inputs from a variety of brainstem and forebrain sources, described above, activate these output systems both in an integrative and differentiated manner, depending on the stimulus input. e. Suprachiasmatic Nucleus The hypothalamic suprachiasmatic nucleus (SCN) contains only pvc VP-ergic neurons and the target sites of their projections include the thalamic paraventricular nucleus (thalamic PVN) and several hypothalamic nuclei. These latter include the periventricular nucleus (PeVN) and dorsomedial nucleus (DMN) (Hoorneman and Buijs, 1982), the contralateral SCN (De Vries et al., 1985), and the OVLT (Hoorneman and Buijs, 1982). Hoorneman and Buijs (1982) have speculated that the VP fibers of the SCN (the vertebrate biological clock) may contribute to the circadian rhythms in feeding (projections to the DMN) and drinking (projections to the OVLT and PeVN). 4. Extrahypothalamic VP-ergic and/or OT-ergic Cell Systems a. Overview Histological identification of the pvc neurons lying outside the hypothalamic cell groups requires pretreatment with colchicine. Because colchicine pretreatment blocks axonal transport of proteins

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Barbara B. McEwen

FIGURE 10 (A) Organization of ascending, predominantly noradrenergic projections (asterisks) to the PVN; the SON has been omitted for clarity. Note the interconnections between the three noradrenergic cell groups that project to the PVN and the fact that only the A1 group substantially innervates both the magnocellular and parvocellular divisions of the PVN. (B) Organization of efferent projections from the PVN, which appear to be involved in the central control of cardiovascular function. The parvocellular division projects to preganglionic cell groups of both divisions of the autonomic nervous system in the brainstem and spinal cord; to the locus coeruleus, a nucleus that appears to influence the intracerebral microvasculature; and to the median eminence. Neurons in the magnocellular division of the PVN (and the SON) project directly to the posterior lobe of the pituitary gland, where they release vasopressin into the general circulation. Abbreviations: A1, catecholaminergic cell group; DVC, dorsal vagal complex; ME, median eminence; IML, intermediolateral cell column of spinal cord; IX, glossopharyngeal nerve; X, vagus nerve; LC, locus coeruleus; PP, posterior pituitary; pc, parvocellular division of the PVN; O and V, oxytocin- and vasopressin-containing portions of the magnocellular division of the PVN. Reprinted with permission from Sawchenko, P. E. & Swanson, L. W., 1981 (Fig. 1, p. 686). Copyright ß 1981 by the American Association for the Advancement of Science.

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FIGURE 11 A drawing to show in a schematic way relationships between the cytoarchitectonic and functional subdivisions of the PVH. The caudal half of the nucleus is shown, collapsed upon itself, in the frontal plane (the third ventricle is to the left). Three major functional groups of cells have been identified on the basis of double retrograde transport studies; they project to the median eminence (ME), to the posterior lobe of the pituitary gland (PP), and to autonomic preganglionic cell groups in the brainstem and spinal cord (ANS). These cell groups are centered in (although not exclusively restricted to) the cytoarchitectonically distinct parts of the nucleus indicated. Source: Sawchenko & Swanson, 1983 (Fig. 1, p. 124). Copyright ß 1983 by Alan R. Liss, Inc. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

synthesized in the cell body, it is frequently used to identify those cells synthesizing small amounts of proteins (Sofroniew, 1985b). This procedure has permitted localization of VP-ergic neurons in certain brainstem sites [e.g., locus coeruleus (LC)], and in a number of limbic forebrain sites [medial amygdala (MA), bed nucleus of the stria terminalis (BNST), septal area and nucleus of the diagonal band of Broca (DBB) (Sofroniew, 1985b) (see Fig. 12). The VP-containing cells originating in the MA project to the ventral hippocampus (VH) and the lateral septum (LS) (Caffe et al., 1989). The BNST projects to the olfactory tubercle, DBB, LS, anterior amygdala, and lateral habenula and to a number of brainstem sites including the LC (Caffe et al., 1989). An in vitro study by Ingram and Moos (1992) combined ICC staining for OT with electrophysiological recording from the lactating rat brain, and demonstrated OT-ergic innervation of the BNST from a continuum of OT cell bodies extending from an anterior pvc division of the PVN through the anterior commissural nucleus and perifornical region of the hypothalamus. These researchers suggested that this limbic system pathway is

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Barbara B. McEwen

FIGURE 12 Drawings of frontal sections at various levels through the rat brain, which show the distribution of magnocellular (mgc) and parvocellular (pvc) vasopressin (VP) and oxytocin (OT) neurons in colchicine-treated rats. Large solid dots depict mgc VP neurons. Small solid

General Introduction to VP and OT

21

involved in mediating the facilitatory effect of OT on the milk ejection reflex of lactating rats. b. Sexually Dimorphic and Gonad-Dependent Circuitry A number of findings by De Vries and colleagues have demonstrated the presence of sexual dimorphic and gonad-dependent VP-ergic circuitry in the rat brain: (1) beginning in early development (i.e., postnatal day 12) the density of VP-ergic fibers in certain limbic system structures (e.g., the lateral septum and lateral habenula) are markedly denser in male than in female rats (De Vries et al., 1981) and this sexual dimorphy is androgen dependent (De Vries et al., 1983); (2) this sexually dimorphic circuitry in the adult is dependent on circulating gonadotropic hormones because castration in male rats, and ovariectomy in female rats, progressively diminishes the presence of VP in dimorphic fiber pathways over successive weeks and hormonal replacement therapy, carried out in male rats, restored VP synthesis in the fiber network (De Vries et al., 1984); and (3) the source of the dimorphic VP-ergic fibers innervating the septal and habenula nuclei is cell bodies in the bed nucleus of the stria terminalis (BNST) and the medial amygdala (MA) because VP was no longer manifest in these cellular groups 15 weeks after castration whereas no obvious changes in VP synthesis occurred in other VP-ergic cell groups such as the hypothalamic PVN or SCN (De Vries et al., 1985). Additional support for the gonadal dependence and limbic system localization of this sexually dimorphic VP-ergic circuitry includes the following findings: (1) AVP neurons in the BNST and MA contain estrogen receptors (Axelson and Van Leeuwen, 1990); (2) mRNA synthesis in these pathways is affected by circulating gonadal steroids because gonadectomy leads to a greater decrease in AVP synthesis in the cell body than in the axon terminals (Miller et al., 1992); and (3) testosterone implants in the castrated male rat restore AVP staining in the various steroid-dependent projections of the BNST (De Vries et al., 1986; Miller et al., 1989a). The exact function of this sexually dimorphic VP circuitry is not known at this time. It may also be noted that, although SON VP-ergic cells projecting to the posterior pituitary lobe are larger in male rats than in female rats, this is correlated with sex differences in body weight and is independent of circulating gonadal hormones, signifying an adaptation that provides a greater dots depict pvc VP neurons. Large open circles depict mgc OT neurons. Small open circles depict pvc OT neurons. Each dot represents from 1 to 6 neurons as seen in 50 m sections. Abbreviations: a, amygdala; AC, anterior cingulate cortex; bnst, bed nucleus of stria terminalis; cpvn, caudal paraventricular nucleus; db, diagonal band of Broca; F, fornix; lc, locus coeruleus; lpo, lateral preoptic area; ls, lateral septum; m5, motor trigeminal nucleus; mpo, medial preoptic area; ms, medial septum; SCN, suprachiasmatic nucleus; sh, suprachiasmatic nucleus; SON, supraoptic nucleus; subc, subcoeruleus nucleus. Figure reprinted from, and legend adapted from, Sofroniew, 1985b (Fig. 1, pp. 348–349). Copyright ß 1985 by Pergamon Press.

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Barbara B. McEwen

amount of hormonal VP to meet the fluid regulation requirements for the larger male rodent (Madeira et al., 1993). 5. VP-ergic and OT-ergic Fibers and Terminals of Unknown Origin There is still much to learn concerning the central distribution of VPergic cells and their fibers. In some cases knowledge is lacking about the projected terminals of localized VP cell clusters (e.g., VP cells in the hypothalamic dorsomedial nucleus). In other cases sites of VP-ergic fibers and/or terminals are known but not their cells of origin. A number of structures in the brain and spinal cord with VP fibers and terminals both of known and uncertain origin are listed in Table II. VP fiber terminals of unknown origin have been localized in certain CVOs (subfornical organ, area postrema, and subcommissural organ) and in a variety of brain sites in the telencephalon (e.g., the BNST, ventral hippocampus, and amygdala), diencephalon (e.g., certain thalamic nuclei), and brainstem (e.g., the substantia nigra and interpeduncular nucleus). These VP-containing neurons synapse on the dendrites or cell bodies of their target sites, providing a neuroanatomical basis for their neural transmitter/modulator actions (Weindl and Sofroniew, 1985).

B. Vasopressin and Oxytocin Receptors 1. Distinction between Ionotropic and Metabotropic Receptor Transmission Two types of intercellular signaling can be distinguished: ionotropic, mediated by a superfamily of receptors known as ligand-gated channels (LGCs), and metabotropic, mediated by G protein-coupled receptors (GPCRs). Both types of receptors are proteins embedded within and spanning the cell membrane. These two types of transmission are discussed by Feldman et al. (1997, pp. 208–227). a. Ionotropic Receptor Transmission In ionotropic receptor transmission, the target cell response depends on selective ionic permeability. When complexed with the ligand or chemical signal (e.g., appropriate neurotransmitter), the LGC exhibits a conformational (three-dimensional structural) change, in this case causing the opening of a specific ionic channel, which results in an excitatory (opening of a Naþ channel) or inhibitory (opening of a Cl channel) response in the postsynaptic cell. LGCs are fast signaling receptors (millisecond latency) because of the direct linkage between receptor activation and channel opening. There is also rapid response termination because the chemical transmitter rapidly dissociates from the receptor, an action that closes the channel. Many well-known neurotransmitters contain receptor subtypes that belong to the LGC superfamily. These include

23

General Introduction to VP and OT

TABLE II Distribution of Vasopressin and Oxytocin Fibers in Brain and Spinal Corda Area innervated

Origin A. Vasopressin Fibers

Telencephalon Cerebral cortex (prefrontal, piriform, cingulate, entorhinal) (*) Claustrum (N) Olfactory bulb (external plexiform layer) (N) Olfactory tubercle (N) Olfactory nucleus (N) Diagonal band of Broca (þþþ) Lateral septum (þþþþ) BNST (þ) Nucleus of the diagonal band of Broca (þþ) Cortical–amygdala transition zone (N) Central, anterior, basal, cortical, and lateral nuclei of the amygdala (þ) MA (þþ) Basal nucleus of Meynert (N) Ventral pallidum (N) Accumbens nucleus (N) Hippocampus (ventral part—CA1, CA3) (þ Subiculum (N) Dentate gyrus (ventral part) (N) Diencephalon OVLT (N) Hypothalamic/preoptic area (N) Posterior hypothalamus (N) Arcuate nucleus (N) Premammillary nuclei (N) Supramammillary nucleus (*) PVN (þþþ) Paraventricular thalamic nucleus/ventral part (N) Thalamic periventricular nucleus (N) Thalamic rhomboid nucleus (N) Thalamic intermediodorsal nucleus (N) Thalamic mediodorsal nucleus (þþþ) Thalamic intralaminar nucleus (including parafascicular and centrolateral nucleus) (N) Dorsomedial hypothalamic nucleus (area surrounding fasciculus retroflexus and dorsal part of zona incerta) (N) Lateral habenula (medial part) (þþþþ) Mesencephalon Central gray (þ) Ventral tegmental area (þ) Substantia nigra, pars compacta (þþ) Substantia nigra, pars reticulata (N) Superior colliculus (N) Dorsal raphe nucleus (þ)

BNST PVN BNST and MA BNST

SCN

SCN SCN SCN

SCN

SCN BNST

(Continues)

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Barbara B. McEwen

TABLE II (Continued) Area innervated

Origin

Ventral parabrachial nucleus (þ) Dorsal parabrachial nucleus (*) Locus coeruleus (þ) Cuneiform nucleus (*) Interpeduncular nucleus (*) Lateral tegmental RF (N) Metencephalon Central gray of the pons (N) Raphe obscuris nucleus (*) Median raphe nucleus (N) Raphe pontis (*) Myelencephalon Raphe magnus (þ) Nucleus solitary tract (þþ) Dorsal motor nucleus of the vagus nerve (þþ) Lateral reticular nucleus (þ) Ambiguus nucleus (N) Nucleus of spinal trigeminal nerve (*) A1 and A2 regions in medulla (N) Spinal cord Laminae I–III (*) Lamina X (þ) Intermediolateral nucleus (*)

PPT

PVN PVN PVN PVN

PVN

B. Oxytocin Fibers Telencephalon Cerebral cortex (including frontal, piriform, cingulate, and entorhinal) (*) Claustrum (N) Olfactory bulb (N) Olfactory tubercle (including islands of Calleja) (N) Anterior olfactory nucleus (N) BNST (N) Interstitial nucleus stria terminalis (*) Lateral septum (*) Amygdala (over most of its rostrocaudal extent) (N) Central, anterior, basal, cortical, and lateral nucleus of amygdala (þ) Ventral hippocampus (þ) Dentate gyrus (N) CA1–CA3 hippocampus (N) Nucleus accumbens (*) Striatum (*) Ventral pallidum (þ) Basal nucleus of Meynert (þ) Substantia innominata (þ) (Continues)

General Introduction to VP and OT

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TABLE II (Continued) Area innervated Diencephalon Supramammillary nucleus (þ) Thalamus (various regions) (N) Subfornical organ (around fenestrated capillaries) (N) Mediodorsal thalamus (*) Mesencephalon Substantia nigra (pars compacta) (þþþþ) Substantia nigra (pars reticulata) (N) PPT (cuneiform nucleus) (þ) Interpeduncular nucleus (þ) Midbrain central gray (þ) Ventral tegmental area (þþ) Raphe dorsalis (þ) Raphe magnus (N) Locus coeruleus (N) Metencephalon Pontine central gray (N) Raphe pontis (N) Parabrachial nucleus (dorsal) (þþ) Parabrachial nucleus (ventral) (þ) Cerebellum (N) Myelencephalon Raphe obscurus (*) Raphe pallidus (N) Raphe magnus (þþ) Lateral reticular nucleus in ventrolateral medulla (þþ) Nucleus of solitary tract (þþþþ) Dorsal motor nucleus vagus (þþþþ) Substantia gelatinosa of trigeminal nerve (rostral part) (þ) Spinal cordb Spinal cord (all segmental levels) (N) Substantia gelatinosa (all levels) (N) Laminae I–III (þ) Lamina X (þþ) Intermediolateral cell column (þ) Lateral funiculus of spinal cord (N) Central gray of spinal cord (N) a

Origin

PVN PVN

PVN PVN PVN PVN PVN

PVN PVN PVN

Key used to indicate density: Few fibers and possibly terminals are present (*); regular presence of a number of fibers and/or terminals (þ); increasing density of fibers and/or terminals (þþ through þþþþ); no information (N). Abbreviations: BNST, bed nucleus of the stria terminalis; MA, medial amygdala; PVN, hypothalamic paraventricular nucleus; PPT, pedunculopontine tegmental nucleus; RF, reticular formation; SCN, suprachiasmatic nucleus. Data based on Buijs (1987); Caffe et al. (1989); Hoorneman and Buijs (1982); Nilaver et al. (1980); Sofroniew (1983, 1985a). b OT predominates over VP in most regions of the spinal cord.

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cholinergic (nicotinic), serotoninergic (5HT3), histaminergic (H1 and H3). -aminobutyric acid (GABA)-ergic (GABAA), glycine, and excitatory amino acid [-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, and N-methyl-d-aspartate (NMDA)] receptors. b. Metabotropic Receptor Transmission Metabotropic receptor transmission is a type of indirect transmission because a second messenger typically mediates the cellular response to the chemical signal (first messenger). Response onset is also slower, its rate depending on the degree of metabolic processing that intervenes between receptor activation and cellular response. A GPCR mediates this type of transmission. All peptidergic, muscarinic, cholinergic, dopaminergic, adrenergic [noradrenaline (NA) and epinephrine (Epi)], serotoninergic (except 5HT3), and certain glutamatergic, GABA-ergic, and histaminergic receptors belong to the GPCR superfamily. In general terms, when the ligand binds to the receptor, the ligand– receptor complex activates a membrane protein (G protein). In its inactivated state the G protein has GDP (guanyl nucleotide diphosphate) bound to it, but the ligand-activated receptor complex catalyzes the replacement of GDP by GTP (guanyl nucleotide triphosphate) and activates the G protein. The activated G protein, in turn, interacts with and stimulates a specific enzyme (e.g., adenylate cyclase), which in turn produces a second messenger [in this case, cyclic AMP (cAMP)]. There are several types of G proteins. For example, depending on the G protein to which the receptor is coupled, the activated G protein may stimulate (Gs) or inhibit (Gi) the enzyme that produces the second messenger. In addition to acting via second messengers, the G protein may itself combine with and activate a channel, illustrating the dual-action capability of the G protein. The G protein contains , , and subunits; the subunit contains a guanyl nucleotide-binding site. In the inactive state the subunit binds GDP. On receptor stimulation, the transmitter–receptor complex activates the G protein [the subunit dissociates from the and subunits and GDP is phosphorylated to form GTP. Transmitter removal from the receptor site (by enzymatic degradation or reuptake) restores the G protein to its inactive state (GTP is dephosphorylated to GDP and the subunit is reassociated with the and subunits). A number of second messengers have been identified [cAMP, cGMP, Ca2þ, diacylglycerol (DAG), and inositol 1,4,5-triphosphate (IP3)]. Of special interest to this discussion are the second messengers DAG and IP3, whose formation is generated, in part, by a G protein coupled to vasopressin V1 receptors, as well as to ACh muscarinic, -adrenergic, 5HT2, and metabotropic glutamate receptor subtypes. DAG and IP3 are formed by a G protein-activated phospholipase, which hydrolyzes the membrane phospholipid, phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 can be hydrolyzed by each of two forms of the phospholipase enzyme. Second

General Introduction to VP and OT

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messengers typically function by activating enzymes (protein kinases) that catalyze phosphorylation of various intracellular proteins. The phosphorylation alters conformation of the affected proteins, with an associated change in biological activity. For example, if the protein is an enzyme, the phosphorylation may increase or decrease its catalytic activity. A phosphoprotein, phosphatase, returns the protein to its nonphosphorylated state by the process of dephosphorylation, which ends the cellular effects of the second messenger. In addition to directly or indirectly gating a selective ion channel, G protein activation may initiate a cascade of intracellular metabolic actions involving a sequence of intracellular messengers, enzymes, and protein substrates that lead to short-term or long-term modulatory processes. In neural transmission, the long-term modulatory processing involves gene activation in the nucleus of the postsynaptic neuron (Fig. 13). It consists of two phases: an initial phase characterized by the synaptic induction of immediate-early

FIGURE 13 Summary of transcription regulation by neurotransmitters. This diagram summarizes the overall biochemical cascade by which neurotransmitters and other firstmessenger substances alter gene transcription in the nervous system. Source: Feldman et al., 1997 (Fig. 6.37, p. 226). Copyright ß 1997 by Sinauer Associates, Inc. Reprinted with permission.

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genes (IEGs), followed by a second phase of synaptically mediated induction of ‘‘late-onset genes.’’ This synaptically induced regulation of gene expression is involved in synthesizing proteins that form neuronal structures such as ion channels, receptors, intracellular messengers, and presumably synaptic structures necessary to forming long-term memories (see Fig. 14). For further discussion see Feldman et al. (1997, pp. 208–227). 2. Characterizing and Localizing V1 and V2 types of VP Receptors in Peripheral and Neural Tissues Although all VP receptors, whether localized within peripheral tissues or in the central nervous system, are GPCRs mediating metabotropic transmission, they can be further categorized as V1 or V2 receptors, depending on the second messenger activated. The second messenger for the V1 type of receptor is the product of membrane phospholipid metabolism and intracellular liberated calcium ions, whereas that for the V2 type of receptor is cAMP. This differentiation was based on study of peripheral tissues (Mitchell et al., 1979). The observation that AVP receptors in the anterior pituitary gland have a slightly different pharmacological profile of binding interactions with AVP analogs led Jard et al. (1986) to suggest that there are two major subtypes of V1 receptors: the V1 receptors in the anterior pituitary (V1b) and the remaining V1 receptors (V1a receptors). a. Vasopressin VIa Receptor i. Localization Tritium-labeled ([3H] AVP, [3H] LVP) VP ligands, were the first radioligands used to identify VP V1 receptors, and have detected these receptors in cells (freshly isolated or cultured) of various tissues and organs in both rats (liver, blood vessels, anterior pituitary gland, medulla of adrenal gland, reproductive organs, and peripheral and central nervous systems) and humans (lymphocytes, monocytes, and platelets) (Brinton et al., 1984; Jard et al., 1986; Thibonnier, 1992). Neuronal VP receptors have long been considered to be mainly, if not exclusively, of the V1a subtype (Tribollet et al., 1988), and a more recent review has reaffirmed the lack of any clear support for the presence of either V2 or V1b receptors in the CNS (Barberis and Tribollet, 1996). Many central VP receptors have been observed in brain sites in which there are no known VP axonal terminals, suggesting that VP molecules activating these receptors are transported by means other than synaptic junctions [e.g., by means of volume conduction (action at a more distant neuronal target than the postsynaptic cell)]. On the other hand, a substantial number of V1a receptors have been found in brain areas that are innervated by VP terminals. Table III provides a list of the CNS sites in which VP V1a-binding sites have been detected in the rat brain by in vitro receptor-binding autoradiography, with a highly sensitive linear radioiodinated V1a antagonist

General Introduction to VP and OT

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FIGURE 14 General mechanism of second-messenger action. Second messengers are intracellular molecules or ions that are regulated by extracellular signaling agents such as neurotransmitters and hormones (first messengers). Second messengers typically operate by activating protein kinases that phosphorylate various target proteins, thereby altering the functioning of these proteins. Such functional effects are subsequently reversed by protein phosphatase-mediated dephosphorylation. Second messengers modulate a wide range of both rapid and long-term neuronal processes. Source: Feldman et al., 1997 (Fig. 6.24, p. 213). Copyright ß 1997 by Sinauer Associates, Inc. Reprinted with permission.

30 TABLE III

Barbara B. McEwen

VP-ergic Binding Sites in the CNS of the Laboratory Rata

Site

Binding site density Telencephalon

Olfactory system/related cortex Anterior olfactory nucleus Olfactory bulb (internal plexiform and glomerulosa layers) Olfactory cortical area Piriform cortex Neocortex Layer IV Layer VI (area 2 in the parietal cortex) Basal ganglia Nucleus accumbens (highly concentrated in restricted areas between core and shell, ventral to LS) Caudate putamen (at ventrolateral edge) Horizontal limb of the diagonal band of Broca Limbic system Lateral septum (density somewhat higher in the intermediate than in the dorsal part) Bed nucleus of the stria terminalis (restricted areas of its anterior part located around the anterior commissure) Hippocampus (dentate gyrus as well as in parts of the fields of Ammon’s horn) Amygdala (amygdalostriatal transition area between the central nucleus and the ventral striatum, but not in amygdala proper)

**** * *** *** ** to *** * {

{

** **** **** *** ****

Diencephalon Thalamus Anteroventral nucleus Ventromedial nucleus Ventrolateral nucleus Posterior nuclear group Mediodorsal nucleus Suprafascicular nucleus Hypothalamus Stigmoid nucleus Zona incerta Tuber cinereum Suprachiasmatic nucleus Arcuate nucleus Magnocellular preoptic Lateral hypothalamic area

*** *** *** ** ** ** **** **** **** *** *** ** **

Midbrain and More Caudal Regions of Brain Mesencephalic central gray (diffusely distributed in all parts of this area) Edinger–Westphal Nucleus Darskchewitsch Raphe nucleus (rostral linear part)

{

*** *** *** (Continues)

General Introduction to VP and OT

31

TABLE III (Continued) Site

Binding site density

Superior colliculus (superficial zonal area and optic layers) Locus coeruleus Interpeduncular nucleus Substantia nigra Ventral tegmental area Nucleus of solitary tract Inferior olive (medial component) Sensory spinal trigeminal nucleus (interpolar part) Parvocellular reticular nucleus Hypoglossal nucleus

*** *** **** ** ** **** **** **** ** **

Spinal Cord Gray matter (distributed evenly in the gray matter)

{

Circumventricular Organs Area postrema (neuroglial part) Subfornical area (associated with the vascularized area of this structure) Pineal gland (mostly in the vascular shell of the gland rather than in its main body) Choroid flexus

****

{

{

****

Blood Vessels of the Brain Large superficial arteries supplying the brain (the anterior cerebral artery, internal carotid area, and the basal artery; consistent with suggestion that AVP plays a role in the regulation of cerebral blood flow) a

{

Data based on in vitro receptor-binding autoradiographic studies with radiolabeled VP antagonists (Barberis and Tribollet, 1996). Key for binding site density: *, just detectable; **, low; ***, moderate; ****, high; {, not indicated.

developed by Barberis et al. (1995). The sensitivity of the radioiodinated AVP antagonist is indicated by the fact it detected V1 AVP receptors in numerous CNS sites that were either faintly or not labeled with [3H]AVP (Barberis and Tribollet, 1996). ii. Receptor-mediated signaling pathway The following discussion, based on the minireview by Thibonnier (1992), is consistent with the general overview provided in the previous discussion of metabotropic neurotransmission (Section III.B.1). A graphic illustration of the major points discussed below is given in Fig. 15. The AVP–receptor complex interacts with G proteins within the plasma membrane. The two activated G proteins stimulate a number of membrane phospholipase enzymes [e.g., phospholipase C (PLC), phospholipase

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Barbara B. McEwen

FIGURE 15 Immediate and secondary signals activated by V1 vascular AVP receptors. Abbreviations: PLC, phospholipase C; PLD, phospholipase D; PLA2, phospholipase A2; PA, phosphatidic acid; AA, arachidonic acid; PC, phosphatidylcholine; PKC, protein kinase C; ER, endoplasmic reticulum; CO, cyclooxygenase pathway; EPO, epoxygenase pathway; IP3, 1,4,5-inositol triphosphate; DAG, 1,2-diacylglycerol; Gq, G protein. Source: Thibonnier, 1992 (Fig. 1, p. 9). Copyright ß 1992 by Elsevier Science Publishers B.V.

D (PLD), or phospholipase A2 (PLA2)] that catalyze the breakdown of membrane phospholipids whose metabolic products enter the cytoplasm, where they act as second messengers. For example, PLC hydrolyzes polyphosphoinositides (PIP2) and forms the second messengers inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 mobilizes calcium ions (Ca2þ) from its intracellular storage sites in the endoplasmic reticulum (ER), and DAG activates protein kinase C (PKC). PLD hydrolyzes phosphatidic acid, which also produces DAG. PLA2 hydrolyzes phosphatidylcholine to form arachidonic acid (AA), which in turn potentiates AVP-induced influx of extracellular Ca2þ and its mobilization from storage in the ER. The Ca2þ ions are also second-messenger molecules. Most second-messenger functions of Ca2þ require binding with a cytoplasmic protein called calmodulin. The Ca2þ–calmodulin complex activates a Ca2þ–calmodulin-dependent protein kinase. Also noteworthy is the alteration in the pH status of the cell. The intracellular increase in Ca2þ produces an initial acidification of the cell interior as a result of a

General Introduction to VP and OT

33

unidirectional (Hþ) influx followed by alkalinization (entry of Naþ), presumably as a result of activation of the Naþ/Hþ exchanger. This dual pH effect appears to be important in coupling receptor excitation with smooth muscle contraction in blood vessels, and also in VP-induced mitogenic effects in the role of VP as a ‘‘cellular growth factor’’ (Thibonnier, 1992). Once activated by the second messengers, the protein kinases phosphorylate proteins that bring about physiological alterations that result in the response of the cell to the original ligand (first messenger). These cell responses may produce relatively short- or long-term effects. Cell responses involving short-term effects include vasopressin V1 receptor-mediated vascular smooth muscle contraction, platelet aggregation, hepatic glucose production, anterior pituitary hormonal release, postsynaptic neuronal activation, and neuromodulation. The cellular responses that produce long-term effects involve the role of AVP as a cellular growth factor involved in embryonic development and in tissue regeneration and proliferation in the adult (e.g., liver regeneration after partial hepatectomy; Hocevar and Fields, 1991). The vasopressin-induced neurotrophic action in cultured hippocampal neurons (e.g., increased number of neurites, and increase in length and neurite diameter as well as in morphological complexity, number of branches, and branch bifurcation points) reported by Brinton et al. (1994) provides another example of the role of VP as a cellular growth factor (see Brinton, 1998, for a discussion of this research and its relevance for a VP role in memory processing). These long-term response effects undoubtedly involve intracellular pathways by which AVP activates gene expression and associated protein synthesis. As Thibonnier notes, little is known at this point concerning the specific intracellular pathways and mechanisms by which these effects are accomplished. Nevertheless, relevant discussions by Schwartz and Kandel (1991), Feldman et al. (1997, pp. 208–225), and Thibonnier (1992) suggest involvement of the following major steps: (1) DAG-activated PKC, or its catalytic components, enter the nucleus and phosphorylate ‘‘transcription factors’’ that stimulate transcription of immediate-early genes (IEGs, or proto-oncogenes) such as c-fos and c-jun; (2) these IEGs transcribe mRNAs, which enter the cytoplasm and synthesize the proteins Fos and Jun (transcription regulatory proteins); (3) these transcription regulatory proteins are then translocated to the nucleus, where they link together to form homodimers (Fos–Fos) or heterodimers (Fos–Jun); (4) these dimers bind to a so-called AP-1 site in the promoter region of the target gene (late onset gene), thereby inducing transcription of the target gene into mRNA; and (5) the mRNA then enters the cytoplasm and synthesizes the protein in the cytoplasm, and these newly formed proteins are incorporated into cellular structural alterations necessary for the long-term effects induced by AVP activation of the V1 receptor.

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b. Vasopressin V2 Receptor To date, the V2 receptor has been localized with assurance only on kidney tubule cells, where it mediates the antidiuretic effect of the peptide. The operation of this receptor and its postulated role in increasing the permeability to water in renal tubular cells is discussed in Norman and Litwack (1987) and diagrammed in Fig. 16. The tubular cells form the epithelial lining of the collecting duct, a component of the renal tubule (nephron, the functional unit of the kidney). Because of tight junctions between the cells of this epithelial lining, this portion of the renal tubule is relatively impermeable to the passage of water. Tight junctions occur at the apical–lateral corners of the interconnected cells, whereas the basolateral surfaces are separated by a lateral intracellular space that is continuous with the interstitial fluid surround. The basal surface of the cell, which faces away from the lumen of the tube, houses the V2 receptor sites whereas the apical (lumenal) surfaces that face the lumen of the collecting duct are the sites at which VP influences cell permeability (see Fig. 16). The VP–V2 receptor complex activates the attached G protein (GDP bound to the subunit is phosphorylated to GTP). The G protein in turn activates the intramembrane enzyme adenylate cyclase. This enzyme catalyzes a reaction in the cytoplasm that forms cAMP from ATP. cAMP acts as a second messenger and activates a protein kinase that phosphorylates integral membrane proteins on the lumenal surface of the cell. When phosphorylated these membrane proteins form the pores, by which water molecules enter the

FIGURE 16 Postulated mechanism of vasopressin action on the renal tubule cell. Abbreviations: AC, adenylate cyclase; TJ, tight junction; MF, microfilament; I, cytoplasmic tubule with water conduction particles; II, delivery of particles of lumenal membrane following fusion; III, particles aggregated on lumenal membrane. Source: Forsling and Grossman, 1986 (Fig. 12.6, p. 164). Published by Croom Helm and Charles Press. Copyright ß 1986 by M. L. Forsling and A. Grossman, and reprinted with their kind permission.

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35

cell, and the vesicles, by which the water molecules are transported to the basolateral surface of the cell. From this location the water molecules exit into the adjacent interstitial fluid and then into the capillaries surrounding the renal tubular system. The net result is water retention in the body and reduced output in the urine (antidiuresis). 3. Classification and Localization of OT Receptors Like the V1 receptor, the OT receptor appears to utilize IP3 and Ca2þ as second-messenger systems, but unlike VP, separate types of OT receptors have not been observed (De Kloet et al., 1990). OT receptors or receptorbinding sites (may respond to both VP and OT ligands) have been localized in several peripheral tissues [e.g., myometrial cells in uterus, myoepithelial cells in mammary gland, pancreatic islet cells (Gao and Henquin, 1993), liver (Hems and Whitton, 1980), adrenal gland (Gibbs, 1986c), and numerous CNS sites (Barberis and Tribollet, 1996). Table IV lists the CNS sites in which OT-binding sites have been detected. This OT receptor mapping was based on in vitro receptor-binding autoradiography with a radioiodinated OT antagonist carried out by Tribollet (1992). Comparison of the data listed in Tables III and IV indicates that the binding sites for VP and OT are widely distributed in the rat brain. However, with the exception of that in the anterior olfactory nucleus, their specific mapping distributions do not overlap (Barberis and Tribollet, 1996). The information presented in Tables III and IV is based directly on the review by Barberis and Tribollet (1996). Sex differences are not present with respect to OT receptor topography or number. However, OT receptors in certain brain sites are gonadally dependent (i.e., inducible). Thus, estrogen (De Kloet et al., 1990) and testosterone (Barberis and Tribollet, 1996) modulate the density of OT receptors in certain brain regions such as the hypothalamic ventromedial nucleus (VMH), olfactory tubercle, central amygdala (CEA), and the BNST (Barberis and Tribollet, 1996). OT receptors in certain areas of the CNS may respond to both VP and OT, but in opposing ways. Thus, an OT receptor in the ventral hippocampus appears to mediate both a VP-facilitating and an OT-attenuating influence on memory for a learned passive avoidance response (De Wied et al., 1993; and see Chapter 5), and an OT receptor in the VMH mediates an OTstimulatory and a VP-inhibitory influence on lordosis behavior in estrogen/ progesterone-primed female rats (De Kloet et al., 1990). Species differences have been noted with respect to VP and OT receptor topography and site-specific gonadal dependency (Barberis and Tribollet, 1996; Tribollet et al., 1992a,b). For example, both the guinea pig and the rat exhibit a high concentration of OT receptors in the ventromedial hypothalamus, but unlike the rat, these receptors are not influenced by gonadectomy in the guinea pig. Barberis and Tribollet (1996) have suggested

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TABLE IV OT-ergic Binding Sites in the CNS of the Laboratory Rata Site

Binding site density

Telencephalon Olfactory system Anterior olfactory nucleus Cerebral cortex Dorsal peduncular cortex Insular cortex Perirhinal cortex Basal ganglia Nucleus accumbens (restricted areas, different from those containing AVP-binding sites) Caudate putamen (dorsomedial and caudolateral parts) Caudate putamen (ventral edge of this complex) Limbic system Bed nucleus of stria terminalis (medially in a region dorsal and posterior to the anterior commissure; an area that differs from that containing AVP receptors) Lateral septum (intermediate part of the lateral septum) Hippocampal ventral subiculum Hippocampal dorsal subiculum Hippocampal CA1 field of Ammon’s horn Hippocampal parasubiculum Hippocampal presubiculum Amygdalohippocampal area Central amygdaloid nucleus Medial amygdaloid nucleus Basolateral amygdaloid nucleus

**** **** ** ** ** *** **** ****

** **** ** ** ** ** ** **** ** **

Diencephalon Thalamus Paraventricular nucleus (anterior part, only area of thalamus that contains OT-binding sites) Hypothalamus Ventromedial nucleus (ventrolateral part and adjacent neuropil) Medial preoptic nucleus Medial tuberal nucleus Mammillary complex (a few nuclei) Midbrain and more caudal regions of the brain Dorsal motor nucleus of vagus nerve Inferior olive nucleus Spinal trigeminal nucleus (substantia gelatinosa portion)

{

**** ** ** ** { { {

Spinal Cord Substantia gelatinosa (at all levels of the spinal cord) Intermediolateral cell column (thoracic levels—preganglionic sympathetic nervous system neurons that innervate the adrenal medulla)

{ {

(Continues)

General Introduction to VP and OT

37

that those VP and OT receptors displaying species-related differences may be involved in mediating species-typical behaviors.

C. Vasopressin and Oxytocin as Neurotransmitters and Neuromodulators in the CNS The concept of neuromodulation continues to be refined and modified with ever increasing knowledge of the cellular effects mediated by metabotropic receptors of amines, acetycholine, and peptide systems in the brain (see Hasselmo, 1995, for a discussion of neuromodulation in the cerebral cortex). For purposes of this discussion, a neuromodulatory action of a neurotransmitter (e.g., VP) enhances or diminishes the effect of another neurotransmitter (e.g., noradrenaline, glutamate) on a given target neuron by means of an interaction between the two neurotransmitters (e.g., VP and noradrenaline). One distinction between a neurotransmitter and a neuromodulatory action can be related to the actions mediated by ionotropic and metabotropic receptors. A neurotransmitter action can be mediated by both types of receptors, but a neuromodulatory action is mediated only by metabotropic receptors. A neurotransmitter action produces an excitatory or inhibitory postsynaptic potential of rapid onset and short duration in the target neuron. Rather than producing a direct excitatory or inhibitory response of rapid onset, a neuromodulatory effect is of slow onset and results in a longer lasting metabolic and/or structural effect that alters the transmitter output or responsivity of the target neuron to other transmitter inputs. Tribollet et al. (1990) have cited several lines of evidence indicating neurotransmitter/neuromodulator actions for VP and OT synthesized by certain cell systems in the hypothalamus and also by numerous VP-ergic systems localized in extrahypothalamic regions of the brain. First, these peptides are synthesized by cells other than the hormone-synthesizing neuroendocrine cells that release VP or OT to the portal and systemic circulations. Second, these VP-ergic and OT-ergic neuropeptide-containing cells project to a variety of sites within the brain and spinal cord and release VP and OT at axonal terminals by the same secretory mechanisms that operate with classic neurotransmitters (Buijs, 1987). Third, specific VP- and OTbinding sites are present in the CNS (Freund-Mercier et al., 1987; Tribollet et al., 1988), and at least some of these sites are functional receptors because VP or OT microinjected into them alters the rate of firing of the neurons containing them (Raggenbass et al., 1988, 1989). Neuromodulatory actions of VP and OT in relation to MP are discussed in Chapters 5 and 15 (also see Brinton, 1990, 1998). a

Data based on in vitro receptor-binding autoradiographic studies with radiolabeled OT antagonists (Barberis and Tribollet, 1996). Key for binding site density: *, just detectable; **, low; *** moderate; ****, high; {, not indicated.

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IV. Functional Aspects Relevant to Memory Processing: Actions at Nonneural Receptor Sites

__________________________________________________________

As noted in an earlier section of this chapter, VP and/or OT axonal terminals and receptors (or receptor-binding sites) have been localized in cells of several extraneural tissues, indicating that these neuropeptides are functionally active at these sites. Below are presented a variety of VP and/ or OT actions at nonneural cell sites, associated with their nonmnemonic functions, which, in this author’s opinion, may also provide mechanisms by which they influence one or more phases of memory processing.

A. Hormonal VP Actions on V1 Receptors in the Peripheral Vasculature and Memory Processing It is well known that there is an increased release of hormonal VP from the posterior pituitary gland in response to a number of conditions that threaten normal plasma fluid levels (dehydration, hyperosmolality, and hypovolemia), (Bourque et al., 1994; Dunn et al., 1973; Feuerstein et al., 1991). The antidiuretic action of hormonal VP at V2 receptors in kidney tubule cells, and pressor action at V1 receptors on smooth muscle cells of peripheral blood vessels, provide corrective actions that help ensure adequate blood perfusion to the vascular beds of vital organs, including the CNS. Koob et al. (1985a, 1989; and see Chapter 6) report that (1) an osmotic stimulus (intraperitoneal injected hypertonic saline), known to release VP in the brain and in the periphery, enhanced retention of a learned avoidance response; (2) pretreatment with a peripherally injected V1 receptor antagonist, known to antagonize the pressor action of peripherally injected AVP, prevented the osmotically induced retention effect; and (3) in contrast to AVP itself, the V1 antagonist appears able to easily penetrate the blood–brain barrier (Chapter 6), indicating that the pretreatment antagonized V1 receptors in the brain and peripheral circulation. Taken together, these findings are consistent with the thesis that the actions of VP at V1 receptors in both the brain and periphery influence memory retention in a homologous manner, but by different mechanisms (Koob et al., 1985b, 1989; and see Chapter 6).

B. VP and OT Interactions with Blood Vessels in the Brain: Role in Memory Processing? Hormonally and neuronally released VP and OT exert both general and specific influences on the cerebrovascular activity that is required for adequate brain functions, including that of learning and memory. Table V cites evidence and references supporting a role for the influence of VP and OT in regulating cerebrovascular activity (both vascular perfusion of the

39

General Introduction to VP and OT

TABLE V Vasopressin and Oxytocin Influences in Cerebrovascular Activity and in Promoting Nutrient Exchanges across the Blood–Brain Barrier Influence

Ref. Vasopressin

1. Hormonal VP regulation of vascular perfusion of the brain and cerebral blood flow (CBF) Hormonal VP provides for adequate blood supply to the brain and normal CBF in response to certain pathological conditions (e.g., hemorrhagic hypotension, shock, subarachnoid hemorrhage). This is accomplished in part by a VP interaction with the capillary endothelium, which causes the secretion of nitric oxide and results in an increase in the diameter of the large cerebral arteries. This in turn decreases microvascular pressure and results in an autoregulatory constriction of arterioles further upstream—the net effect is a maintenance of normal CBF 2. Neuronally released VP innervates brain microvessels: Influence on regional cerebral blood flow? VP-ergic neurons directly innervate brain microvessels, suggesting a means by which central VP systems can directly regulate regional CBF. Central VP-ergic innervation of the brain microvasculature is also consistent with VP control of local vascular tonus

3. VP and regional uptake of nutrients at the blood–brain barrier that indirectly influence memory processing Hormonal VP acting on the lumenal side of the blood–brain barrier may regulate nutrient supply to the brain. This includes an AVP-induced increase in brain uptake of nutrient substances in certain structures (e.g., hippocampus) that indirectly enhance memory processing. For example, VP enhances uptake of orotic acid (RNA precursor needed for protein synthesis in the cerebrum) and of tyrosine (precursor needed for the synthesis of catecholamines). Orotic acid has been implicated in enhancing memory processing as have catecholamines

Armstead et al. (1989, 1990); Faraci et al. (1988); Katusic (1992); Katusic et al. (1984); Kim et al. (1988); Kozniewska and Szczepanska-Sadowska (1990); Suzuki et al. (1992); Takayasu et al. (1993); Tsugane et al. (1994); van Zwieten et al. (1988)

Albeck and Smock (1988); Cach et al. (1989); Itakura et al. (1988); Jojart et al. (1984); Pearlmutter et al. (1988); Ray and Choudhury (1990); Smock et al. (1987)

Ermisch (1992); Ermisch et al. (1982); Kretzschmar et al. (1986); Landgraf et al. (1978); Reichel et al. (1996); Ruthrich et al. (1982)

Oxytocin 1. Hormonal OT regulation of vascular perfusion of the brain and CBF a. Infusion of OT, as well as VP, into the vertebral artery of dogs dilated the major resistance vessels of the brain and constricted the smaller arteries and arterioles supplied by the vertebral artery. Moreover, this OT influence was mediated by a V1 receptor, because a V1 antagonist blocked the effect of both hormones

Suzuki et al. (1992)

(Continues)

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TABLE V (Continued) Influence

Ref.

b. OT, like VP, may regulate CBF during traumatic states (e.g., hemorrhagic hypotension, footshock). This may occur via an OT interaction with the endothelium, similar to that observed for VP (causes secretion of nitric oxide, which in turn dilates the large resistance arteries, thereby supplying adequate blood to the brain and normal CBF) 2. Neuronally released OT innervates brain microvessels: Influence on regional cerebral blood flow? OT terminals are present on large intracerebral arteries, where the peptide may regulate CBF in rats and in humans 3. OT regulation of nutrient exchange across the blood–brain barrier a. Hormonally released OT: Hormonal OT, like VP, enhances uptake of orotic acid (RNA precursor) across the brain capillary endothelium b. Centrally released OT: Central OT-ergic fibers, like VP-ergic fibers, may innervate brain blood vessels

Bari et al. (1987); Katusic et al. (1986); Kim et al. (1988)

Abrams et al. (1985); Muchlinski et al. (1988); Zimmerman et al. (1984)

Landgraf et al. (1977)

Landgraf et al. (1977)

brain and regulation of cerebral blood flow), and in the promotion of nutrient exchanges across the blood–brain barrier. In general, VP and OT help protect, especially under pathological conditions, an adequate supply of blood to the brain and normal regional blood flow. It is therefore possible that these peptides are also involved in directing local blood flow to memory processing brain sites and enhancing nutrient exchanges across the blood–brain barrier at these sites. Regional cerebral blood flow (rCBF), measured by positron emission tomography (PET) scanning, is not an infrequently used method of mapping the cerebral brain areas involved in various types of short-term and long-term MP (e.g., Bonda et al., 1996; Dade et al., 1998; Gur et al., 1993; Honda et al., 1998). 1. VP–OT Interactions with Extracerebral and Intracerebral Vasculature a. Roles of Extracerebral and Intracerebral Vasculature in Brain Perfusion and Circulation Cohen et al. (1996) have distinguished extracerebral from intracerebral blood vessels on the basis of their location, their neuronal innervation, and their functional roles in cerebral circulation. The extracerebral brain vessels are the large cerebral arteries that overlie the ventral and dorsal surfaces of the brain, and their ramifications as small pial vessels that run in the subarachnoid space (space filled with cerebrospinal fluid between

General Introduction to VP and OT

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arachnoid and pial brain coverings) and perforate the cortical mantle to the border of the Virchow–Robin space [the perivascular space formed by the sleeve of pia mater that accompanies the pial vessel into the brain tissue (parenchyma)]. These blood vessels are innervated by nerve endings whose cell bodies originate in the sensory or autonomic ganglia of the peripheral nervous system. Functionally, they are the resistance vessels in the vascular bed of the brain, ensuring an adequate supply of blood to the brain, and maintaining CBF within normal limits. The intracerebral (intraparenchymal) vessels are the microvessels (microarterioles, capillaries, and venules) that form the microvasculature of the brain. These microvessels are embedded within the brain substance below the Virchow–Robin border and are innervated by various neuronal systems, and their functions include the regulation of local blood flow in the immediate surround, as well as the capillary exchanges that occur between the brain and the blood. Peripheral and/or central neurons, as well as hormones, can interact with these vessels and thereby influence their functioning. Selected research literature examining the contribution of these neuropeptides to the functional roles of the extracerebral and intracerebral vasculature is referenced in Table V. The relevance of these functional roles for memory processing is briefly given below. b. Interaction with Extracerebral Vasculature: Homeostatic Regulation of Brain Perfusion Extensive experimental evidence suggests that hormonal VP, and perhaps OT (VP/OT), help maintain normal brain perfusion and cerebral blood flow (CBF). According to Katusic et al. (1984), this is accomplished by the following sequence of events: (1) increased VP/OT, produced in response to certain pathological conditions (e.g., hemorrhagic hypotension), interacts with the endothelium of large cerebral arteries to produce nitric oxide that dilates these vessels, reducing their resistance and increasing blood flow into the brain; and (2) the increased flow through these large resistance vessels results in an increase in blood pressure in pial vessels and this, in turn, causes an autoregulatory dilation of microvessels (arterioles, capillaries, and venules) that maintain normal CBF in the localized brain region. The VP/OT regulation described above, although helping to ensure overall maintenance of an adequate blood perfusion to the brain, and rCBF, is unlikely to be the mechanism of local shifts in blood flow that accommodate increased metabolic demands in brain sites engaged in information processing (including memory formation and retrieval). The VP (and OT) influence on the overall regulation of blood flow in the brain appears to occur only when plasma VP levels reach values that occur under severe threats to adequate brain perfusion (e.g., moderate to severe hemorrhage, shock, intracranial hypertension, or severe hypoxia) (Faraci et al., 1988).

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c. Interaction with Intracerebral Vasculature i. Regulation of local blood flow Local shifts in cerebral blood flow that occur in connection with nutrient and oxygen uptake in metabolically active brain sites (e.g., those engaged in memory formation or retrieval) depend on the microvasculature of the brain. It is also possible that in learning situations the transmitters released in the central neuronal systems innervating these small blood vessels might simultaneously modulate blood flow in the microvasculature serving these metabolically active brain sites. Previous researchers have (1) related brain function to the regulation of cerebral blood flow (Lou et al., 1987; Villringer and Dirnagl, 1995) and (2) provided support for a central neurogenic control of cerebral microcirculation (Cohen et al., 1996; Reis and Iadecola, 1989). The diameter of this microvasculature might be controlled directly by central VP-ergic and/or OT-ergic neural innervation, or indirectly by central VP and/or OT systems interacting with neurotransmitter output from one or more centrally projecting aminergic [e.g., serotonin (5-hydroxytryptamine 5-HT), dopamine (DA), noradrenaline (NA), and/or cholinergic (ACh)] systems that terminate on these microvessels (Cohen et al., 1996; Kalaria et al., 1989; Krimer et al., 1998; Vaucher and Hamel, 1995). A direct influence of these neurohypophysial peptides on brain microvessels is suggested by histochemical studies investigating the presence of binding sites or neuronal terminals of VP-ergic (e.g., Jojart et al., 1984; van Zwieten et al., 1988) and/or OT-ergic (Martin and Landis, 1981; Recht et al., 1982) neurons in close proximity to small cerebral blood vessels, and by an in vitro electrophysiological study suggesting a VP influence on both microvascular and neuronal activity in the hippocampus of the rat (Albeck and Smock, 1988; Smock et al., 1987). The histochemical studies have demonstrated (1) the presence of VPbinding sites in (a) endothelial cells of capillaries, especially dense in the hippocampus, striatum, and locus coeruleus, but much less so in the septum and cerebral cortex, and (b) endothelial cells and pericyte-like cells of larger blood vessels in the striatum and locus coeruleus of Brattleboro HODI (homozygous diabetes insipidus) rats (van Zwieten et al., 1988). Pericytes can contract, and may participate in regulating blood flow in small vessels (Stensaas, 1975); and (2) VP-ergic neural connections with brain microvessels in the rat are supported by immunoelectron–histochemical evidence obtained by Jojart et al. (1984). They found contact between VP cell bodies and processes (mainly dendrites) and small blood vessels in extrahypothalamic regions, and suggested that their findings provided ‘‘immunocytochemical evidence that vasopressinergic neuronal elements can directly innervate microvessels in the brain and thereby participate in regulating the local permeability of and the flow through the cerebral microvessels’’ (p. 259).

General Introduction to VP and OT

43

The in vitro study used electrophysiological methods to investigate the neuronal and microvascular effects of micromolar concentrations of AVP directly applied to brain microvessels in a hippocampal brain slice preparation from the rat (Albeck and Smock, 1988; Smock et al., 1987). This treatment both increased excitability of the sampled pyramidal neurons, and constricted the microvessels penetrating the tissue. These results are consistent with the thesis that VP has a role in regulating microcirculation in brain sites concerned with memory processing. The possibility that central VP-ergic and/or OT-ergic systems modulate memory processing by interacting with classic transmitter systems (i.e., NA, DA, 5-HT, and ACh) in their regulation of cerebral blood flow receives support from a number of lines of evidence: (1) each of these classic transmitter systems appears to influence cerebral blood flow (Bonvento et al., 1989; Cohen et al., 1996; Dacey and Bassett, 1987; Hertz, 1992); (2) central catecholaminergic, serotonergic, and cholinergic systems exert important influences in one or more phases of memory processing (acquisition, storage, or retrieval) (Beninger, 1983; Deutsch, 1983; Ferry et al., 1999; Hagan and Morris, 1988; Harley, 1987; Lee and Ma, 1995; Major and White, 1978; McEntee and Crook, 1991; Ploeger et al., 1992; Steckler and Sahgal, 1995); and (3) evidence discussed in subsequent chapters of this text suggests that the influence of VP and OT on memory processing may be mediated, at least in part, by interactions with classic neurotransmitter monoaminergic (Chapter 4), and cholinergic (Chapter 10) systems that project to the limbic structures (septal area, hippocampus, and amygdala), which have been implicated in mediating the influence of VP and/or OT in some phase of memory processing (Chapters 4 and 10). In summary, the observations reported above are consistent with the speculation that central VP-ergic and OT-ergic systems, acting on their own and/or by interacting with one or more aminergic or cholinergic neurotransmitter systems, may modulate (enhance or inhibit) memory formation and retrieval by neurogenically influencing microvascular blood flow and metabolism in memory-processing sites. ii. Nutrient exchange across the blood–brain barrier Ermisch and colleagues (e.g., Landgraf et al., 1977) have developed the view (discussed at length in Chapter 14) that hormonal VP and OT could contribute to memory processing by influencing the transport of required nutrients across the endothelium (blood–brain barrier) of capillaries perfusing memory-processing brain sites. The ability of VP to influence nutrient transport across the endothelium may be evolutionally related to its ability to influence water permeability and electrolyte exchanges across brain capillaries, which is its main action in the regulation of brain osmolality (see Table V). To date, the above possibility has been extensively investigated only in the case of vasopressin (Chapter 14).

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C. Actions of VP and OT in Nonneural Tissue Sites and Glucose Regulation: Role in Memory Processing 1. Functional Role for VP and OT in Glucose Storage and Utilization VP and OT actions in nonneural tissues and within the brain have an important role in glucose storage and utilization. VP promotes eating behavior and/or increased hepatic production of glucose after hypoglycemia (Baylis and Robertson, 1980; Keppens and de Wulf, 1974), and inhibits food intake in association with food satiation (Kow and Pfaff, 1986). Similarly, an OT neuronal system of PVN origin projects to brainstem ANS centers and regulates feeding, digestion, and metabolism (McCann and Rogers, 1990; Sofroniew and Schrell, 1981; Tribollet et al., 1988). This OT pathway appears to inhibit feeding and associated digestive processing in response to food satiation or gastric distress (Olson et al., 1991; Verbalis et al., 1986), but promotes feeding, digestion, and anabolic metabolism (tissue growth and fat storage) during pregnancy and lactation (Uvnas-Moberg, 1989, 1994). 2. Stress-Associated Actions of VP and OT at Nonneural Tissue Sites and Enhanced Glucose Availability to the Brain: Role in Memory Processing? In the context of the present discussion this author suggests that during stress VP and OT may directly or indirectly enhance glucose availability to the brain, which provides energy for brain sites engaged in neuronal activity mediating adaptive stress response, including any memory processing inherent in that responding. Relevant evidence is discussed below. a. VP and OT Action in the Pancreas and Liver: Direct Influence on Glucose Metabolism Evidence that peripherally circulating VP and/or OT influences glucose metabolism in the pancreas and liver has been obtained from in vivo and in vitro studies with a variety of species, including rats, mice, dogs, sheep, and humans (Altszuler et al., 1992; Gao et al., 1991, 1992; Mineo et al., 1997; Paolisso et al., 1988; Yibchok-Anun et al., 1999). These studies have shown that (1) VP and OT receptor-binding sites are present in pancreatic islet beta cells, which synthesize and release insulin (promotes glucose storage in liver and adipose tissue), and in pancreatic islet alpha cells, which synthesize and release glucagon, which promotes glucose release from storage and availability as energy fuel for metabolic activity both peripherally (e.g., muscles) and centrally (CNS); (2) peripherally applied VP and/or OT releases insulin from pancreatic beta cells (Gao and Henquin, 1993; Lee et al., 1995) and glucagon from pancreatic alpha cells (Mineo et al., 1997); and (3) peripherally circulating VP and OT can act at receptorbinding sites in hepatic cells, where they promote the breakdown of liver

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glycogen and, concomitantly increase the blood glucose level (Hems and Whitton, 1980; Hems et al., 1978). b. VP and OT Influences on Stress Hormones: Indirect Influence on Glucose Metabolism A VP/OT-mediated increase in blood glucose level during stress could occur (1) directly by a VP/OT hormonal action on glucose metabolism in the pancreas and liver as described above, or (2) indirectly by the glucosegenerating actions of glucocorticoids (Guyton, 1985) and adrenaline (Smythe et al., 1984). A number of physiological stressors (also known as homeostatic stressors) and emotional stressors release VP and/or OT from the posterior pituitary into the systemic circulation. Hormonally circulating VP and/or OT could, in turn, increase the blood glucose level by their actions in the liver and pancreas. The main stressors that release VP into the systemic circulation are homeostatic stressors such as hemorrhage (Plotsky et al., 1985a), hypoglycemia (Chiodera et al., 1992; Plotsky et al., 1985b), hyperosmolality (Dunn et al., 1973), and hypoxia (Kelestimur et al., 1997). Except for a highly emotionally/motivationally arousing noxious stimulus such as repetitive high-frequency footshocks (Onaka, 2000), less noxious emotional stressors do not release VP from the posterior pituitary lobe. On the other hand, hormonal OT is released into the systemic circulation by many of the homeostatic stressors that release VP (Gibbs, 1984, 1986a,b), and in addition by certain emotional stressors as well [e.g., conditioned fear stimuli, and intermittent low-frequency footshocks (Onaka, 2000), immobilization restraint (Gibbs, 1984; Lang et al., 1983), and forced swimming (Lang et al., 1983)]. A variety of stressors activate hypothalamic VP and/or OT cell groups that release hormones into the portal circulation, which in turn stimulate the pituitary–adrenocortical axis, and thereby the release of glucocorticoids from the adrenal cortex. The same or different stressors may activate VP and/or OT, synthesized within the adrenal medulla (Ang and Jenkins, 1984), which in turn promotes the release of adrenaline from the adrenal medulla. Evidence for a stress-associated VP and/or OT influence on the pituitary–adrenocortical axis, and on epinephrine release from the adrenal medulla in response to stress, is provided in a subsequent section of this discussion (Section IV. D.2). c. Relation between Glucose-Induced Enhancement of Memory Processing, Stress, and VP- and/or OT-Induced Release of Stress Hormones that Enhance Glucose Availability to the Brain Glucose-mediated enhancement of retention in a variety of aversive and appetitive learning paradigms has been demonstrated in the research studies of Messier, White, and colleagues. For example, posttraining injections of glucose enhanced retention on learned aversive and appetitive learning tasks in rats (Gold, 1986; Messier and White, 1987)

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and in mice (Messier and Destrade, 1988). Moreover, these learning paradigms involve some type of stress factor (e.g., footshock, food or water deprivation) that releases VP and/or OT hormones into the portal circulation, thereby activating the pituitary–adrenocortical axis and subsequent release of glucocorticoids. These stress factors may also activate the VP- and/or OT-induced release of adrenaline from the adrenal medulla. Taken together, these observations indirectly support the speculation that VP and OT actions on receptor sites in nonneural tissues (anterior pituitary gland and adrenal medulla) during stress provide a contribution to memory processing via an indirectly generated increase in glucose availability to brain sites engaged in such processing during stress.

D. Stress-Induced VP/OT Effects on Receptors in the Adrenal Medulla and Anterior Pituitary Gland: Relevance for Memory Processing Evidence cited below is relevant to yet another means by which VP/OT actions on nonneural tissue receptor sites may affect memory processing. Namely, it refers to the fact that during a stress response, these neurohypophyseal peptides release other stress hormones, which in turn may influence memory processing. These VP/OT effects derive from their actions at receptor sites in adrenal medullary cells that release epinephrine, or in cells of the anterior pituitary gland that release ACTH and -endorphin (an opioid). ACTH, in turn, stimulates the release of corticoids from the adrenal cortex. Relevant evidence is discussed below. 1. VP/OT Actions on Receptors in the Adrenal Medulla and Memory Processing Ang and Jenkins (1984) reported the presence of VP and OT in the adrenal medulla and noted that neither plasma levels, nor VP and OT derived from via hypothalamic PVN-activated sympathetic neural output, could account for their concentrations in this tissue. They suggested that these peptides are locally present in this tissue and may contribute to the regulation of catecholamine (adrenaline) output from this structure. Grazzini et al. (1998) provided supportive evidence indicating that AVP and OT are synthesized and released from the adrenal cortex and medulla where, in rats, they contribute to the release of aldosterone from the adrenal cortex, and of adrenaline from the adrenal medulla. Moreover, the peptideinduced release of adrenaline is mediated by V1a or V1b vasopressin receptors when activated under basal or stress conditions (Grazzini et al., 1998). McGaugh and colleagues have consistently demonstrated that peripherally circulating adrenaline modulates MP in a passive avoidance (PA) paradigm. In their more recent research studies (McGaugh, 2000; McGaugh

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et al., 1996; Williams et al., 1998), they have further clarified the mechanisms by which this catecholamine acts on central target sites that modulate the memory formation of emotional experiences, such as occurs in the passive avoidance paradigm. Their research findings are consistent with the suggestion that the epinephrine released by an action of VP at adrenal medulla receptor sites may mediate an indirect contribution of VP to memory processing. 2. Support for Stress-Associated VP and OT Influence on the Pituitary–Adrenocortical Axis a. Hypothalamic VP and OT Activation of the Pituitary–Adrenocortical Axis during a Stress Response The following lines of evidence support a role for hypothalamic VP and OT activation of the pituitary–adrenocortical axis during stress: (1) a subset of parvocellular cells in the PVN costores and coreleases AVP and corticotropin-releasing hormone (CRH) in response to certain stressors (Aguilera, 1994; Sawchenko et al., 1984, 1992). CRH is the major neurohormone of influence in the stress-induced release of ACTH and related stress hormones from the anterior pituitary gland (Plotsky, 1991; Stout et al., 1995); (2) VP and OT act synergistically with CRH in their influence on the anterior pituitary–adrenocortical axis in response to certain stressors (Antoni, 1986, 1993; Gibbs et al., 1984); (3) homozygous Brattleboro rats (genetically deficient in AVP) exhibit less than normal adrenocortical responses to a variety of physiological (McCann et al., 1966; Yates et al., 1971) and emotional stressors [e.g., restraint and handling (Wiley et al., 1974), and noise (Yates et al., 1971)]; (4) OT in portal blood vessels is present at concentrations that can potentiate CRH-induced ACTH secretion in vitro (Gibbs, 1985, 1986b). Portal blood vessels conduct pituitary releasing hormones from the neuroendocrine cell terminals in the median eminence to the pituitary cells that synthesize and release pituitary hormones; and (5) AVP, released into the portal blood by various physical stressors, is increased in parallel with ACTH in the general circulation (Gibbs, 1985; Plotsky et al., 1985a). There is little dispute concerning a VP influence on the release of ACTH and other pituitary stress hormones from the anterior pituitary gland in selected stress situations. Kjaer (1996), however, has more recently reviewed evidence that questions the physiological significance of a similar role played by OT during stress. b. Types of Stressors that Activate VP and OT Cell Groups Involved in the Regulation of Hormonal Release from the Anterior Pituitary Buijs and Van Eden (2000) have differentiated two types of stressors—homeostatic and emotional stressors—that activate those hypothalamic VP and OT cell groups that control neuroendocrine and autonomic nervous system effector activity, as well as neural processing in higher brain centers engaged in stress response. Homeostatic stressors are stimuli that threaten homeostatically

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regulated physiological functions. They are processed mainly within brainstem circuitry and reach their hypothalamic VP and OT target sites via prescribed brainstem pathways. Emotional stressors are emotionally and motivationally arousing stimuli, which are processed primarily in forebrain limbic circuitry, and reach their hypothalamic VP and OT target sites via selected brain pathways engaged in this processing. Homeostatic stressors that activate the pituitary–adrenocortical axis via the release of hypothalamic VP and/or OT into the portal circulation include insulin-induced hypoglycemia (Whitnall, 1989), hemorrhage (Plotsky, 1989; Plotsky et al. 1985a), food and water deprivation (El Fazaa et al., 2000), and hypertonic saline injections (Koob et al., 1985a). Emotional stressors that produce this effect include acute immobilization (Bartanusz et al., 1993; Murphy et al., 1995), long-term immobilization (De Goeij et al., 1992a), tail-hang stress (Gibbs, 1986b), presentation of repetitive footshocks (Onaka, 2000), shaker stress (Nishioka et al., 1998), social defeat (Engelmann et al., 2000; Wotjak et al., 1996), chronic social stress in highly structured rat colonies (De Goeij et al., 1992b), and encounters with stranger conspecifics in unfamiliar, but not familiar, settings (e.g., home cage) (Romero et al., 1995). c. Stress Hormones Released from the Anterior Pituitary and the Adrenal Cortex: Effects on Learning and Memory i. ACTH Numerous studies with intact rats have indicated that systemically administered ACTH improves learning or retention in tasks using positive reinforcers (Gray, 1971; Guth et al., 1971; Leonard, 1969; Murphy and Miller, 1955), and in those using footshock-motivated escape (Mirsky et al., 1953), active avoidance (Bohus et al., 1968; De Wied, 1966, Murphy and Miller, 1955), and passive avoidance [Leshner et al., 1975; Levine and Jones, 1965; Lissak and Bohus, 1972; Pappas and Gray, 1971; Roche and Leshner, 1979 (cited in De Wied and Jolles, 1982)]. Gold and van Buskirk (1976a) and Martinez et al. (1991) reported that in a passive avoidance (PA) paradigm, the influence of ACTH on memory depended on its level in the bloodstream, which in turn was influenced by the dose level of injected ACTH or the intensity of the training footshock (i.e., low levels of circulating ACTH enhanced, whereas high levels impaired, PA retention). These latter findings suggest that ACTH exerts a modulating influence on learning and memory and may also indicate a primary effect on the arousal system, which secondarily influences memory processing. ii. b-Endorphin The effect of peripherally circulating -endorphin on memory processing in tasks involving positive and negative reinforcers has been tested by pharmacological treatments that enhance or deter the effectiveness of the hormonal action on tested behavior (i.e., peripherally administered opioid agonists and antagonists, respectively). Opioid antagonists

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(e.g., naloxone and naltrexone) or agonists (morphine), peripherally administered, are nonselective in their effects on the different classes of opioids and influence the actions of enkephalins (secreted by the adrenal medulla during stress) as well as -endorphin (secreted by the anterior pituitary). A number of researchers reported that peripherally injected naloxone and naltrexone improved memory processing in a variety of learning tasks (Gallagher and Kapp, 1978; Messing et al., 1979), and that pretreatment with morphine blocked these effects (McGaugh and Gold, 1989). These findings suggested that opioids exert an amnestic action in memory processing. However, this conclusion may be premature, because other researchers using peripherally and or intracerebroventricularly injected opioids have reported memory facilitation (De Wied, 1977; Kovacs et al., 1981), attenuation of carbon dioxide-induced amnesia (Rigter, 1978), no effect (Bohus, 1986; Martinez and Rigter, 1980), or dose-dependent dual effects on memory retrieval (Bohus, 1980). Taken together, these discrepant findings indicate that although -endorphin influences memory processing, the specific nature of its influence remains to be clarified. iii. Corticosterone The adrenal glucocorticoid (cortisol in humans, corticosterone in rodents) enters the brain and activates two types of receptors: glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs). Both receptors are involved in information processing, but each influences a different aspect of this processing. MR activation appears important for evaluation of the stimulus situation and selection of appropriate response strategies, and GR activation is involved in short-term (Conrad et al., 1999) and long-term (Oitzl and de Kloet, 1992) memory processing. Although this stress hormone undoubtedly influences various types of memory, research attention has focused primarily on its role in hippocampus-dependent memory: short-term memory (STM) and/or long-term memory (LTM) involved in spatial orientation and navigation, and LTM assessed in avoidance learning paradigms. Experimental findings from this line of research inquiry indicate that (1) this stress hormone has an important role in mediating STM tested in the eight-arm radial maze (Luine et al., 1996), and in acquisition, storage (Oitzl and de Kloet, 1992; Roozendaal et al., 1996b), and retrieval (de Quervain et al., 1995) stages of LTM involved in spatial navigation of a water maze and in the step-through PA paradigm; (2) the degree and duration of the stressor are important parameters affecting adaptive performance in learning encounters. The former is directly related to the amount of glucocorticoid released into the circulation and hence the number of central GRs occupied by the hormone. Experimental studies cited by Roozendaal (2000) indicate that a moderate amount of administered glucocorticoid, or degree of imposed stress, facilitates PA memory consolidation, but extremely low or high levels impair it; (3) this curvilinear dose–response effect is in

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accord with the observation that memory processing is impaired by surgically induced depletion of circulating glucocorticoid (Oitzl and De Kloet, 1992; Roozendaal and McGaugh, 1996a,b; Roozendaal et al., 1996b,c), as well as by severe stress that results in the release of an inordinate amount of circulating glucocorticoid (Roozendaal et al., 1996a); (4) prolonged stress (continuous or repeated episodes over a long duration) can compromise hippocampus-dependent STM and LTM, and lead to atrophy in selected hippocampal neuronal structures (McEwen, 2001; McEwen and Sapolsky, 1995). This is demonstrated by the following experimental observations: (a) long-term restraint stress (6 h/day for 3 weeks) reversibly impaired spatial STM in a Y-maze, which paralleled the reversible stressinduced atrophy of dendrites of hippocampal neurons (Conrad et al., 1996; Luine et al., 1994), and (b) chronically injected corticosterone (3 months of stress-equivalent concentration of released glucocorticoids) impaired spatial memory tested in the Barnes maze (McLay et al., 1998); and (5) corticosterone activation of GR sites in the basolateral amygdala interacts with noradrenaline in its modulation of hippocampus-dependent memory processing (Roozendaal, 2000).

Barbara B. McEwen

Part II

De Wied and Colleagues I: Evidence for a VP and an OT Influence on MP: Launching the ‘‘VP/OT Central Memory Theory’’

I. Chapter Overview

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This chapter begins with a brief description of the active and passive avoidance tasks that were the primary paradigms used by De Wied and colleagues in their vasopressin (VP)/oxytocin (OT) research on memory processing. It then discusses early studies that led, and later studies that continued, to support the speculation that aside from their well-known actions as peripheral hormones, these neurohypophysial peptides have central nervous system effects that play a significant role in memory processing. More specifically, the studies reviewed herein have been interpreted as support for two major ideas about the roles of VP and OT in memory processing: (1) VP facilitates this processing, an effect that appears to be far more consistently observed during the stages of consolidation and retrieval, than during learning; and (2) OT serves as a natural amnestic that impairs memory consolidation and retrieval, at least in avoidance paradigms. The Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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chapter concludes with the formulation of these basic ideas as three theoretical propositions together with relevant evidence from the studies reviewed in this chapter.

II. Major Task Paradigms Used by De Wied and Associates in Their VP/OT Memory Research

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The behavioral pharmacological research conducted by De Wied and colleagues used laboratory rats trained and tested primarily in multitrial active avoidance tasks (pole jump task and shuttlebox task), and the single-trial passive avoidance task.

A. Multitrial Two-Way Active Avoidance Task: The Shuttlebox Task The two-way shuttlebox is an elongated conditioning box divided into two chambers by a central barrier, housed in a sound- and light-attenuating chamber lit by a single overhead house light (see Fig. 1). At the onset of each trial, the conditioned stimulus (a loud buzzer placed behind the shuttlebox) is presented and 10 s later it is accompanied by the unconditioned stimulus [footshock (FS) delivered through the grid floor]. The rat avoids the FS if it jumps over the barrier in the 10 s during which the buzzer is presented alone. If the crossing occurs while the grid floor is ‘‘hot,’’ the electric current is switched off and an escape response is scored. In both cases responding also terminates the buzzer. To avoid excessive exposure to FS, no trial is allowed to exceed 20 s. Each trial begins with the onset of the buzzer every 60 s. If the rat crosses the hurdle in the absence of the buzzer, an intertrial response is scored. Use of the two-way shuttlebox eliminates handling between trials and thereby reduces the potential influence of this source of experimenter bias. The training/testing procedure and peptide treatment schedule for this task is essentially the same as discussed for the pole jump avoidance task (described below).

B. Multitrial One-Way Active Avoidance Task: The Pole Jump Task In the pole jump task rats are trained to avoid an unconditioned stimulus (FS delivered through a grid floor at some specified intensity, e.g., 0.2 mA) by performing the avoidance response (jumping onto a ridged pole) within 5 s of the onset of the conditioned stimulus (illumination of the lamp located at the top of the pole) (see Fig. 2). The ridged pole enables the rat to maintain its position after the jump, thereby precluding multiple jump

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FIGURE 1 Shuttlebox footshock avoidance test. Source: Van Wimersma Greidanus and De Wied, 1977 (Fig. 1, p. 227). Copyright ß 1976 by Academic Press.

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FIGURE 2 Pole-jumping footshock avoidance test. Source: Van Wimersma Greidanus and De Wied, 1977 (Fig. 2, p. 228). Copyright ß 1976 by Academic Press.

responses. Failure to make the avoidance response results in an FS delivered through the grid floor. Once the avoidance response occurs, the light and FS are terminated. The rat is placed on the floor before each trial and is removed from the pole at the end of the trial. The maximal trial length is 5 s and the intertrial interval (ITI) is, on average, 60 s (King and De Wied, 1974). Typically, the rat receives 10 training trials/day for a designated number of days or until it reaches the learning criterion. In the former case, the subjects are tested for achievement of the learning criterion in the first 10-trial extinction session. Subjects failing to reach the learning criterion are discarded from the experiment. Extinction testing begins the day after acquisition training with 10 extinction trials/day for a specified number of

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days. The extinction procedure is the same as that for training except that the footshock is absent. Depending on the experimental protocol, a single treatment may be given (e.g., a single injection either after achievement of the learning criterion or before the first extinction session), or a chronic treatment regimen (two or more injections during either acquisition and/or extinction training) may be given.

C. Single-Trial Step-Through Passive (Inhibitory) Avoidance Task The task, described in Ader and De Wied (1972), is based on the speciesspecific preference of the rat for darkened as opposed to brightly lighted spaces. The apparatus, placed in a dark, sound-attenuated room, consists of a mesh-covered elevated runway, illuminated by a 25-W lamp placed over the center of the runway, attached to a guillotine-operated door of a Lucite box with black walls and a grid floor. On day 1 the rat is placed inside the dark box for 2 min and it is then given one or two trials in which it is placed on the platform, facing away from the door, and allowed to enter the box. The latency to enter the box is recorded. On day 2 the rat is placed on the platform, as on day 1, and reentry latency is recorded for each of three trials, separated by a 2-min intertrial interval. The rat remains in the box for 10 s. Entry into the box on trial three results in an FS (duration and intensity depend on the experimental design) delivered through the grid floor of the dark box. Subjects with an average response latency exceeding 30 s on the three trials given on day 2 are eliminated from further testing. Twenty-four hours after the single acquisition trial, the rat is placed on the platform and the reentry latency to the dark compartment is recorded. For this 24-h retention test, as well as for any subsequent retention tests, FS is omitted. The peptide is typically injected either immediately after the FS trial (memory consolidation design) or 1 h before the 24-h retention test (memory retrieval design). Depending on the protocol, a delay interval may be inserted between the acquisition trial and the peptide treatment.

III. Role for Vasopressin in Facilitating Memory Consolidation and Retrieval

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Research conducted primarily from the mid-1960s through the end of the 1970s laid the basis for assigning a role to VP in memory processing and for designating the basic nature of that role. Before performing behavioral studies on the posterior pituitary hormones VP and OT, De Wied had focused on the potential role played by the anterior pituitary hormone, adrenocorticotropic hormone (ACTH), in learning and memory. Despite

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turning to the roles played by VP and OT in this behavior, interest has been maintained in ACTH, its noncorticotropic fragment ACTH(4–10), and the related peptides -melanocyte-stimulating hormone (-MSH) and -MSH. As becomes evident in subsequent discussion, many studies on VP and/or OT by De Wied and colleagues have included experiments using ACTH-like peptides for comparisons. The interaction of these anterior and posterior pituitary peptides in many stress situations (Chapter 1) is a sufficient rationale for this common interest.

A. Avoidance Conditioning Paradigms: Selected Studies 1. Selected Studies a. De Wied (1965) De Wied (1965) reported on a series of experiments designed to investigate the effect of a posterior pituitary lobectomy (removal of the posterior and intermediate lobes), and the consequent loss of associated hormones, on the maintenance of a conditioned shuttlebox response in an inbred strain of male Wistar rat. The procedures for behavioral testing and treatment schedules were the same for all experiments using the shuttlebox avoidance task. After recovery from surgery, the experimental operates and the sham operates (controls) were trained on the shuttlebox task for 14 days (10 conditioning trials per day), using a progressively diminished intertrial interval procedure designed to increase resistance to extinction in normal rats (Murphy and Miller, 1956). Subjects that achieved the learning criterion (averaged 80% correct responses during the last 3 days of conditioning) were maintained for further testing, whereas the nonlearners were dropped from the experiment. Extinction training (10 trials per day), which began on day 15, was identical in procedure (except for absence of the FS) and ITI schedule to that used during training. Extinction testing was carried out for 9, 10, or 14 days. Depending on the experiment, placebo (solution medium) or longacting preparations of microgram quantities of one of the following replacement hormones was administered every other day throughout extinction testing: pitressin tannate suspended in arachis oil (10 IU/ml), purified lysine vasopressin (LVP, 300 IU/mg), ACTH (163 IU/mg), and synthetic -MSH. The test substance was subcutaneously injected 2 h before daily testing, beginning on the last day of conditioning and every other day throughout extinction testing. The first experiment tested the effect of posterior pituitary lobectomy on avoidance behavior and water intake, and the effect of pitressin tannate replacement therapy on extinction behavior. The results indicated that (1) the lobectomy produced diabetes insipidus, inducing average daily water intake two to three times that for the sham operates, and that pitressin tannate replacement therapy corrected the endocrine disorder; and (2) the

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lobectomy failed to alter acquisition but significantly increased the rate of extinction of the conditioned response relative to that for sham operates, and that pitressin tannate therapy prevented this abnormally rapid rate of response extinction. Pitressin is a relatively crude extract prepared from posterior and intermediate lobes, and therefore includes the posterior pituitary hormones (VP and OT) as well as the intermediate lobe hormone, -MSH. Experiment 2 examined the effect of using either purified LVP or synthetic -MSH on shuttlebox avoidance extinction in posterior pituitarylobectomized (posterior lobectomized) rats. Each of these peptides was given in two dose levels under the treatment schedule used in experiment 1. LVP significantly decreased water intake, and significantly inhibited the rapid rate of extinction observed in the placebo-treated posterior lobectomized rats. -MSH treatment produced the same behavioral effect as LVP, but did not correct the diabetes insipidus. Experiment 3 tested the time required to escape footshock in a simple runway task to determine whether depressed sensory and/or motor capacities may have caused the rapid extinction in the posterior lobectomized subjects. After 2 days of training, the subjects were tested for 24 days (five trials per day) to determine the time required to traverse a 240-cm-long runway to escape FS delivered through the grid floor. There was no significant difference in performance of this task between the posterior lobectomized and the sham-operated rats; both groups rapidly increased their escape speed to similar levels of asymptotic performance that were maintained throughout the 24-day observation period. Experiment 4 examined the effect of shuttlebox avoidance training on ACTH release in sham-operated and posterior lobectomized rats. For this experimental test, the plasma level of corticosterone was measured 10 min after completion of the 10-trial conditioning session on days 1, 4, 7, 10, and 13 of shuttlebox avoidance conditioning. Relative to the sham operates, the rise of plasma corticosterone during shuttlebox conditioning was significantly lower in the lobectomized rats. This result is consistent with other studies demonstrating that posterior lobectomy reduces ACTH release in response to emotional stress (De Wied, 1961; Smelik, 1960; Smelik et al., 1962). Experiment 5 demonstrated that ACTH treatment significantly increased avoidance responding during extinction on the shuttlebox task, but did not influence daily water intake. Experiment 6 tested the possibility that the anterior pituitary hormone, ACTH, mediated the effect of the intermediate/posterior pituitary hormones on avoidance response observed in experiments 1 and 2. In this experiment, the entire pituitary gland was removed (hypophysectomy), and the rats were maintained on thyroxine, cortisone, and testosterone replacement therapy to enable them to achieve the learning criterion on this task (De Wied, 1964). Independent groups of

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hypophysectomized subjects received placebo, LVP, or -MSH treatment during extinction. Unlike the placebo-treated rats, both LVP- and -MSHtreated rats maintained avoidance response during extinction (i.e., exhibited a significantly higher number of avoidance responses than did the placebotreated rats). Thus, ACTH did not mediate the behavioral effects of these posterior/intermediate lobe hormones. Experiment 7 tested whether ACTH has a critical role in maintaining the conditioned avoidance response. Shuttlebox avoidance behavior of anterior lobectomized rats was compared with that of the sham operates. Rats in the former group were maintained on cortisone, testosterone, and thyroxine to permit their avoidance conditioning. Eight of the 14 anterior lobectomized rats, and 8 of the 10 sham operates, reached the conditioning criterion and the rate of extinction was similar for both of the trained groups, indicating that ACTH was not critical for maintaining the conditioned avoidance response. It was concluded that the peptides present in the posterior lobe as well as the intermediate lobe of the pituitary gland are physiologically involved in maintaining, but not acquiring, the learned avoidance response. Moreover, this influence is independent of the roles of these peptides in releasing ACTH, which by itself is not critical for maintenance of this learned behavior. Because purified LVP may have been contaminated with other peptides, De Wied could not rule out the possibility that peptides not related to vasopressin were responsible for the behavioral effect observed. Finally, it was noted that the effect of these pituitary peptides on the maintenance of conditioned avoidance behavior points to a physiological function in the central nervous system, although it was acknowledged that this speculation required future experimental support. b. De Wied and Bohus (1966) De Wied and Bohus (1966) tested the effects of long-acting zinc phosphate preparations of pitressin tannate and synthetic -MSH on acquisition and extinction of a shuttlebox avoidance response in normal (physically intact) inbred Wistar male rats. This study was stimulated by observations that ACTH analogs administered during avoidance learning delayed extinction of the response (De Wied, 1966), and that either ACTH, -MSH, or pitressin, given during extinction, prevented the rapid extinction of the conditioned avoidance response observed in posterior lobectomized rats (De Wied, 1965). A major objective of this study was to investigate whether the similar influences of pitressin and -MSH on extinction occur by means of a common mechanism. Acquisition training, consisting of 10 trials/day, using an intertrial interval sequence designed to increase extinction response as described by De Wied (1965), continued until the subject attained the learning criterion of 80% or more avoidance responses in three consecutive daily sessions. The first extinction period began on the day after the rat attained the learning

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criterion and continued for 14 days (10 trials per day). A second extinction period began 21 days after completion of the first and lasted for 3 sessions (10 trials/day). Depending on the treatment group, the subject received an injection of pitressin tannate (1 IU/rat, subcutaneous), synthetic -MSH (10 g/rat, subcutaneous) as a long-acting zinc phosphate preparation, or placebo (the long-acting zinc phosphate complex by itself) either during acquisition or during the first extinction period. Rats treated during acquisition received the first injection 18 h before the first session and thereafter every other day until the learning criterion was achieved. Rats treated during the first extinction period had the first injection immediately after the last session of acquisition and thereafter every other day during the 14-day extinction period. When the two peptides were administered only during acquisition, neither affected the rate of acquisition, and only pitressin inhibited extinction of the conditioned avoidance response; that is, the pitressin-treated subjects did not exhibit response extinction, unlike those given -MSH. When the peptides were given throughout the first extinction period, both maintained the avoidance response, in contrast to the placebo controls. When tested in the second extinction period, at which time treatment had been discontinued for 3–5 weeks, extinction was still inhibited for the rats given pitressin (retention effect) but not for those given -MSH or vehicle during the first extinction period. Thus, only pitressin had a long-term effect on extinction behavior. The authors concluded that -MSH inhibits extinction (preserves retention) only during the course of treatment (when the peptide is present in the body), whereas the effect observed for pitressin persisted beyond the duration of its presence in the body. It was concluded that one or more of the peptides contained in the pitressin solution was instrumental in preserving the conditioned response, that is, in forming a long-term memory of the experience. c. De Wied (1971) De Wied (1971) compared the effects of various peptides on acquisition and extinction of a pole jump active avoidance response in intact rats. The peptides included ACTH(4–10), an ACTH fragment that lacks the adrenal cortical activity of the parent peptide (100 g/rat, subcutaneous); lysine vasopressin (1 g/rat, subcutaneous); oxytocin (1 g/rat, subcutaneous); and angiotensin II (1 g/rat, subcutaneous). The latter two were included because of their structural or physiological similarity to vasopressin. Because the preparation of long-acting vasopressin or oxytocin was not feasible, only the other two peptides were prepared for long-lasting activity. Experiment 1 tested the effects of a single injection of ACTH(4–10), lysine vasopressin (LVP), or physiological saline (placebo) on extinction of the pole jump avoidance response. Training on the pole-jump task consisted

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of 10 trials/day for 3 days with an ITI averaging 60 s. Those animals that made 10 or more avoidance responses during the 30 conditioning trials were selected for extinction training, which began on day 4. Rats that made 80% or more avoidance responses on the first day of extinction (10 trials/day) were used for further extinction testing. Independent groups of rats received a single injection of a given peptide immediately after the last trial of the first extinction session. Subsequent extinction sessions occurred 2, 4, 24, 48, and 72 h later. ACTH(4–10) significantly inhibited extinction in the 2- and 4-h extinction sessions whereas LVP significantly inhibited extinction in all extinction sessions. Experiment 2 tested the effects on extinction of a single injection of the following peptides: oxytocin (1 g/rat, subcutaneous), angiotensin II (1 g/ rat, subcutaneous), insulin (1 g/rat, subcutaneous), and growth hormone (1 g/rat, subcutaneous), using the same training procedure and injection schedule as for experiment 1. Extinction testing occurred 2, 4, and 24 h after the injection. None of these peptides, at this dose level, influenced extinction of the avoidance response. Experiment 3 tested the possibility of a critical period for the effect of LVP (1 g/rat, subcutaneous) on the maintenance of the avoidance response. Rats were injected with placebo (physiological saline) or the test solution either immediately, or 1 or 6 h after the last trial of the first extinction session. Extinction sessions were run 24 and 48 h after the first extinction session. The effect of ACTH(4–10) (100 g/rat, subcutaneous) was measured for comparative purposes. LVP inhibited extinction of the response when the injection was given immediately or 1 h after completion of the first extinction session, although the effect of the latter was not as complete as when LVP was immediately administered. At a delay of 6 h, LVP did not significantly influence extinction relative to placebo controls. As in the first experiment, ACTH(4–10) did not influence extinction at 24 or 48 h after injection. The results of this experiment indicated a critical period for the influence of vasopressin on extinction. Taken together, the results of this study led to the following conclusions: (1) vasopressin is the peptide in the pitressin extract that facilitated retention (i.e., prolonged extinction) of the conditioned avoidance response; (2) in contrast to the immediate and relatively brief effects of ACTH(4–10), the long-term effect of vasopressin on the maintenance of the avoidance response suggests an influence on memory consolidation; (3) the effect is highly specific because, at the same dose level, the structurally related peptide oxytocin did not prolong extinction of the avoidance response; and (4) preservation of the learned response appears not to be related to the effect of vasopressin on blood pressure or carbohydrate metabolism because, at the same dose level, neither the pressor substance, angiotensin II, nor insulin and growth hormone, which influence carbohydrate metabolism, influenced extinction of the avoidance response.

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d. Ader and De Wied (1972) Ader and De Wied (1972) used a passive (inhibitory) avoidance task to test the effects of vasopressin on memory processing in normal male inbred Wistar rats. The single-trial step-through passive avoidance (PA) task is well suited to dissociate the effects of an experimental treatment on memory consolidation and retrieval. The rats were trained with a 1-s footshock (FS) of either a low (0.125 mA) or a high (0.250 mA) intensity level. These FS parameters permitted sufficient latitude to observe either an increase or a decrease in reentry latency (Ader et al., 1972). Depending on the experiment, various doses of lysine vasopressin (LVP) or saline (placebo) were subcutaneously injected either immediately after the learning trial (consolidation design) or 1 h before the 24-h retention test (retrieval design). Depending on the experiment, reentry latency was tested 24, 48, and/or 72 h after the learning (FS) trial. Prolonged reentry latency (PA behavior) was interpreted as indicating retention of the avoidance learning. In experiment 1, the subjects received a 1-s, 0.250-mA FS and, depending on the group, a posttraining injection of saline or LVP (0.15, 0.49, or 1.35 g/rat, subcutaneous) or a preretention injection of saline or LVP (0.49 or 1.35 g/rat, subcutaneous). In addition, nonshocked subjects were used to test for nonspecific effects of LVP on reentry latency. Retention was tested both 24 and 48 h after the learning trial. Neither the salinetreated rats nor the LVP-treated nonshocked rats showed an increase in reentry latency. The rats that received the high-intensity FS increased reentry latency on both retention tests. Relative to saline controls, the subjects that received the two higher doses of LVP, either immediately after the FS trial or 1 h before the 24-h retention test, exhibited significantly higher reentry latencies on the 48-h but not the 24-h retention test. In experiment 2, the rats were tested with the low (0.125-mA) footshock intensity level and received posttraining placebo or LVP (1.35 g/rat, subcutaneous). The results indicated that, at this low FS level, LVP had little or no effect on reentry latencies, and although the LVP-treated rats showed somewhat longer reentry latencies than saline controls at the 48-h retention test, the high variability precluded statistical significance. Experiment 3 was conducted to determine whether the significant difference between the placebo- and LVP-treated groups observed in experiment 1 was due to the posttraining treatment, or represented a ‘‘cumulative effect of testing at 24 hr’’ (Ader and De Wied, 1972, p. 47). To this end, the subjects were tested at the 0.250-mA FS level, received a posttraining injection of placebo or LVP (1.35 g/rat, subcutaneous), and were tested for retention 48 and 72 h after the FS trial, without the intervening 24-h test. The saline- and LVP-treated animals showed preshock median response latencies of 7 and 7.2 s, respectively. When tested on the 48- and 72-h retention tests, the latencies for the saline group were 11 and 21 s, respectively, and for the vasopressin-treated group the latencies were 36.5

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and 102.0 s, respectively. However, because of the small number per group, these differences were not statistically significant. Nevertheless, these findings were interpreted as indicating that the differences in PA response between the LVP- and saline-treated subjects were due to the posttraining treatment, and not to effects associated with the intervening 24-h test. In conclusion, this study extended the findings that vasopressin induced long-term strengthened maintenance of a conditioned avoidance response in multitrial active avoidance paradigms (De Wied, 1965, 1971; De Wied and Bohus, 1966) to the single-trial passive avoidance paradigm. In addition, the results of this study support the proposal that the effects of vasopressin on avoidance behavior reflect the ability of the peptide to facilitate both memory consolidation and retrieval. e. Bohus and Colleagues (1972) Bohus et al. (1972) investigated the temporal effects of LVP treatment on retention. As De Wied (1971) demonstrated, vasopressin treatment delayed 6 h after training is no longer effective in facilitating long-term maintenance of that response. Bohus et al. (1972) also investigated whether the effect of a pretraining injection of LVP on retention of an avoidance response learned when the individual is under maximal influence of the peptide treatment will generalize to facilitate retention of a second avoidance response learned 6 h later. This question regarding the generality of the effect of LVP on behavior was stimulated by the report by Nash (1971) that vasopressin is released in response to specific and nonspecific stimuli and emotional stress. The observation that LVP prolongs extinction of a conditioned passive response (Ader and De Wied, 1972) as well as an active avoidance response (De Wied, 1971) made it possible to study generalization effects of vasopressin across two fear-motivated learning situations in which only one of the learning tasks is presented under the maximal influence of the peptide treatment. Male rats of an inbred Wistar strain were trained and tested on either or both (depending on the experiment) the pole-jump active avoidance (AA) task and the single-trial step-through passive avoidance (PA) task. Training on these tasks was separated by a 6-h interval between morning and afternoon training sessions. Pole-jump acquisition training was given daily for 3 days (10 trials/day) with ITIs averaging 60 s. Extinction training (10 nonreinforced extinction trials/day) occurred in the two successive days following the completion of learning. Subjects that failed to meet the learning criterion (five or more avoidances on day 2 of training) were dropped from the study. PA training occurred over 2 days, with FS occurring once the subject entered the dark (shock) box on trial 3 of day 2. ITIs for PA training were approximately 2 min. Animals with a response latency of 30 s or more on any of the three trials given on day 2 were eliminated from the study.

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Retention was tested 24, 48, and (in one experiment) 72 h after the FS trial, with reentry latency recorded to a maximum of 300 s. In experiment 1, two independent training groups were formed with one group trained on the pole-jump task in the morning, and the other in the afternoon. The subjects in both training groups received a single subcutaneous injection of placebo (saline) or synthetic LVP (1 g/rat) on day 3 (last day of acquisition training). Depending on the training group, the injection was given 1 h before the morning session, or 6 h before the afternoon session. The results of experiment 1 indicated that rats trained on the AA task and injected with LVP 1 h before the final acquisition session showed significantly more avoidance responses than saline controls on both day 4 and 5 of AA extinction. However, there were no differences in extinction between the LVP and saline-treated subjects tested in the afternoon, that is, 6 h after treatment. There was no effect of training time per se on retention, that is, saline controls trained and tested in the morning did not differ in extinction behavior from those trained and tested in the afternoon. In experiment 2, two groups of rats were trained on the PA task, either in the morning or in the afternoon session. A single subcutaneous injection of LVP (1 g/rat) or placebo (saline) was given on the morning of day 3, at either 1 or 6 h before the 24-h retention test (i.e., 23 or 18 h after the FS trial). A second retention test was run on day 4 (48 h). The results of experiment 2 indicated no significant increment in reentry latencies after the FS trial in the saline-treated subjects relative to preshock latencies. Significant and substantial increments in reentry latencies in subjects given LVP 1 h before the 24-h retention test, occurred for both retention tests, whether reentry latencies were compared with preshock data or with reentry latencies of the saline controls. Subjects given an LVP injection 6 h before the 24-h retention test did not differ in PA retention from saline controls. Again, training time per se did not influence PA retention. In experiment 3, the subjects were trained in both the AA and PA tasks. Half the subjects were trained on the AA task in the morning session, and the PA task in the afternoon; the remaining subjects received the reversed training regimen. On day 3 of the experiment, the subjects received either placebo (saline, subcutaneous) or synthetic LVP (1 g/rat, subcutaneous) 1 h before the morning session. Subsequently the subjects were run in the afternoon session, 6 h after the injections, and tested for extinction on days 4 and 5. The results of experiment 3 indicated that (1) when LVP was injected 1 h before the final acquisition session on the AA task, the LVP-treated rats made significantly more avoidance responses than saline controls on both days of extinction (Fig. 3A, left). No such difference was found for reentry latency on the PA retention task given 6 h later; neither LVP- nor salinetreated subjects exhibited increased reentry latency during retention test

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FIGURE 3 Effects of a single injection of LVP on active and passive avoidance behavior. The rats were trained in active avoidance in the morning session and then in passive avoidance in the afternoon (A) or in passive avoidance in the morning and active avoidance in the afternoon (B). The arrows indicate the time of injection of LVP (solid lines) or saline (broken lines). Source: Bohus et al., 1972 (Fig. 1, p. 195). Copyright ß 1972 by Academic Press.

trials (Fig. 3A, right); and (2) when LVP was injected 1 h before the first PA retention test, a significant increase in reentry latency relative to saline controls was observed (Fig. 3B, left). Conversely, the AA behavior of the LVP-treated and saline-treated rats did not differ when studied in the afternoon (Fig. 3B, right).

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One interpretation of the results of this study was that whether LVP treatment prolongs resistance to extinction on an AA task depends on the interval between the final acquisition session and the vasopressin treatment. Taken together, the findings of De Wied (1971) and those of experiment 1 suggest that treatment delayed as much as 6 h before or after the final avoidance training session will not influence AA extinction behavior. A second interpretation was that a similar temporal relationship may hold for the PA task, as suggested by the findings in experiment 2. However, the failure of the saline controls to show an increase in reentry latencies after the FS trial raises the question of whether the FS intensity in this study was sufficient to promote PA conditioning, despite the fact that this FS level was successful in PA conditioning in previous studies (i.e., Ader and De Wied, 1972). A third interpretation, derived from the findings of experiment 3, is that the influence of vasopressin on avoidance behavior is specific for the response occurring during the period of optimal peptide influence and does not generalize to other fear-motivated behaviors. Presumably there was an insufficient amount of the peptide present in the CNS 6 h after treatment when a different training task was introduced. f. King and De Wied (1974) King and De Wied (1974) designed several experiments (1) to help identify the onset of the long-term effects of LVP by determining the point during training when the long-term effect of the peptide on extinction can occur, and then (2) to help dissociate the operant and respondent components of the conditioned avoidance response in acquisition, and to establish whether either component may act as a substrate for the long-term effects of vasopressin on extinction. Male inbred Wistar rats were trained and tested for extinction in a pole-jump avoidance task. Depending on the treatment groups, the subjects received synthetic LVP (1 g/rat, subcutaneous) or an equal volume of placebo (physiological saline, subcutaneous). The timing of the treatments varied within and between experiments. Experiment 1 tested whether LVP, given during acquisition training, can maintain a learned avoidance response even if the peptide is systemically absent when the subject makes the first avoidance response. If it cannot, how many avoidance responses must be emitted while the peptide treatment is still effective, to result in a treatment retention effect? For this experiment several groups of rats were tested, and the probability of emitting at least one successful avoidance response in the presence of LVP was systematically varied. In experiment 1a, four groups of LVP-treated subjects and four groups of saline-treated subjects were given 10 training trials on day 1 with a 6-h delay between the initial and later block of training trials, selected because previous findings (De Wied, 1971) indicated that posttraining, LVP exerts no influence on retention (response extinction) at this delay interval. It was

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reasoned, therefore, that any effect of the peptide should be limited to those trials given 1 h after injection. For the 1:9 group, the delay was between trial 1 and the remaining nine trials. For the remaining three training groups, the delay occurred after the third (3:7 group), fifth (5:5 group), or tenth (10:0) acquisition trial. The single LVP or saline injection was given 1 h before the initial block of training trials. The subjects in each group were brought to the test room after the 6-h delay, but the 10:0 group received no further acquisition trials on day 1. A 60-s ITI was used on each day of acquisition training (10 trials/ day). No injections were given on days 2 and 3 of acquisition or on the two days of extinction testing (days 4 and 5, 10 trials/day; ITI ¼ 60 s). Experiment 1b was carried out to replicate and extend the findings of experiment 1a and used the identical procedure, with the single exception that the animals were trained in the first block of acquisition trials up to the first correct avoidance response before the 6-h delay was imposed. The results of experiments 1a and 1b indicated no difference between any of the saline- and LVP-treated groups, either in number of trials to the first correct avoidance or total number of correct avoidances during avoidance learning. There were no significant differences between any of the LVPand saline-treated subjects on the first extinction session. However, LVP treatment significantly enhanced resistance to extinction on the second extinction session for all training groups except the 1:9 group. Moreover, the greater the number of acquisition trials given before the 6-h delay, the greater the resistance to extinction. Because the subjects in groups 3:7 and 5:5 had not made any avoidance response in the presence of LVP, it was reasoned that the conditioned stimulus–unconditioned stimulus (CS–US; light–FS) pairing elicited a classically conditioned fear response that may have acted as a weak behavioral substrate mediating the LVP-induced extinction effect observed in these groups. This possibility was tested in a subsequent experiment (experiment 3). Experiment 2 investigated whether the long-term effect of LVP on extinction requires that LVP be present at the time a successful avoidance is made or whether the effect will occur if LVP is injected after the response has been made. In experiment 2a, four independent groups were formed. After the first avoidance response on day 1 of acquisition, groups 1 and 2 received LVP or saline, respectively, without delay, and groups 3 and 4 received these treatments after a 6-h delay interval. On day 2 the subjects received 10 additional acquisition trials plus the remaining trials that they failed to receive on day 1 to make up the initial 10-trial session. A final 10 acquisition trials were given on day 3, the last day of training. Extinction trials (10/day) were given on days 4 and 5. In experiment 2b, four groups were formed, with two groups receiving a single injection of LVP immediately after the third or fourth trial, and two control groups receiving a saline injection after these trials. The remainder of

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the trials needed to make up 10 trials for day 1, and an additional 20 acquisition trials, were equally distributed across days 2 and 3 of acquisition training. Extinction trials (10/day) were given on days 4 and 5. LVP treatment did not influence avoidance learning relative to the saline controls for any of the groups tested in experiments 2a and 2b. In experiment 2a, subjects treated with LVP immediately, but not 6 h after making the first correct avoidance response, exhibited prolonged extinction relative to the saline controls (i.e., showed no tendency for response extinction in session 2 of extinction, whereas the saline controls and the subjects treated with LVP 6 h after the correct avoidance response did extinguish). In experiment 2b, subjects treated with LVP immediately after either the third or fifth acquisition trials, in the absence of a correct avoidance response, exhibited a somewhat greater degree of resistance to extinction than the saline control groups, but these differences were not statistically significant. Again, classic conditioning may have served as the substrate mediating the slight LVP extinction effect observed in experiment 2b. Experiment 3 investigated the role of classical conditioning in mediating the LVP-induced extinction effects observed in experiments 1a and 2b. Subjects received 10 trials of classical conditioning (light–FS pairings) without the opportunity to make pole-jump avoidance responses (i.e., pole absent); light and FS terminated together on each trial and an ITI of 60 s was used. LVP (1 g/rat) or saline was injected subcutaneously, 1 h before the classical conditioning trials. This classical conditioning experience was followed, 24 h later, by 3 days (10 trials/day) of normal pole-jump avoidance training and thereafter by 2 days (10 trials/day) of extinction. In this experiment, LVP treatment before classical conditioning reliably enhanced subsequent acquisition of the pole-jump response, an influence more prominent in the earlier than in the later sessions of learning. This LVP treatment was also effective in maintaining the later-learned instrumental avoidance response, as indicated by a greater degree of avoidance response in the LVPtreated than in the saline-treated subjects in session 2 of extinction. These findings indicated that classical conditioning can serve as an effective substrate mediating the long-term behavioral effects of LVP. However, the authors noted that comparisons between the extinction data for experiment 1a and 3 suggest that classical conditioning alone is less effective than instrumental conditioning (both components present) as a substrate mediating the effect of a peptide on retention (prolonged extinction). Experiment 4 tested the effects of LVP treatment on extinction of a polejump avoidance response, when the treated subjects were given a series of CS-only/response prevention trials interpolated between avoidance training and the extinction test. This procedure, during which the FS and the central pole are absent, typically accelerates extinction of the avoidance response (Berch and Paynter, 1972), presumably because the subject, forced to remain in the situation, learns that the CS (light) is no longer followed by the US (FS)

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(Seligman and Johnston, 1973). In this experiment, the subjects received 3 days (10 trials/day) of pole-jump avoidance training and were formed into four independent groups. Groups 1 and 2 received a subcutaneous injection of LVP (1 g/rat) and physiological saline, respectively, 6 h after the completion of avoidance training and 1 h before the CS-only/response prevention trials. The response prevention trials consisted of 10 presentations of CS (5 s of light) with ITIs of 60 s. Groups 3 and 4 were similarly treated with LVP and saline, respectively, but returned to their home cages immediately thereafter without response prevention trials. The next day extinction testing was initiated and carried out in two consecutive days (10 trials/day). The results of experiment 4 were as follows: (1) for the saline-treated subjects, comparisons between those receiving and not receiving response prevention trials indicated that response prevention accelerated response extinction in both sessions, although it was statistically significant only for session 1; (2) however, for subjects given the response prevention trials, comparisons between the LVP- and saline-treated subjects indicated that LVP treatment increased resistance to extinction (statistically significant in session 2); and (3) in addition, comparisons between the LVP- and salinetreated subjects that had not received response prevention trials indicated no significant differences in resistance to extinction for either extinction session. Several conclusions can be drawn from this study: (1) some degree of associative strength must be present for vasopressin treatment to increase long-term maintenance of the learned behavior. This was indicated by the findings that the later in the 10-trial acquisition session the 6-h delay was inserted on ‘‘the day’’ of acquisition, the stronger was the effect of LVP on extinction behavior (experiment 1a); in addition, the effect of LVP on retention occurred even when treatment was restricted to the first emission of the avoidance response (experiment 1b); (2) the classically conditioned component of avoidance conditioning is by itself a sufficient substrate mediating the effect of LVP on extinction; however, it is not as effective as the instrumentally conditioned avoidance response, which includes both the classical and instrumental components (experiment 3); (3) this study confirmed time-dependent effects previously observed for the influence of vasopressin on retention (extinction) (Bohus et al., 1972; De Wied, 1971). These observations indicated that the closer in time the vasopressin treatment is to the learning experience, the more effective is the peptide in prolonging the learned behavior. Thus, LVP effectively prolonged avoidance extinction when injected 1 h before, or immediately after, the learned behavior, but not when the injection was 6 h before that behavior (experiments 1a, 1b, and 2a); (4) the accelerated extinction effect after response prevention trials in the saline-treated subjects was consistent with the proposal that the interpolated trials permitted the new learning that the CS (light) would not be followed by the US (FS), and confirmed previous findings with this experimental technique (Bersh and Paynter, 1972); and

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(5) the authors suggested that the failure of LVP to accelerate extinction in subjects given the response prevention trials might indicate that the peptide has no important role in facilitating the short-term memory processing involved in the new learning occurring during this interpolated experience. Whatever the basis for this effect, a subsequent replication study, with the exception that LVP was given after rather than before the interpolated experience, obtained the same results (Hagan, 1982; discussed in Chapter 9). g. Hagan and Colleagues (1982) Hagan et al. (1982) investigated timeand dose-dependent effects of posttraining treatment with LVP on shuttlebox avoidance extinction in adult males of an inbred strain of Wistar rat. Experiment 1 examined the effects of five posttraining doses of LVP on response extinction. The subjects were returned to the home cage immediately after reaching the learning criterion (10 consecutive correct avoidance responses) and 30 min later received a single subcutaneous injection of placebo or LVP (0.11, 0.33, or 0.99 g/rat), Two additional dose levels, 0.036 and 2.97 g/rat, were given in a second independent phase of the experiment. Two measures of extinction responding were used: the number of avoidance responses and the number of intertrial shuttle responses. The effect of the five treatments, relative to the saline controls, on these measures is diagrammed in Fig. 4. The mean avoidance response levels (Fig. 4, left) and intertrial response levels (Fig. 4, right) are expressed as a percentage of the saline controls for each dosage group. Relative to saline

FIGURE 4 The effects of ﬁve doses of LVP, injected subcutaneously 30 min after training, on extinction response 24 h later. Mean response levels for LVP-treated rats are expressed as a percentage of their saline controls. þ, p ¼ 0.05; þþ, p ¼ 0.01; ANOVA followed by Neuman– Keuls test. Source: Hagan et al., 1982 (Fig. 1, p. 217). Copyright ß 1982 by Academic Press.

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controls, avoidance responding during extinction was significantly increased by the intermediate dose levels (0.11, 0.33, and 0.99 g) and significantly reduced by the lowest dose (0.036 g) and the highest dose (2.97 g). The 0.11-g dose was most potent in inhibiting extinction (subjects treated with this dose level made significantly more avoidances than those treated with either the 0.33- or 0.99-g dose), and was the only dose level to significantly increase intertrial responding. Of those that decreased avoidance responding, the 2.97-g dose level was significantly more effective than the 0.036-g dose level in facilitating extinction of the learned response. The results of this experiment indicate an inverted U-shaped dose– response function for extinction of the avoidance response after posttraining vasopressin at a 30-min delay interval. Gold and Van Buskirk (1976a) observed a similar inverted U-shaped dose response curve relating ACTH to passive avoidance behavior and, in addition, noted a strong interaction between dose and FS levels (i.e., a high dose facilitates retention at a low FS intensity but disrupts retention at an intermediate or high FS intensity, whereas a low dose facilitates retention at both a low and intermediate FS level); they suggested that ACTH treatment may modulate normal hormonal responses to FS, thereby mimicking the effect of the higher FS level during training. For example, FS during PA training increases plasma levels of catecholamines (epinephrine and norepinephrine) (Gold and McCarty, 1981). The possibility that the interaction of vasopressin with FS levels may be similar to that proposed for ACTH was tested in experiment 2, which presented low and high dose levels of LVP under a higher FS intensity than that used in experiment 1. The training and treatment procedures in experiment 2 were identical to those for experiment 1, except that the FS level was increased from an intensity of 0.15 to 0.45 mA and, depending on the treatment group, the subjects received a single posttraining injection of placebo or one of two dose levels (0.11 or 2.97 g) of LVP. The results replicated the findings in experiment 1. Relative to the saline controls, the 0.11-g dose of LVP significantly increased, whereas the 2.97-g dose level significantly decreased, avoidance response during extinction. Intertrial response, which was almost totally suppressed during extinction, was not affected by the peptide treatments. Because the effectiveness of the low and high dose levels of LVP on avoidance response remained essentially the same when the subjects were trained with either high or low FS intensities, it was thought unlikely that LVP mediated its effect on extinction by altering the hormonal consequences of FS. This conclusion was considered tentative, however, until peripheral catecholamine levels are directly measured after posttraining VP treatment in an avoidance paradigm. Time-dependent effects were tested with the 0.11- and 2.97-g dose levels of LVP in experiment 3. The training and treatment procedure was the

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same as in experiment 2, except that placebo or the peptides were injected either immediately or 60 min after training. The results were as follows: (1) when treated immediately after training, the subjects receiving the 0.11-g dose of LVP made significantly more avoidance responses during extinction than those receiving either saline or the 2.97-g dose of the peptide; moreover, the high dose of the peptide did not affect avoidance responding relative to the saline controls; (2) when treated 60 min after training, the subjects given the low (0.11 g) and high (2.97 g) dose levels both significantly increased avoidance responding during extinction relative to saline controls, and there were no significant effects on intertrial responses. Figure 5 shows the avoidance and intertrial responses made during extinction testing when the two dose levels were given 0, 30, or 60 min after training. These findings indicated that the lower (0.11 g) dose of LVP prolonged extinction when injected within 1 h of completion of training, although this dose was most effective when given at the 30-min delay interval. In contrast, the effect of the higher (2.97 g) dose of LVP on avoidance extinction depended on the time of injection, exerting no effect when given immediately after learning, facilitating extinction when given 30 min after training, and prolonging extinction when given 60 min after training. The authors concluded that the interaction between dose level and injection interval requires an explanation more complex than that the peptide simply facilitates or inhibits memory consolidation. Taken together, the results of this study (1) further supported a vasopressin facilitation of retention (prolonged extinction) in aversive learning

FIGURE 5 The effects of 0.11 and 2.97 g of LVP, injected subcutaneously 0, 30, or 60 min after training, on extinction responding 24 h later (see Fig. 4 for explanation). þþ, p ¼ 0.01. Source: Hagan et al., 1982 (Fig. 2, p. 223). Copyright ß 1982 by Academic Press.

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situations; (2) indicated an inverted U-shaped dose–response curve similar to that observed for ACTH analogs in aversive paradigms, although the underlying mechanism appears to differ for the two peptides; and (3) revealed time-dependent effects that complexly interact with low and high dose levels of the peptide. Evidence that vasopressin is involved in releasing corticotropin in response to certain stressors (Gillies and Lowry, 1979), and interacts with brain monoaminergic neurotransmission (Kovacs et al., 1980a,c; see discussion in Chapter 4), suggested that a vasopressin interaction with other neuropeptides of brain and/or pituitary origin may be of significance for the complex interactions observed in this study.

B. Appetitive Learning Paradigms 1. Selected Studies a. Garrud and Colleagues (1974) Garrud et al. (1974) designed several experiments to examine the effects of various ACTH analogs [ACTH(1–39), ACTH(1–24), and ACTH(1–10)], corticosterone, and the neurohypophysial peptides, desglycinamide lysine vasopressin (DG-LVP) and OT, on retention of a food-rewarded runway response. The ACTH experiments are not discussed here except to note that each of the ACTH analogs, given during extinction testing, increased the maintenance of the learned runway response relative to placebo controls as they do in FS avoidance paradigms, and that this ACTH effect is independent of its adrenocortical hormonereleasing function (De Wied, 1966). Two experiments examined the effect of the neurohypophyseal hormones on retention of this learned runway response. In the first experiment, the subjects were subdivided into four independent groups and respectively received: placebo, DG-LVP (2 g/rat, subcutaneous), or OT (2 g/rat, subcutaneous) as chronic treatment (1 h before each day of extinction testing), or DG-LVP (2 g/rat, subcutaneous) as a single treatment (1 h before day 1 of extinction testing). The DG-LVP treatment effect was retested in experiment 2. The dependent measure was alley-running time (i.e., time from the opening of the start box door until entry into the goal box). Before assignment to the various treatment groups for extinction testing, the subjects were matched for average asymptotic running time for the last day of the 5-day acquisition period (8 trials/day). Extinction testing (6 trials/day for 4 days) began the following day. Neither VP nor OT influenced extinction under any treatment regimen used during extinction testing, and the lack of a vasopressin effect on extinction was replicated in the second experimental test. The failure of VP to influence extinction on this appetitive task was inconsistent with previous findings, indicating the ability of the peptide to retard extinction on active and passive avoidance tasks (Bohus et al., 1972; De Wied, 1971).

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Moreover, the effects of the peptide on avoidance behavior occurred with smaller dose levels than those used in the present study. Bohus gave a possible rationale for this negative finding in the discussion of his findings on a sexually motivated discrimination task with the same peptide analog (described below). b. Bohus (1977) Bohus (1977) examined the effect of DG-LVP (lacks the endocrinological effects of the parent peptide) on acquisition and retention of a sexually rewarded spatial discrimination response in a T-maze apparatus, using male Wistar rats. The T-maze contained a start box, a runway, and left and right L-shaped arms at the end of which were the goal boxes. The shape of the arms prevented any view of the goal compartments from the choice point. Plastic sliding doors separated the start box, the arms, and the goal compartments. Each goal box was divided into two identical compartments by a wire mesh sliding door. Three goal box groups were run: (1) the female copulation reward group, where the males were allowed to copulate with the receptive female rat; (2) the female noncopulation group, where the males were prevented from contacting the female by a wire mesh partition; and (3) the male goal group, where a male of similar age and weight to the female was placed in the goal box. The procedure had four phases. In phase 1, the males in the female copulation group (CP) were permitted to copulate with a receptive female in one of the two goal boxes. The males in the remaining goal box groups were placed in the other goal box with the female behind the partition (female noncopulation group, NCP) or with a male (male goal group, MG). Phase 2 provided a test for goal preference by means of three runs in the maze during which the goal box was empty. Phase 3 consisted of 4 days of acquisition training (1 session/day, 4 trials/session). All trials of the first session, and trials 2 and 3 of the remaining three sessions, were forced choices [one of the two sliding doors was closed, forcing the rat to run to the correct (nonpreferred) goal box, which contained the goal animal]. Subjects in the CP group were allowed to copulate until the first ejaculation occurred or were removed after 5 min; incorrect choices during the free choice trials resulted in confinement in the empty goal box for 5 min. The rats in the other two groups remained in the goal box for 5 min even after making a correct choice. The 20-min ITI was spent in waiting cages outside the test room. Phase 4 consisted of 2 days of retention testing (1 session/day, 4 free trials/session); the procedures were the same as that of the free trials during the acquisition phase. A chronic treatment regimen was used during acquisition training in which the subjects in each goal box group received a single subcutaneous injection of either DG-LVP (1 g/rat) or physiological saline immediately after the last trial of each daily session. The dependent measures were (1) the total number of correct choices during acquisition (free trials) and retention

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and (2) response running time (i.e., time from opening of the start box until the entry of the rat into the goal compartment). The choice behavior results were as follows: (1) choice behavior was affected by the training conditions. As acquisition training progressed an increasing percentage of sexually rewarded (CP) rats displayed correct choice behavior, whereas the nonrewarded (NCP and MG) rats failed to show a regular choice pattern and the percentage of these subjects displaying a correct choice rarely exceeded 50%; (2) DG-LVP treatment significantly enhanced retention but not acquisition in the rewarded (CP) group. Although the peptide-treated rats did make slightly more correct choices than their saline controls during acquisition training, this did not reach statistical significance; and (3) DG-LVP did not influence choice behavior in the sexually nonrewarded (NCP or MG) groups during either acquisition training or retention testing. The results for the response running times were as follows: (1) running time was related to the training condition. CP rats, constant in their running times, always ran faster than NCP and MG rats. In contrast, as training continued, running times progressively increased among the NCP and MG rats, as indicated by a significant training by trial interaction for this measure in both groups during forced and free trials; and (2) there was no DG-LVP influence on running time during acquisition or retention for any of the three goal box groups. These results were interpreted as follows: (1) the facilitation of retention by DG-LVP was a long-term effect (i.e., lasted beyond the actual presence of the peptide within the body), and this replicated and extended earlier findings by De Wied and colleagues with active (Bohus et al., 1973; De Wied, 1971; De Wied and Bohus, 1966) and passive (Ader and De Wied, 1972) avoidance tasks; (2) learning this discrimination depended on the presence of the sexual reward. In this connection, DG-LVP influenced correct performance level during the retention test only in the CP group, indicating that the daily posttraining injections of DG-LVP facilitated retention of what had been learned during that session, and its better maintenance during retention testing; (3) although not commented on by the author, a ‘‘memory consolidation effect’’ produced by the daily postacquisition peptide treatments may have contributed to the modest, although nonsignificant, improvement observed during acquisition in the peptide-treated CP subjects relative to their saline controls; and (4) the relation between running time and training condition suggests that incentive motivation may have been an important parameter affecting running time. If so, the absence of a DG-LVP influence on running time may indicate a lack of peptide effect on motivation. This finding may be related to the failure of Garrud et al. (1974), who also used a running time measure, to obtain a vasopressin effect on retention of the food-rewarded runway response, which, in turn, raises a question about the desirability of using this response measure in tests of vasopressin effects on learning and memory.

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c. Vawter and Van Ree (1995) Vawter and Van Ree (1995) investigated the effects of peripherally administered DG-AVP on memory processing in an open field spatial learning task [the hole board search task (HBST)] in male Wistar rats. In each of three experiments, separate groups of rats received a subcutaneous injection of physiological saline or DG-AVP: 0.17 or 0.5 g (experiment 1), 1.0 or 3.0 g (experiment 2), or 10.0 g (experiment 3). This pretreatment was administered 1 h before each daily acquisition session. The test apparatus for the HBST is an open-top Plexiglas square box with a start box attached along one wall and separated from the main arena by a sliding door. The raised Plexiglas floor of the main arena contains 16 holes overlying food wells. The test procedure involved a 5-day period of habituation to the apparatus directly followed by 6 days of acquisition training. Food was absent in the apparatus during the first 2 days of habituation, but in the last 3 days each of the 16 food wells was baited with a single food pellet that the rat was allowed to eat in the 15-min session. During acquisition (10 trials/day), one food pellet was placed in the same four food wells. After each daily testing session the rats were returned to food cages for the daily food ration. An acquisition trial was ended once all four food pellets were consumed. A visit to the food well was defined as a head dip into the hole overlying a food well. The number of visits and revisits to baited and to nonbaited food wells was recorded for each trial. Three of the five dependent variables tested in this study were included for discussion in this review: learning, working memory (WM), and reference memory (RM). Learning (error reduction) was evaluated by tabulating errors per session (total number of wells visited and revisited until 4 food pellets were consumed in 10 consecutive trials minus 40 for the total food well visits; a score of 0 indicated errorless performance). WM scores per session equaled the total number of revisits to baited or nonbaited food wells during the 10-trial session (the lower the score, the better the WM). RM scores per session equaled the ratio of the daily total number of visits and revisits to food-baited wells divided by the total number of visits and revisits to nonbaited wells (the higher the ratio the better the RM). Because of initial differences in body weight, the food deprivation schedule resulted in greater percentage body weight loss in the saline controls in experiments 1 and 2 than in experiment 3. Preliminary statistical analyses indicated significant differences in task performance between the controls tested in experiment 1 versus 3 and in experiment 2 versus 3, but not for those tested in experiment 1 versus 2. Accordingly, one multivariate analysis of variance (MANOVA) was computed for the pooled experiment 1 and 2 data, the other for the data of experiment 3. The MANOVA program for the pooled experiment 1 and 2 data used a treatment factor (five treatment groups: DG-AVP dose levels of 0, 0.17, 0.5, 1.0, and 3.0 g), a sessions factor (six testing sessions) and repeated measures on error, WM and RM scores. Percentage of body weight loss was

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entered as a daily covariant for each rat. Significant multivariate treatment by session interactions were followed up by t-test comparisons. The results obtained for the pooled experiment 1 and 2 data indicated (1) a highly significant sessions multivariate factor, indicating that task performance significantly changed between sessions for each dependent measure. Data plots for each dependent measure, showed that both saline controls and each treatment group exhibited improved learning proficiency, WM, and RM over successive test sessions; (2) a nonsignificant main treatment effect, indicating that the overall improvement for each dependent variable over the 6-day testing period did not differ from that exhibited by saline controls; (3) the regression of body weight loss on between-group multivariate measures was not significant, indicating that differential body weight loss was not a causal influence on performance differences obtained in this analysis; (4) the multivariate DG-AVP treatment by session interaction was significant for the comparison between saline controls and rats pretreated with the 1.0-g dose of DG-AVP. Follow-up t-test comparisons indicated that, compared with saline controls, rats pretreated with the 1-g dose of DG-AVP showed enhanced learning proficiency in sessions 2 and 5, enhanced WM in session 5, and enhanced RM in sessions 2 and 5; and (5) two sets of univariate interactions were significant. The first pertained to learning and included two significant univariate interactions for errors: DGAVP 0.17 g sessions, and DG-AVP 0.50 g sessions. Follow-up t-test comparisons revealed that relative to pretreatment with saline, learning errors were increased in sessions 3 and 4 (nearly significant) after a 0.17g dose of DG-AVP, and in session 2 (nearly significant) and session 4 (significant) after pretreatment with a 0.50-g dose of the peptide. The second set of univariate interactions pertained to WM and indicated a significant DG-AVP 0.5 g sessions interaction. Follow-up t-test comparisons indicated that relative to saline controls, rats pretreated with a 0.5-g dose of DG-AVP were impaired in WM (higher WM scores) in test session 2 (nearly significant) and test session 4 (significant). The MANOVA computed for the data of experiment 3 (i.e., compared effects of pretreatment with saline versus that with a 10.0-g dose of DG-AVP on the three dependent measures) yielded neither a significant main treatment effect nor a significant multivariate interaction of treatment sessions. Two types of deductions seem warranted from the results of this study. The first is that food deprivation and its sequelae (e.g., body weight loss) can exert an important influence on behavioral performance in the HBST. Vawter and Van Ree (1989) drew this conclusion from results obtained in a study with the HBST in which they manipulated the food deprivation level and showed that higher levels of deprivation improved test performance. In the present study, preliminary analysis used to determine the feasibility of pooling data of the three experiments indicated that differences in behavioral performance among the saline controls were associated with differential

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percentage of body weight loss. This finding led these researchers to partial out the effects of body weight loss on test performance by using food deprivation as a covariant in their analyses of the pooled experiment 1 and 2 data. The second point pertains to the dose-dependent effect of DG-AVP on test performance in this task. Specifically, the statistical analyses indicated no significant effects of DG-AVP with respect to the rate of learning or of general improvement in WM and RM over the 6 days of behavioral testing. However, the results of the analyses performed on the pooled experiment 1 and 2 data yielded a significant dose sessions interaction and follow-up statistical comparisons suggested a curvilinear dose–response relationship between DG-AVP and test performance on the dependent measures during a test session located in the early part (session 2) and later part (session 4 or 5) of the 6-day test period. Specifically, during these test sessions, dose levels at the low end of the range (0.17 and 0.5 g) impaired, those in the middle of the dose range (1.0 g) improved, and those at the high end (3.0 g) did not influence learning, WM, or RM in this appetitive spatial memory task. d. Vawter and Colleagues (1997) Vawter et al. (1997) investigated the effect of AVP(4–8) on learning, WM, and RM in male Wistar rats tested in the HBST with the same habituation-acquisition training protocol as used by Vawter and van Ree (1995). AVP(4–8) is a metabolite of AVP that is more potent than the parent peptide in its memory-facilitating effects in avoidance learning paradigms (Burbach et al., 1983b; see Chapter 5). The subjects were subdivided into three groups and injected subcutaneously with either physiological saline or with 0.3 or 1.0 g of [pGlu4Cyt6]AVP(4–8) [AVP(4–8)] 1 h before the day’s testing session. Comparisons across the three treatment groups [0.0, 0.3, and 1.0 g of AVP(4–8)] (treatment factor) for each dependent measure were statistically analyzed with a computerized MANOVA program that applied a repeated measures design to the sessions factor. Significant sessions treatment interactions were followed up by post hoc individual t-test comparisons. The results of the statistical analyses were as follows: (1) there was no significant difference between the controls and the two AVP(4–8) treatment groups in mean percentage of body weight loss after the second training session, indicating that the motivational level was similar for all three treatment groups; (2) the MANOVA yielded no significant main treatment effect for error scores (i.e., no significant difference in overall learning proficiency among the three treatment groups). However, the treatment sessions interaction was significant for this measure, and post hoc t-test comparisons indicated that during the first test session rats pretreated with the low dose of AVP(4–8) made significantly fewer errors than either saline controls or rats pretreated with the high dose of the AVP metabolite; (3) the MANOVA yielded no significant main treatment effect for the WM scores, indicating that as in the case of learning, pretreatment with the dose range of AVP(4–8)

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used in this study did not differentially affect overall WM performance on the HBST. But here again, the treatment sessions interaction was significant for WM and follow-up t-test comparisons indicated that during the first test session, WM performance in rats pretreated with the low dose of AVP(4–8) was significantly improved over that of rats pretreated with the high dose of the peptide, or given placebo (saline); and (4) the MANOVA did yield a main treatment effect for RM indicating overall significant differences between the groups on this factor. The treatment sessions interaction was also significant for this measure, and post hoc t-test comparisons showed that relative to the saline controls and rats given the 1.0-g dose level, pretreatment with the low dose (0.3 g) of the peptide significantly improved RM during test sessions 4, 5, and 6, a nonsignificant trend already present during session 3. The results of this study with AVP(4–8) can be related to those of the previously discussed study by Vawter and Van Ree (1995), which examined the effects of various doses of DG-AVP [AVP(1–8)] on these three dependent measures. First, both peptides dose-dependently influenced learning, WM, and RM during one or more daily training sessions. In the case of DG-AVP these dose-related effects indicated that low doses (0.17 and 0.5 g) impaired (significant or near significant), a mid-level dose (1.0 g) significantly improved, and dose levels at the high end of the tested range (3.0 and 10.0 g) were without effect. Of the two dose levels tested for AVP(4–8), the low dose (0.3 g) significantly improved and the high dose (1.0 g) was without effect. Second, the MANOVAs in both studies indicated that treatment was not significant for any of the dependent measures except for RM in the present study. Taken together, these findings are in accord with findings by De Wied and colleagues on avoidance learning (Bohus et al., 1978b; De Wied, 1965; De Wied and Bohus, 1966), which suggest that the effect of AVP-related peptides on learning is not as consistent or as important as their effect on long-term memory (reference memory). Third, the finding of a significant main treatment effect for RM in the present study, but not in the study by Vawter and Van Ree (1995), indicates that AVP(4–8) is more potent than DG-AVP in its effects on long-term memory as measured on this test.

IV. Oxytocin: A Natural Amnestic in Aversive Learning Situations

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A. Introduction In an early study with these neurohypophysial peptides, De Wied (1971) found no effect of a posttraining injection of OT (1.0 g/rat, subcutaneous) on rate of extinction of a conditioned pole-jump avoidance response and concluded that oxytocin, unlike vasopressin (VP), had no influence on

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memory processing. Subsequent observations were interpreted as confirmation of this view, because Bigl et al. (1977, as cited in Kovacs and Telegdy, 1982), injecting a high dose of OT 1 h before the last acquisition session of a pole-jump task, also found no peptide effect on subsequent extinction, and Walter et al. (1975) reported that OT did not influence puromycin-induced amnesia in mice. However, this latter finding is subject to reinterpretation in light of subsequent findings. Schultz et al. (1974) were the first investigators to suggest that OT may attenuate long-term memory in aversive learning situations. They compared the effects of daily pretesting injections of physiological doses of VP and OT on retention (rate of extinction) or a learned AA and PA response. Neither peptide influenced the rate of learning of the AA response in a platformjump shock avoidance task, but in contrast to AVP, OT impaired retention (significantly accelerated response extinction) relative to its placebo control group. However, chronic treatment with the same dose concentrations did not produce the expected effects of these peptides on the PA task. In this task, the dependent variable was the amount of time in a 3-min session that the rat remained in the lighted compartment over successive test days after administration of a severe footshock on entry through a small hole into the adjacent dark compartment. With this measure of PA behavior, VP accelerated the rate of extinction, and OT-treated rats did not significantly differ in PA behavior from their placebo-treated counterparts. The amnestic action of OT in the AA task paradigm, together with the opponent effects on retention exerted by the two peptides on both tasks, was followed by subsequent studies designed to clarify the comparative effects of these peptides on memory processing in avoidance learning tasks. A potential amnestic effect of OT was subsequently assessed using active and passive avoidance paradigms, and central as well as peripheral routes of administration. Moreover, additional studies examined dose- and time-dependent effects of exogenous oxytocin and sought to verify a role for endogenous oxytocin in the modulation of memory. These studies are described in more detail below. 1. Selected Studies a. Walter and Colleagues (1975) Walter et al. (1975) studied the ability of LVP, AVP, and OT, as well as numerous metabolic derivatives of these peptides, to prevent puromycin-induced amnesia in an inbred strain of Swiss-Webster mice. These mice were trained, in a single session, to avoid FS by traversing the stem of a Y-maze and turning left into the safe goal box within a 5-s time limit, to a criterion of 9 of 10 correct responses. A single peripheral injection of the peptide (0.1 mg dissolved in 0.2 ml of solution) was given immediately after training. Twenty-four hours later, an intracerebral injection of puromycin was applied bitemporally for primary influence on the hippocampi and entorhinal cortices. Savings on the retention

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test given 1 week later indicated that AVP, LVP, DG-LVP, and several metabolic fragments of the neurohypophyseal hormones (including PLG, the C-terminal tripeptide of OT) significantly attenuated the puromycin-induced amnesia. The failure of the parent peptide, OT(1–9), to influence savings in these subjects may be interpreted as an expected failure to protect against the amnesia, if this peptide functions as a natural amnestic in the brain. b. Kovacs and Colleagues (1978) Kovacs et al. (1978) tested the effect of VP, and OT, on acquisition and retention of a passive avoidance response in which rats were trained to escape footshock delivered through a grid floor by jumping onto a bench; stepping down onto the grid floor produced further footshock. For the training trial, the rat was placed on the floor of the test box and, after the onset of FS, the experimenter recorded the time required for the subject to jump to the safe bench and remain there for 180 s. This response measure comprised the step-on latency and operationally defined the time to learn the PA response. Twenty-fours later the rat was returned to the test box and placed on the bench for the retention test, with the FS absent. The latency to step down from the bench (step-down latency) was measured to a maximum of 180 s. LVP (300 mU/kg, subcutaneous), oxytocin (300 mU/kg, subcutaneous), or placebo (vehicle used for each peptide) was injected 10 min before the PA acquisition session on day 1 and 10 min before the PA retention session on day 2. The results are diagrammed in Fig. 6. Neither peptide influenced acquisition (i.e., step-on latency), but both significantly influenced retention. Vasopressin facilitated it (lengthened step-down latency) whereas oxytocin impaired it (shortened step-down latency). c. Bohus and Colleagues (1978a) Bohus et al. (1978a) investigated time gradient effects of centrally (intracerebroventricularly) administered OT and VP on retention of a PA response. The rats were trained on the single-trial step-through passive avoidance task with an FS intensity of 0.25 mA lasting for 2 s. Physiological saline, VP (1.0 ng/rat, intracerebroventricular), or OT (1.0 ng/rat, intracerebroventricular) was administered in a single dose either immediately or 3, 6, or 23 h after the single learning trial. Retention (reentry latency) was tested 24 h after the learning trial. OT significantly reduced reentry latencies relative to controls at all posttraining delays with the exception of the 6-h delay interval. Attenuation of PA retention was strongest when OT was given immediately posttraining (consolidation effect) or 1 h before the retention test (retrieval effect). AVP treatment produced a mirror image of this result and demonstrated a timedependent facilitation of PA retention (i.e., significantly prolonged reentry latencies relative to controls). Like OT, VP was most effective when injected immediately posttraining (consolidation effect) or 1 h before the 24-h retention test (retrieval effect). VP exerted a weak facilitation at a 3-h delay and no effect at a 6-h delay.

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FIGURE 6 Effect of vasopressin and oxytocin on passive avoidance behavior. The number in each column represents the number of animals used. Source: Kovacs et al., 1978 (Fig. 1, p. 802). Copyright ß 1978 by Pergamon Press.

The authors argued that because consolidation (memory storage) is a time-dependent process and amnesic treatments show typical time gradient effects on retention of a learned response, the effect shown by OT in this experiment supports its role as an amnesic neuropeptide. The failure of an OT effect when injected 6 h after the learning trial rules out a proactive influence on retrieval. The OT-induced attenuation and VP-induced facilitation of retention when the peptides were injected intracerebroventricularly 1 h before the 24-h retention test demonstrate the ability of these peptides to modulate memory retrieval as well as consolidation. d. Bohus and Colleagues (1978b) Bohus et al. (1978b), in a series of behavioral experiments, compared the effects of both peripherally and centrally administered OT and VP, or their antisera, on acquisition and retention of a pole-jump shock avoidance task, and on memory consolidation and retrieval on a single-trial step-through passive avoidance task. A final set of electrophysiological experiments examined the effect of intracerebroventricularly injected OT or OT antiserum on hippocampal theta rhythm during paradoxical (REM) sleep. The behavioral experiments are described below; the electrophysiological data are discussed in Chapter 5.

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FIGURE 7 Effects of various doses of oxytocin (OXT) administered subcutaneously 1 h before the ﬁrst three extinction sessions on the maintenance of a conditioned pole-jumping avoidance response. SAL, saline; CAR, conditioned avoidance response. Source: Bohus et al., 1978b (Fig. 1, p. 241). Copyright ß 1978 by Pergamon Press.

An initial set of experiments assessed the effect of peripherally administered OT and/or VP on retention of a pole-jump active avoidance response (experiments 1 and 2) and their effect on a step-through passive avoidance task (experiment 3). In experiments 1 and 2 the rats were trained (5 days, 10 trials/day) and tested for extinction (5 days, 10 trials/day) on a pole-jump avoidance task. In experiment 1, a randomly selected group of rats was peripherally injected with placebo (physiological saline) or with one of three doses of OT (0.002, 0.02, or 0.2 g/rat, subcutaneous) 1 h before the first three daily extinction sessions. It was found that OT prolonged extinction at the higher (0.2 g) dose level, but had no effect at the lower (0.002 and 0.02 g) dose levels. (see Fig. 7). In experiment 2, similarly selected independent groups of rats received placebo, AVP (0.02 g/rat, subcutaneous), or OT (0.02 g/rat, subcutaneous) 1 h before each acquisition and extinction session. The results showed that when administered throughout acquisition and extinction, AVP had no effect on acquisition but delayed extinction, whereas OT influenced neither acquisition nor retention of the active avoidance response at this dose level (see Fig. 8). In experiment 3, independent groups of rats, trained on the step-through passive avoidance (PA) task, received a single peripheral injection of saline,

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FIGURE 8 Comparative effects of arginine vasopressin (AVP) and oxytocin (OXT) administered subcutaneously during both acquisition and extinction on conditioned polejumping avoidance behavior. Source: Bohus et al., 1978b (Fig. 2, p. 242). Copyright ß 1978 by Pergamon Press.

one of two doses of OT (0.1 or 0.5 g/rat, subcutaneous), or AVP (0.5 g/ rat, subcutaneous) immediately after the FS learning trial. Retention was tested 24 and 48 h after the learning trial. Posttraining AVP facilitated PA retention (i.e., significantly prolonged median reentry latency) at both the 24- and 48-h retention tests. OT did not influence median reentry at either dose level, but the lower dose (0.1 g) produced a bimodal effect on the distribution of reentry latencies, with the majority of the rats showing longer or shorter reentry latencies than the controls. There was no bimodal distribution apparent in the AVP-treated rats, because 90% of these animals showed reentry latencies in the highest category (greater than 120 s). The results of this first set of experiments corroborate documented observations that peripherally administered VP generally has no influence on acquisition but facilitates retention behavior on both active and passive avoidance tasks (experiments 2 and 3). The results of the experiments that tested for the purported amnesic effect of peripherally administered OT were less straightforward. When this treatment did influence retention, it either mimicked the effects of AVP on AA extinction (experiment 1) or produced a bimodal distribution of PA reentry scores in which some of the subjects showed facilitated PA retention relative to controls (experiment 3). It was suggested that these complex behavioral effects of OT may have been due, in

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part, to its rapid metabolism in the blood and biotransformation into shorter peptides, some of which (e.g., the C-terminal peptide, PLG) have AVP-like retention effects when given in high quantities (De Wied, 1976). A second set of experiments tested the effect of centrally (intracerebroventricularly) administered OT and VP on the same active (experiment 4) and passive (experiment 5) avoidance tasks used in the earlier set of experiments. In experiment 4, the rats received 3 days of training (10 trials/day) on the pole-jump task followed by 2 days of extinction testing (10 trials/ day). Independent groups of rats received either saline, AVP (1.0 ng/rat, intracerebroventricular), or OT (1.0 ng/rat, intracerebroventricular) immediately after each acquisition session. Centrally administered AVP had no effect on acquisition but significantly delayed extinction, especially on the second day of extinction (Fig. 9). Centrally injected OT significantly retarded learning on day 3 of acquisition, and exhibited a nonsignificant tendency to facilitate response extinction on the first day of extinction testing (see Fig. 9). In experiment 5, an intracerebroventricular injection of either placebo, OT (0.05, 0.1, 1.0, or 10.0 ng/rat, intracerebroventricular), or AVP (0.05, 0.1, 1.0, or 10.0 ng/rat, intracerebroventricular) was administered immediately after the single learning trial on the step-through passive avoidance task. Retention was tested 24 and 48 h after the learning trial. Centrally

FIGURE 9 Pole-jumping avoidance behavior of rats after intraventricular administration of oxytocin (OXT) or arginine vasopressin (AVP) immediately after each acquisition session. Source: Bohus et al., 1978b (Fig. 3, p. 243). Copyright ß 1978 by Pergamon Press.

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injected AVP at the 1-ng dose level facilitated PA retention (prolonged median reentry latency) on the 24-h retention test only, and at the highest dose level (10 ng) facilitated retention on both 24- and 48-h tests. Posttraining OT influenced PA retention on the 24-h but not the 48-h retention test. It retarded PA retention (shortened median reentry latency) at the two middose levels (0.1 and 1.0 ng) but not at the lower (0.05 ng) dose level. At the high dose of 10 ng, OT did not affect median reentry latency but did produce a bimodal distribution, with the majority of reentry latencies being shorter (60%) or longer (30%) relative to the controls. The results of this second set of experiments confirm previous reports that centrally administered VP facilitates retention on both active (experiment 4) and passive (experiment 5) avoidance tasks. The observation that these retention effects of VP occur at much lower dose levels for central compared with peripheral administration is consistent with the notion that VP exerts its effect on active and passive avoidance behavior by influencing central rather than peripheral receptor sites. As with peripherally injected OT, centrally administered OT produced less consistent effects on avoidance retention than occurred with AVP. At a dose level (1 ng/rat) at which VP clearly facilitated retention on both avoidance paradigms, OT retarded PA retention (experiment 5) but did not produce the expected ‘‘amnestic’’ influence on the active avoidance task (experiment 4). The bimodal effect on PA retention observed for the 10-ng dose level again suggested some tendency for high doses of OT to mimic the influence of AVP on retention. A third set of experiments tested the effects on active (experiment 6) and passive (experiment 7) avoidance behavior of centrally administered anti-VP or anti-OT serum. In experiment 6, independent groups of rats received either placebo (normal rabbit serum, 2 l/rat, intracerebroventricular), anti-VP serum (1 l/rat, intracerebroventricular), or anti-OT serum (2 l/ rat, intracerebroventricular) 30 min before each acquisition session on the pole-jump active avoidance task. Both antisera facilitated acquisition but had opposing effects on extinction of this response. Reducing endogenous brain levels of OT with anti-OT serum resulted in a prolonged extinction (i.e., produced a higher number of avoidance responses compared with saline controls) whereas reducing central VP levels with anti-VP serum hastened the rate of extinction (i.e., produced fewer avoidance responses than saline controls) (see Fig. 10). In experiment 7, independent groups of rats were centrally injected with either placebo (rabbit serum, 1 l/rat, intracerebroventricular), antiOT serum (1 l/rat, intracerebroventricular), or anti-VP serum (1 l/rat, intracerebroventricular) immediately after a single passive avoidance learning trial. Retention was tested 24 and 48 h later. The centrally injected anti-VP serum impaired PA retention (shortened median reentry latencies) whereas the anti-OT serum facilitated it (prolonged reentry latency).

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FIGURE 10 Effects of intraventricular administration of oxytocin (OXT) or arginine vasopressin antiserum 30 min before each acquisition session on pole-jumping avoidance behavior of the rat. Se, serum. Source: Bohus et al., 1978b (Fig. 4, p. 243). Copyright ß 1978 by Pergamon Press.

Taken together, the results of this third set of experiments support the suggestion that endogenous VP and OT systems in the brain exert opposing effects on memory consolidation, with the VP system facilitating, and the OT system retarding, retention on active (experiment 6) and passive (experiment 7) avoidance tasks. e. Gaffori and De Wied (1988) Gaffori and De Wied (1988) examined the effects of varying doses of OT(1–9) and OT fragments on spontaneous behavior in the open field, and on memory consolidation and/or retrieval in avoidance learning tasks. A major purpose of this study was to investigate potential factors that may have contributed to discrepant findings reported in the OT/memory processing literature. As noted above, OT has been observed to facilitate (Walter et al., 1975, 1978), attenuate (Bohus et al., 1978b; Schultz et al., 1974), produce a bimodal action (Bohus et al., 1978b), or exert no effect (e.g., Bohus et al., 1978b; De Wied, 1971) on this retention. The reasons for these discrepant findings are not clear, although they could be related to any of a number of factors on which these studies differed (e.g., task paradigms, test procedures, OT analogs, dose levels, routes of treatment delivery). In an attempt to shed light on this problem, the present study tested the effects of various doses of OT (1–9) and OT fragments on active and passive avoidance behavior in male Wistar rats. Because these OT peptides differed in their molecular structure,

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comparisons of their behavioral effects permitted an application of a structure–activity analysis to this problem. Assessment of treatment effects on behavioral performance was based on comparisons between each dose/ peptide experimental treatment group and its own saline control group. Spontaneous behavior (locomotion, rearing, self-grooming, and fecal boluses) was measured during a 3-min test session in a circular open field. Depending on its assigned dose/peptide treatment group, each rat received a single subcutaneous injection of (1) a 0.3- or 3.0-g dose of OT(1–9) or DGOT [OT(1–8)]; or (2) a 0.3-g dose of either OT(4–9), OT(4–8), OT(5–9), or OT(5–8). This treatment was given 15 or 60 min before behavioral testing. Compared with saline controls, the high dose (3.0 g) but not the low dose (0.3 g) of OT(1–9) significantly reduced all measures of general activity 15 min but not 60 min after treatment. Neither dose of DGOT, nor the 0.3 g dose any of the other OT metabolites, significantly affected any measure of spontaneous behavior assessed at either treatment–test time interval. In the PA paradigm, depending on the experimental treatment group, each rat received a subcutaneous injection of (1) a 0.3-, 1.0-, or 3.0-g dose of OT(1–9) or DGOT; (2) a 0.1- or 0.3-g dose of OT(4–9) or OT(4–8); (3) a 0.03-, 0.1-, 0.3-, 1.0-, or 3.0-g dose of OT(5–9); or (4) a 0.1-, 0.3-, or 1.0-g dose of OT(5–8). These treatments were given either immediately after the footshock learning trial (consolidation design) or 1 h before the 24-h retention test (retrieval design). The strength of the PA behavior was measured by the reentry latency (i.e., the longer the reentry latency, the stronger the PA behavior and, by inference, the stronger the memory). The results demonstrated that relative to saline controls, (1) OT(1–9) and DGOT attenuated consolidation and retrieval at the 1.0- and 3.0-g dose levels, but were without effect at the 0.3-g dose level; (2) OT(4–9) and OT(4–8) attenuated memory consolidation and retrieval at the 0.3-g dose level, but not at the 0.1-g dose level; (3) OT(5–9) attenuated memory consolidation at the 3.0-g dose level, and memory retrieval at both the 0.1- and 0.3-g dose levels, the highest dose levels used for testing each of these measures; and (4) OT(5–8) attenuated consolidation at a dose level of 0.3 g, and retrieval at a dose level of 1.0 g, the highest dose levels used for the these tests. Taken together, these results indicated that the parent peptide, OT(1–9), and each of its tested fragments produced a dose-dependent amnestic action on PA behavior. No bimodal effect was evident because lower doses had no influence on either PA memory consolidation or retrieval. An additional finding was that, with the exception of DGOT, the OT fragments were generally more potent than the parent peptide, producing the same behavioral effect at far lower dose levels. In the AA paradigm, rats that reached the learning criterion (7 of 10 correct avoidances) at the conclusion of the last acquisition session (day 3 of training) were randomly assigned to a given treatment group and

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were subcutaneously injected with saline or with a selected dose of one of the OT peptides. These subjects were then given extinction training (10 nonreinforced trials/day) on days 4, 5, and 8 of behavioral testing. The rats in each experimental dose/peptide treatment group received a single subcutaneous injection of a specific dose level for each tested OT peptide. Dose levels tested for each of the tested OT peptides were as follows: (1) five dose levels were tested for OT(1–9) (0.001, 0.01, 0.1, 1.0, and 3.0 g) and for OT(4–9) and OT(5–9) (0.0001, 0.001, 0.01, 0.1, and 0.3 g); (2) three dose levels (0.01, 0.10, and 0.30 g) were tested for OT(4–8) and OT(5–8); (3) two dose levels (1.0 and 3.0 g) were tested for DGOT; and (4) one dose (3.0 g/rat) was tested for each of the two C-terminal peptides, PLG (Pro-Leu-Gly-NH2) and LG (Leu-Gly-NH2). The results indicated that compared with the saline control condition, (1) OT(1–9) at doses of 1.0 and 3.0 g significantly facilitated retention (inhibited extinction), whereas a lower dose (0.1 g) produced a similar but nonsignificant effect, and the two lowest doses (0.01 and 0.001 g) tended (not statistically significant) to attenuate this retention (facilitated extinction); (2) the OT fragments OT(4–9) and OT(5–9) produced an even more potent bimodal effect, significantly impairing retention (accelerating extinction) at the lower dose range (0.001 and 0.01 g) and facilitating it (retarding extinction) at the highest end of the tested dose range (0.1 and/or 0.3 g); (3) the removal of the terminal glycinamide residue of the OT molecule eliminated the bimodal action of the peptide. Thus DGOT, OT(4–8), and OT(5–8) attenuated retention (facilitated extinction) only when tested at their higher tested dose level(s), but had no influence on retention when tested at their lowest tested dose level; and (4) the two Cterminal di- and tripeptides significantly facilitated retention (inhibited extinction) at their only tested dose level (3 g). These findings on the AA task can be summarized as follows. First, an OT dose-related bimodal effect was observed whereby low doses of the peptide attenuated retention (facilitated response extinction), and high doses enhanced it (prolonged response extinction). Second, this bimodal effect was eliminated by removal of the C-terminal amino acid residue, glycinamide (position 9). Third, compared with the parent peptide, OT(1–9), the smaller fragments, OT(4–9) and OT (5–9) demonstrated a more pronounced bimodal effect that was more effective in both directions. The comments made below are in accord with those offered by the authors in their discussion of the results of this study. 1. Consistent with studies by Burbach et al. (1983b) and De Wied et al. (1987), these findings indicated that with the exception of DGOT, the OT fragments were in general far more behaviorally potent than OT(1–9) in both the AA and PA tasks. Thus, a nanogram dose of the former produced the same behavioral effect as a microgram dose of the latter.

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2. The dose-related bimodal effect that was observed in the AA, but not the PA, paradigm was not readily explained. One possibility is that the dose levels selected for study in the PA paradigm were insufficient to demonstrate the bimodal action that was observed in the AA paradigm. 3. The bimodal action of OT(1–9) observed in the AA task may also account for its effect in the open field test. Thus, the same high dose level of OT(1–9) that facilitated retention on the AA task exerted an initial suppression of general behavior assessed in the open field test. This type of behavioral suppression has also been reported for peripherally administered AVP (e.g., Ettenberg et al., 1983a [Chapter 6]; and Gaffori and De Wied, 1986 [Chapter 5]). Koob and colleagues (Chapter 6) have postulated that high levels of peripherally circulating AVP induce an aversive pressor-associated arousal action that is responsible for its facilitative effect on memory processing, and for its observed behavioral suppressive effects (e.g., Ettenberg et al., 1983a). It is therefore possible that the VP-like effects observed on AA retention and on open field behavior after high doses of peripherally administered OT were attributable to an action at vasoactive VP receptor sites. If so, the failure of a high dose of the OT fragments to influence open field behavior is consistent with findings that, unlike OT(1–9), the C-terminal OT fragments act selectively at central sites and do not share the endocrine actions of the parent peptide (Burbach et al., 1983b). 4. On the other hand, the bimodal effects of OT as well as of its fragments on the AA task may have been due to a central action of these peptides, and to an ability to activate central V1 receptor sites. This proposal is consistent with the demonstrated affinity of OT for putative central V1 receptor sites (Jard et al., 1987) and with the observation that OT, microinjected into the dorsal septal nucleus, exerts a VP-like effect on PA behavior (Kovacs et al., 1979a; see Chapter 4). 5. The additional finding that PLG and LG facilitated retention in the AA paradigm is consistent with a similar finding by Kovacs and Telegdy (1986), and suggests that the terminal part of the OT molecule contains a second ‘‘message’’ that opposes the more prevalent amnestic action of the peptide. This evidence is consistent with the proposal that after its administration, metabolic breakdown of OT in the blood and in the brain might generate a sufficient number of metabolites that oppose the amnestic action of OT, and produce a VP-like action on memory processing. In conclusion, although the dose-related bimodal action of OT demonstrated in this study was an important finding and clearly relevant to the controversial nature of the OT memory processing research literature, the results of this study make it equally clear that the ‘‘the amnesic effect apparently represents the main influence of OXT and related peptides on memory processes, when one assumes that the lower doses are more physiological than the higher doses’’ (Gaffori and De Wied, 1988, p. 161).

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V. Effects of VP on Retrograde Amnesia: Effect on Memory Retrieval?

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A. Introductory Comments: Retrograde Amnesia and Memory Retrieval Generally speaking, retrograde amnesia (RA) refers to the inability to remember experiences that occurred just before a temporary but severe disturbance of the normal physiological activity of the brain. In the animal laboratory, RA can be produced by postlearning or preretention application of any of a variety of treatments that disrupt neural activity (e.g., carbon dioxide, electroconvulsive shock, or pentylenetetrazole) or inhibit protein synthesis (e.g., puromycin). Traditionally, the amnesia produced by immediate postlearning application of one of these amnesic agents has been attributed to a disruption of memory consolidation, the process by which fragile, temporary short-term memory is changed into resistant, permanent long-term memory. However, certain researchers have challenged this consolidation-based explanation and favored instead one that attributes RA to impairment of memory retrieval (Miller and Springer, 1973). This explanation is based on the assumption that memory consolidation occurs so rapidly (within fractions of a second) that even immediate application of an amnestic agent is unable to disrupt it (Miller and Springer, 1973). Experimental support for the retrieval impairment explanation of RA comes from demonstrations of spontaneous recovery from this amnesia (Nielson, 1968; Zinkin and Miller, 1967), and from studies in which the animal subject is presented with a noncontingent reminder stimulus (e.g., the training apparatus minus the reinforcer, or the reinforcer placed in a different context) during the interval between the amnesic treatment and the retention test (Miller and Springer, 1973). Moreover, certain pharmaceutical agents (e.g., scopolamine) injected before the retention test also help to reinstate the lost memory (Davis et al., 1971). 1. Selected Studies a. Rigter and Colleagues (1974) Rigter et al. (1974) tested the ability of DG-LVP to attenuate RA induced by carbon dioxide (CO2) treatment, using a single-trial step-through passive avoidance (PA) task. Two other experiments tested the effects of ACTH(1–10) and ACTH(11–24) on this behavior, but except for brief comparative statements these are not described in the present discussion. Three pretraining trials were run on day 1. The subject was placed at the end of the runway facing the entrance to the darkened box; after entering the box, the door was closed, and the rat was retained there for 10 s. Latency to enter the box on all three trials was recorded and the ITI was 2 h. On day 2,

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a single acquisition trial was run, identical to the pretraining trials except that a 3-s, 0.5-mA footshock (FS) was given through the grid floor of the box to the FS groups. The subjects assigned to the non-FS groups remained in the box for another 3 s. Immediately after the acquisition trial, the subjects were given either the amnestic treatment (CO2 groups) or the sham amnestic treatment (no CO2 groups). Vehicle solution (saline) or DG-LVP (10 g/rat, subcutaneous) was administered either 1 h before the learning trial, 1 h before the 24-h retention test, or at both times in rats made amnesic by posttrial CO2 treatment (FS–CO2 groups). Depending on the combinations of FS, amnestic, and peptide treatments given, the subjects were allocated to 12 groups and the specific treatments for each group are given in Table I. The results were as follows: (1) DG-LVP given 1 h before the acquisition trial had no influence on the step-through latencies on that trial; (2) PA behavior was not exhibited in the non-FS groups, nor were reentry latencies

TABLE I Combination of Foot Shock, Amnestic, and Drug Treatments Characterizing Each of 12 Groups of Subjects for Each Experiment Conducted in Study a: Design of Experiments 1, 2, and 3 Treatment 1 h before: Group Control No FS–No CO2 No FS–No CO2 Experiment 1 No FS–CO2 No FS–CO2 Experiment 2 FS–CO2 FS–CO2 FS–CO2 FS–CO2 Experiment 3 FS–No CO2 FS–No CO2 FS–No CO2 FS–No CO2

Foot shock

CO2

Acquisition

Test

— —

— —

Saline Saline

Saline Drug

— —

þ þ

Saline Saline

Saline Drug

þ þ þ þ

þ þ þ þ

Saline Drug Saline Drug

Saline Saline Drug Drug

þ þ þ þ

— — — —

Saline Drug Saline Drug

Saline Saline Drug Drug

Abbreviations: FS, footshock; no FS, no footshock. CO2, CO2 treatment; no CO2, no CO2 treatment. a Depending on the experiment, the drug was ACTH (4–10) (experiment 1), ACTH (11–24) (experiment 2), or DG-LVP (experiment 3). Source: Rigter et al., 1974 (Table 1, p. 382). Copyright ß 1974 by Brain Research Publications, Inc. Table reprinted and legend adapted by courtesy of Elsevier Science, present publisher of this journal.

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FIGURE 11 Effect of desglycinamide-lysine vasopressin on CO2-induced amnesia for a passive avoidance response. Indicated are the latencies at the test trial. The scores were divided into three classes: (1) 0–10 s (no avoidance); (2) 10.1–299.9 s (nonoptimal avoidance); and (3) 300.0 s (optimal avoidance). Saline: 1 ml of saline per rat, administered subcutaneously 1 h before trial; DGLVP: 10 g of desglycinamide-lysine vasopressin per rat, administered subcutaneously 1 h before trial. FS, foot shock; No FS, no foot shock; CO2, CO2 treatment; No CO2, no CO2. Source: Rigter et al., 1974 (Fig. 3, p. 386). Copyright ß 1974 by Brain Research Publications Inc. Figure reprinted and legend modiﬁed by courtesy of Elsevier Science, present publisher of this journal.

influenced in these subjects by posttraining CO2 treatment (Fig. 11, No FS–No CO2 and No FS–CO2 columns); (3) DG-LVP given 1 h before the test trial did not influence reentry latencies in the non-FS groups, whether or not they received CO2 treatment (Fig. 11, No FS–No CO2 and No FS–CO2 columns); (4) CO2 treatment produced almost complete amnesia in the saline-treated groups which received FS (i.e., no significant difference in reentry latencies between these subjects and the saline-treated subjects in the No FS–No CO2 and the No FS–CO2 groups, respectively); (5) this amnesia was reduced by treatment with DG-LVP given before the training trial, the test trial, or both (i.e., significantly increased reentry latencies in each of these three groups relative to the saline-treated FS–CO2 groups). In addition, the three DG-LVP groups did not significantly differ in reduction of amnesia (see Fig. 11, FS–CO2 columns); (6) the reduction in amnesia was not complete, as indicated by significantly greater reentry latencies in each of the DG-LVP-treated FS–No CO2 groups and the comparably

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DG-LVP-treated FS–CO2 group (Fig. 11, FS–CO2 and FS–No CO2 columns); and (7) the absence of significant differences in PA response between the saline- and DG-LVP-treated subjects in the FS–No CO2 groups was probably due to ceiling effects because the reentry latencies in the saline controls were near maximal levels (see Fig. 11, FS–No CO2 columns). The ACTH fragment, ACTH(4–10), did not significantly reduce RA when given 1 h before the acquisition trial, but did so when given 1 h before the test trial or before both acquisition and test trials. The ability of ACTH(4–10) to reduce RA further supports findings that the ability of ACTH to facilitate retention does not depend on its adrenocortical activity (De Wied, 1969). The finding that ACTH(4–10), unlike DG-LVP, reduced RA when given either before the test trial or at both the acquisition and test trial, but not when given before the acquisition trial alone, supports previous findings suggesting that ACTH(4–10) has short-term but not longterm effects on memory retrieval (i.e., the peptide is most effective when present in the body) (Bohus et al., 1972; De Wied and Bohus, 1966). The antiamnestic effect of DG-LVP when administered before the training trial suggested to the authors that the peptide was able to protect memory consolidation from the adverse effects of amnestic treatment. However, this vasopressin protection against the amnestic agent could indicate protection against the disruption of retrieval because the peptide also had an antiamnestic effect when injected before the test trial. b. Bohus and Colleagues (1982) Bohus et al. (1982) compared various routes of AVP administration when testing the ability of the peptide to reverse pentylenetetrazole-induced amnesia in a single-trial passive avoidance (PA) task. AVP was injected peripherally (2 g/rat, subcutaneous), intracerebroventricularly (10 ng/rat), or into selected limbic system brain sites (100 pg/rat per left and right side of each bilateral structure; 200 pg/rat into the midline dorsal raphe nucleus). The amnesic agent was injected intraperitoneally immediately after the PA learning trial and AVP was injected shortly before the 24-h retention test. Intracerebroventricularly injected AVP mimicked the antiamnestic effect of subcutaneously injected AVP. The much lower dose level required for central versus peripheral administration once again suggested a central site of action for the antiamnestic action of the peptide. Moreover, an AVPinduced reversal of RA also occurred when the peptide was microinjected into either the dentate gyrus of the hippocampus or the central amygdala, but not into the dorsal raphe nucleus or dorsal septum, indicating that the VP antiamnestic effect is mediated by some but not all limbic system structures implicated in memory processing. Insofar as the antiamnestic effect reflects a retrieval effect of the peptide action, this finding suggests that the dentate gyrus of the hippocampus and the central amygdala mediate the retrieval effect of VP.

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In addition to the studies carried out by De Wied and colleagues, other laboratories have reported findings that support the view that the antiamnestic property of AVP is achieved by effects on retrieval. These findings include the following demonstrations: (1) LVP, peripherally administered 1 h before either the PA learning trial or the 24-h retention test, attenuates RA induced by a posttraining convulsive dose of pentylenetetrazole (Bookin and Pfeifer, 1977); (2) LVP, given 1 h before either the PA learning trial or the 24h retention test, exerted long-term attenuation (present at 12 days after learning) of RA induced by posttraining electroconvulsive shock treatment (Pfeifer and Bookin, 1978); and (3) LVP given 1 h before the 48-h retention test, but not at the PA learning trial, significantly reduced RA produced by an inhibitor of noradrenaline synthesis (diethyldithiocarbamate) (Asin, 1979).

VI. VP and OT Appear to Have No Important Effects on the Learning Phase of Memory Processing

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Numerous studies by De Wied and colleagues led them to conclude that VP and OT exert a major influence on the consolidation of long-term memory and on its subsequent retrieval, but not on the learning phase of memory processing. However, the generalization that these neurohypophyseal peptides do not influence learning needs some qualification because under certain conditions they have been observed to do so. Thus, VP may influence the rate of acquiring an active avoidance response when learning is slow or impaired as in completely hypophysectomized rats (Bohus et al., 1973; Lande et al., 1971); or at dose levels that are especially aversive (Gaffori and De Wied, 1985; see Chapter 3); or through its ability to interact with the subject’s arousal level (Skopkova et al., 1991; see Chapter 3). OT has been observed to attenuate acquisition of active avoidance behavior in intact rats after intracerebroventricular administration (Bohus et al., 1978b). The conclusion that neither VP nor OT makes a major contribution to the learning phase of memory processing is based on avoidance paradigms with normal rats receiving VP or OT treatment, as well as with rats deficient in VP and/or OT because of surgical intervention (posterior pituitary lobectomy), genetic deficiency [Brattleboro diabetes insipidus (DI) rats], or experimental neutralization of these peptides (treatment with VP or OT antiserum). The failure of VP to influence avoidance learning was originally observed by De Wied (1965), who reported that rats deprived of the posterior/ intermediate lobes of the pituitary exhibited abnormally rapid extinction of an active avoidance response, but acquired the response as readily as the sham-operated controls. AVP also failed to influence avoidance learning when peripherally or centrally injected into normal rats. De Wied and

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Bohus (1966) reported no effect on shuttlebox avoidance learning in intact rats peripherally injected with VP (pitressin). Bohus et al. (1978b) observed that peripherally injected AVP throughout acquisition and extinction in a pole-jump shock avoidance task failed to influence acquisition of the response but markedly delayed its extinction, and similar results were obtained when the peptide was injected into a lateral ventricle (intracerebroventricularly injected). These findings have been described earlier in this chapter. Studies by De Wied and colleagues have also indicated that, although experimentally or genetically induced depletion of endogenous VP severely impairs memory storage on active and passive avoidance conditioning tasks, it does not appear to prevent successful acquisition of the avoidance responses. Thus, Van Wimersma Greidanus and De Wied (1976a) neutralized endogenous VP in normal rats by injecting anti-VP serum immediately after a PA learning trial and tested PA behavior at various time intervals. Because PA behavior was evident when tested at less than 2 h, but not after a delay of 6 h or more, it it was concluded that VP neutralization had prevented memory consolidation but not original learning per se. Moreover, as discussed earlier, their studies with the Brattleboro DI rat led De Wied and colleagues to conclude that VP deficiency severely impaired retention behavior, but did not prevent normal learning of a passive avoidance response nor attainment of a shuttlebox avoidance response, a pole-jump avoidance response, or an aversively motivated T-maze discrimination. (Bohus et al., 1975; De Wied et al., 1975, 1988). A failure of OT to influence acquisition behavior has also been demonstrated for active and passive avoidance tasks. Schultz et al. (1974, 1976) found that OT, injected peripherally 15 to 25 min before daily sessions of a bench-jumping active avoidance task, did not influence the rate of learning. Peripherally administered OT also failed to influence acquisition of a conditioned pole-jump avoidance response (Bohus et al., 1978b) or a step-down passive avoidance response (Kovacs et al., 1978). As noted above, however, there are occasions when VP can influence learning. Lande et al. (1971) studied the effect of several products obtained by fractionation of porcine pituitary extract on acquisition of a shuttlebox conditioned avoidance response in completely hypophysectomized Wistar rats. In comparison with sham-operates, the hypophysectomized rats were sharply diminished in their rate of acquisition, producing only 16 successful avoidance responses compared with the 114 responses of the sham-operates during 140 acquisition trials. The fractionated products were tested for their ability to influence the rate of conditioning and for hormonal activity. Further study isolated and identified a peptide, tentatively identified as DG-LVP, that acted to normalize acquisition behavior without influencing the diminished tropic effects resulting from the removal of the anterior pituitary hormones: ACTH, thyroxine, and testosterone. Bohus et al.

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(1973) verified that LVP significantly facilitates the rate of conditioning relative to placebo controls. In this study, the subjects were given a subcutaneous injection of placebo (saline), LVP (1 g/rat), or ACTH(4–10) (20 g/ rat) during the first half of the 14-day period of training. Both LVP and ACTH(4–10) significantly improved acquisition to an equal degree, but unlike ACTH(4–10), the LVP-induced gains in conditioning were maintained throughout the training period even after discontinuation of treatment on day 7, indicating that ACTH analogs have ‘‘short-term’’ but not ‘‘long-term’’ effects similar to those of vasopressin in these learning-impaired subjects. Aversive and arousal effects of AVP treatment can influence learning, as has been indicated by Gaffori and De Wied [1985; aversive effect (Chapter 3)] and Skopkova et al. [1991; arousal effect (Chapter 3)]. These VP influences on learning represent immediate effects on behavior but are interpreted as separate from the mechanism responsible for the long-term effect of the peptide on retention. Gaffori and De Wied (1985) observed facilitated avoidance learning on a pole-jump task when vasopressin, at a dose level (3 g/rat, subcutaneous) that produced aversive effects, was injected before training. Moreover, the learning effect occurred when AVP was injected 20 min before the training session, by which time it would produce maximal discomfort. The aversive effects of the peptide were inferred from the depressed grooming and exploratory behavior that resulted when vasopressin was injected 15, but not 60, min before the open field test. The hypothesis that the aversive effects of the high dose of AVP were responsible for the learning enhancement was supported by a subsequent observation that indicated that DG-AVP, which lacks the aversion-associated endocrine effects of the large dose of AVP, had no effect on either open field behavior or on acquisition of the pole-jump avoidance response (Gaffori and De Wied, 1985). Skopkova et al. (1991) also demonstrated a vasopressin effect on learning by means of an interaction with the arousal level. In this study subjects chosen from a genetically nonselected strain of Wistar rat were prerated for low and high levels of nonspecific excitability (behavioral arousal) on the basis of exploratory activity observed in an open field test. DG-AVP, which had earlier been observed to increase exploratory activity in the open field (Skopkova et al., 1987), was used in this study to manipulate arousal level in these low- and high-excitable rats. The low dose (0.1 g/rat) and the high dose (1.0 g/rat) of DG-AVP, given 40 min before the first acquisition session, interacted with the prerated excitability levels to influence learning in accordance with the proposed inverted-U relation between arousal and performance (Hebb and Donderi, 1987). This was interpreted as an immediate, arousal-associated effect of peptide treatment on learning that was not related to its long-term effect on extinction. Both these studies are discussed more fully in Chapter 3.

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VII. Theoretical Propositions of the ‘‘VP/OT Central Memory Theory’’ and Relevant Evidence

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What is herein referred to as the ‘‘VP/OT Central Memory Theory’’ comprises an evolving viewpoint as to how these neurohypophysial peptides operate to influence learning and memory. Underpinning this viewpoint is acceptance of the memory consolidation theory, which suggests that longterm memory is formed by consolidation, a time-dependent process that alters the memory trace from a temporary, fragile form to one that is durable and resistant to disruption by agents influencing neural activity and metabolism (e.g., electroshock, deep anesthesia, and protein synthesis inhibition). De Wied and colleagues also appear to accept the view that the short-term memory trace systems operating during learning are independent of those representing long-term memory, insofar as a given treatment can affect one without influencing the other. (see McGaugh, 1966, 2000, for further discussion of memory consolidation as a time-dependent process and the question of whether or not short-term and long-term memory systems are independent of one another).

A. Proposition 1: VP Facilitates Memory Consolidation and Retrieval Initial evidence relevant to proposition 1 was obtained from experiments with rats, which found that surgical removal of the intermediate/ posterior lobes of the pituitary gland led to abnormally rapid rates of extinction of a learned shuttlebox shock avoidance response (De Wied, 1965). The facilitated extinction could not be adequately accounted for by the associated disturbance in water metabolism, and it was corrected by replacement therapy with pitressin as well as purified LVP. Moreover, the normalization of extinction induced by posterior pituitary hormone replacement therapy was not mediated by a VP-induced release of ACTH (De Wied, 1965). An investigation of the effects of ACTH-like peptides and VP on intact rats showed that both delayed extinction on active avoidance tasks, but unlike ACTH, which needed to be present in the body for full effectiveness, VP prolonged extinction for weeks after termination of treatment (De Wied, 1971; De Wied and Bohus, 1966). Clearly, the mechanisms responsible for these diverse effects differ, and the proposal that the long-term effect of VP on extinction reflected an influence on formation of long-term memory (i.e., memory consolidation) was launched. Subsequent evidence relevant to this proposition came from studies assessing the effects of either exogenous or endogenous VP in physiologically intact rats, tested in numerous active or passive avoidance paradigms, and in a few appetitive tasks. Peripherally administered VP-like peptides [pitressin tannate, DG-AVP, AVP(4–8)], chronically injected during acquisition

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training, have been shown to facilitate memory consolidation in a shuttlebox avoidance task (De Wied and Bohus, 1966), a sex-rewarded spatial discrimination task in a T-maze (Bohus, 1977), and a hole board search task (Vawter et al., 1997). Similarly, a single peripheral VP treatment, when given after the completion of training, facilitated memory consolidation on a multitrial active avoidance task (Ader and De Wied, 1972; De Wied, 1971; Hagan et al., 1982), and on a step-through passive avoidance task when given after a single learning trial (Bohus et al., 1978b). Centrally (intracerebroventricularly) injected VP has also been shown to facilitate memory consolidation after chronic treatment during acquisition training on a polejump shock avoidance task, and after a posttraining treatment on the step-through passive avoidance task (Bohus et al., 1978a). Time-dependent effects are also consistent with the proposal that VP influences memory consolidation. These studies have shown that the closer in time VP treatment is to the completion of task acquisition, the more effective is its facilitation of retention—with full effectiveness achieved with treatment within 1 h of training, partial effectiveness at a 3-h delay, and no effect when treatment is delayed by 6 h (Bohus et al., 1972, 1978a; De Wied, 1971; Hagan et al., 1982). Also consistent with a VP influence on memory consolidation is the finding that some degree of associative strength is necessary to demonstrate the influence of VP on retention (King and De Wied, 1974). The single-trial step-through passive avoidance paradigm has been particularly useful in demonstrating that AVP, injected peripherally (Ader and De Wied, 1972) and centrally (intracerebroventricularly) (Bohus et al., 1978a), facilitates memory retrieval (prolonged reentry latency after AVP treatment given 1 h before the 24-h retention test) as well as memory consolidation (prolonged reentry latency after posttraining AVP treatment). The influence of VP on memory retrieval has also been studied by using the retrograde amnesic (RA) paradigm. De Wied and associates have demonstrated that peripherally administered VP reverses experimentally induced RA (taken as evidence of the effect of VP on memory retrieval) in a passive avoidance task, whether injected immediately after the learning trial (Rigter et al., 1974) or 1 h before the 24-h retention test (Bohus et al., 1982; Rigter et al., 1974). Bohus et al. (1982) reported that AVP, injected intracerebroventricularly, or microinjected into the hippocampus or the central nucleus of the amygdala, fully reversed severe experimentally induced RA. The ability of VP to attenuate or reverse RA on a PA task has also been observed by other investigative teams (e.g., Asin, 1979; Bookin and Pfeifer, 1977; Pfeifer and Bookin, 1978). A role for endogenous VP in memory processing has been demonstrated in rats genetically deficient in VP, and in normal rats depleted of VP by peripherally or centrally injected anti-VP serum (discussed in Chapters 3 and 4). Studies described in this chapter and the next indicate that intracerebroventricularly injected anti-VP serum attenuates memory

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consolidation in both active (Bohus et al., 1978b) and passive [Van Wimersma Greidanus et al., 1975a (Chapter 3); Bohus et al., 1978b] avoidance paradigms.

B. Proposition 2: OT Attenuates Memory Consolidation and Retrieval The evidence relevant to proposition 2 is somewhat inconsistent, especially when OT is peripherally administered. Early studies indicated that peripherally administered OT had no influence on extinction of a conditioned pole-jump shock avoidance response, whether the injection was given just after the completion of training (De Wied, 1971) or 1 h before the last acquisition session (Bigl et al., 1977). Subsequent observations by both Schultz et al. (1974), using a platform-jump active avoidance task, and Kovacs et al. (1978), using a bench-jump passive avoidance task, demonstrated that peripherally administered OT had no influence on acquisition but did attenuate memory on these paradigms. These results led to the view that OT might serve as a natural amnestic in the brain. In a later series of experiments, Bohus et al. (1978b) studied the effect of various dose levels of peripherally administered OT on retention in both an active (multitrial pole-jump task) and passive (single-trial step-through task) avoidance paradigm. Depending on the dose level, peripherally administered OT either had no effect (0.002 or 0.02 g) or prolonged (0.2 g) extinction of a conditioned pole-jump avoidance response. At a dose level of 0.1 or 0.5 g, OT had no effect on median reentry latency, but at the 0.1-g dose level OT produced a bimodal effect on the distribution of reentry latencies in a PA paradigm. On the other hand, peripherally administered AVP, at dose levels ineffective for OT, significantly prolonged extinction of the conditioned active avoidance response and increased reentry latency on the passive avoidance task. Gaffori and De Wied (1988) observed that peripherally administered OT, and certain OT fragments, produced a dose-related bimodal effect, whereby high dose levels facilitated, and low dose levels attenuated, retention for a learned pole-jump shock avoidance response. This bimodal action depended on the presence of the C-terminal glycinamide amino acid residue in the molecule. Noting that the low doses more closely resemble physiological levels of the peptide, it was suggested that the amnestic action of OT is its primary effect in aversive learning encounters. This suggestion was supported by the observation that peripherally administered OT and its fragments produced a dose-dependent amnestic action on PA behavior. The absence of a bimodal effect in this paradigm may have been due to the fact that a sufficiently high dose level of OT was not tested in this paradigm. The dose-related bimodal effect, together with the finding that certain of the tested OT fragments (PLG and LG) produced a VP-like effect in the AA

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paradigm, led Gaffori and De Wied (1988) to suggest two plausible explanations for the discrepant results obtained after peripherally administered OT in aversive learning paradigms. First, one contributing cause might be that OT metabolism occurs at a faster rate in the blood than in the brain and that some of the metabolites of the peptide (e.g., PLG) mimic the influence of VP on retention. Second, considering the affinity of OT for central VP receptors (Jard et al., 1987), high doses of peripherally administered OT might have a better chance of reaching and activating central VP receptor sites. More consistent evidence in support of the proposal that OT attenuates memory consolidation and retrieval in avoidance paradigms comes from studies using central administration of the peptide, either intracerebroventricularly or locally injected into selective brain sites. Bohus et al. (1978a) observed that posttraining, intracerebroventricularly administered OT slightly attenuated retention on a pole-jump avoidance task and severely retarded memory consolidation in a PA paradigm. Bohus et al. (1978b) observed that a posttraining and preretention intracerebroventricular injection of OT attenuated PA memory consolidation and retrieval, respectively, and further indicated that these influences are time dependent, with weaker effects occurring with increased delay between OT administrations and the learning or retention trial. It has also been shown that posttraining OT attenuates PA memory consolidation when microinjected into the hippocampal dentate gyrus and dorsal raphe nucleus (Bohus et al., 1982). Another demonstration of the putative amnestic role for OT has been the effects on memory processing produced by neutralization of endogenous levels of OT via peripheral or central injections of anti-OT serum. When peripherally or intracerebroventricularly administered, anti-OT serum significantly prolonged extinction on the pole-jump task and significantly lengthened reentry latency on the step-through PA task (Bohus et al., 1978a). Both observations indicate that reducing endogenous levels of this amnestic peptide improves memory consolidation on these avoidance tasks.

C. Proposition 3: VP and OT Do Not Have a Major Role in the Learning Phase of Memory Processing According to proposition 3, under normal circumstances neither VP nor OT affects learning and therefore they have little, if any, consequence for factors that support this initial phase of memory processing (i.e., attention, arousal, and motivation). However, under special conditions, as when the subject’s ability to process information is impaired (De Wied, 1965; and see De Wied and Gispen, 1977), or when arousal effects of the peptide are enhanced (Gaffori and De Wied, 1985; Skopkova et al., 1991), the peptide can produce an influence on learning behavior.

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Several experimental protocols have provided evidence supporting this proposition. One has been the study of rats deprived of hormonal VP and OT through surgical removal of the intermediate/posterior lobes of the pituitary gland. These rats exhibit normal acquisition on a shuttlebox avoidance task (De Wied, 1965). A second protocol is the administration of VP or OT during acquisition of an avoidance or appetitive response in normal, physically intact rats. Supportive evidence obtained from studies on avoidance behavior was as follows: (1) Bohus et al. (1978b) observed no effect of chronic treatment with either peripherally injected AVP or OT, or with intracerebroventricularly administered AVP, on the rate of acquiring the pole-jump shock avoidance response; (2) De Wied and Bohus (1966) observed no learning effect of chronic treatment with peripherally injected pitressin tannate on a shuttlebox avoidance task; and (3) Kovacs et al. (1978) observed no influence of peripherally injected LVP or OT given 10 min before the acquisition session on rate of acquiring a bench-jump passive avoidance response. Relevant findings from appetitive learning tasks were as follows: (1) Bohus (1977) observed that subcutaneous injections of DG-AVP (1 g) given immediately after completion of daily training sessions improved retention but had no effect on acquisition of a learned spatial discrimination in sexually rewarded rats; and (2) Vawter and colleagues found that, relative to saline controls, chronic pretreatment with optimal doses of DG-AVP (1.0 g) (Vawter and van Ree, 1995) or AVP 4–8 (0.3 g) (Vawter et al., 1997) did not reliably improve learning over the course of 6 days of training on the hole board search task. A third protocol is the neutralizing of central levels of VP or OT by intracerebroventricular injection of either anti-VP or anti-OT serum during acquisition of the avoidance response. Research with this protocol, however, has not provided consistent support for the proposition. Van Wimersma Greidanus et al. (1975a; Chapter 3) observed no learning deficit on a step-through PA task in rats in which central VP levels had been neutralized by intracerebroventricularly injected anti-VP serum. On the other hand, data obtained by Bohus et al. (1978b) suggested that brain levels of OT, and perhaps VP, may normally exert an inhibitory role in some process affecting the rate of learning, because neutralizing the brain levels of both peptides facilitated learning of a pole-jump avoidance response. Further, whereas intracerebroventricularly injected VP did not influence this learning, a central injection of OT at the same dose level facilitated it (Bohus et al., 1978b). A fourth and final protocol providing information relevant to this proposition has been the study of learning and memory in rats genetically deficient in brain vasopressin (discussed in Chapter 3).

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De Wied and Colleagues II: Further Clarification of the Roles of Vasopressin and Oxytocin in Memory Processing

I. Chapter Overview

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The research presented in Chapter 2 led De Wied and colleagues to conclude that vasopressin (VP) and oxytocin (OT) have important roles in modulating the formation of long-term memory, but are not importantly involved in the early learning phase of memory processing. Tested in both aversive and appetitive paradigms, peripherally administered VP consistently facilitated memory consolidation. The inﬂuence of OT was examined only in aversive paradigms in which, in general, it produced an amnestic effect. However, dose level and route of administration (central versus peripheral) were important parameters determining the speciﬁc type of memory modulation produced by this peptide. Although this early phase of their research launched the view herein referred to as the ‘‘VP/OT Central Memory Theory,’’ the heavy reliance on the peripheral route of administration opened the door for a number of alternative explanations of the means by Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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which these injected neuropeptides exerted their behavioral effects on learning paradigms. The evidence presented in this chapter pertains to four major questions: (1) Are VP and OT physiologically involved in memory processing, or could the ﬁndings presented in Chapter 2 be due to some nonphysiological pharmacological effect of the peptides? (2) If endogenous VP and OT inﬂuence memory processing, is this inﬂuence exerted by their peripheral (hormonal) and/or central (neurogenic) activities? (3) How important is a VP interaction with the central arousal system for the learning and memory effects induced by peripheral administration of the peptide? (4) Do peripherally administered VP and OT affect memory storage by an action exerted at peripheral and/or central receptor sites? As in Chapter 2, propositions formulated to deﬁne the major components of the ‘‘VP/OT Central Memory Theory’’ are presented at the end of this chapter along with relevant experimental support.

II. Establishing the Roles of Endogenous VP in Memory Processing: The Brattleboro Rat Model

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A. Introductory Comments De Wied and colleagues used three experimental models to study the physiological involvement of VP and/or OT in memory processing: (1) posterior pituitary lobectomized subjects are deprived of VP, OT, and other hormones present in the posterior/intermediate lobes of the pituitary gland, but not of central VP-ergic and OT-ergic circuitry. In an early series of experiments, De Wied (1965) compared these subjects with sham operates on learning and memory tasks before and after hormonal replacement therapy. These experiments were discussed in Chapter 2; (2) the Brattleboro rat, derived from the Long-Evans strain of hooded rats, is an inbred strain of rat that exhibits a genetic mutation at the single-gene locus encoding synthesis of the VP precursor (Schmale and Richter, 1984). Rats homozygous for the mutation (HODI rats) lack VP and suffer from diabetes insipidus (a disorder of water metabolism characterized by excessive ingestion of water, and urination). Rats heterozygous at the gene site (HEDI rats) are partially deprived of VP and do not have diabetes insipidus (Van Wimersma Greidanus and De Wied, 1977). Numerous studies have compared HODI, HEDI, and Long-Evans normal (LENO) rats on learning and memory tasks and the results of these studies and their controversial interpretations are discussed below; and (3) selective neutralization of peripheral and central endogenous VP or OT by antisera allows comparison of the relative contribution of peripheral and central stores of the peptide(s) to memory processing; this research is discussed in a subsequent section of this chapter.

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B. Early Research Studies by De Wied and Colleagues with the Brattleboro Rat 1. Selected Studies a. De Wied et al. (1975) De Wied et al. (1975) compared HODI and HEDI rats on learning and retention of a passive avoidance (PA) response, using a single-trial step-through passive avoidance task. HODI rats were differentiated from HEDI rats by measuring water intake over a 2-day period before experimental testing. Examination of VP in the posterior pituitary lobe indicated nondetectable levels in HODI rats, and the level was signiﬁcantly lower (about one-half the amount) in HEDI rats than in Wistar rats. In experiment 1, subjects were randomly assigned to the nonshock group or to shock groups that received a 3-s footshock (FS) at one of three levels of intensity (0.25, 0.50, or 1.0 mA). The reentry latencies of these subjects were tested 24, 48, and 120 h after the single PA training trial. For the HODI rats, none of the FS intensity levels produced signiﬁcant PA behavior relative to the nonshock controls (i.e., none of the FS groups differed from the nonshock controls in median reentry latency on any of the three retention tests). Although the reentry latency for the highest FS level was slightly above preshock levels, the difference was not statistically signiﬁcant. For the HEDI rats, PA behavior was observed for the 0.5- and 1.0-mA FS levels (reentry latencies signiﬁcantly longer than the nonshock controls for the 0.5-mA FS in the 48- and 120-h retention test and in all three retention tests for the 1.0-mA FS level). Moreover, the 1.0-mA FS produced maximum PA behavior (median reentry latency of 300 s) in the HEDI rats for all three retention tests. When the unconditioned stimulus (UCS) was made more aversive (i.e., the 1.0-mA FS was given for 10 s rather than then 3 s), the HODI rats still failed to exhibit PA behavior. However, when they were exposed to a 3-s FS of 1.0 mA, and injected with either arginine vasopressin (AVP) or desglycinamide-lysine vasopressin (DG-LVP; 1 g/rat) immediately after the FS trial, they did show normal PA behavior on all three retention tests (i.e., their reentry latencies were signiﬁcantly longer than those of the nonshocked controls, and highly similar to the PA behavior of HEDI rats assessed under identical test conditions). These latter observations indicated that the effect of the parent peptide on PA behavior was not due to its ability to normalize water metabolism in these VP-deﬁcient rats, because the DG-LVP analog lacks endocrinological effects. Experiment 2 used a 3-s FS of 1.0-mA intensity, and tested reentry latency either immediately or 3 h after the FS training trial. At both test times the median reentry latency for the HEDI rats was at the maximum value (300 s). For the HODI rats, reentry latency tested immediately after the FS trial was near the maximal value, and although the reentry latency was markedly reduced at the 3-h test, it was still signiﬁcantly different from

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preshock values. These ﬁndings suggest that, unlike retention, PA learning (reentry latency at zero delay) is normal in HODI rats. b. Bohus et al. (1975) Bohus et al. (1975) compared Brattleboro HODI, HEDI, and Wistar normal (WNO) rats for acquisition and extinction on two multitrial active avoidance-learning tasks, an open ﬁeld test of exploratory and emotional reactivity, and a threshold reactivity test for various FS intensities. HODI and HEDI rats were also tested for acquisition/retention, and for pituitary–adrenal reactivity during retention testing in a single-trial passive avoidance task. Independent groups of subjects were used for each of the avoidance tasks, the open ﬁeld test, and for assessing sensitivity to FS. Shuttlebox avoidance training lasted 12 days (10 trials/day) and was followed by 7 sessions of extinction (10 trials/session). The results are diagrammed in Fig. 1, and were as follows: (1) HODI rats reached the learning criterion (80% avoidance responses in 12 days), although their rate of acquisition was slower, and their total number of avoidance responses was signiﬁcantly lower, than that for HEDI and WNO rats; acquisition performance was similar for the latter two groups; and (2) avoidance responding extinguished more rapidly in HODI rats than in either HEDI or WNO rats. Acquisition training in the pole-jump avoidance task occurred over a 6-day period (10 trials/day) and was followed by 4 days of extinction (10 trials/day). The results, given in Fig. 2, were as follows: (1) HODI and HEDI rats were similar in learning performance (both groups reached an approximately 75% performance level at the end of the 6-day acquisition period), and they were both deﬁcient (slower in reaching the avoidance criterion and

FIGURE 1 Acquisition and extinction of a shuttlebox avoidance response in homozygous (Ho-DI) and heterozygous (He-DI) diabetes insipidus rats and in Wistar (Wi) rats. Source: Bohus et al., 1975 (Fig. 1, p. 611). Copyright ß 1975 by Brain Research Publications Inc. Reprinted by courtesy of Elsevier Science, present publisher of this journal. CAR, conditioned avoidance response.

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FIGURE 2 Acquisition and extinction of a conditioned avoidance response in a pole-jumping situation in homozygous (Ho-DI) and heterozygous (He-DI) diabetes insipidus rats and in Wister (Wi) rats. Source: Bohus et al., 1975 (Fig. 2, p. 611). Copyright ß 1975 by Brain Research Publications Inc. Reprinted by courtesy of Elsevier Science, present publisher of this journal.

made signiﬁcantly fewer correct avoidance responses) relative to WNO rats; and (2) WNO rats were signiﬁcantly more resistant to response extinction than HEDI rats, which, in turn, made signiﬁcantly more avoidance responses than their HODI counterparts. Independent groups of HODI and HEDI rats were randomly assigned to shock (3 s, 1.0-mA FS) and nonshock conditions, and after a single learning trial were tested for PA retention at 1 min (0-h delay), 3 h, and 24 h. Pituitary–adrenal activity (plasma corticosterone levels) was assessed 15 min after the beginning of each retention test. The behavioral results indicated the following: (1) full PA behavior (maximum reentry latency of 300-s duration) occurred in both groups of Brattleboro rats when tested 0 h after the learning trial; and (2) in contrast to HEDI rats, which continued to exhibit full avoidance behavior in the retention tests 3 and 24 h after the learning trial, HODI rats showed partial passive avoidance at 3 h (much reduced but signiﬁcantly different from nonshock controls) and none at 24 h (no difference from nonshock controls). Thus HODI rats were strongly and signiﬁcantly impaired in PA retention tested 24 h after the learning trial. Measures of endocrine activity indicated that the plasma corticosterone level during retention was positively related to PA behavior. On each retention test, HEDI rats showed full PA behavior and corticosterone levels that were consistently signiﬁcantly higher than those of nonshocked controls. Plasma corticosterone levels in HODI rats decreased over the longer retention intervals, as did PA behavior. At 0-h retention, when full PA behavior occurred in HODI rats, plasma corticosterone levels did not differ from HEDI levels. At the 3-h retention test, when HODI rats showed partial PA behavior, plasma corticosterone levels were drastically reduced relative to the

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HEDI level, although still higher than that of nonshocked controls. At the 24-h retention test, when there was no evidence of PA behavior in HODI rats, plasma corticosterone levels remained signiﬁcantly lower than those of HEDI rats and did not differ from nonshocked control levels. These observations indicate that in the absence of VP there is an impairment of the psychological mechanisms underlying PA behavior and a correlated deﬁciency in the pituitary–adrenal endocrine response to the fear-provoking environment. Painted lines marked off a number of central and peripheral squares on the ﬂoor of the circular open ﬁeld. Subjects were placed in the center of the arena at the beginning of each daily 3-min session over four consecutive days and observed for ambulation (number of squares entered), rearing (both in the center of the ﬁeld and near the walls), grooming (face washing, etc.), and defecation (number of boluses deposited). Results of the open ﬁeld test were as follows: (1) ambulation (number of squares entered) was higher for both Brattleboro rat groups relative to WNO rats in both sessions 3 and 4 because of the failure of the former groups to show exploratory behavior habituation over the course of testing; (2) relative to WNO rats, Brattleboro rats showed a greater incidence of rearing in the middle of the arena in session 1, and near the wall in sessions 2 and 3 (HODI rats) or in sessions 3 and 4 (HEDI rats); and (3) except for session 1, during which defecation was performed more frequently by Brattleboro rats, and sessions 3 or 4, during which both HODI and HEDI rats groomed more frequently than did WNO rats, the groups were similar in these measures of open ﬁeld behavior. Taken together, these ﬁndings suggest that, relative to WNO rats, Brattleboro rats evidenced more exploratory activity (ambulation), an initially higher level of autonomic reactivity (defecation), and a slower rate of habituation to the novel environment of the open ﬁeld test. The test for sensitivity to FS pain used 2 sets of 12 FS intensities varying between 33 and 300 A. The index of threshold responsiveness was deﬁned as the lowest intensity of FS that elicited a reaction such as ﬂinch and jerk and/or jump and run. Pituitary–adrenal responsiveness was also determined in this test. The results of these tests indicated that the threshold currents for behavioral reactivity were similar in magnitude for HODI and HEDI rats but less for WNO rats. Pituitary–adrenal activity, indicated by plasma corticosterone levels after presentation of an FS series, did not differ between HODI and HEDI or between HODI and WNO rats, although HEDI rats exhibited larger increases in plasma corticosterone than did WNO rats. In summary, the results indicated that (1) learning may take place in the partial (HEDI) or total (HODI) absence of endogenous VP, because both groups learned both pole-jump and shuttlebox avoidance responses and exhibited full PA behavior when tested 1 min after the FS learning trial. Nevertheless, learning deﬁcits were observed in HODI rats on both multitrial avoidance tasks. Relative to WNO rats, they were slower to acquire successful avoidance responses, and made fewer correct avoidance responses

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during the learning period; (2) a consistent retention deﬁcit occurred in both HODI and HEDI rats, compared with WNO rats, on both active avoidance tasks. Of the two, HODI rats were more profoundly impaired in all three active and passive avoidance behavior retention tasks; (3) among the Brattleboro rats, pituitary–adrenal endocrine reactivity appears to be positively related to retention performance in the passive avoidance task; this is consistent with the suggestion that the memory of the FS mediates the pituitary–adrenal reaction elicited during the PA retention test. This is further supported by the lack of difference between HODI and WNO rats in pituitary–adrenal endocrine reactivity in response to a pain stimulus (FS); (4) relative to WNO rats, both Brattleboro groups showed more exploratory activity (ambulation, rearing) and autonomic reactivity (defecation) and slower habituation when placed in a novel environment (open ﬁeld test results); and (5) because HODI and HEDI rats did not differ in open ﬁeld behavior or pain sensitivity, yet did differ in both active and passive avoidance retention behavior, it was concluded that the former factors were not causal inﬂuences on the differences in retention between the two groups.

C. Inconsistencies in the Research Literature Regarding the Putative Brattleboro Diabetes Insipidus Retention Deficit Because De Wied and colleagues propose that vasopressin has an important role in memory storage and retrieval but not in the processes underpinning learning per se, this discussion has been limited to evidence relating the genetically associated VP deﬁciency to performance on tasks of retention. Subsequent to the early studies by De Wied and colleagues (Bohus et al., 1975; De Wied et al., 1975), a number of investigators reported that Brattleboro rats were equal to, or in some cases superior to, Long-Evans normal (LENO) rats in various tests of retention. Brito (1983) and Williams et al. (1983a) observed that HODI rats were equivalent to LENO rats in PA retention in a step-through PA task, and Miller et al. (1976) observed no signiﬁcant differences among HODI, HEDI, and LENO rats in extinction performance in a shuttlebox shock avoidance task. Although Bailey and Weiss (1979) observed that HODI rats were poorer in PA retention than HEDI rats, both groups did exhibit memory for the shock experience because latencies to enter the dark chamber were sharply increased in postshock relative to preshock trials; moreover, reentry latencies in both Brattleboro groups were higher than those observed in LENO rats tested in a separate experiment. Bailey and Weiss (1978) found that PA retention was equivalent for HODI and HEDI rats and was superior to LENO rats throughout the 4-day postshock observation period. Failure to observe a retention deﬁcit in Brattleboro VP-deﬁcient rats has also been reported by researchers using other types of retention tests. Brito

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(1983) observed no impairment in working memory in HODI relative to LENO rats in a T-maze alternation task tested with a short (8 s) delay between the forced-choice (information) and the free-choice run. Moreover, Brito and colleagues (Brito et al., 1981, 1982) observed that HODI rats retained a punishment-induced inhibition of a food-approach response longer than did LENO rats. On the other hand, several investigators have obtained evidence suggesting a retention deﬁcit in HODI rats. Ambrogi Lorenzini et al. (1985) and Drago and Bohus (1986) reported that Brattleboro DI rats extinguished a conditioned one-way shock avoidance response more rapidly than did LENO rats. Stoehr et al. (1993) tested the effect of vasopressin on conditioned freezing behavior to aversive shock treatment. HODI, HEDI, and LENO rats received three footshocks in a sound-attenuated box on the training day. The following day, and for 3 days thereafter, the rats were returned to the box without further shock treatment and evaluated for spontaneous freezing behavior. HODI rats showed signiﬁcantly less freezing behavior than did HEDI or LENO rats on each of the four test days. The authors interpreted these data in support of the notion that vasopressin has an important role in mediating appropriate autonomic and emotional responsivity in fear-conditioning paradigms. However, the reduced fear could also have been mediated, in part, by poorer retention in the HODI rats. Colombo et al. (1992) used a delayed alternation T-maze task, with varying intertrial intervals, to assess ability to retain spatial information in memory using a recently developed VP-deﬁcient strain of rat. Relative to M520/NO rats, M520/DI rats exhibited diabetes insipidus and were signiﬁcantly impaired in memory on this task. M520/HZ rats did not display diabetes insipidus and their task performance was intermediate between that of M520/DI and M520/NO rats.

D. Colony-Specific Heritable Traits and Inconsistent Findings Concerning a Retention Deficit in the Brattleboro DI Rat These contradictory ﬁndings may have been due, in part, to heritable behavioral characteristics, other than VP deﬁciency, that distinguish different laboratory colonies of Brattleboro rats. Although various groups of HODI rats share the VP gene mutation, differences between them in genetic background may be a more critical determinant of their behavior abnormalities (Brito, 1983; Williams et al., 1983a). For example, Gash et al. (1982) have pointed out that because small numbers of Brattleboro rats have been dispersed to found the various established colonies, it is possible that colony behavioral differences, independent of AVP deﬁciency, are due to differences in characteristics of the various colony founders.

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1. Selected Studies a. Herman et al. (1986a) Herman et al. (1986a) subsequently tested this hypothesis. Brattleboro DI (DI) and normal Long-Evans (NO) rats were obtained from different laboratory colonies and tested for timidity in an open ﬁeld emergence task, and for learning and memory in a simple runway approach/avoidance task and in a delayed nonmatching-to-sample (DNMS) task. DI and NO rats were obtained from colonies maintained at Charing Cross Hospital in London (CC/DI and CC/NO) and colonies of American suppliers in Rochester, New York (i.e., RO/DI and RO/NO). These researchers found that (1) DI rats from both colonies exhibited a high degree of neophobia (only 3 of the 10 subjects left the home cage to enter the open ﬁeld arena over the 4-day test period). In contrast, both NO groups showed a strong tendency to enter the open ﬁeld with decreasing emergence latency as a function of testing experience; (2) acquisition of the food-rewarded runway response was equivalent for the CC/DI, RO/DI, and RO/NO groups but slower for the CC/NO group; (3) the mouthshocks given on the last day of training severely disrupted the food-approach habit in all groups in the ﬁrst postshock trial, indicating memory for the shock in all the subjects; (4) recovery from the punishment-induced response inhibition was quite variable among the groups, with the RO/DI group showing accelerated recovery of approach compared with the CC/DI group and both NO groups; (5) all four groups evidenced dispositional memory (acquired the nonmatching-to-sample contingency in the DNMS task), but RO/DI rats learned the contingency more quickly than either the CC/DI group or the CC/NO group; and (6) RO/DI rats also outperformed CC/DI rats on representational memory as indicated by the greater accuracy of their responses, which depended on trial-speciﬁc information, and by a lesser tendency to adopt a position habit, which does not require representational memory. The results were interpreted as indicating that the VP deﬁciency accounted for the ‘‘fearfulness’’ observed in the open ﬁeld emergence test, but that colony-speciﬁc genetic and/or early experiential factors, rather than a VP deﬁciency per se, contributed to the differences observed in long-term memory in the approach/avoidance task (i.e., rate of recovery from the mouthshock experience) and in dispositional and representational memory in the DNMS task. Conﬁrmation of the Herman et al. hypothesis about colony differences in emotional and cognitive behavior led these researchers to develop an inbred strain of rats homozygous for the VP deﬁciency (Roman high avoidance [RHA]:di/di) but otherwise identical to a normal Roman high avoidance (RHA:þ/þ) inbred strain of rats in heritable behavioral traits. The RHA:di/di strain was derived from an original mating between Brattleboro HODI rats and RHA:þ/þ rats and subsequent backcrosses between F2 generations of offspring homozygous for the VP deﬁciency and the RHA:þ/þ parent strain.

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b. Herman et al. (1986b) Herman et al. (1986b) compared RHA:di/di rats with RHA:þ/þ rats in the same tasks used by Herman et al. (1986a), and included a test of PA behavior and additional tests of open ﬁeld emotional behaviors. The results indicated that (1) in the approach/avoidance task the groups were equivalent in adapting to the maze (eating in the goal box) and in acquiring the runway response, and both showed memory for the shock experience, although di/di rats showed shorter postshock latency to approach the goal box. The main difference between the groups concerned performance recovery from the shock experience: di/di rats gradually recovered their preshock goal approach speed during successive postshock sessions whereas þ/þ rats showed no strong tendency to enter the goal box during any of the postshock sessions; (2) in the DNMS task, both groups were able to use representational and dispositional memory to solve the problem, but relative to þ/þ rats, di/di rats were less accurate over all sessions and required signiﬁcantly more trials to acquire the DNMS contingency, suggesting impaired dispositional memory; and (3) in the PA task, the two groups did not differ on latency to enter the dark (shock) compartment, either before or 24 h after the shock trial. Reentry latencies tended to be distributed in a bimodal fashion for both groups. The open ﬁeld test results indicated that di/di rats habituated more slowly to the open ﬁeld than did þ/þ rats. The two groups did not differ in ambulation scores on trial 1, but did so on subsequent trials. In comparison with þ/þ rats, di/di rats showed higher rates and slower habituation of rearing, higher defecation scores, and a greater tendency to ambulate in the central squares; there were no differences in freezing at the center of the open ﬁeld or in grooming behavior. These ﬁndings indicated that di/di rats were able to learn the simple runway goal-approach response in a normal fashion and evidenced no absolute memory impairment because they were able to use dispositional and representational memory to solve the DNMS problem, showed inhibited goal box approach 24 h after the shock experience, and PA retention equivalent to that of þ/þ rats. Nevertheless, poorer performance in the cognitive tasks indicated by a signiﬁcantly shorter postshock approach latency, greater recovery of the goal-approach response in the runway, more trials required to master the DNMS contingency, and reduced accuracy in performing this memory task could suggest relative memory impairment of di/di rats. An alternative interpretation is suggested by the open ﬁeld behavior of di/di rats; subsequent to day 1 in the open ﬁeld arena, di/di rats showed greater frequency in ambulation and rearing and slower habituation of this behavior than did þ/þ rats. Moreover, the heightened autonomic reactivity (defecation) of di/di rats relative to þ/þ rats was present on all test days. This difference in open ﬁeld behavior suggests a heightened emotional/ arousal level among þ/þ subjects that, in turn, could have inﬂuenced

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performance in the cognitive tasks. Herman et al. (1986b) were inclined to favor this latter interpretation. They hypothesized that VP deﬁciency may, directly or indirectly, result in greater than normal increases in arousal and/or arousability in certain situations (e.g., under food deprivation), which can then affect attentional selectivity and other cognitive functions necessary for normal performance on behavioral tasks, thereby indirectly inﬂuencing memory processes.

E. Brattleboro Rat Retention Deficit I: A Primary or Secondary Effect of VP Deficiency? Bailey and Weiss (1981) suggest that the HODI retention deﬁcits that have been observed in avoidance tasks may represent secondary rather than primary effects of chronic VP deﬁciency. These include increased plasma levels of OT (released by the chronic state of mild dehydration), deﬁciency in growth hormone (may be responsible for the smaller size of HODI rats), and decreased responsiveness of the pituitary–adrenocortical system to some stressors [associated with decreased release of corticotropin-releasing factor (CRF) and reduced anterior pituitary responsiveness to CRF, both of which are reversed by VP treatment]. For example, a PA retention deﬁcit could be due to a reduced pituitary–adrenal response to FS in the learning trial (Weiss et al., 1969, 1970). However, the ﬁnding that a single injection of DG-AVP promptly reversed the PA retention deﬁcit without correcting the diabetes insipidus disorder suggested that the VP deﬁciency had a primary effect on retention (De Wied et al., 1975).

F. Brattleboro Rat Retention Deficit II: An Arousal-Mediated Phenomenon? The study of Brattleboro rats is also pertinent to determining the mechanism by which exogenous vasopressin may inﬂuence retention in avoidance tasks. For example, arousal level has been suggested as an indirect mechanism by which vasopressin alters retention behavior in avoidance tasks (discussed in Chapters 6 and 7). Apropos to this idea, several investigators have suggested that differences in retention between Brattleboro and LENO rats may be due to alterations in emotionality, temperament, or arousal level produced directly or indirectly by VP deﬁciency. As previously discussed, Herman et al. (1986a,b) share this viewpoint. Several researchers have suggested that Brattleboro rats are more fearful than LENO rats. This view has been supported by the following ﬁndings: (1) Bailey and Weiss (1981) observed that compared with LENO rats, HODI and HEDI rats showed less exploratory behavior (crossings) in the open ﬁeld arena and remained frozen or inactive rather than running from the experimenter’s approach at the end of the test. This behavior of the Brattleboro

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rats was interpreted as indicating greater fear, which in turn was considered to be consistent with the longer PA reentry latencies observed in these rats (Bailey and Weiss, 1978, 1979); and (2) Brito et al. (1981) and Brito (1983) judged HODI rats to be more timid than LENO rats as indicated by (a) their slower emergence from the home cage into an open ﬁeld, (b) the smaller amount eaten when fed in a novel environment, and (c) their slower adaptation to a T-maze apparatus used for various cognitive tests (Brito, 1983; Brito et al., 1981). Some researchers, although failing to observe evidence of greater fear and timidity, nevertheless have obtained data suggestive of an altered arousal level in HODI and HEDI rats. Williams et al. (1983a) observed that compared with normal LE rats, HODI rats (both males and females) exhibited signiﬁcantly higher activity scores (crossing and rearing activity) during the initial 15 trials in an open ﬁeld test. Williams et al. (1983b) showed that peripherally administered AVP (5 g/rat, subcutaneous) counteracted the early-trial increase in activity level exhibited in HODI rats. Moreover, AVP decreased open ﬁeld activity to a lesser degree in normal rats, indicating their lower sensitivity to the inﬂuence of the hormone on this aspect of behavior. The ﬁnding that the activity level of the VP-deﬁcient rat is elevated during an early observational period and that exogenous vasopressin can counteract this activity was interpreted as indicating that AVP has a direct effect on the processes that underlie this increased activity (presumably the altered emotional, motivational, and/or attentional state of the animal). Still other researchers have reported no evidence of increased timidity in novel environments associated with inherited VP deﬁciency. Laycock and Gartside (1985) reported that Brattleboro DI rats showed less fear and performed signiﬁcantly better than LENO rats in an operant task when ﬁrst placed in the Skinner box and Colombo et al. (1992) reported no signiﬁcant difference among M520/HO, M520/HE, and M520/NO rats in rate of adapting to a novel test environment (the T-maze alternation task), a measure used by Brito et al. (1981) to operationally deﬁne ‘‘timidity.’’

G. Brattleboro Rat Model Revisited: Recent Findings by De Wied and Colleagues 1. Selected Study: De Wied et al. (1988) De Wied et al. (1988) reported a reinvestigation of retention behavior in the Brattleboro rat. HODI, HEDI, Long-Evans normal (LENO), and Wistar normal (WNO) rats were tested for emotional behavior in large and small open ﬁelds, and for acquisition and/or retention in three avoidance tasks (polejump avoidance, step-through PA, and shock-avoidant discrimination in a T-maze) and two appetitive tasks (food-rewarded visual discrimination and the hole board search task).

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The open ﬁeld test results veriﬁed earlier reports of an altered state of emotionality or motivation in HODI rats (Bohus et al., 1975). In both the small and large open ﬁelds, HODI rats exhibited a higher level of activity and slower habituation than the other groups. With the exception of normal learning in the original and reversed discrimination of the aversive T-maze task, HODI and the HEDI rats exhibited learning and retention deﬁcits in all the remaining behavioral tests. Thus, in comparison with LENO and WNO rats, both HODI and HEDI rats were signiﬁcantly impaired in acquisition of the black–white discrimination in the food-rewarded Y-maze, learning, working and reference memory in the hole board food search task, acquisition and maintenance of the pole-jump shock avoidance response, and retention in the PA task. Moreover, the HEDI rats tested in this study were more impaired in performing active and passive avoidance tasks than was observed earlier (Bohus et al., 1975) and did not differ from HODI rats in the other tests of learning and retention. The observation that the DI rats in these more recent studies appeared to be even more impaired in learning and retention than those used earlier supported the hypothesis that genetic differences exist for various colonies of this inbred strain. On the other hand, the authors noted that while there were differences in degree, the VP-deﬁcient rats were consistently impaired in avoidance task retention in both their earlier and more recent investigations.

H. Section Summary and Concluding Remarks The Brattleboro DI rat model would seem to be a highly valuable asset to the investigation of the putative role of vasopressin in memory processing. Theoretically, if endogenous vasopressin has an important role in memory storage and retrieval, even if limited to stressful learning conditions (e.g., avoidance paradigms), VP-depleted subjects should exhibit poorer memory than their VP-repleted counterparts in such learning environments. Early ﬁndings by De Wied and colleagues (Bohus et al., 1975; De Wied et al., 1975) did indicate that HODI rats were signiﬁcantly impaired in memory in active, and especially passive, avoidance tasks. Subsequent research from other laboratories, however, questioned such a memory impairment (e.g., Bailey and Weiss, 1978, 1979; Brito, 1983; Miller et al., 1976; Williams et al., 1983a). The research of Herman and colleagues (1986a) suggested that variation in genetic backgrounds of different laboratory colonies of Brattleboro rats could produce inherited variations in behavior tendencies that affect performance in many studies. This could contribute to the inconsistent retention effects observed in various studies and also obscure which of the behavioral results were due to the VP deﬁciency rather than to colony-speciﬁc genetic background inﬂuences.

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De Wied and colleagues (1988) acknowledged the inﬂuence of colonyspeciﬁc genetic differences on performance when comparing the results of their earlier and later studies with the Brattleboro rat model, but noted that retention impairment was a consistent ﬁnding in both sets of studies. Aside from the inconsistent results regarding a Brattleboro-associated retention deﬁcit, there is the problem of interpreting the cause of this deﬁcit when it does occur. A number of investigators disagree with De Wied and colleagues’ proposal that the absence of vasopressin at memory-processing sites is the major cause of the retention impairment observed in Brattleboro DI rats. It has been pointed out that any or some combination of hormonal and/or metabolic secondary effects of diabetes insipidus may inﬂuence cognitive performance in these animals (Bailey and Weiss, 1981). In addition, alterations in emotional, temperamental, and/or arousal levels, directly or indirectly caused by the VP deﬁciency, are viewed by many researchers as chief mediators of the cognitive effects observed in these Brattleboro rats (e.g., Bailey and Weiss, 1981; Brito et al., 1981; Herman et al., 1986b; Williams et al., 1983a,b). When considering the secondary effects associated with the genetic VP deﬁciency, together with heritable trait differences among localized colonies of Brattleboro or other inbred strains of VP-deﬁcient rats, it seems an inescapable conclusion that this experimental model is of limited value in settling issues raised by alternative theories regarding the nature of the contribution of vasopressin to memory processing.

III. Further Study of the Role of Endogenous VP and OT in Memory Processing: Peripheral and/or Central Mechanisms?

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A. Introductory Comments Because VP and OT are present in the body as both hormones and neural transmitters, and are released centrally and peripherally by a variety of stress stimuli (Chapter 1), it is important to learn the sources of these peptides that have putative effects on retention. De Wied and colleagues theorize that it is the central, and not the peripheral, systems that physiologically modulate memory consolidation and retrieval, and mediate the retention effects observed after peripheral injection of these peptides. Participation of central VP and OT circuitry in memory processing is quite feasible given the presence of VP-ergic and OT-ergic ﬁber terminals and binding sites within brain structures implicated in memory processing (Buijs, 1987; Buijs et al., 1978; Caffe et al., 1987; De Vries and Buijs, 1983; Sofroniew, 1983; Sofroniew and Weindl, 1978; Van Leeuwen and Caffe, 1983). De Wied and colleagues have used experimental and correlational techniques in their studies of physiological involvement of peripheral and central

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VP and OT systems in memory storage, in both active and passive avoidance paradigms. The experimental technique discussed below temporarily removed the inﬂuence of a centrally or peripherally localized neuropeptide by neutralizing it through selective injection of antiserum to the peptide. Their correlational studies examined levels of VP or OT within the plasma, cerebrospinal ﬂuid (CSF), or speciﬁc brain sites at designated times during learning or retention testing and correlated these levels with measures of learning and/or retention.

B. Neutralizing Peripheral or Centrally Circulating VP or OT by Antiserum Treatment: Effect on Memory Processing 1. Selected Study: Van Wimersma Greidanus et al. (1975a) Van Wimersma Greidanus et al. (1975a) neutralized central or peripheral levels of vasopressin by injection of VP antiserum, and studied the effect on passive avoidance (PA) retention in male Wistar rats. They were tested in a single-trial step-through PA task during which they received either no FS or a 3-s FS of 0.75-mA intensity. Retention testing occurred either 2 min, 1 h, 4 h, 24 h, or 48 h after the learning trial, with 300 s allowed for maximal reentry latency. One group of rats was injected intracerebroventricularly with 1 l of anti-VP serum or control rabbit serum. This procedure neutralized central but not hormonal levels of VP because it had no effect on urine production, water consumption, or urine vasopressin levels. Another group was injected intravenously with 100 l of anti-VP serum or control rabbit serum. Injections were given either 0.5 h before, or immediately after, the PA learning trial. Neutralization of central VP levels did not affect PA learning because reentry latencies were maximal (300 s) in the rats injected intracerebroventricularly with anti-VP serum, as they were in the controls, when tested 2 min and 1 h after the learning trial. However, PA retention was severely retarded in the anti-VP-treated subjects, as indicated by reentry latencies at 4 h that were signiﬁcantly reduced, relative to those of the controls, and even more markedly reduced at 24 and 48 h after the learning trial. Retention, however, was not affected when peripherally circulating AVP was neutralized by an intravenous injection of 100 times the amount of antiserum used for intracerebroventricular injection. For these peripheral VP-neutralized rats, median reentry latency was maximal, as it was for the rats given control rabbit serum at both the 24- and 48-h retention tests. A role for limbic VP-ergic systems in PA retention has been indicated by the observation that anti-VP serum more efﬁciently neutralizes central vasopressin when it is injected into limbic system structures rather than into a lateral ventricle. Thus, PA retention impairment can be achieved by a more

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diluted antiserum solution if injected into the various limbic system structures rather than into the lateral ventricle (reported in Van Wimersma Greidanus et al., 1986). A role for central OT systems in memory modulation is suggested by the ﬁndings that neutralizing central levels of OT by intracerebroventricularly injected anti-OT serum improved PA retention (Bohus et al., 1978b; and see Chapter 2).

C. Correlational Studies 1: Avoidance Retention and AVP Levels in the Blood Correlational studies have been conducted to further investigate the role of endogenous vasopressin in memory storage and retrieval. Because it is possible that vasopressin released from the brain into peripheral circulation during learning contributes to memory processing, studies have been conducted to relate plasma levels of vasopressin to active and passive avoidance behavior. 1. Selected Studies a. Thompson and De Wied (1973) Thompson and De Wied (1973) tested the proposition that VP is released by environmental cues previously associated with an aversive experience. Antidiuretic (AD) activity was bioassayed from eye plexus blood collected from anesthetized male rats of an inbred Wistar strain. The blood was collected from rats anesthetized with ether for 45 s, immediately after the ﬁrst (24 h), or in some experiments after the second (48 h), PA retention trial. The assay was accomplished by injecting the collected serum into female rats of the same inbred Wistar strain, which were then bioassayed for AD activity by assessing the effect of the injected serum on urine ﬂow rate in water-loaded alcohol-anesthetized rats over consecutive 10-min periods. Eye plexus blood instead of trunk blood was used because of its higher level of AD activity. In addition, AD activity was measured in rats whose PA behavior had been modiﬁed by treatment with either adrenocorticotropic hormone peptide [ACTH(1–10); 30 g/rat, subcutaneous] or DG-LVP (0.5 g/rat, subcutaneous), which lacks the pressor and antidiuretic effects of the parent peptide (De Wied et al., 1972). In the experiment that studied AD activity under nonpeptide treatment conditions, the subjects were assigned to either the nonshocked (NS) control group or received a 3-s FS at an intensity of 0.25, 0.50, or 1.00 mA in the PA learning trial, and were tested for retention 24 h later. In the two experiments that used peptide-treated subjects, PA training and testing was similar except that the shocked groups received an FS of only 0.25-mA intensity and were tested for retention at both 24 and 48 h after the FS trial. The peptides, either ACTH(1–10) (30 g/rat, subcutaneous) or DG-LVP (0.5 g/rat, subcutaneous), or placebo solutions were injected 1 h before the 24-h retention test trial (retrieval design).

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The results were as follows: (1) although the numbers of subjects in the nonpeptide-treated FS groups were too small for statistical analysis, the median reentry latencies in the 24-h retention trial increased in correspondence with increased shock intensities, consistent with previous observations (Ader and De Wied, 1972); (2) AD activity level in the nonpeptide-treated NS group, sampled immediately after the 24-h retention trial, was signiﬁcantly lower than that in each of the FS groups, and AD levels increased incrementally in correspondence with increased levels of shock intensity; (3) relative to placebo treatment, ACTH(4–10) did not inﬂuence AD activity in the NS controls but signiﬁcantly increased it in the shocked subjects. Median reentry latency was also signiﬁcantly higher in the ACTH-treated group than in the placebo-treated FS group. However, neither the increased reentry latency nor the increased AD activity was maintained in the second retention test, 24 h later; (4) DG-LVP, devoid of endocrine activity, unexplainably increased AD activity in the nonshocked controls after the 24-h retention trial; nevertheless, AD activity in the DG-LVP-treated shocked subjects was signiﬁcantly higher than that for the DG-LVP-treated nonshocked controls; and (5) DG-LVP treatment markedly increased reentry latency and signiﬁcantly increased AD activity in the shocked subjects relative to the placebotreated controls, and these measures remained largely unchanged in the second retention test trial. These results led to the following conclusions: (1) the proposal that ACTH-like peptides and vasopressin inﬂuence memory retrieval by different mechanisms was again supported by the results indicating a short-term effect of ACTH(4–10) versus a long-term effect of DG-LVP on PA behavior; (2) conditioned cues, which can elicit memory retrieval for the footshock experience, can also cause a release of VP into the peripheral circulation, and these effects are related to severity of the aversive experience; and (3) when reentry latency was increased by either DG-LVP or ACTH(1–10) the release of AVP into the peripheral circulation also increased. Although this could imply that the VP released into the circulation may have fed back to inﬂuence the behavioral response, the authors pointed out the need for further experiments on this question. Information relevant to this issue is provided by experimental ﬁndings discussed below. b. Van Wimersma Greidanus et al. (1979a) Van Wimersma Greidanus et al. (1979a) examined AVP levels in the peripheral circulation (trunk blood) in male inbred Wistar rats tested in active (pole-jump) and passive (single-trial, step-through) avoidance tasks. AVP levels in trunk blood were measured in independent groups of rats killed at various points in time during the course of active or passive avoidance testing. For the active avoidance task, the animals received acquisition training (10 trials/day) on days 1–3 and extinction testing (10 trials) on day 4. Plasma levels of AVP were assessed from trunk blood collected either before (i.e.,

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after a 3-min exposure to the pole-jump apparatus) or immediately after each day’s session of acquisition and extinction. The results were as follows: (1) successful avoidance responding increased over the 3-day acquisition period, reached the criterion performance level on day 3, and, as expected, decreased below asymptotic performance level during extinction testing on day 4; and (2) there was no signiﬁcant change in plasma AVP level measured before or after the behavioral test session on any day of acquisition or during extinction. Moreover, neither pre- nor postsession AVP levels during extinction differed signiﬁcantly from any values observed during acquisition training. Thus plasma AVP levels were not related to behavioral changes that occurred during acquisition or extinction testing in this active avoidance task. In the passive avoidance task, depending on the group, the rats were not shocked (NS group) or were given a 3-s FS of low intensity (0.25 mA; LS group) or high intensity (1.0 mA; HS group). Trunk blood was collected either 5, 60, or 300 s (maximal duration for observing reentry latency) after the onset of the 24-h retention test. In addition, trunk blood was collected 300 s after the learning trial for one group of subjects tested with the highintensity FS. The results indicated that (1) reentry latency increased with increasing FS intensity, with all subjects in the HS group exhibiting a maximal PA response (300-s reentry latency); (2) when trunk blood was collected 5, 60, or 300 s after the onset of the retention test trial, there was no signiﬁcant difference between the LS group and its NS control group in plasma level of AVP; (3) the HS group did not differ from its NS control group in plasma AVP when blood was collected 5 or 60 s after the onset of the retention test. However, when blood was collected at the end of the test trial, plasma AVP level in the HS group (median reentry latency, 300 s) was signiﬁcantly higher than that of either the NS or LS group; and (4) plasma AVP levels did not differ from basal levels in rats killed 300 s after receiving the 1.00-mA FS, in accordance with a report by Husain et al. (1976) that noxious stimuli do not necessarily cause a rise in plasma AVP levels. The lack of relation between learning performance and plasma AVP levels (assessed either during the 3 days of multitrial pole-jump avoidance learning, or during the single PA learning trial with the 1.00-mA FS) indicated that plasma AVP is an unlikely route by which endogenous vasopressin inﬂuences retention in these tasks. The ﬁndings that plasma AVP levels in the HS group increased after 300 s, but not after 5 or 60 s in the postshock test environment, suggests that the longer the exposure to this environment, the greater the hormonal AVP released by the conditioned fear stimuli. c. Mens et al. (1982a) Mens et al. (1982a) determined plasma levels of oxytocin (OT) and vasopressin (VP) during acquisition and retention of passive avoidance (PA) behavior in male inbred Wistar rats. Rats in the OT assessment groups were given either no shock (NS group) or a 3-s FS

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of low intensity (0.25 mA) or high intensity (0.75 mA). Blood was collected in these subjects either before the behavior test (basal level assessment), 20 min after the learning trial, or 180 s (maximal duration for retention trial) after the 24-h retention test. Rats in the VP assessment groups received either no shock or a 3-s FS of 0.50-mA intensity. In these rats, blood was collected after the 2-min adaptation trial on day 1 of PA training, 20 min after the learning trial, or 180 s after the 24-h retention test. Plasma levels of the peptides were measured by a radioimmunoassay (RIA) procedure. These tests permitted a measure of peptide release into peripheral circulation by the noxious unconditioned stimulus (FS) during the PA learning trial, and by the conditioned stimuli present during the PA retention trial. The results for subjects in the OT assessment groups were as follows: (1) reentry latencies were positively related to shock intensity, with a maximal median reentry latency (180 s) for the rats given the highest FS level; (2) when measured 20 min after the learning trial, mean plasma OT in the nonshocked group did not differ signiﬁcantly from the basal value, nor did the nonshocked group signiﬁcantly differ from the 0.75-mA shock group in this measure. However, the 0.25-mA shock group unexpectedly exhibited a signiﬁcant decrease in plasma OT relative to the nonshocked group; and (3) after the retention trial, there were no signiﬁcant differences between the nonshocked controls and either of the shocked groups in mean plasma OT levels, nor was there any relationship between plasma OT levels and reentry latencies. The results for the VP assessment groups were as follows: (1) reentry latencies were bimodally distributed and subjects were classiﬁed as avoiders (median reentry latency, 180 s) or nonavoiders (median reentry latency, 68 s); (2) plasma levels of AVP assessed 20 min after the FS trial or 3 min after onset of the retention trial did not differ from levels obtained during adaptation to the task; and (3) there was no relationship between reentry latency and plasma levels of AVP. Summarizing, barring the exception of the plasma OT result, which needs replication, this study indicates, for the FS levels used, no change in plasma levels of VP or OT during PA learning or retention; nor were these levels related to reentry latencies. These results further support the view that hormonal VP or OT feedback during PA acquisition does not contribute to memory processing in this task. d. Laczi et al. (1983c) Laczi et al. (1983c) investigated vasopressin levels in eye plexus blood in relation to passive avoidance (PA) acquisition and retention in normal Wistar (WNO) rats, Brattleboro HODI and HEDI rats, and Brattleboro HONO variant rats, normal for the gene locus encoding the VP precursor. Because only female HONO rats were available, their data were compared with data obtained from female WNO rats. Male rats were used in all other experimental tests. Depending on the experiment, eye plexus blood was collected from anesthetized rats at various times after the

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PA learning trial and/or the 24-h retention trial. The collected blood samples were used to assess the plasma level of antidiuretic (AD) activity and/or immunoreactive AVP (irAVP). Experiment 1 was designed to replicate and extend the experimental study of Thompson and De Wied (1973), and assessed both AD activity and irAVP in eye plexus blood withdrawn either 30 s after the PA learning trial or 30 s after the 24-h retention trial. The WNO subjects were assigned to either a nonshock (NS) group or to one of three FS groups (a 3-s FS at an intensity of 0.25, 0.50, or 1.0 mA). The results of experiment 1 were as follows: (1) each of the FS groups displayed signiﬁcantly longer reentry latencies than did the NS controls; (2) reentry latencies increased with FS intensity, but maximum latency (300 s) was already present in rats given the 0.50-mA FS; (3) reentry latency was not affected by the procedure of collecting eye plexus blood after the training trial because there was no signiﬁcant difference in this measure between subjects from which the blood was taken after the learning trial and those from which it was taken after the retention trial; (4) plasma AD activity and irAVP levels were high in nonshocked as well as in shocked rats assessed after the training trial, and these levels were not signiﬁcantly correlated with FS intensity. This ﬁnding suggested that handling and the test environment itself were sufﬁcient to release VP into the circulation and FS stress failed to add to this effect; (5) when assessed after the retention trial, AD activity was slightly but signiﬁcantly higher in the 0.25-mA FS than in the nonshocked group, and marked and signiﬁcant differences in this measure occurred between the subjects given higher FS levels and NS controls; and (6) AD activity after the retention trial was positively and signiﬁcantly correlated with FS intensity used in the learning trial, indicating that the noxious conditioned stimuli caused a release of VP into the peripheral circulation during the retention trial. The results of this experiment corroborated the observations of Thompson and De Wied (1973), which showed that PA performance is related to AD activity in eye plexus blood (i.e., the higher the reentry latencies the higher the levels of AD activity in eye plexus blood collected immediately after the 24-h retention trial). Experiment 2 was designed to replicate the unexpected ﬁnding in experiment 1 that FS did not further increase the release of VP into the peripheral circulation. Plasma VP was collected for RIA assessment 30 s (0 min), 10 min, and 30 min after the PA learning trial to compare WNO groups receiving no shock, a low FS (0.25 mA), or a high FS (1.00 mA). The results indicated that (1) no signiﬁcant differences among the three FS groups in irAVP levels were observed for any of the three collection times; (2) nevertheless, reentry latency in the 24-h retention trial was signiﬁcantly related to FS intensity; and (3) irAVP levels 30 min after the PA learning trial were signiﬁcantly reduced compared with those determined 0 min after the learning trial for the NS and shocked groups.

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In experiment 3, male HEDI and HODI subjects and female HONO and WNO rats received either no FS or a 1.0-mA FS during the PA learning trial, and were tested for retention 24 h later. Eye plexus blood, collected 30 s after the learning trial, was tested by RIA, and samples obtained 30 s after the 24-h retention test were measured by both AD assay and RIA. The results were as follows: (1) for the HEDI subjects: reentry latencies were maximal (300 s) for the 1.0-mA FS group, irAVP levels did not signiﬁcantly differ between NS and FS groups whether sampled after the learning or the retention trial, and irAVP was lower in comparison with NS WNO rats; (2) none of the HODI rats showed PA behavior, and irAVP levels were under the limit of detection in both NS controls and shocked rats; and (3) comparisons between HONO and WNO female rats indicated signiﬁcantly longer reentry latencies for the FS relative to the NS groups for both strains of rat; for blood collected after the learning trial there were no differences in AD and irAVP activity between the FS and NS groups of either strain. When sampled after the retention trial, however, AD and irAVP activity were signiﬁcantly higher in the FS than in the NS groups in both strains of rat. In experiment 4, VP levels in eye plexus blood and PA behavior were tested in WNO rats pretreated with intracerebroventricularly injected antiVP serum (1 l of undiluted serum) or control (normal rabbit serum) serum. Injections were given 0.5 h before the PA learning trial (consolidation design) or 0.5 h before the 24-h retention trial (retrieval design). The subjects were assigned to either the nonshock condition or the high FS (1.0 mA) condition. Eye plexus blood was collected 30 s after the learning trial or 30 s after the retention trial. The results indicated that (1) intracerebroventricular injection of anti-VP serum, given before either the learning or the retention trial, markedly reduced reentry latency compared with rats treated with control serum, indicating a marked impairment of both consolidation and retrieval; (2) there were no differences in irAVP levels, assessed after the training trial, between rats pretreated with anti-VP or control serum; and (3) irVP levels assessed after the retention trial were signiﬁcantly lower in rats pretreated with anti-VP serum than in controls, whether injections were given before PA training or retention testing; moreover, the decrease in irAVP was more marked when anti-VP serum was given before the retention trial rather than the training trial. Summarizing the results of this study: (1) plasma AVP assessed after the 24-h retention trial was signiﬁcantly higher in the FS than in the NS groups for WNO males (experiments 1 and 2) and for both HONO and WNO females (experiment 3), and this level was positively associated with reentry latency scores (experiments 1 and 2), which in turn reﬂected the FS intensity used in the learning trial (experiments 1 and 2); (2) plasma AVP assessed after the PA learning trial was not inﬂuenced by FS in WNO males (experiments 1 and 2), HEDI males (experiment 3), or HONO and WNO females (experiment 3); nor was there a relationship between this level of plasma

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AVP and reentry latency scores (experiments 1 and 2); (3) anti-VP serum, given 0.5 h before the learning and the retention trial, signiﬁcantly impaired memory consolidation and retrieval, respectively (experiment 4); and (4) moreover, anti-VP serum injected 0.5 h before either the training or retention trial impaired the ability of conditioned stimuli, encountered in the retention test, to release VP into the circulation (experiment 4). The authors concluded that the results of this study are in accord with the interpretation that although hormonal AVP is released by memorymediated fear of the test environment, the painful FS itself does not signiﬁcantly inﬂuence this release. More importantly, the results indicate that there is no basis for concluding that peripherally circulating VP during the learning trial contributes to memory storage in this task.

D. Correlational Studies 2: Avoidance Retention and AVP Levels in the CSF The presence of central VP-ergic and OT-ergic binding sites in various limbic structures suggests that central VP and OT systems may play important roles in memory storage. If so, one might expect that CSF levels of these peptides would correlate with retention behavior, whether CSF serves as a medium for transporting the peptide from sites of synthesis to sites of memory processing or whether it merely contains the spillover effects of VP and OT secreted at activated neural terminals. Several studies, described below, were carried out to determine whether CSF levels of VP and/or OT are related to PA retention. 1. Selected Studies a. Van Wimersma Greidanus et al. (1979a) Van Wimersma Greidanus et al. (1979a) examined irAVP in the CSF of rats tested in a single-trial, step-through passive avoidance task. The rats received either no FS, a lowlevel FS (0.25 mA, 3 s) or a high-level FS (1.0 mA, 3 s). CSF in a lateral ventricle was collected from freely moving rats via a previously implanted cannula, 5 min after the 24-h retention test but not after the FS training trial. The results indicated that whereas median reentry latency scores increased in accordance with increased FS intensity, the no-shock and two footshock groups did not signiﬁcantly differ in CSF level of irAVP. b. Mens et al. (1982a) Mens et al. (1982a) assessed irVP and irOT levels in the CSF at various times during PA testing. CSF was withdrawn from the cisterna magnum of freely moving rats via previously implanted cannulas. Samples were collected during the adaptation trial, 20 min after the FS learning trial, and 3 min after the onset of the 24-h retention test. Subjects in the OT-tested groups received no shock (NS group) or an FS of 0.25- or 0.75-mA intensity for 3 s. Subjects in the VP-tested groups received no shock or a single 0.50-mA FS for 3 s.

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The results for the irOT-tested subjects indicated (1) no signiﬁcant difference in CSF levels of irOT between the NS group and either the lowor high-FS groups when tested after the learning trial, or after the retention test trial; (2) no signiﬁcant correlation between reentry latencies and OT levels in CSF measured after the retention test; and (3) marked differences in reentry latencies among the different shock groups, with maximum PA behavior (median reentry latency, 180 s) occurring at the 0.75-mA FS level. The results for the irAVP-tested subjects indicated: (1) no signiﬁcant effect of the FS experience on CSF levels of irAVP and (2) a bimodal distribution of reentry latency scores for the shocked rats, with good avoiders exhibiting a median reentry latency of maximal duration (180 s) and nonavoiders exhibiting a median reentry latency of 68 s. In addition, (3) CSF levels of irAVP in avoiders were not signiﬁcantly different from those in nonavoiders, and individual CSF levels of AVP were not correlated with avoidance reentry latencies in the 24-h retention test. c. Laczi et al. (1984) Laczi et al. (1984) determined CSF levels of irVP and irOT in male Wistar rats tested in a single-trial passive avoidance task. The rats received either no FS, a low-level FS (0.25 mA), or a high-level FS (1.00 mA) for 3 s. Retention test trials were given at both 24 and 120 h (5 days) after the learning trial. CSF was collected from the cisterna magnum in cannulated, anesthetized rats 30 s after the learning trial and the 24-h and 120-h retention test trials. Compared with the nonshocked rats, shocked rats exhibited longer reentry latencies in both retention tests, although the latencies were slightly lower in the 5-day test than in the 24-h test. Reentry latencies were positively correlated with FS intensity. When measured after the learning trial, CSF AVP levels were increased in shocked rats relative to the nonshocked controls, but this increase was statistically signiﬁcant only for rats that received the 1.0-mA FS. When measured after the 24-h retention test, CSF irAVP was signiﬁcantly increased for the low-level FS animals but was below the detection limit for the high-level FS-treated animals. After completion of the 5-day retention test, CSF irAVP was signiﬁcantly increased in both FS groups and the amount of increase was related to reentry latencies, which in turn reﬂected intensity of the FS in the PA learning trial. The ﬁnding that irAVP in the CSF was below the detection limits in the 24-h retention test in rats that received a high level of FS required explanation. The authors suggested that the accessible pool of VP in the terminals of stimulated VP-ergic neurons may have been exhausted by the intense FS experience, and this prevented overspill in the CSF 24 h later. When tested 5 days later, the releasable pool of AVP was fully recovered, so that a signiﬁcant increase in CSF irAVP was in evidence. This explanation rests on the assumption that AVP in the CSF represents spillover from central VPergic networks that deliver the peptide to memory storage sites in the brain.

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Although all three studies described above found a positive relation between PA behavior (reentry latencies) and FS intensity, they were not consistent with respect to a relationship between CSF levels and PA behavior. Of the two studies that observed CSF levels of VP and/or OT after the learning trial, Mens et al. (1982a) found no effect of an FS intensity of 0.25 or 0.75 mA on irOT, or of 0.50 mA on irVP level in CSF when it was sampled 20 min after the PA learning trial. On the other hand, Laczi et al. (1984), who assessed irVP level in the CSF immediately (30 s) after the learning trial, observed a signiﬁcant increase in this level in the FS groups, the magnitude of which was positively related to the FS intensity. This discrepancy may have been due, in part, to differences in behavioral test procedure: Laczi et al. (1984) took precautions to habituate the subjects to handling and to the test conditions before experimental testing, because these factors could potentially mask the effects of the FS experience on irAVP levels in activated limbic sites and hence in the CSF (Laczi et al., 1983b). The absence of a relation between PA behavior and CSF level of VP in the studies by Van Wimersma Greidanus et al. (1979a) and Mens et al. (1982a), when tested in the 24-h retention test, were both interpreted as evidence of the argument that CSF is not involved as a medium of transport for VP and OT from sites of synthesis to sites involved in memory processing. The results obtained by Laczi et al. (1984), although differing in their speciﬁcs from those of the former two studies, nevertheless suggest a similar interpretation: that is, CSF appears not to function as a medium for transporting AVP to sites engaged in memory processing; rather, the changes in CSF AVP observed at different times during PA testing reﬂect spillover effects from VP-ergic circuitry activated during the testing process.

E. Correlational Studies 3: Avoidance Retention and AVP Levels in Selected Brain Structures The presence of central VP-ergic and OT-ergic ﬁbers terminating in brain sites implicated in memory processing suggests the possibility that brain vasopressin may function as a neurotransmitter or neuromodulator to inﬂuence memory processing; in this case, a positive relationship between CSF levels of vasopressin and avoidance retention would signify diffusion of vasopressin from activated VP-ergic terminals into the CSF. To investigate the role of brain VP-ergic terminals in memory processing, studies have examined the relationship between avoidance retention and vasopressin levels in dissected brain structures. 1. Selected Studies a. Laczi et al. (1983a) Laczi et al. (1983a) measured the irAVP content in various midbrain–limbic system sites after a 24-h PA retention trial in male Wistar rats. The subjects received either no shock or a 3-s FS of 1.0-mA

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intensity. Reentry latencies were observed to a maximum of 300 s in the 24-h retention test. Immediately thereafter each animal was killed and the brain was removed and dissected. irAVP content was measured in several extrahypothalamic brain sites (medial and dorsal raphe nucleus, locus coeruleus, lateral septum, and central amygdala nucleus) believed to mediate the effects of AVP on memory processing (Bohus et al., 1982; Kovacs et al., 1979a; Van Wimersma Greidanus et al., 1979b; see Chapter 4). The results demonstrated that (1) reentry latencies were signiﬁcantly higher in shocked rats than in nonshocked controls; (2) no signiﬁcant differences in irAVP levels between shocked rats and nonshocked controls occurred in the medial and dorsal raphe nuclei; and (3) relative to controls, irAVP levels in the shocked rats were signiﬁcantly decreased in the lateral septum, and increased in the central amygdala and locus coeruleus of the limbic–midbrain areas. Suggested interpretations for these results were as follows: (1) the postretention decrease in septal irAVP, coupled with the similar decrease in hippocampal irAVP reported by Laczi et al. (1983b; see below), could indicate unrecovered low levels in these brain sites caused by AVP activation during memory consolidation. These researchers further noted that several lesion and microinjection studies (Kovacs et al., 1979a; Van Wimersma Greidanus and De Wied, 1976b; see Chapter 4) also support a role for septal and hippocampal sites in mediating the inﬂuence of VP on memory consolidation; and (2) the increase in irAVP that occurred in the central amygdala nucleus and the locus coeruleus could have been related to the activation of neurons in these areas during the PA retention trial itself. That is, activation of VP circuitry in these areas may relate to a role in retrieval (amygdala; Van Wimersma Greidanus et al., 1979b, described in Chapter 4) and in arousal (stress)-related activation (locus coeruleus; Mason and Iversen, 1978). b. Laczi et al. (1983b) Laczi et al. (1983b) measured irAVP in the hippocampus and amygdala (i.e., amygdala plus overlying pyriform cortex) of male Wistar rats in association with various types of stress stimulation (handling, novelty, and anesthesia) as well as during acquisition and retention of a PA response. Four experiments were conducted. Experiment 1 tested the effects of each of the following stressors on irVP content in the hippocampus: handling the subjects, ether anesthesia (45 s duration), novelty (exposure to the PA apparatus without previous habituation and in the absence of FS), and pain (3-s FS at an intensity of 1.0 mA after entry into the dark chamber of a PA apparatus). The rats were killed immediately after these experiences. Hippocampal irAVP content was not signiﬁcantly affected by handling alone or by handling followed by anesthetic treatment. Initial exposure to the PA test box alone signiﬁcantly decreased the irVP level in the hippocampus, but the addition of a painful FS

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to this novel exposure produced no further change in hippocampal irVP content. Experiment 2 tested the effects of a 1.0-mA FS experience on hippocampal and amygdalar irAVP levels in rats previously adapted to the PA test apparatus. When killed immediately after the PA learning trial, hippocampal but not amygdalar irAVP was signiﬁcantly decreased in shocked, relative to nonshocked, rats. In experiment 3, hippocampal irAVP was measured in rats killed immediately after the 24-h PA retention session. Approximately half of these animals were given ether anesthesia for 45 s before sacriﬁce. For statistical comparisons, the animals that received an FS (1.0 mA) in the PA learning trial were classiﬁed as either poor avoiders (reentry latencies less than 100 s) or good avoiders (reentry latencies greater than 100 s). Compared with the nonshocked controls, there was signiﬁcantly less hippocampal irVP content in the good, but not the poor, avoiders. In experiment 4, hippocampal and amygdalar irAVP contents were measured in rats killed either immediately before or after the 120-h (5-day) retention test of a PA task. The rats received either no FS or a 3-s FS of 1.0-mA intensity during the learning trial. All shocked animals were classiﬁed as good and poor avoiders, as in experiment 3, and compared with nonshocked controls for irAVP content. When measured after the 5-day retention test, hippocampal irAVP was signiﬁcantly decreased in good but not in the poor avoiders, whereas irAVP level in the amygdala was not signiﬁcantly affected in either the good or poor avoiders. When killed immediately before the 5-day retention test, there were no signiﬁcant differences between the shocked and nonshocked rats for either hippocampal or amygdalar irAVP. The overall results of this study were interpreted as indicating that (1) hippocampal irAVP is inﬂuenced by some forms of stress (footshock and novelty) but not by others (ether anesthesia); (2) the FS stress-induced change in hippocampal irAVP appears to be transient because it was not present 5 days later in rats killed before the retention test; (3) hippocampal irAVP content was related to avoidance performance because good avoiders exhibited a reduced level of hippocampal irAVP at both the 24- and 120-h retention trials; and (4) immunoreactive VP content in the amygdala was not associated with PA performance. The failure of this latter study (Laczi et al., 1983b) to observe a change in amygdalar irVP content during retention testing differed from the results of their former study (Laczi et al., 1983a), which indicated an increase in amygdalar AVP level during PA retention testing. It is possible that the more extensive fragment of the amygdala (i.e., including the overlying pyriform cortex), tested in the later study (1983b), masked the increased irAVP in the more restricted central amygdaloid nucleus observed in the former study (1983a).

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The opposing directional changes in irAVP content in the two sets of limbic structures indicated by the studies of Laczi et al. (1983a, 1983b) may be related to the observation that AVP in the septal–hippocampal area has been observed to play a role in memory consolidation whereas that in the amygdala appears to be implicated in memory retrieval (Bohus et al., 1982).

F. Section Summary Taken together, the studies reported in this section support the view that central VP-ergic and OT-ergic circuitry, not peripherally circulating hormonal VP and OT, is physiologically involved in memory storage and retrieval. Speciﬁc lines of supportive evidence are as follows: ﬁrst, even though intensity of the aversive unconditioned stimulus is signiﬁcantly correlated with passive avoidance behavior, there is no signiﬁcant relationship between intensity of the aversive stimulus and amount of VP and/or OT released into the blood, or between reentry latencies and plasma levels of VP and/or OT (Laczi et al., 1984; Mens et al., 1982a; Van Wimersma Greidanus et al., 1979a); second, the studies relating CSF levels of VP and/or OT to PA behavior (Laczi et al., 1984; Mens et al., 1982a; Van Wimersma Greidanus et al., 1979a) are perhaps best interpreted as indicating that ﬂuctuation in CSF levels of these peptides observed during PA acquisition and retention reﬂect spillover effects from activated central VP-ergic and OT-ergic circuitry; and third, studies that have assessed levels of VP content in limbic areas implicated in memory processing have demonstrated altered levels of irAVP content in these structures in shocked but not unshocked subjects during PA retention (Laczi et al., 1983a,b).

IV. Vasopressin-Induced Increase in Behavioral Arousal Is Not Essential for Its Effect on Memory Processing

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A. Introductory Comments Peripherally injected VP, in quantities used in behavioral experiments, produces pressor effects. It has been theorized by Koob and associates (Chapter 6) that the pressor-induced arousal effects of peripherally injected AVP and related peptides account for the retention effects observed in various conditioning tasks. De Wied and colleagues argue that, whether peripherally or centrally injected, AVP inﬂuences memory processing by a direct inﬂuence on central memory sites. They have used two procedural strategies to demonstrate that the pressor and behavioral arousal effects accompanying peripherally administered vasopressin may contribute, but are not essential to, the ability of the peptide to facilitate memory processing.

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In one experimental strategy, De Wied and colleagues use metabolic derivatives that are virtually devoid of the antidiuretic, pressor, and corticotrophic effects of the parent peptide but nevertheless produce the behavioral effect of the parent peptide. These AVP derivatives include desglycinamide vasopressin (DG-AVP and DG-LVP) and a number of C-terminal metabolic fragments such as [pGlu4,Cys6]AVP(4–8), [Cyt6]AVP(5–8), and [Cyt6]AVP(5–9). The second strategy has been to show that on those occasions when a vasopressin analog does increase behavioral arousal, this arousal effect may inﬂuence the short-term memory processes involved in learning but is not essential for the effect of the peptide on long-term memory storage. 1. Strategy 1: Dissociation of the Behavioral and Endocrine Effects of Peripherally Administered Vasopressin—Use of DG-AVP and Other C-Terminal VP Metabolites a. Selected Study: Gaffori and De Wied (1985) Gaffori and De Wied (1985) tested the effects of AVP and DG-AVP on a measure of behavioral arousal (open ﬁeld behavior) and on acquisition and extinction in a pole-jump active avoidance task performed by male inbred Wistar rats. On day 1 of testing, the rats were placed in the open ﬁeld for 3 min, and 5 min later were given 10 acquisition trials in the pole-jump shock avoidance task. Acquisition training was continued on days 2 and 3 (10 acquisition trials/day), and followed by 3 days of extinction testing (days 4, 5, and 8; 10 trials/day). Depending on the group, the subjects received a single subcutaneous injection of AVP (3 g/rat), DG-AVP (3 g/rat), or an equal volume of physiological saline (saline) either 15 or 60 min before the open ﬁeld test on day 1 of training (i.e., 20 or 65 min before the ﬁrst trial of avoidance training). When injected 15 min before testing, AVP but not DG-AVP signiﬁcantly inﬂuenced open ﬁeld behavior whereas neither peptide affected this behavior when injected 60 min before testing. The AVP-induced modiﬁcations in open ﬁeld behavior patterns indicated that except for locomotor/rearing activity in the center of the ﬁeld, which was signiﬁcantly increased by AVP, all other ﬁeld behaviors (grooming, fecal boluses, and locomotor/rearing activity in the arena periphery) were signiﬁcantly decreased by the peptide. Subjects treated with AVP 20 min before the ﬁrst acquisition trial made signiﬁcantly more avoidance responses on day 2 of training compared with saline-treated controls. This did not occur if AVP was given 60 min before training, nor did it occur after DG-AVP treatment. Regardless of the time of injection, AVP and DG-AVP each exerted a long-term effect on maintenance of the conditioned avoidance response (i.e., signiﬁcantly increased resistance to extinction on days 5 and 8). In discussing these results the authors noted that (1) although a peripheral injection of 3 g of AVP altered open ﬁeld activity 15 min after

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treatment, it is not clear that this can be interpreted as an increase in behavioral arousal. Thus, an increase in locomotor activity in the center of the arena combined with an overall decrease in grooming behavior and defecation is generally interpreted as indicative of decreased emotionality (Gispen and Isaacson, 1981; Hall, 1934; Whimbey and Denenberg, 1967); (2) the decreased overall level of general activity could have been related to an increase in blood pressure (BP) and bradycardia, which have been observed 10 and 30 min after AVP injection (e.g., De Wied et al., 1984a), but these physiological measures were not taken in this study; (3) it is also possible that the AVP-induced changes in physiology that contributed to the open ﬁeld behavior were also responsible for the improved learning behavior of the subjects treated with AVP 20 min before day 1 of acquisition behavior; (4) however, the observation that resistance to extinction was equally effective in subjects treated with AVP 65 min before training (when the effects of AVP on open ﬁeld behavior had disappeared), or with DG-AVP (which lacks the endocrinological effects of AVP), indicates that any arousal inﬂuence of this peptide is independent of its long-term effect on behavior. The authors, concluded that ‘‘ although peripheral effects of AVP may contribute to its memory-modulating effects, such peripheral effects are not essential’’ (Gaffori and De Wied, 1985, p. 443). DG-LVP-induced facilitation of memory consolidation has also been observed in an active avoidance task by De Wied et al. (1972) and in a sexually rewarded appetitive task by Bohus (1977; see Chapter 2). In addition, several studies have provided data on the comparative memory-facilitating effectiveness of AVP(1–9) and other C-terminal metabolites in active and passive conditioned avoidance paradigms. Examples of these ﬁndings include the following: (1) De Wied et al. (1987) administered C-terminal fragments that were more potent than AVP(1–9) in facilitating passive avoidance memory consolidation and retrieval, memory consolidation (posttraining peptide effect on response extinction) in a conditioned polejump avoidance task, and memory retrieval (i.e., ability to inhibit experimentally induced retrograde amnesia); (2) Gaffori and De Wied (1986) observed that peripherally injected C-terminal fragments were more potent than AVP(1–9) in their ability to facilitate PA memory consolidation and retrieval; (3) Kovacs et al. (1986; see Chapter 5) observed that C-terminal fragments are more potent than AVP(1–9) in facilitating PA memory consolidation, whether injected peripherally, intracerebroventricularly, or intracerebrally into selective limbic system brain sites; (4) Burbach et al. (1983a; see Chapter 5) demonstrated that when intracerebroventricularly administered, C-terminal fragments [pGlu4,Cyt6]AVP(4–9) and [pGlu4,Cyt6]AVP(4–8) were far more potent than the parent peptide in facilitating PA memory consolidation and retrieval. Further, unlike AVP(1–9), these fragments showed no pressor activity when peripherally administered (intravenously)

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over the entire dose range tested; and (5) De Wied et al. (1984a) reported a similar high degree of behavioral potency when the C-terminal fragment [pGlu4,Cyt6]AVP(4–8) was peripherally administered in a test of PA memory consolidation. 2. Strategy 2: Evidence That the Arousal Effect of Peripherally Injected VP Is Not Essential for Its Influence on Memory Storage a. Selected Study: Skopkova et al. (1991) Skopkova et al. (1991) investigated whether the long-term behavioral effects of DG-AVP are the result of an initial increase in behavioral arousal during the learning phase. The subjects, male Wistar rats of a genetically nonselected strain, were rated as high or low in nonspeciﬁc excitability on the basis of exploratory activity displayed in an open ﬁeld test. The subjects received 3 days of acquisition training in a shuttlebox avoidance task (10 trials/day), followed by 2 days of extinction testing (10 trials/day). A single injection of placebo or DGAVP was administered subcutaneously 40 min before the ﬁrst acquisition trial at a dose of either 0.1, 0.3, or 1.0 g/rat. The low and high doses of DGAVP were given to manipulate the arousal level of the subject during learning. The inference that DG-AVP inﬂuences arousal level was based on a previous observation that it stimulated exploratory behavior in an open ﬁeld test (Skopkova et al., 1987). Results indicated that the major peptide effects on performance occurred on the ﬁrst day of acquisition and on both days of extinction, and involved both the lowest (0.1 g) and highest (1.0 g) dose levels of the peptide. Of interest to this discussion were the interactional effects between these dose levels and performance of the high- and low-activity subjects. Acquisition of the shuttlebox avoidance response was facilitated in low-activity rats given the lowest dose of DG-AVP and impaired in high-activity rats given the highest dose of the peptide. During extinction, however, the peptide produced a dose-dependent facilitation of retention in both high- and low-activity rats. Moreover, the 0.1-g dose of DG-AVP, which had facilitated acquisition in the low-activity subjects, resulted in faster extinction in these subjects than in the highactivity group, indicating that the short-acting arousal effect of the peptide on acquisition behavior did not exert a long-term inﬂuence on this behavior. Interpretation of these results was based on proposals that arousal level is an important aspect of learning, and is related to performance efﬁciency by an inverted U-shaped function. Accordingly, it was theorized that in low-activity subjects the low dose of DG-AVP increased arousal to a level that greatly facilitated learning performance, whereas in high-activity subjects the high dose of the peptide increased arousal to a level that impeded it. The observations on extinction (retention) behavior were interpreted as indicating that the direct inﬂuence of the peptide on long-term memory was independent of its short-term arousal effect.

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V. Peripherally Administered Neurohypophysial Peptides and Central Memory Processing

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A. Does Peripherally Injected VP or OT Reach Central Memory-Processing Sites? An important question in behavior pharmacology is whether peripherally injected peptides such as VP and OT can produce centrally mediated behavioral effects, given a blood–brain barrier (BBB) that presumably prevents these peptides from entering the brain. The BBB issue is discussed in detail in Chapter 14. At this point we can note two opposing theoretical interpretations of the means by which peripherally administered vasopressin inﬂuences memory storage and retrieval. De Wied and colleagues argue that some tiny amount of the pharmacological dose of the parent peptide, or one of its behaviorally active metabolic fragments, enters the brain and directly activates central vasopressin receptors that mediate the inﬂuence of the peptide on memory storage and retrieval. In contrast, Koob and colleagues argue against the likelihood that vasopressin can penetrate the BBB and suggest instead that the pressor response induced by the increased level of the peripherally circulating peptide raises the subject’s behavioral arousal, which is then responsible for the observed effect on retention behavior (this view is discussed in Chapter 6). De Wied and colleagues cite neuroanatomical observations as well as behavioral ﬁndings from several of their studies in support of their theoretical position, as described below. De Wied and colleagues have presented several lines of evidence consistent with the view that peripherally administered neurohypophysial peptides modulate memory storage and retrieval by a direct inﬂuence on central memory-processing sites. First, for a given effect on retention much smaller doses of the neurohypophysial peptides are required when injected intracerebrally than peripherally (De Wied, 1976; Kovacs et al., 1979a,b, 1986). Microgram dose levels are required for peripheral injection (e.g., Ader and De Wied, 1972; Bohus et al., 1972; De Wied et al., 1984a), nanogram amounts are needed for intracerebroventricular injections (e.g., Bohus et al., 1978b; Kovacs et al., 1978), and picogram amounts are required when directly microinjected into speciﬁc brain sites (e.g., Kovacs et al., 1979a). Second, De Wied and colleagues have consistently demonstrated that the endocrine-related (pressor/aversive) functions of peripherally injected vasopressin are not required for its effects on retention, because metabolic derivatives lacking these functions nevertheless facilitate memory storage and/or retrieval in appetitive (Bohus, 1977; Vawter et al., 1997) as well as in active (De Wied et al., 1972; Gaffori and De Wied, 1985; Skopkova et al., 1991) and passive (e.g., De Wied et al., 1991) avoidance paradigms. A third line of experimental support derives from studies that have utilized vasopressin agonists and antagonists to test the proposition that

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peripheral VP directly inﬂuences central VP receptors that mediate its effect on memory processing. Two of these studies are described below. The ﬁrst (De Wied et al., 1984a) used agonist–antagonist interactions to investigate whether peripheral VP receptors mediate the pressor but not the behavioral actions of the peptide. The second (De Wied et al., 1991) used agonist– antagonist interactions to further characterize the central VP receptor and to distinguish it from peripheral V1,V2, and OT receptors. 1. Selected Study: De Wied et al. (1984a) De Wied et al. (1984a) conducted a study to differentiate between pressor and behavioral effects induced by peripherally administered VP. The study tested the interaction between the potent VP antagonist d(CH2)5[Tyr (Me)]AVP and the parent peptide AVP(1–9) and between the antagonist and the metabolic fragment [pGlu4,Cyt6]AVP(4–8) on both blood pressure and passive avoidance retention. Results indicated that peripherally administered AVP(1–9) produced a pressor effect and facilitated PA retention whereas the VP fragment produced only the PA retention effect. When centrally (intracerebroventricularly) administered, both the parent peptide and the VP fragment facilitated PA retention whereas neither increased blood pressure. When peripherally administered, the antagonist blocked both the pressor and the retention effect of peripherally administered AVP(1–9) as well as the retention effect of peripherally administered VP fragment. When centrally administered, the VP antagonist blocked the retention effect of the centrally administered peptides and, in addition, the retention but not the pressor effect of peripherally administered AVP(1–9). The results were interpreted as follows. First, the relatively large dose of VP antagonist used for peripheral administration (3 g/rat, subcutaneous) penetrated the BBB sufﬁciently to inﬂuence the central VP receptor, which is structurally similar to the peripheral VP receptor. Second, the very small dose of VP antagonist used for central injection (3 ng/rat, intracerebroventricular), although large enough to diffuse from the ventricle to central VP memory receptor sites, was not sufﬁcient to reach the peripheral VP pressor receptors. Thus the centrally injected antagonist was able to block the behavioral but not the pressor effects exerted by peripherally administered AVP. It was concluded that the receptors mediating the behavioral effects of peripherally administered VP are in the brain, and those involved in the pressor response are in the periphery.

VI. Theoretical Propositions of the ‘‘VP/OT Central Memory Theory’’: Continued

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This section continues the discussion of the theoretical propositions codifying the views of De Wied and colleagues on the roles of vasopressin and oxytocin in memory processing. The studies described in this chapter

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provided ﬁndings relevant to propositions 1 and 3, delineated in Chapter 2, and to propositions 4, 5, and 6 which are added here.

A. Proposition 1: VP Facilitates Memory Consolidation and Retrieval The study of subjects deﬁcient in endogenous vasopressin (e.g., Brattleboro rats and normal rats injected with anti-VP serum) is especially pertinent to the physiological role of vasopressin in memory processing. Preliminary studies by De Wied and colleagues with Brattleboro rats led them to conclude that central levels of endogenous vasopressin directly contribute to memory processing: (1) Brattleboro HODI rats were severely disturbed in long-term retention in a single-trial passive avoidance task (Bohus et al., 1975; De Wied et al., 1975) and in multitrial shuttlebox and pole-jump shock avoidance tasks (Bohus et al., 1975); (2) this disturbance was interpreted as due to a deﬁciency of VP at central memory-processing sites, and not a secondary result of the diabetes insipidus, because DG-LVP treatment normalized PA retention without correcting the diabetes insipidus (De Wied et al., 1975); (3) the retention impairment was reﬂected in the absence of a pituitary–adrenal axis stress response evoked by the conditioned stimuli present at the 24-h PA retention test (Bohus et al., 1975); (4) experimental testing for threshold responsiveness to a range of FS intensities ruled out pain sensitivity deﬁcit as a cause for the retention impairment (Bohus et al., 1975; De Wied et al., 1975); (5) moreover, there was no deﬁciency in the normal pituitary–adrenal axis stress response evoked by the painful FS stimuli (Bohus et al., 1975). Later work with Brattleboro rats by De Wied and associates (De Wied et al., 1988) replicated the earlier-found deﬁcits in retention in the active and passive avoidance tasks and, in addition, found signiﬁcant impairment in retention of an aversive reversal discrimination problem in a T-maze, appetitive black–white discrimination in a Y-maze, and impairment in both working and reference memory in the hole board food search task. The Brattleboro rat model does not provide a straightforward means by which to learn about the nature of the contribution of VP to memory processing. A number of hormonal and metabolic secondary effects of diabetes insipidus (Bailey and Weiss, 1981) and alterations in temperament, emotion, and arousal level have been observed in these rats (Brito, 1983; Williams et al., 1983a,b). These factors themselves may inﬂuence behavioral performance in a variety of learning situations (Bailey and Weiss, 1981). For example, an altered baseline arousal level in HODI rats, inferred from study of their open ﬁeld behavior (Bohus et al., 1975; De Wied et al., 1988; Williams et al., 1983a,b), has been suggested by some researchers as the cause of the impaired retention observed in VP-deﬁcient rats (e.g., Herman et al., 1986b). Although De Wied and colleagues accept that an altered

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arousal level may disrupt attention and the short-term memory processing involved in a variety of cognitive tasks, they maintain that the vasopressin deﬁciency at central memory-processing sites is responsible for the memory storage deﬁcits that they have consistently observed in HODI rats (De Wied et al., 1988).

B. Proposition 3: VP and OT Have No Major Role in the Learning Phase of Memory Processing De Wied and colleagues have reported that contrary to the memory deﬁcit, Brattleboro rats do not exhibit persistent impairment in the attentional and short-term memory processes required for successful learning. Their early research showed that Brattleboro HODI rats successfully learned the PA response, because testing immediately after the learning trial indicated reentry latencies at near maximal value. When tested 3 h later, the reentry latencies, although shorter, were still signiﬁcantly longer than those of nonshocked controls (Bohus et al., 1975; De Wied et al., 1975). Moreover, corticosterone activity, interpreted as a stress response to the conditioned fear stimuli, was positively correlated with the reentry latency data (Bohus et al., 1975). HODI rats also attained the designated learning criterion during the acquisition period in multitrial shuttlebox and polejump shock avoidance tasks, although at a slower rate than normal Wistar rats (Bohus et al., 1975). They also exhibited normal learning in the original and reversed discrimination of an aversive T-maze task (De Wied et al., 1988). However, De Wied and colleagues have observed signiﬁcant impairment in HODI and HEDI rats in acquisition of an appetitive black–white discrimination, the pole-jump task, and in working memory in a hole board food search task (De Wied et al., 1988). Because arousal effects associated with exogenous AVP may inﬂuence learning behavior (e.g., Gaffori and De Wied, 1985; Skopkova et al., 1991), the alteration in arousal level inferred from open ﬁeld test behavior in Brattleboro DI rats (De Wied et al., 1988; Herman et al., 1986b) may be the basis for the learning deﬁcits that are inconsistently observed in these VP-deﬁcient rats.

C. Proposition 4: Central VP-ergic and OT-ergic Circuitry and Not Peripherally Circulating Hormones Are the Primary Means by Which Neurohypophysial Peptides Influence Memory Processing According to proposition 4, feedback from peripherally circulating neurohypophysial peptides does not play an important role in memory storage; rather, it is the central VP-ergic and OT-ergic circuitry that mediates this function.

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Three lines of experimental evidence support this proposition, one of which derives from studies that have neutralized peripheral and central stores of vasopressin. For example, Van Wimersma Greidanus et al. (1975a) observed that an intracerebroventricular injection of anti-VP serum that neutralized central levels of vasopressin signiﬁcantly impaired passive avoidance memory consolidation, whereas a peripheral injection that neutralized plasma VP levels had no effect. A second line of support derives from the correlation of avoidance retention behavior with endogenous VP or OT localized either peripherally (in the plasma) or centrally (collected from cerebral spinal ﬂuid or selected dissected brain sites). A number of studies have found that plasma AVP levels, assessed after daily learning sessions in a multitrial active avoidance task or after a single passive avoidance learning trial, are related neither to rate of extinction in the active avoidance task (Van Wimersma Greidanus et al., 1979a) nor to reentry latency scores in the passive avoidance task (Laczi et al., 1983c; Mens et al., 1982a; Van Wimersma Greidanus et al., 1979a). Moreover, there were no signiﬁcant differences in postlearning plasma AVP levels between shocked and nonshocked groups in a passive avoidance learning trial (Laczi et al., 1983c). Similar results have been obtained in studies that assessed plasma OT level during passive avoidance learning (Mens et al., 1982a). Because hormonal levels of these neurohypophysial peptides were not differentially increased from control values during the learning trials it is unlikely that hormonal feedback at this time could contribute to memory consolidation. Thus, these ﬁndings support the proposition that peripheral levels of these peptides do not contribute to memory storage in these tasks. Studies that assessed CSF levels of AVP or OT after a PA learning trial, and again after retention test trials given 24 and/or 120 h later, suggest that CSF does not transport these peptides from sites of synthesis to sites of activity in the brain. Instead, the peptide levels in CSF represent peptides that have been released in the brain by activated VP-ergic and OT-ergic synapses (Laczi et al., 1984; Mens et al., 1982a). Laczi et al. (1983a,b) interpreted signiﬁcant changes that occurred in irAVP levels in midbrain–limbic sites in shocked animals in a 24-h PA retention trial as support for putative roles of this VP-ergic circuitry in memory consolidation (septal/hippocampal area) and retrieval (central amygdala). A third line of support for this proposition derives from observations suggesting that peripheral and central vasopressin receptors have separate functions, with the former mediating only the endocrinological effects of the peptide, and the latter only the retention effects. This support includes observations that (1) VP-binding sites are present in memory-processing limbic system structures (Chapter 1); (2) centrally (intracerebroventricularly) injected AVP(1–9), at a dose level that facilitates avoidance retention, lacks the pressor/bradycardial effects characterizing peripherally (subcutaneously) administered AVP(1–9), suggesting that the central receptors that

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mediate the behavioral effects of the peptide are not involved in producing its peripheral pressor effects (De Wied et al., 1984a); (3) subcutaneously or intracerebroventricularly injected C-terminal VP fragments produce potent behavioral but no endocrinological effects, suggesting that these fragments activate central/behavior-mediating, but not peripheral/endocrinologically mediating VP-ergic mechanisms (De Wied et al., 1984a); and (4) a neurohypophysial peptide–receptor complex in the brain differs in structural/ functional character from V1, V2, and OT receptors in the body periphery (De Wied et al., 1991; see Chapter 5). Although the preceding discussion indicates substantial support for proposition 4, there is the puzzling ﬁnding that surgical removal of the posterior/intermediate lobes of the pituitary gland (neurohypophysectomy) impairs retention in avoidance paradigms (De Wied, 1965; see Chapter 2). If hormonal vasopressin has no important role in memory processing, why are neurohypophysectomized animals impaired in conditioned avoidance response retention and why is the impairment normalized by vasopressin replacement therapy? One possible answer may come from the observation that this surgery results in substantial structural damage to the hypothalamic nuclei (supraoptic and paraventricular) that produce the neurohypophysial hormones (Moll and De Wied, 1962). Because cells in these nuclei send neural output to extensive brain sites, especially in the brainstem, it is possible that some of these neural outputs serve a direct or indirect role in memory processing. As a result, damage to these hypothalamic nuclei may result in reduced VP transmitter output in this circuitry, which would then be increased by VP treatment.

D. Proposition 5: VP and OT Modulate Memory Processing Directly and Not by an Indirect Influence on Behavioral Arousal The idea that a vasopressin effect on retention is secondary to an inﬂuence on the behavioral arousal level of the subject is prominent in the theoretical views of both Koob and colleagues (Chapter 6) and Sahgal and colleagues (Chapter 7). However, early in their behavioral research with neurohypophysial peptides, De Wied and colleagues [De Wied, 1971 (Chapter 2); Van Wimersma Greidanus et al., 1975a] observed that although both vasopressin and ACTH [including ACTH(4–10)] prolonged extinction of a conditioned response, ACTH had only a short-term inﬂuence (while the peptide was present in the system) whereas AVP exerted a long-term effect, lasting for days and weeks after AVP treatment was discontinued. These observations led the authors to the tentative conclusion that whereas ACTH and related peptides may inﬂuence memory processing via an effect on temporary supportive functions such as behavioral arousal, neurohypophysial peptides were more likely to act on consolidation processes and

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therefore instigate a more permanent change in the central nervous system (also see Van Wimersma Greidanus et al., 1975b; Chapter 4). Subsequent research was designed to respond directly to the challenges offered by arousal-based theories of vasopressin and memory processing. One such line of investigation included studies using vasopressin analogs lacking the pressor effects that, according to Koob and associates, produce the arousal changes responsible for the memory effects of the peripherally administered peptide. Numerous studies have reported that peripheral administration of the peptide analog DG-LVP (DG-AVP), which lacks the endocrine (hence pressor) effects of the parent peptide, can effectively facilitate memory consolidation in an active avoidance task (De Wied et al., 1972; Gaffori and De Wied, 1985; this chapter) and a sexually rewarded appetitive task (Bohus, 1977; see Chapter 2). Moreover, peripherally administered C-terminal fragments, demonstrated to lack pressor effects over a wide range of dose levels (Burbach et al., 1983a; see Chapter 5), have been shown to be far more potent than the parent peptide in facilitating memory consolidation in a pole-jump avoidance task (De Wied et al., 1987; see Chapter 5) and memory consolidation and retrieval in a passive avoidance task [De Wied et al., 1984a, 1987 (Chapter 5); Gaffori and De Wied, 1986 (Chapter 5)], and in protecting against experimentally induced retrograde amnesia (De Wied et al., 1987; see Chapter 5). More recently, Skopkova et al. (1991) showed that although DG-AVP apparently does have arousing properties that can exert an immediate effect on acquisition, its inﬂuence on extinction is due to an effect on memory consolidation. Further, because of the virtual lack of pressor effects of this VP analog, the behavioral effect cannot be ascribed to peripheral inﬂuences as theorized by Koob and associates.

E. Proposition 6: The Effect of Exogenously Administered VP and OT on Memory Processing Is Due to Action Exerted at Central and Not Peripheral Receptor Sites Proposition 6 represents a second key difference between the theoretical view of De Wied and colleagues and that of Koob and colleagues. According to the De Wied group, when vasopressin (or oxytocin) is peripherally injected (typically with a supraphysiological dose), a sufﬁcient fraction of the injected peptide, or one of its behaviorally active metabolites, is able to cross the blood–brain barrier (BBB), enter the brain, and directly access central receptors involved in memory processing. This proposition is rejected by Koob and associates (Chapter 6), who propose instead that the injected vasopressin acts at peripheral vascular receptors that mediate the memory effect via pressor-induced changes in behavioral arousal. The issue of the ability of the peptide to cross the BBB remains to be settled (see

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Chapter 14), but De Wied and colleagues have offered indirect evidence in support of this proposition. Their argument that only a tiny fraction of the injected peptide needs to penetrate the BBB and reach appropriate brain receptors is supported by data demonstrating that increasingly smaller quantities of the parent peptide [AVP(LVP)(1–9) or OT(1–9)] are required for a retention effect as the treatment route proceeds from peripheral to intracerebroventricular to intracerebral (local brain site) injection. Thus, for a comparable effect on avoidance behavior, a microgram quantity is needed for peripheral injection [e.g., Ader and De Wied, 1972; Bohus et al., 1972 (Chapter 2); De Wied et al., 1984a], a nanogram (0.001 g) quantity is required for intracerebroventricular injection (e.g., Bohus et al., 1978a,b; see Chapter 2), and a picogram (0.001 ng) quantity is needed for intracerebral injection (Bohus et al., 1982; Kovacs et al., 1979a,b; see Chapter 4). Although this proposition admits the possibility that sufﬁcient amounts of behaviorally active metabolites of the parent peptide may cross the BBB into the brain, there has been no direct evidence that the endogenous metabolite fragments observed from in vitro and in vivo studies of the rat brain are also produced in peripheral tissues. However, in a more recent publication, these authors did report evidence of the presence of endogenous DG-AVP in the plasma of Wistar rats (Laczi et al., 1991). Also consistent with this proposition is evidence of dissociation between the peripheral receptors that mediate the endocrine effects of vasopressin and the central receptors that are involved in its memory-processing effects. Thus, as reported in a previous section of this chapter, peripheral injections of DG-AVP and other metabolites lacking the endocrine effects of the parent peptide effectively enhanced memory consolidation for both avoidance (Gaffori and De Wied, 1985) and appetitive (Bohus, 1977; see Chapter 2) learned behaviors, and memory retrieval of a conditioned passive avoidance response (e.g., De Wied et al., 1991). Moreover, ﬁndings of studies that employed combinations of peripherally and centrally injected vasopressin agonists and antagonists to produce interactional effects support the theory that a VP receptor localized in the body periphery mediates its effects on blood pressure while a receptor of similar structure, but localized within the brain, mediates its effects on memory storage (De Wied et al., 1984a,b). De Wied and colleagues (1991) have also used agonist–antagonist interactions to further characterize and distinguish central from peripheral neurohypophysial peptide receptors as described in Chapter 5.

Barbara B. McEwen

De Wied and Colleagues III: Brain Sites and Transmitter Systems Involved in the Vasopressin and Oxytocin Influence on Memory Processing

I. Introductory Remarks

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This chapter discusses evidence relevant to the brain structures that may be target sites for the effects of vasopressin (VP) and oxytocin (OT) on memory processing, as well as the monoamine classic transmitter systems with which these neuropeptides may interact to produce these effects.

II. Localizing Central Sites for the Memory-Modulating Effects of VP and OT by Means of Lesioning and Microinjection Techniques

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Lesion and microinjection techniques have been used to localize the central sites at which VP and OT exert their effects on memory storage and retrieval. It is rationalized that lesioning (injuring or destroying) a Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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structure (e.g., nucleus or pathway) important to the inﬂuence of the peptide on storage or retrieval will attenuate or prohibit the relevant retention behavior on subsequent peptide treatment of the lesioned subjects. The lesion technique is not without problems of interpretation because failures to produce impairment do not necessarily indicate that the structure is irrelevant to the effect of the peptide on retention. The failure may simply be due to insufﬁcient tissue damage from the lesion, or other participating sites that can substitute for the damaged component. Further, demonstration of lesion-induced impairment could also be misinterpreted because ‘‘ﬁbers of passage’’ (i.e., ﬁbers in the damaged area deriving from cell bodies originating elsewhere in the brain), rather than the cell bodies in the damaged brain nucleus, may be the basis of the observed behavioral deﬁcit. Microinjection of a peptide or its antiserum into a speciﬁc brain site is justiﬁed on the basis that increasing or reducing its level in a brain site that normally mediates the inﬂuence of the peptide on retention should enhance and impair retention behavior, respectively. Studies utilizing this technique are also not without interpretive problems. For example, microinjection of the peptide or its antiserum could fail to modulate memory because of an insufﬁcient dose level. This may account for several early failures in studies that used unilateral injections in bilaterally distributed brain structures (De Wied et al., 1976). Despite these caveats, lesion and microinjection studies have provided evidence of the localization of a number of brain sites involved in mediating the effects of the neurohypophysial peptides on memory consolidation and/or retrieval. These include forebrain limbic structures implicated in memory processing, and brainstem sites that give rise to catecholamine and serotonin projections to these forebrain structures.

A. Thalamic–Limbic Lesions: Influence on the Memory-Modulating Effects of Adrenocorticotropic Hormone-Like Peptides and of VP and/or OT De Wied and colleagues had earlier reported that adrenocorticotropic hormone (ACTH)-like peptides, as well as vasopressin, prolonged extinction of conditioned avoidance behavior, although the former produced short-term, the latter, long-term effects on this behavior (De Wied, 1971; De Wied and Bohus, 1966; see Chapter 2). ‘‘ACTH-like peptides’’ refer to ACTH(1–39) derivatives, such as ACTH(1–10), as well as hormones such as -melanocyte-stimulating hormone (-MSH), which are derived from the same prohormone as ACTH. A series of studies conducted by Van Wimersma Greidanus and associates, described below, examined the possibility that the different behavioral effects of the two neuropeptides involve different brain structures, or the same structures functioning in different operative modes.

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1. Selected Studies a. Van Wimersma Greidanus et al. (1974) Van Wimersma Greidanus et al. (1974) tested the ability of lesions in the thalamic parafascicular nucleus (pfc nucleus) of male Wistar rats to interfere with the effects on extinction behavior usually induced by lysine vasopressin (LVP) (experiment 1) and ACTH(4–10) (experiment 2). The pfc nucleus is part of the diffuse thalamic projection system, and it has been shown that microinjection of LVP (Van Wimersma Greidanus et al., 1973) and implantation of ACTH(1–10) (Van Wimersma Greidanus and De Wied, 1971) into this nucleus help maintain a conditioned avoidance response. After recovery from surgery, the lesioned animals and the sham operates in both experiments were trained in a pole-jump avoidance task (days 1–4, 10 trials/day), tested for extinction (days 5 and 8, 10 trials/day), given reacquisition training (10 trials on day 9), and tested for extinction on days 10, 11, 12, and 15 (10 trials/day). In experiment 1, all subjects received a subcutaneous injection of placebo (physiological saline) or LVP (1.8 or 5.4 g/rat) immediately after the extinction session on day 10. In experiment 2, the pfc-lesioned rats received placebo or ACTH(4–10) (1.0 or 9.0 g/rat) and the sham operates were given placebo or ACTH(4–10) (1.0 or 3.0 g/rat), 1 h before each extinction session on days 10, 11, 12, and 15. After completion of behavioral testing, histological examination was carried out to determine the exact location and size of the lesion. The results were as follows: (1) the lesion in experiment 1 destroyed the pfc nucleus and extended into the mediodorsal thalamus; (2) this lesion impaired acquisition and hastened extinction of the avoidance response in placebo-treated rats; (3) the single injection of LVP (1.8 or 5.4 g/rat, subcutaneous) dose dependently reversed the effects of the lesion on extinction. The low dose of LVP preserved the learned response for the ﬁrst 2 days of the 4-day extinction period whereas the high dose preserved it for the duration of extinction testing; (4) the thalamic pfc lesion in experiment 2 was more restricted than in experiment 1 and did not affect acquisition, but did reduce response during extinction relative to the sham operates; and (5) the chronically injected ACTH(4–10), which lacks the corticotrophic effects of ACTH(1–39), dose dependently inhibited extinction in the sham operates; however, neither the low dose nor the extremely high dose of the peptide was able to reverse the effect of the lesion on extinction. The results were interpreted as follows: (1) although the thalamic pfc nucleus is normally involved, along with other brain sites, in mediating the effect of vasopressin on extinction, it is not essential for that effect to occur. Thus, whereas low amounts of the peptide preserved the pole-jumping response in sham operates throughout extinction, it did not do so for pfclesioned rats. However, higher doses were able to overcome the effects of the lesion; (2) on the other hand, the pfc nucleus must be intact for

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ACTH(4–10) to exert its retrieval effect on avoidance retention, because neither the low dose nor the high dose of this peptide was able to overcome the lesion-induced reduction of avoidance response during extinction. b. Van Wimersma Greidanus et al. (1975b) Van Wimersma Greidanus et al. (1975b; and cited in Van Wimersma Greidanus et al., 1983) reported the effects of extensive bilateral lesions in the rostral septal area on the ability of ACTH-like peptides and LVP to inﬂuence extinction of a polejump avoidance response. The lesion almost completely destroyed the medial septal nucleus and partially destroyed the lateral septal nucleus and the nucleus accumbens. This region proved to be another brain site essential for the short-term behavioral action of ACTH-like peptides, because ACTH(1–10) treatment, given during extinction of the pole-jump task, was unable to maintain the learned response in these septally lesioned animals. This limbic site also appears to be essential for mediating the ability of vasopressin to preserve a learned avoidance response because a single subcutaneous injection of a 9-g dose of LVP, given before extinction, failed to maintain avoidance responding in lesioned subjects even when the much smaller, 3-g dose did so for the sham operates. c. Van Wimersma Greidanus and De Wied (1976b) Van Wimersma Greidanus and De Wied (1976b) studied the effect of an anterodorsal hippocampal lesion on extinction of a pole-jump shock avoidance response in male inbred Wistar rats after treatment with either LVP (experiment 1) or ACTH(4–10) (experiment 2). In experiment 1, the subjects received either sham operations, small, or large bilateral lesions. After recovery from surgery, 10 training trials were given on each day of acquisition: days 1–4 for sham operates and the small-lesioned operates, and days 1–5 and day 8 for the large-lesioned operates. After reaching the learning criterion (eight or more correct avoidances), they received a single subcutaneous injection of either placebo or LVP (1.0 or 3.0 g/rat for sham operates, 1.0, 3.0, or 9.0 g/rat for the small-lesion groups, and 1.0 or 9.0 g/rat for the largelesion groups). Extinction testing occurred on days 5, 8–12 and 17 for sham operates; on days 6, 8, 9, and 11 for small-lesioned groups; and on days 9–12 and 17 for the large-lesioned groups. The results of experiment 1 were as follows: (1) for the sham operates, LVP produced a long-term, dose-dependent inhibition of extinction. Thus, LVP-treated rats continued to exhibit a high rate of avoidance responding on the last day of extinction, when placebo controls no longer responded; (2) the small lesion did not interfere with acquisition, but tended to accelerate the rate of extinction among the placebo controls. This lesion prevented the LVP-induced inhibition of extinction in subjects given the 1.0- or 3.0-g dose, but not in those given the 9.0-g dose; and (3) the large lesion retarded acquisition of the avoidance response and prevented LVP-induced inhibition

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of avoidance extinction whether the subjects received the 1.0-g or the 9.0-g dose level. This ﬁnding suggests that an intact anterodorsal hippocampus is essential for mediating the LVP-induced prolonged extinction effect in this task. In experiment 2, the subjects received either sham operations or large bilateral lesions in the anterodorsal hippocampus. After recovery from surgery, the sham operates were trained on days 1–3 (10 trials/day) and, having attained criterion performance, were tested for extinction on days 4, 5, and 8 (10 trials/day). These sham operates received a subcutaneous injection of placebo or ACTH(4–10) (3.0 g/rat, subcutaneous) 1 h before each day of extinction. The lesioned subjects were given 6 days of training (days 1–5 and day 8; 10 trials/day) and 3 days of extinction (days 9, 10, and 11; 10 trials/day), provided they reached criterion performance. These subjects received a subcutaneous injection of placebo or ACTH(4–10) (9 g/rat, subcutaneous) 1 h before each day of extinction. The results of experiment 2 were as follows: (1) relative to the placebo-treated subjects, a 3.0-g dose of ACTH(4–10) inhibited extinction of the avoidance response in the sham operates; and (2) the large lesion retarded acquisition of the avoidance response as was observed in experiment 1, and prevented ACTH(4–10) from producing its inhibitory effect on response extinction, even though the large (9.0 g) dose of the peptide was used. Taken together, the results of this study indicated that an intact anterolateral hippocampus is as essential for the short-term behavioral effects of ACTH-like peptides as it is for the long-term behavioral effects of vasopressin treatment. d. Van Wimersma Greidanus et al. (1979b) Van Wimersma Greidanus et al. (1979b) observed the effect of bilateral lesions of the amygdala complex on the ability of peripherally administered ACTH(4–10) and desglycinamide-lysine vasopressin (DG-LVP) to prolong active avoidance extinction in male inbred Wistar rats. Experiment 1 tested the effect of DG-LVP treatment on pole-jump shock avoidance behavior in sham operates and amygdala-lesioned subjects. After recovery from surgery, the subjects were given 4 days of acquisition training (10 trials/day) followed by 4 extinction sessions (10 trials/session): 2 on day 5, and 1 each on days 6 and 8. Immediately after the extinction session on day 5, the subjects received a subcutaneous injection of placebo (physiological saline) or DG-LVP (3 g/rat for the sham operates, and up to 5 g/rat for the lesioned rats). The effect of this single injection was observed 4 h later in a second 10-trial extinction session on day 5 and thereafter in the extinction sessions on days 6 and 8. Experiment 2 employed a similar training/extinction procedure to test the effect of ACTH(4–10) on response extinction in amygdala-lesioned and sham-operate subjects. However, in this experiment the rats were tested for

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extinction on days 5 and 6 (10 trials/day), and received an injection of placebo or ACTH(4–10) (3.0 g for sham operates and 9.0 g for lesioned rats) 1 h before each 10-trial extinction session. The results were as follows: (1) the lesion damaged the central and basolateral parts of the amygdaloid complex; (2) the lesion did not inﬂuence acquisition or extinction of the conditioned avoidance response in the placebo-treated rats; (3) DG-LVP treatment induced a long-term inhibitory effect on extinction in the sham operates but failed to do so in the amygdala-lesioned subjects (Fig. 1); and (4) ACTH(4–10) produced its short-term inhibitory effect on extinction in the sham operates on both days of extinction testing but failed to inﬂuence extinction, at either dose level, in the amygdala-lesioned subjects (Fig. 2). e. Van Wimersma Greidanus et al. (1979c) Van Wimersma Greidanus et al. (1979c) investigated whether the various limbic system structures act individually, or as an integrated system in mediating the inhibitory effect on avoidance extinction exerted by ACTH(4–10) and DG-LVP. Fibers of the fornix (connects hippocampus with other limbic sites and with the hypothalamus) and the stria terminalis (connects amygdala with other limbic sites and with the hypothalamus) were transected. The effect of this on extinction of a pole-jump avoidance task, after treatment with placebo or each of

FIGURE 1 Effect of DG-LVP on extinction of a pole-jumping avoidance response in rats with lesions in the amygdaloid complex and in sham-operated animals. AM, amygdaloid; CAR, conditioned avoidance response; s.c., subcutaneous. Source: Van Wimersma Greidanus et al., 1979b (Fig. 2, p.294). Copyright ß 1979 by Pergamon Press and Brain Research Publications Inc.

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FIGURE 2 Effect of ACTH (4–10) on extinction of a pole-jumping avoidance response in rats with lesions in the amygdaloid complex and in sham-operated rats. Source: Van Wimersma Greidanus et al., 1979b (Fig. 3, p.294). Copyright ß 1979 by Pergamon Press and Brain Research Publications Inc.

the peptides, was tested in male Wistar rats. The surgery left intact the individual limbic system sites but prevented operation of the limbic system as an integrated unit. The subjects received 10 acquisition trials per day until they reached a learning criterion of 75% correct avoidance responses in a single 10-trial session. Extinction testing (10 trials/day) began the day after the ﬁnal day of training. The long-term effect of DG-LVP treatment on response maintenance was tested by administering a single subcutaneous injection of placebo (physiological saline) or DG-LVP (3 g/rat for sham operates and 3 or 9 g/rat for operates) immediately after the last acquisition trial, and was followed by response extinction carried out for three consecutive days. The short-term effect of ACTH(4–10) on response maintenance was evaluated

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by chronic subcutaneous injections of placebo or ACTH(4–10) (3 g/rat for sham operates; 3.0 or 9.0 g/rat for operates) administered 1 h before each of the two consecutive days of extinction testing. The fornix–stria terminalis transection hastened acquisition of the avoidance response, a result previously obtained in avoidance paradigms after damage of the fornix pathway (Alvarez-Palaez, 1973; De Castro and Hall, 1975). This enhanced acquisition may be related to the decrease in freezing behavior and increase in spontaneous motor activity previously noted in similarly lesioned rats (Alvarez-Palaez, 1973; Liss, 1968). The transection did not interfere with maintenance by vasopressin of the avoidance response, because DG-LVP dose dependently inhibited extinction in both the lesioned and sham-operate rats. In contrast, the inhibitory action of ACTH(4–10) on extinction, observed in the sham operates, was prevented by the lesion even when a high dose of the peptide was employed. The overall results were interpreted to suggest that the limbic system acts as an integrated functional unit when mediating the proposed attention/arousal/retrieval effects of ACTH, but as separate and independent substrates for the proposed consolidation/retrieval effects of vasopressin on avoidance extinction. 2. Summary: Lesion Studies Taken together, the results of these studies indicated that (1) the thalamic pfc nucleus, part of the diffuse thalamic arousal system, is involved in mediating the effects on avoidance retention produced by both sets of peptides. However, although it is essential for the retrieval effect induced by the ACTH-like peptides, the pfc nucleus is not as important for the effect of vasopressin on memory consolidation (Van Wimersma Greidanus et al., 1974); (2) other limbic system structures such as the septal region (Van Wimersma Greidanus et al., 1975b), hippocampus (Van Wimersma Greidanus and De Wied, 1976b), and amygdala (Van Wimersma Greidanus et al., 1979b) do appear to play a crucial role in mediating the effects of ACTH-like peptides as well as vasopressin on retention behavior; and (3) transections of pathways interlinking components of the limbic system are more detrimental to ACTH-like peptide effects on avoidance retention than effects induced by vasopressin (Van Wimersma Greidanus et al., 1979c). This latter ﬁnding is in accord with the hypothesis that the septal region, hippocampal area, and amygdala complex act as separate substrates in mediating the effects of vasopressin on extinction and therefore when engaged in the process of memory consolidation. On the other hand, these limbic system components function as an integrated unit mediating the effects of ACTH on behavior, and therefore when engaged in the neuronal activity that contributes to attention, motivation, and retrieval (Van Wimersma Greidanus et al., 1979c).

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B. Microinjection of VP, OT, or Their Antisera into Discrete Brain Sites 1. Microinjections of VP or OT into Selected Brain Sites a. Van Wimersma Greidanus et al. (1973) Van Wimersma Greidanus et al. (1973) demonstrated prolonged maintenance of a conditioned pole-jump shock avoidance response after posttraining unilateral microinjections of arginine vasopressin (AVP) into the parafascicular nucleus of the thalamus, but not in numerous other structures that may be involved in memory processing (e.g., the reticular formation, midbrain gray, substantia nigra, putamen, dentate gyrus of the hippocampus, and cortex). The authors suggested that an insufﬁcient amount of peptide administered by the unilateral injection procedure may have been responsible for some of these failures. This was supported by bilateral microinjection studies that employed the passive avoidance paradigm, as described below. b. Kovacs et al. (1979a) Kovacs et al. (1979a), noting the evidence suggesting that vasopressin and oxytocin produce opposing effects on CNS mechanisms involved in memory processing (Bohus et al., 1978a,b; see Chapter 2), designed several experiments to study the brain sites activated by these peptides in producing their effects on avoidance behavior. Using male inbred Wistar rats and a passive avoidance (PA) memory consolidation design, these peptides were microinjected into midbrain/limbic sites that former lesion studies suggested were involved in mediating the effect of vasopressin on avoidance extinction (see previous section of this chapter). The dose levels for both peptides were 0 (saline controls) or 20–25 pg for bilateral injections into the hippocampus, dorsal septal area, and amygdaloid nuclei, and 0 (saline controls) or 50 pg for midline injection into the dorsal raphe nucleus; these dose levels were behaviorally ineffective for both peptides when injected into the lateral ventricle (Bohus et al., 1978b). PA behavior was tested 24 and 48 h after the single footshock (FS) trial. The results were as follows: (1) when microinjected into the hippocampal dentate gyri, AVP signiﬁcantly increased and OT signiﬁcantly decreased median reentry latency relative to saline controls; (2) when microinjected into the dorsal septal nuclei, both peptides signiﬁcantly increased median reentry latency relative to the saline controls in both retention tests; (3) relative to the saline controls, neither AVP nor OT inﬂuenced median reentry latency on either retention test when injected into the central amygdaloid nuclei; and (4) when microinjected into the dorsal raphe nucleus, AVP signiﬁcantly increased, and OT signiﬁcantly decreased, median reentry latencies relative to saline controls in the ﬁrst, but not the second, retention test. Taken together, these results suggest that all the brain sites tested, except the central nucleus of the amygdala, are sites at which VP and/or OT inﬂuence memory consolidation. The failure of either AVP or OT, microinjected

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into this area, to inﬂuence memory consolidation suggests that this amygdala nucleus is not involved in the inﬂuence of these peptides on memory consolidation. The ﬁndings that, when microinjected into the hippocampal dentate gyri or the dorsal raphe nucleus, AVP facilitated and OT inhibited PA memory consolidation, agree with the effects produced by much larger quantities of these peptides injected into a lateral ventricle (Bohus et al., 1978a,b). The ﬁnding that OT microinjected into the septal nuclei mimicked the effect of VP when microinjected into this region, although unexpected, has been observed in biochemical studies (Van Heuven-Nolsen et al., 1984a; this chapter), and in electrophysiological studies (Joels and Urban, 1982; Urban, 1981; see Chapter 5) conducted by De Wied’s colleagues. The authors suggested two reasons why OT facilitated PA memory consolidation when injected into the septal nuclei. Noting that peripherally injected OT, especially at high doses, sometimes produces VP-like effects on active and passive avoidance retention (Bohus et al., 1978b), it was suggested that the septal area may be the target site for these effects of OT. Alternatively, the observation that the C-terminal fragment of OT [Pro-Leu-Gly (PLG)] attenuates puromycin-induced amnesia (Walter et al., 1975) and prolongs extinction in a pole-jump shock avoidance task (Walter et al., 1978) led to the suggestion that brain region differences in degradation of this peptide (Burbach et al., 1980a) may have produced this effect. c. Kovacs et al. (1979b) Kovacs et al. (1979b) tested the effect of AVP microinjected into either the dorsal raphe nucleus or the locus coeruleus on memory consolidation in a step-through passive avoidance paradigm. The subjects, male inbred Wistar rats, were assigned to various groups: an AVP/ locus coeruleus group (25 pg bilaterally injected into the locus coeruleus on each side of the brain); a saline/locus coeruleus control group (bilateral injection of an equal volume of physiological saline); an AVP/dorsal raphe group (50 pg unilaterally injected into the dorsal raphe nucleus at midline); and a saline/dorsal raphe control group (a unilateral injection of an equal volume of physiological saline). The single treatment was given immediately after the FS learning trial, and median reentry latency was tested 24 and 48 h later. The results demonstrated that (1) AVP microinjected into the locus coeruleus had no effect on memory consolidation (no signiﬁcant difference between VP-treated and saline-treated controls on reentry latency scores) and (2) AVP microinjected into the dorsal raphe nucleus improved memory consolidation at the 24-h retention test (reentry latency was signiﬁcantly greater in the VP-treated subjects than in the saline-treated subjects). d. Bohus et al. (1982) Bohus et al. (1982) reported the outcomes of several experimental tests carried out to determine brain sites mediating the effects of VP on memory retrieval, in a passive avoidance (PA) paradigm. Three experiments tested the ability of peripheral, intracerebroventricular,

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and localized injections of AVP into speciﬁc brain structures to reverse retrograde amnesia (RA) in a step-through passive avoidance task. RA was induced by an intraperitoneal injection of pentylenetetrazole (PT, 45 mg/kg) given immediately after the single learning trial. The rationale for this paradigm is that amnesia can be viewed as a deﬁcit in memory retrieval (Spear, 1973). In each experiment the subjects received a single treatment of placebo (physiological saline) or AVP 1 h before the 24-h retention test. In experiment 1, PT signiﬁcantly reduced reentry latency relative to the nonamnestic controls, and peripherally injected AVP (2 g/rat, subcutaneous) substantially reversed this amnestic effect (i.e., median reentry latency was signiﬁcantly increased in the AVP-treated subjects relative to the placebo controls). In experiment 2, intracerebroventricularly injected AVP (10 ng/ rat) fully reversed the severe amnestic effect induced by PT treatment. In experiment 3, AVP was microinjected into speciﬁc brain structures, either bilaterally (100 pg on each side of midline) into the hippocampal dentate gyrus, dorsal septum, or central nucleus of the amygdala, or unilaterally (200 pg) along the midline into the dorsal raphe nucleus. In this experiment, pretreatment with PT produced a severe amnestic effect, and AVP induced a relatively small but signiﬁcant reversal of this amnesia when injected into either the central nucleus of the amygdala or the hippocampal dentate gyrus, but not the dorsal septum or dorsal raphe nucleus. The results of the experiments reported above, together with those of Kovacs et al. (1979a,b), suggest that individual brain sites may or may not mediate both consolidation and retrieval effects of these peptides. Thus, according to these ﬁndings the hippocampal dentate gyrus mediates the effects of these peptides in both memory consolidation and retrieval; the central nucleus of the amygdala is involved in the effect of AVP on retrieval, but not in the inﬂuence of either peptide on consolidation; and the dorsal septal nucleus and the dorsal raphe nucleus are involved in the effects of the peptides on consolidation, but not in the inﬂuence of AVP on retrieval. 2. Microinjections of VP or OT Antiserum into Selected Brain Sites a. Kovacs et al. (1980a) Kovacs et al. (1980a) demonstrated that VP antiserum microinjected into the dorsal raphe nucleus immediately after the learning trial attenuates passive avoidance behavior in a 24-h retention test trial. This ﬁnding suggests that endogenous VP localized in this structure is involved in memory consolidation. This study is discussed later in this chapter because of its relevance to interactional effects between VP and brain catecholamines. b. Kovacs et al. (1982a) Kovacs et al. (1982a), noting evidence that the dorsal hippocampal complex plays a role in mediating the retention effects of peripherally (Van Wimersma Greidanus and De Wied, 1976b, described above) and centrally (Kovacs et al., 1979a, described above) injected

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vasopressin, tested for a role of endogenous hippocampal VP in memory processing. Using male inbred Wistar rats in a PA consolidation design, hippocampal VP was neutralized by a bilateral microinjection of VP antiserum (1 l of a 1:50 dilution) into the dentate gyrus in the dorsal part of the hippocampal complex. For comparison, VP antiserum (2 l of a 1:50 or 1:10 dilution) was injected into a lateral ventricle (intracerebroventricular injection) in two other groups of subjects. Normal rabbit serum was injected into the control groups. All solutions were delivered via previously implanted cannulas in freely moving subjects immediately after the PA learning trial, and retention was tested 24 h later. Histological study at the conclusion of behavioral testing permitted localization of the tip of the cannulas as well as immunocytochemical detection of spread of the injected antiserum in the brain. Results indicated that the intracerebroventricularly injected antiserum, at the stronger (1:10) but not the weaker (1:50) dilution, signiﬁcantly inhibited PA retention in the 24-h retention test. However, microinjection of the weaker solution into the hippocampal dentate gyrus did severely impair PA behavior. Because the 1:50 dilution of the antiserum had no effect when injected directly into the cerebrospinal ﬂuid, it was concluded that the microinjected antiserum had its effect in the brain and did not result from leakage into the ventricular ﬂuid. Histological ﬁndings indicated that the antiserum microinjected into the dorsal hippocampus, although maximally concentrated in a 1-mm surround of the cannula tract, also spread rostrally toward the dorsolateral septum and ventrocaudally into a small region of the ventral hippocampus. Because of the uptake of the antiserum in the septal area and ventral hippocampus, it is possible that a combination of these sites mediated the effect observed in this study. c. Veldhuis et al. (1987) Veldhuis et al. (1987) used a memory consolidation and retrieval design with a step-through PA task to investigate the effects of VP antiserum injected intracerebroventricularly or locally into each of several brain sites (dorsal hippocampus, ventral hippocampus, dorsolateral septum, or caudate nucleus). Injections of antiserum (1:50 dilution) or normal rabbit serum (control serum) were administered via preimplanted cannulas into freely moving male Wistar rats. The single injection was given either immediately after the PA learning trial (consolidation design) or 1 h before the 24-h retention test (retrieval design). Retention (reentry latency) was measured 24 and 48 h after the learning trial. For the antiserum dilution used, the results of the consolidation design indicated the following: (1) there was no signiﬁcant difference between subjects receiving an intracerebroventricular injection of antiserum or control serum in median reentry latency on either retention test; (2) subjects injected via either the ventral or dorsal hippocampus with anti-VP serum

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had a sharply and signiﬁcantly reduced median reentry latency in both retention tests relative to those injected with control serum; and (3) there was no signiﬁcant difference in median reentry latency for either retention test between those subjects receiving VP antiserum in the dorsolateral septum or the caudate nucleus and those receiving the control serum. The results for the retrieval design indicated that (1) the subjects receiving an intracerebroventricular injection of either anti-VP serum or control serum exhibited maximum PA behavior (median latency, 300 s in duration) in both retention tests; (2) anti-VP serum, injected into the dorsal or ventral hippocampus or into the dorsolateral septum, signiﬁcantly reduced reentry latencies in both retention tests relative to serum controls; and (3) median reentry latency in subjects microinjected with anti-VP serum in the caudate nucleus did not differ signiﬁcantly from that of their serum controls. These results were interpreted as indicating that endogenous vasopressin in the dorsal and ventral hippocampus is important for both memory consolidation and retrieval, whereas that in the dorsolateral septum appears to be functionally involved only in memory retrieval. The failure to obtain evidence of a role of caudate nuclear VP in memory consolidation or retrieval is not surprising given the sparse and diffuse input into this region from extrahypothalamic VP-ergic projections (Buijs, 1983) and failure to detect VP-receptor sites in this region (Ostrowski et al., 1994). d. Van Wimersma Greidanus and Baars (1988) Van Wimersma Greidanus and Baars (1988) tested the effects of locally injected OT antiserum on PA behavior in male inbred Wistar rats. Either normal rabbit serum (control group) or OT antiserum was microinjected into the dorsolateral septum or the ventral hippocampus immediately after the PA learning trial or 1 h before the 24-h retention test. Reentry latencies were measured (to a maximum of 300 s) both 24 and 48 h after the PA learning (FS) trial. Results indicated that, relative to the control groups, reentry latencies were signiﬁcantly increased (1) in both retention tests after a posttraining injection of anti-OT serum (1:100 dilution) into the dorsolateral septum or the ventral hippocampus, and (2) in the 24-h but not the 48-h retention test after a preretention injection of OT antiserum (1:50 dilution) into the dorsolateral septum or the ventral hippocampus. These results suggest that endogenous OT within the ventral hippocampus and dorsolateral septum is normally involved in attenuating memory consolidation and retrieval in this passive avoidance paradigm. 3. Summary: Microinjection Studies These microinjection studies have extended and been generally consistent with the previously discussed lesion research. One potential source of inconsistency pertains to the role of the amygdala in memory consolidation assessed in the microinjection study of Kovacs et al. (1979a), and the lesion

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study of Van Wimersma Greidanus et al. (1979b). The microinjection study found no support, and the lesion study positive evidence, of a role for the amygdala in mediating the inﬂuence of VP in memory consolidation. However, the two studies are not directly comparable because a fairly restricted area (central nucleus of the amygdala) was affected in the microinjection study whereas extensive damage (central and basolateral nuclei of the amygdala) was inﬂicted in the lesion study. The microinjection studies also support the hypothesis of an amnesic effect of OT on PA retention because the effects of locally injected OT typically oppose those of VP, whereas neutralizing endogenous levels of OT in selective brain sites has effects mimicking those of VP treatment. The studies that used the active avoidance (AA) or PA experimental paradigm pointed to roles for the parafascicular thalamus (AA) and various limbic system structures (PA) in mediating the effects of locally microinjected VP and/or OT on memory consolidation or retrieval. Speciﬁcally, these studies suggested the following: (1) VP improved memory consolidation when injected into the thalamic pfc nucleus, dorsal septal area, hippocampal dentate gyri, or dorsal raphe nucleus, but had no effect when injected into the central nucleus of the amygdala or the locus coeruleus; (2) VP improved memory retrieval when injected into the hippocampal dentate gyri or the central nucleus of the amygdala, but had no effect on retrieval when injected into the dorsal septal area, or the dorsal raphe nucleus; and (3) OT attenuated memory consolidation when injected into the hippocampal dentate gyrus or the dorsal raphe nucleus, improved it when injected into the dorsal septal nucleus, and had no effect when injected into the central nucleus of the amygdala. The unexpected ﬁnding that OT mimicked, rather than opposed, the behavioral effect of VP when microinjected into the septal area may be related to the ability of OT or the C-terminal tripeptide derivative of OT (PLG) to stimulate VP receptors in this brain site and thereby produce a VP-like effect. The observation that VP receptors are abundant, whereas OT receptors are sparse, in the lateral septal area (Barberis and Tribollet, 1996) is consistent with this possibility. The VP antiserum studies demonstrated that neutralizing endogenous AVP in some limbic system structures implicated in memory processing produced effects opposite to those observed after microinjection of the peptide itself. Speciﬁcally, these studies suggested that endogenous VP released in the dorsal or ventral hippocampus enhances both memory storage and retrieval, that in the dorsal raphe nucleus enhances consolidation, and that in the dorsolateral septum enhances retrieval. The caudate nucleus, however, appears not to be involved in endogenous vasopressin effects on either memory consolidation or retrieval. Neutralization of endogenous OT has suggested that both dorsal septal and ventral hippocampal OT has a physiological role in modulating (attenuating) PA memory consolidation and retrieval (Van Wimersma Greidanus and Baars, 1988).

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III. Interaction between VP/OT Peptides and Brain Catecholamines in Memory Processing

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There has been much theoretical speculation and research on the possibility that the three brainstem–telencephalic monoaminergic projection systems [noradrenergic (NA-ergic), dopaminergic (DA-ergic), and serotonergic (5HTergic)] modulate memory processing. These monoaminergic projection systems originate in cell populations localized in the locus coeruleus of the pons (NA-ergic projection), substantia nigra and ventral tegmental areas (DA-ergic projections), and dorsal raphe nucleus (5HT-ergic projection) of the midbrain, and they project to telencephalic structures implicated in memory processing (i.e., the hippocampus, septum, amygdala, and striatum). These same structures also receive terminals from extrahypothalmic VP- or OT-containing ﬁber projections or contain receptors for these peptides (see Chapter 1). The presence of this anatomical substrate is in accord with a putative neurohypophysial peptide–monoaminergic interaction in memory processing. Although the animal literature is not entirely consistent, there is some degree of support for the propositions that NA-ergic (Ellis, 1985; Lee and Ma, 1995; Sara, 1985) and DA-ergic (Beninger, 1983; Packard and White, 1991) projections facilitate, and that the 5HT-ergic projection inhibits (Altman and Normile, 1988; McEntee and Crook, 1991; Ogren, 1985), memory processing, at least in certain types of learning and memory tasks. De Wied and colleagues, and related groups (e.g., Kovacs and colleagues), have investigated the possibility that the central neurohypophysial peptidergic ﬁber systems modulate memory processing by inﬂuencing neurotransmission in these brainstem–telencephalic monoaminergic projections. This research has particularly focused on the ﬁber projections containing the catecholamine neurotransmitters [i.e., noradrenaline (NA) or dopamine (DA)], and has employed three research protocols: (1) the behavioral/biochemical protocol includes experiments that investigate the ability of neurohypophysial peptides or their antisera to inﬂuence PA retention as well as catecholaminergic (CA-ergic) neurotransmission; (2) the behavioral protocol examines VP or OT modulation of avoidance retention after experimental lesions in a selected catecholamine-containing projection system; and (3) the biochemical protocol examines the ability of neurohypophysial peptides to modulate neurotransmission in CA-ergic terminals innervating selective brain sites implicated in memory processing. CA-ergic neurotransmission is assessed by determining the rate at which the neurotransmitter disappears (rate of utilization) once further synthesis of the catecholamine is prevented by pretreatment with -methylparatyrosine (-MPT), an inhibitor of the enzyme necessary for synthesis of either NA or DA. Thus, if VP activates the neurotransmitter-containing ﬁber, the rate of neurotransmitter disappearance (utilization) should be increased; if the peptide inhibits it, the disappearance rate will be decreased.

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A. The Behavioral/Biochemical Protocol: Influence of VP, OT, or Their Antisera on PA Behavior and Catecholaminergic Neurotransmission 1. Selected Studies a. Kovacs et al. (1977) Kovacs et al. (1977) carried out behavioral and biochemical experiments to investigate an AVP/CA-ergic interaction in memory processing. The behavioral experiment determined the effect of CA synthesis inhibition on the ability of peripherally administered LVP to inﬂuence PA learning and retention in male CFY rats. The biochemical experiment investigated the effect of peripherally administered LVP on -MPT-induced utilization of NA and DA in selective brain structures. In the behavioral experiment, the CA-synthesizing enzyme inhibitor -MPT was injected alone or in combination with peripherally administered LVP. The effect of these treatments on learning and retention of a benchjump passive avoidance (PA) task was assessed. The rats were trained to jump onto a bench and to remain there for 180 s (PA component), and total time required for them to remain on the bench for 180 s (PA learning criterion) was scored (step-on latency). Retention was tested 24 h later when the rats were returned to the apparatus and placed on the bench; the latency to step down onto the grid ﬂoor was used as the measure of PA retention. On both training and retention test days, the subjects received either vehicle control, LVP (300 mU/kg, intraperitoneal), -MPT (80 mg/ kg, intraperitoneal), or LVP and -MPT. The -MPT injection was given 3 h before, and the vehicle or LVP injection 10 min before, the behavioral session. Neither LVP nor -MPT treatment alone or in combination inﬂuenced learning (i.e., no signiﬁcant difference in step-on latency between the vehicle controls and the three treatment groups). On its own, -MPT did not inﬂuence PA retention, whereas LVP alone facilitated PA response (signiﬁcantly increased step-down latency relative to the vehicle control). When combined with LVP treatment, the enzyme inhibitor prevented the signiﬁcant retention effect induced by LVP, indicating the importance of an intact CA-ergic projection system for mediating the inﬂuence of vasopressin on PA retention. The biochemical experiments tested the ability of LVP to inﬂuence NA and DA: (1) levels in the hypothalamus, septum, and striatum and (2) turnover rate (utilization) in these structures. Naive rats were used in these experiments to avoid an interaction of LVP and behavioral training effects on NA and DA metabolism. The subjects received either no treatment, LVP (300 mU/kg, intraperitoneal), or vehicle control solution 10 min before sacriﬁce, and the effects of these treatments on NA and DA levels in the hypothalamus, septum, and striatum were assessed. Results indicated that, relative to intact and vehicle controls, peripherally administered LVP

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decreased DA content in the hypothalamus, septum, and striatum but had no signiﬁcant effect on NA content in any of these brain structures. In biochemical testing of the effect of LVP on NA and DA turnover in these structures, the subjects were either nontreated, or received -MPT injected together with either LVP (300 mU/kg, intraperitoneal) or vehicle control solution 4 h before sacriﬁce. The results demonstrated that LVP increased the -MPT-induced disappearance of DA in the septum and the striatum, and of NA in the hypothalamus. The results of this study conﬁrm earlier ﬁndings that vasopressin, although not importantly involved in avoidance learning, does play a role in its retention (see Chapter 2), and also suggested that brain catecholamines (CAs) appear to be involved in mediating the VP retention effect because inhibiting their synthesis prevented the effect. Moreover, the observation that LVP inﬂuenced CA utilization in some areas of the brain is consistent with this suggestion. b. Kovacs et al. (1979a) Kovacs et al. (1979a) further investigated the effects of AVP and OT on memory consolidation in a PA task, and on CA neurotransmission. The behavioral experiments, discussed earlier in this chapter, demonstrated that VP and OT signiﬁcantly modulated memory consolidation when microinjected into the hippocampal dentate gyrus, the dorsal septal nucleus, and the dorsal raphe nucleus, but not the central nucleus of the amygdala. The biochemical studies were done 1 week later, and used those subjects for which treatment resulted in signiﬁcant PA retention. As in the behavioral experiment, AVP (20–25 pg for bilateral injections or 50 pg for a midline injection) or placebo was microinjected into either the hippocampal dentate gyrus, dorsal septal nucleus, or dorsal raphe nucleus. The microinjection was given 30 min after intraperitoneal injection of -MPT, and 3 h later the rats were killed and the brains dissected. The following nuclei were removed and examined for NA and DA content: dorsal septal nucleus, medial septal nucleus, hippocampal dentate gyrus, thalamic parafascicular nucleus, caudate nucleus, red nucleus, dorsal raphe nucleus, and locus coeruleus. AVP injection into the hippocampal dentate gyrus increased utilization of NA in that structure and also in the red nucleus. AVP injection into the dorsal septal nucleus decreased NA utilization in the septal nucleus itself and increased it in the red nucleus. AVP injected into the dorsal raphe nucleus did not inﬂuence NA utilization in any of the brain nuclei studied but did increase DA disappearance in both the locus coeruleus and the red nucleus. Whether the increased utilization of NA and DA in the red nucleus after VP injection into the septal–hippocampal system and locus coeruleus, respectively, is related to mnemonic and/or motor processing remains to be clariﬁed. The behavioral and biochemical data were interpreted as indicating that (1) VP and OT modulate memory consolidation in limbic system structures

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that also receive input from extrinsic catecholaminergic projection systems, and (2) the ability of AVP to modulate catecholaminergic neurotransmission is related to this behavioral effect. c. Veldhuis et al. (1987) Veldhuis et al. (1987) carried out behavioral and biochemical experiments to investigate the effects of endogenous AVP on PA retention and CA utilization in selected limbic and striatal brain sites of male Wistar rats. The behavioral experiments, described earlier in this chapter, showed that anti-VP serum attenuated PA memory consolidation and retrieval when microinjected into the dorsal and ventral hippocampus, and retrieval when injected into the dorsal septal nucleus. The biochemical experiments were carried out 1 week after completion of the behavioral experiments. Thirty minutes after intraperitoneal injection of -MPT, vehicle or anti-VP serum was microinjected into the same brain structures treated during the behavioral experiments. Three hours later the animals were killed and the injection regions were dissected for study. Anti-VP serum inﬂuenced NA utilization when microinjected into the dorsal or ventral hippocampus (decreased utilization) or into the dorsolateral septum (increased utilization). Inﬂuence on DA utilization was not applicable because, after -MPT administration, the concentration of this catecholamine was below the limit of detection in all three of these brain structures. Microinjection of the antiserum into the caudate nucleus had no effect on DA utilization, and NA concentration was below the limits of detection after -MPT treatment. The biochemical data suggest that endogenous VP modulates NA neurotransmission in each of the three limbic system sites inﬂuenced by the antiserum in the behavioral experiments (i.e., dorsal and ventral hippocampus, and dorsal septal nucleus). The authors noted the discrepancy between their failure to demonstrate an interaction between VP and the nigrostriatal system and the results of several behavioral and biochemical studies that have obtained positive ﬁndings (e.g., Van Heuven-Nolsen and Versteeg, 1985; see Section III.C.1.a). They suggested that the present ﬁndings could be due to the fact that the inﬂuence of VP on DA neurotransmission in the caudate nucleus is indirect (i.e., mediated by VP-modulated neurons that impinge on DA terminals but are located outside the area treated in this study). 2. Summary Each of the studies described above (including both behavioral and biochemical experiments) provided support for a VP/CA-ergic interaction in memory processing. Kovacs et al. (1977) conﬁrmed the importance of CA-ergic neurotransmission for mediating the inﬂuence of VP on PA retention, and also indicated that VP modulates CA-ergic neurotransmission in a number of areas of the brain (e.g., hypothalamus, striatum, and septum). Kovacs et al. (1979a) observed that AVP microinjected into selected limbic

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system structures (hippocampus, septal area, and dorsal raphe nucleus) facilitates PA memory consolidation, whereas OT microinjected into some of these structures (hippocampus and dorsal raphe nucleus) attenuates it. Moreover, AVP modulates NA neurotransmission in both the hippocampus and dorsal septal nuclei. Veldhuis et al. (1987) demonstrated that reducing endogenous VP in the hippocampus and septal area attenuates memory consolidation and/or retrieval and that VP modulates NA neurotransmission in these sites.

B. The Behavioral Protocol: Effect of Selective Lesions in a Catecholaminergic Projection System on VP-Induced Avoidance Retention 1. Selected Studies The two studies described in this section focused on a potential interaction between VP and the NA-ergic system originating in the locus coeruleus, in the upper pontine level of the brainstem, and projecting to the telencephalon (ceruleo–telencephalic NA pathway). This pathway was interrupted by chemical lesions placed in the dorsal noradrenergic bundle, and the effect of this lesion on the ability of the peptide to facilitate PA retention was studied. The rationale for this protocol is that a failure of VP to inﬂuence retention in the absence of the fully functioning transmitter system indicates the necessity for that system in mediating the retention effect of VP. The ﬁrst study employed exogenous vasopressin, and the second manipulated central levels of endogenous vasopressin. Both studies provided support for the proposed VP/CA-ergic interaction in memory processing, and also for a secondary role for the 5HT-ergic projection in mediating the VP inﬂuence on memory processing. a. Kovacs et al. (1979b) Kovacs et al. (1979b) lesioned the dorsal noradrenergic bundle (DNAB, a major part of the ceruleo–telencephalic pathway) and studied the effect on VP-induced PA retention. This pathway innervates structures that have been implicated, by lesion (Van Wimersma Greidanus and De Wied, 1976b) and microinjection studies (Kovacs et al., 1979a), in the effects of vasopressin on avoidance behavior. Because VP has been reported to inﬂuence DA (Kovacs et al., 1977, 1979a) and serotoninergic neurotransmission (Ramaekers et al., 1977), and the DNAB interacts with these neurotransmitter systems (e.g., Anderson et al., 1977; Antelman and Caggiula, 1977), the lesion technique was also applied to a brain structure containing cells producing DA (nucleus accumbens), and serotonin [5HT; dorsal raphe nucleus (DRN)]. The subjects received either sham operations (microinjected physiological saline) or chemical lesions in the DNAB [microinjected 6-hydroxydopamine (6-OHDA) toxin], nucleus accumbens (microinjected 6-OHDA toxin),

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or the DRN [microinjected 5,6-dihydroxytryptamine (5,6-DHT) toxin]. The chemical lesions were made by microinjection of the designated neurotoxic compound via a metal guide cannula acutely implanted in the target structure until completion of the lesion. The chronically implanted cannula(s) permitted a subsequent injection of AVP [50 pg, unilaterally into the DRN; 25 pg, bilaterally into the locus coeruleus (LC)] into freely moving conscious subjects during behavioral testing. Behavioral testing began 10 days after lesioning and cannula implantation. Behavioral testing was carried out in a single-trial step-through passive avoidance task (2-s FS at 0.25-mA intensity). A single postlearning injection of physiological saline or AVP was given either subcutaneously (AVP dose of 5 g/rat, subcutaneous) or intracerebrally via the implanted cannula (AVP at a bilateral dose of 25 pg or 50 pg for midline injection). Retention test trials (up to maximum of 300 s) were given 24 and 48 h after the PA learning trial. The behavioral results were as follows: (1) for the sham operates, the 5-g dose of AVP signiﬁcantly increased reentry latency relative to the saline controls in both retention tests; (2) comparisons between lesioned and shamoperate saline controls indicated that the lesion interfered with PA retention in the 48-h, but not the 24-h, retention test. However, the lesion did prevent the AVP-facilitated retention effect in both tests; (3) neither the nucleus accumbens lesion nor the DRN lesion prevented the facilitation of PA behavior produced by postlearning peripherally administered AVP; (4) the postlearning microinjection of AVP into the LC did not inﬂuence PA behavior in either retention test; (5) a posttraining microinjection of AVP into the dorsal raphe nucleus signiﬁcantly facilitated PA retention in the 24-h retention test. Although chemically lesioning the DRN did not inﬂuence PA behavior per se, it did prevent the facilitated PA retention induced by the AVP microinjection into this region; and (6) injection of the NA toxin into the DRN (destructive of NA terminals synapsing on 5HT cell bodies in the nucleus) did not inﬂuence PA behavior per se but prevented the facilitated PA retention effect induced by postlearning microinjection of AVP into the nucleus. After completion of the behavioral studies, neurochemical techniques applied to dissected brains determined catecholamine (NA and DA) content in the brain as well as serotonin uptake in the midbrain and dorsal hippocampi. Results of the neurochemical analysis indicated that (1) the 6OHDA-induced lesion of the DNAB signiﬁcantly decreased NA content in the LC (site of A6 cell population, the source of the NA ﬁbers of the DNAB) and in the hippocampal dentate gyrus (a target structure of NA ﬁbers in the DNAB). However, this lesion did not affect DA content assessed in the nucleus accumbens or caudate nucleus (sites that receive synaptic input from the DNAB). This ﬁnding is consistent with studies that have shown that 6-OHDA damage to the DNAB depletes NA levels mainly in the forebrain (Mason and Iversen, 1977), but produces no changes in brain

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DA levels (Roberts et al., 1976); and (2) the 5,6-DHT lesion of the dorsal raphe nucleus signiﬁcantly decreased serotonin uptake in the midbrain and in the dorsal hippocampus (target areas receiving serotonin input from the dorsal raphe nucleus). The results of this study led the authors to three major conclusions. First, the DNAB–NA ﬁber pathway is important for expression of the AVPinduced facilitation of PA memory consolidation. This conclusion rests on the observations that the 6-OHDA lesion of the DNAB prevented AVP facilitation of PA memory consolidation, and that this lesion depleted NA content in a brain site implicated in the AVP effect on retention while failing to inﬂuence DA content in the brain. Second, the interaction between AVP and the DNAB–NA ﬁber system appears to occur in the region of the ﬁber terminals of this pathway. This conclusion rests on the ﬁndings that (1) locally applied AVP facilitated PA memory consolidation when microinjected into the DRN, which contains NA terminals synapsing on 5HT cell bodies, but not when microinjected into the LC, which contains the cell bodies of the DNAB–NA ﬁbers, and (2) lesioning the DRN prevented the PA response normally maintained by posttraining microinjection of AVP into this structure. Third, this study indicated that the serotonin (5HT) projection to the telencephalon, which originates in the DRN, is of secondary importance to the VP-induced facilitation of memory, because activity in the 5HT pathway is modulated by NA input from the LC. Figure 3 provides a schematic illustration of the neurotransmitter pathways involved in the inﬂuence of AVP on PA behavior.

FIGURE 3 Neurotransmitter pathways involved in the action of vasopressin. Key: Norepinephrine (NE) pathways, solid arrows; serotonin (5HT) pathways, dashed arrows; HPC, hippocampus; SEPT, septum; LC, locus coeruleus; DNB, dorsal noradrenergic bundle. Source: Kovacs et al., 1979b (Fig. 5, p. 83). Copyright ß 1979 by Elsevier/North-Holland Biomedical Press.

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b. Kovacs et al. (1980a) Kovacs et al. (1980a) obtained further support for an NA–VP interaction mediating the retention effects of the peptide. The DRN contains serotonin cell bodies and also terminals of both NA and DA projection systems. Reducing endogenous AVP in this area by microinjection of anti-VP serum impaired PA behavior in a 24-h retention test. The antiserum interacted with the CA terminals rather than the 5HT cell bodies in the region, as indicated by the failure of the antiserum to inﬂuence PA retention once the CA terminals in the region were destroyed.

C. The Biochemical Protocol: The Effect of VP, OT, or Their Antisera on Catecholaminergic Transmission in Selected Brain Sites Studies examining the effect of the neurohypophysial peptides on catecholaminergic utilization fall into two categories depending on whether vasopressin or oxytocin levels in selected brain sites were elevated by microinjected VP or OT, or reduced by microinjection of their antisera. 1. Effect of Centrally Injected AVP or OT on Catecholamine Neurotransmission a. Selected Studies i. Tanaka et al. (1977a) Tanaka et al. (1977a) studied the effect of intracerebroventricularly administered AVP (30 ng/rat) on catecholaminergic (CA-ergic) utilization in 37 individual brain sites dissected out of the following brain regions: hindbrain, midbrain, hypothalamus, thalamus, preoptic area, amygdala, and hippocampus. Vasopressin inﬂuence on CA-ergic utilization was assessed by evaluating the rate of disappearance of NA and DA after treatment with the CA synthesis enzyme inhibitor, -MPT. An AVP-induced increase in this rate would signify activation by the peptide, whereas a decrease would indicate an inhibitory effect of the peptide. Intracerebroventricularly injected vasopressin signiﬁcantly increased the rate of NA disappearance (increased utilization) in several regions of the hindbrain (locus coeruleus, A1 region, and nucleus of the solitary tract), midbrain (dorsal raphe nucleus), thalamus (parafascicular nucleus), and hypothalamus (anterior hypothalamic nucleus), and in the medial forebrain bundle and the dorsal septal nucleus. It signiﬁcantly decreased the rate of NA disappearance in the red nucleus of the midbrain and in the supraoptic nucleus of the preoptic area. The vasopressin-induced increase in NA utilization in the subiculum and dentate gyrus of the hippocampus was close to statistical signiﬁcance. A vasopressin-induced increase in the rate of DA disappearance (increased utilization) was observed in the basal ganglia (caudate nucleus), hypothalamus (median eminence), and midbrain (dorsal raphe nucleus and in the A8 region, the origin of the DA nigrostriatal projection system).

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Several of the regions in which CA-ergic utilization was increased by AVP have been implicated, by lesion or microinjection studies described earlier, in a vasopressin facilitation of retention in avoidance behavior. These regions include the parafascicular thalamus, dorsal septal nucleus, locus coeruleus, and dorsal raphe nucleus. The nearly signiﬁcant VP increase in NA utilization in the hippocampus is also noteworthy. ii. Van Heuven-Nolsen et al. (1984a) Van Heuven-Nolsen et al. (1984a) studied the effect of intracerebroventricularly administered OT (0, 1, 10, 100, or 1000 ng/rat) on the rate of disappearance of NA and DA after -MPT treatment. OT was administered 30 min after -MPT treatment and the animals were killed 3 h later. Twenty-eight nuclei were dissected from various brain regions for study. At one or more dose levels, intracerebroventricularly injected OT signiﬁcantly modulated NA utilization relative to saline controls in a few restricted brain sites: it increased NA utilization in the supraoptic nucleus, and decreased it in the lateral septal (LS) nucleus, medial septal (MS) nucleus, and the anterior hypothalamic area. OT did not signiﬁcantly inﬂuence DA utilization in any of the brain nuclei studied, although it did exhibit a nonsigniﬁcant tendency to increase it in the caudate nucleus, globus pallidus, and medial septal nucleus. None of the midbrain sites (i.e., red nucleus, central gray, and A8 region) showed an OT effect on DA utilization. The authors concluded that, in comparison with the effects of VP on NA utilization (Tanaka et al., 1977a,b), these results suggest that OT interactions with the catecholamines are less widespread and, where effective, it generally tends to attenuate NA utilization whereas VP tends to increase it. iii. Van Heuven-Nolsen et al. (1984b) Van Heuven-Nolsen et al. (1984b) carried out two experiments to study the effects of intraamygdalainjected AVP and two related peptides (the AVP derivative cyclo[Lys-Gly], and the C-terminal tripeptide of oxytocin, Pro-Leu-Gly-NH2 [PLG]) on CA utilization in various brain structures. In the ﬁrst experiment, vehicle, AVP (0.01, 0.1, or 1 pmol/rat), cyclo[Lys-Gly] (2.7 or 27 pmol/rat), or PLG (1.8 or 18 pmol) was microinjected into the amygdala 30 min after intraperitoneally injected -MPT. The rats were killed 3 h later and the amygdala was dissected and examined for NA and DA utilization. The results indicated that DA utilization in the amygdala was increased by the highest dose of AVP (1 pmol), the lower dose of cyclo[Lys-Gly] (2.7 pmol), and the higher dose of PLG (18 pmol). None of the peptides inﬂuenced NA utilization in the amygdala. In the second experiment, vehicle, cyclo[Lys-Gly] (0.27, 2.7, or 27 pmol), or PLG (0.18, 1.8, or 18 pmol/rat) was microinjected into the amygdala 30 min after intraperitoneal injection of -MPT. The animals were killed 3 h later, the brain was dissected, and NA and DA utilization was assessed in the nucleus

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accumbens, each of ﬁve nuclei of the amygdala complex (basal, central, cortical, lateral, and medial nuclei), the thalamic parafascicular (pfc) nucleus, and the hypothalamic paraventricular nucleus (PVN). The results indicated that the OT fragment PLG, at doses of 1.8 and 18 pmol, signiﬁcantly enhanced DA utilization in the cortical amygdaloid nucleus, and a similar effect in the central amygdaloid nucleus neared statistical signiﬁcance. The AVP residual cyclo[Lys-Gly], at a dose of 2.7 pmol, facilitated DA utilization in the central amygdaloid nucleus, and a similar effect in the cortical amygdaloid nucleus neared statistical signiﬁcance. DA levels in the other nuclei studied were either below the limit of detection after -MPT pretreatment or were not inﬂuenced by these peptides. In summary, the peptides had no inﬂuence on NA utilization in any of the brain nuclei studied. The increase in local DA utilization after their microinjection into various nuclei of the amygdala suggests that VP and related peptides may exert at least some of their effects on retrieval processes via DA-containing terminals in the amygdala. The observation that local microinjection of AVP, cyclo[Lys-Gly], and PLG into the amygdala reverses experimentally induced amnesia (Bohus et al., 1982) is in accord with this suggestion, because retrograde amnesia may involve a deﬁcit in memory retrieval (Dunn, 1980). iv. Van Heuven-Nolsen and Versteeg (1985) Van Heuven-Nolsen and Versteeg (1985) carried out microinjection, push–pull perfusion, and in vitro experiments to test the hypothesis that AVP interacts with the nigrostriatal DA system, and to learn more about the site and nature of this interaction. Two microinjection experiments were conducted. In one experiment, saline or AVP (10, 100, or 1000 pg/rat) was bilaterally injected into the A9 region of the substantia nigra, the cell body region of this pathway, and its effect on DA utilization in the caudate nucleus (CN) and the nucleus accumbens (NA), two terminal regions of the pathway, was assessed. In the other experiment, saline or AVP (10, 100, 1000, or 10,000 pg/rat) was bilaterally microinjected into the CN and evaluated for its effect on DA utilization in that structure. In both experiments the rats received an intraperitoneal injection of -MPT 30 min before the microinjection experiments. The rats were killed 3 h after the microinjections, their brains were removed and sectioned, and cannula placements were histologically veriﬁed. The tissue sections from selected parts of the CN and/or NA were homogenized and DA concentration levels in the homogenates were measured. The results indicated that AVP, at a dose of 100 pg/rat or greater, signiﬁcantly increased DA utilization in the CN when microinjected into that brain site, but had no inﬂuence on DA utilization in either the CN or the NA when microinjected into the A9 region of the substantia nigra.

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In the push–pull experiment, a push–pull cannula was implanted in a site in the CN that previous experiments had indicated increased DA utilization from microinjected AVP. AVP was either added to or omitted from (control procedure) the medium used to perfuse the implanted brain area, permitting an in vivo test of the effect of AVP on the activity of the cells that release the neurotransmitter (DA) into the perfusate. The percentage of DA release during the 30 min of VP perfusion relative to that calculated from the ﬁrst four fractions obtained before VP treatment was determined for each rat. Addition of AVP to the medium at a ﬁnal concentration of 106 M strongly enhanced DA efﬂux from the CN, an effect that was immediate and lasted only during the time that the peptide was present in the perfusion medium. The effect of VP on DA synthesis in the CN was deduced by two experiments that used in vitro assessment of tritiated DA conversion from tritiated tyrosine, its precursor molecule. In the ﬁrst experiment, AVP (concentration range, 108 to 104 M) was added to CN slices in vitro from the beginning of the incubation period. In the second experiment, VP (0.1, 1.0, or 5.0 nmol in 1 l of saline) or 1 l of saline was initially injected in vivo, that is, into a lateral ventricle of rats via a preimplanted cannula. Each rat was decapitated 1 h after the injection, its brain was removed, and the CN was dissected and assessed in vitro for tritiated DA content. The results of the ﬁrst experiment indicated that AVP exerted a biphasic effect, inhibiting DA accumulation at a low concentration (107 M) and progressively increasing it up to a concentration of 105 M. The results of the second experiment indicated no signiﬁcant effect on DA accumulation in CN slices for any of the doses administered in vivo. Taken together, the results of these various experiments suggest that although VP may have a modest effect on DA synthesis, its primary inﬂuence is the enhancement of DA release in the nigrostriatal pathway by an action exerted at the terminus (CN) rather than at the origin (A9 region) of this pathway. This VP effect may involve a direct VP presynaptic action on DA terminals or may inﬂuence other neurons, which in turn modulate this DA activity. An indirect effect on DA release could explain the repeated failures of these researchers to observe an AVP inﬂuence on DA release from the CN, using in vitro preparations that presumably removed circuitry essential for mediating the inﬂuence of VP on this release (unpublished observations cited in Van Heuven-Nolsen and Versteeg, 1985). In addition to the present study, a number of lines of behavioral and biochemical evidence, cited by these authors, also support a VP inﬂuence on DA neurotransmitter activity in the nigrostriatal system: (1) intracerebroventricularly injected LVP produced ipsilateral turning in rats with 6-OHDA-induced lesions in the substantia nigra (Schultz et al., 1979); (2) systemically injected DG-AVP modulated self-stimulation of the substantia nigra and ventral tegmental area in rats preimplanted with electrodes in these brain sites (Dorsa and Van Ree, 1979);

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and (3) DA utilization was decreased in the striatum–caudate nucleus region after intracerebroventricular administration of VP antiserum (Versteeg et al., 1978) and in the VP-deﬁcient Brattleboro HODI (homozygous diabetes insipidus) rat (Kovacs et al., 1980b). b. Summary Centrally (intracerebroventricularly) administered AVP (Tanaka et al., 1977a), and AVP and the OT fragment PLG (Van HeuvenNolsen et al., 1984a), have been shown to inﬂuence NA and/or DA neurotransmission in a number of brain sites, several of which have been implicated in memory processing. Van Heuven-Nolsen et al. (1984a) found that VP and PLG (which has a VP-like effect on memory processing) (Walter et al., 1975; see Chapter 2) produced similar effects on catecholamine neurotransmission (i.e., enhanced DA, but not NA, neurotransmission in the amygdala). This ﬁnding suggested that DA terminals in the amygdala may be modulated by VP input to mediate the inﬂuence of the peptide on memory retrieval. Van Heuven-Nolsen and Versteeg (1985) demonstrated that the inﬂuence of AVP on DA neurotransmission in the nigrostriatal pathway was mediated at its terminal region, and not its cell body region, thereby enhancing the release of DA into the caudate nucleus and nucleus accumbens, two major target regions of this pathway. 2. Intraventricularly Injected VP or OT Antiserum: Effect on Catecholamine Neurotransmission in Selected Brain Sites a. Selected Studies i. Versteeg et al. (1979) Versteeg et al. (1979) studied the effect of intracerebroventricularly injected anti-VP serum on the -MPT-induced disappearance of catecholamines (NA and DA) in selective brain sites of male Wistar rats. Antiserum or control rabbit serum was injected into a lateral ventricle 30 min after intraperitoneal injection of -MPT, and 3 h later the rats were killed and the brain regions dissected for radioenzymatic assay. NA and DA concentrations were measured in various sites in the telencephalon (caudate nucleus, dorsal septal nucleus, and dorsal hippocampus), diencephalon (thalamic parafascicular nucleus and the following hypothalamic areas: paraventricular nucleus, arcuate nucleus, median eminence, and supraoptic nucleus), midbrain (dorsal raphe nucleus), and hindbrain (locus coeruleus, A1 and A2 regions, and nucleus of solitary tract). The results indicated that anti-VP serum signiﬁcantly decreased NA utilization in the dorsal septal nucleus, the thalamic parafascicular nucleus, and the nucleus of the solitary tract and DA utilization in the caudate nucleus and the A2 region of the medulla. In comparison with intracerebroventricularly injected AVP (Tanaka et al., 1977a; described earlier), anti-VP serum effects on catecholamine utilization, although involving fewer brain sites, were in the expected opposite direction. The difference in the number of brain sites

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inﬂuenced in the two studies was attributed to the dose level used in the AVP study, which resulted in a relatively high VP concentration at its sites of action. It was also noted that although the anti-VP serum undoubtedly decreased the bioavailable AVP at these sites, the extent of this decrease is not known. The data were interpreted as supporting the postulate that endogenous AVP exerts a tonic inﬂuence on CA-ergic neurons terminating in various brain regions, several of which have been implicated in memory processing. ii. Kovacs and Telegdy (1983) Kovacs and Telegdy (1983) studied the effect of peripherally and intracerebroventricularly injected OT, and of intracerebroventricularly injected desglycinamide oxytocin (DG-OT) and anti-OT serum, on CA utilization in the brains of an inbred strain of Sprague-Dawley rat. The role of these peptides in CA-ergic neurotransmission was of interest because previous work had indicated that OT may function as a natural amnestic (e.g., Bohus et al., 1978a; Kovacs et al., 1978; see Chapter 2) and that DG-OT, although practically devoid of the endocrine activities of OT, is as potent as the parent peptide in its ability to attenuate avoidance retention (Kovacs et al., 1982b). OT was administered peripherally (0, 0.81, 8.1, or 81.0 g/kg, intraperitoneal) or centrally (1 g/rat, intracerebroventricular). DG-OT (1 g/rat, intracerebroventricular) and anti-OT serum [diluted 1:10 with cerebrospinal ﬂuid (CSF)] were injected into a lateral ventricle in separate groups of rats. Vehicle solution, or normal rabbit serum diluted 1:10 with CSF, served as appropriate controls for the various peptide treatments, and intraperitoneal saline served as the control for -MPT treatment. Peptides and antiserum were injected 30 min after -MPT treatment and the rats were killed 3 h later. The following brain regions were dissected for examination of NA and DA levels: midbrain, hypothalamus, septum, and striatum. On the basis of comparisons with control data, the results indicated that (1) depending on the dose, peripherally administered OT accelerated NA and decreased DA neurotransmission in the midbrain, failed to affect utilization of either neurotransmitter in the hypothalamus or the septum, and had opposing effects on striatal DA utilization depending on whether OT was simultaneously administered, or given 30 min after inhibition of CA synthesis (i.e., inhibited DA disappearance in the former case, and facilitated it in the latter); (2) intracerebroventricularly administered OT failed to inﬂuence NA and decreased DA neurotransmission in the midbrain, increased NA and decreased DA neurotransmission in the hypothalamus, failed to inﬂuence utilization of either transmitter in the septal area, and facilitated DA disappearance in the striatum; (3) intracerebroventricularly administered DG-OT had no inﬂuence on NA and decreased DA utilization in the midbrain, had no inﬂuence on the utilization of either neurotransmitter in the hypothalamus, increased NA and decreased DA

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utilization in the septal area, and had no inﬂuence on DA utilization in the striatum; (4) intracerebroventricularly administered anti-OT serum had no inﬂuence on NA but increased DA utilization in the midbrain, decreased NA but was without inﬂuence on DA utilization in the hypothalamus, and had no inﬂuence on NA but decreased DA utilization in the septum; and (5) in contrast to the facilitation of striatal DA utilization by intracerebroventricularly administered OT (see result 2 above), intracerebroventricularly injected OT antiserum normalized the accelerated -MPT-induced striatal DA utilization that resulted from intracerebroventricularly administered normal rabbit serum. Although the peptides and antiserum inﬂuenced either NA or DA utilization in several of the regions studied, the most interesting ﬁnding relevant to memory processing was the pattern of effects on DA utilization in the midbrain. Central (intracerebroventricular) treatment with OT or DG-OT had opposite effects, compared with anti-OT serum, on DA neurotransmission. This parallels the attenuation of PA retention induced by intracerebroventricular injection of OT (Bohus et al., 1978b) or DG-OT (Kovacs et al., 1982b) and its improvement by intracerebroventricularly injected anti-OT serum (Bohus et al., 1978b). The authors suggested that the pattern of effects on DA utilization in the midbrain might be related to the inﬂuence of OT on DA utilization in memory processing, a suggestion awaiting experimental evidence. They pointed out that monoaminergic-containing cell bodies in this region [e.g., the dorsal raphe nucleus and the DA-containing cells (A9 and A10 groups) that project to limbic and striatal structures, respectively] are known to receive OT terminals of neurons originating in the hypothalamic PVN. It is also noted that OT-induced inhibition of steady state DA levels in the midbrain was previously observed by Schwarzberg et al. (1981). Further study is needed to clarify the role of the OT interaction with DA projections to learn whether this interaction is involved in modulation by the peptide of conditioned avoidance retention behavior. b. Summary The two studies described above conducted experiments that reduced the bioavailability of brain levels of VP or OT and studied the effect on CA-ergic neurotransmission in various brain sites implicated in memory processing. Relevant to the putative role of VP in memory modulation were the ﬁndings by Versteeg et al. (1979) that endogenous VP interacts with NA-ergic neurotransmission in the dorsal septal nucleus and the thalamic parafascicular nucleus, and with DA-ergic neurotransmission in the caudate nucleus. Relevant to the putative role of OT in memory modulation was the pattern of ﬁndings by Kovacs and Telegdy (1983) indicating OT/DA-ergic interactions in the midbrain that paralleled earlier described behavioral ﬁndings of OT effects on retention in avoidance conditioning tasks.

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IV. Interaction between VP and Catecholamines of Peripheral Origin during Memory Processing

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Whereas studies described in the preceding section of this chapter support an interaction between central catecholaminergic systems and the neurohypophysial peptides in memory processing, experiments described below indicate the importance of the adrenal medullary hormone adrenaline (epinephrine) for the contribution of vasopressin to memory processing. The role of peripheral catecholamines in memory processing is documented by a number of lines of evidence: (1) PA retention was enhanced after posttraining peripherally injected adrenaline or noradrenaline (Gold et al., 1977), whereas peripherally administered inhibitors of catecholamine synthesis impaired retention (Palfai and Walsh, 1979); (2) PA retention was signiﬁcantly impaired after surgical removal of the adrenal medulla (the endocrine source of peripherally circulating epinephrine), and epinephrine replacement therapy dose dependently normalized this retention deﬁcit (Borrell et al., 1983c); and (3) more recent evidence indicates that central effects of peripherally administered epinephrine entail the release of noradrenaline in the basolateral medulla, mediated by a pathway that involves activation of vagus nerve endings by peripherally circulating catecholamine (Cahill and McGaugh, 1996; Williams et al., 1998).

A. Selected Studies: Borrell et al. (1983a,b) Experiments by Borrell and colleagues, cited below, investigating a potential interaction between vasopressin and peripherally circulating epinephrine in memory processing were reported in two sources: Borrell et al. (1983a,b). These experiments demonstrate the importance of peripherally circulating epinephrine for expression of (1) the facilitating effects of exogenous AVP on memory consolidation and retrieval (experiments 1–4) and (2) the normal role played by endogenous vasopressin in memory processing (experiments 5 and 6). A single-trial step-through PA task was used for testing retention in both normal Wistar rats and Brattleboro HODI and HEDI (heterozygous diabetes insipidus) rats. Manipulation of hormonal epinephrine was accomplished by surgical removal of either the entire adrenal gland (adrenalectomy, ADX), which depleted the subjects of both adrenocortical hormones and the catecholaminergic hormone, or the adrenal medulla (adrenomedullectomy, ADMX), which deprived the animals only of the hormonal catecholamine, and by the use of hormonal replacement therapy (various doses of epinephrine for ADMX experiments, and of epinephrine and corticosterone for ADX experiments). Experiments testing the effects of exogenous vasopressin on PA retention or retrieval injected vehicle or AVP either peripherally (1 g/rat, subcutaneous) or centrally

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(5 ng/rat, intracerebroventricular), using a memory consolidation or retrieval test design. In experiments with Brattleboro rats, exogenous AVP was used for replacement therapy. In all experiments PA retention was tested 24 h after the PA learning trial. In experiment 1, rats that had received sham operations, adrenalectomies, or adrenomedullectomies were injected either peripherally with physiological saline or AVP (1 g/rat, subcutaneous), or centrally with saline or AVP (5 ng/rat, intracerebroventricular). To test AVP effects on memory consolidation, the injections were given immediately after the learning trial (2-s FS of 0.50-mA intensity); to test the effects of VP on memory retrieval, the injections were given 1 h before the 24-h retention test (reentry latency measured up to a maximum of 300 s). The results demonstrated that (1) ADX and ADXM signiﬁcantly impaired PA retention (reentry latencies in the two operate groups were signiﬁcantly below the 300-s median reentry latency of the sham operates); (2) neither a posttraining nor preretention injection of subcutaneously or intracerebroventricularly administered AVP normalized the retention deﬁcit observed in the ADX and ADXM rats; and (3) the lack of an AVP effect on memory consolidation and retrieval, even when the ADX rats were given corticosterone replacement therapy, indicated that the failure of VP to facilitate PA behavior was not due to the absence of this adrenal cortical hormone. Experiment 2 was designed to determine the degree to which epinephrine replacement therapy in ADX rats could correct the PA retention deﬁcit in both saline- and AVP-treated rats. The ADX rats received a subcutaneous injection of either vehicle control solution or epinephrine (0.0025, 0.05, or 50 g/kg, subcutaneous) immediately after the learning trial. The three doses of epinephrine selected for study were based on the inverted U-shaped dose– response curve relating PA retention to posttraining epinephrine treatment in intact rats (i.e., the facilitatory effect disappears with further increases in dose level; Gold and Van Buskirk, 1975). Accordingly, the three dose levels of epinephrine were judged as least effective (0.0025 g/kg, subcutaneous), submaximally effective (0.05 g/kg, subcutaneous), and as a high, normally amnesia-inducing dose (50.0 g/kg, subcutaneous). Physiological saline or AVP (1 g/rat, subcutaneous) was injected either immediately after the PA learning trial or 1 h before the 24-h retention test. Testing for VP inﬂuence on memory retrieval occurred only in ADX rats given the low (0.0025 g/kg) or high (50 g/kg) dose of epinephrine. Results indicated that ADX rats given posttraining injections of AVP showed no improvement of PA retention after the least effective dose of epinephrine, improved PA retention after the submaximal dose of epinephrine (i.e., signiﬁcantly more rats showed maximal avoidance behavior in the AVP-treated group), and signiﬁcantly improved PA retention after the normally amnesia-inducing high dose of epinephrine. On the other hand, the ADX rats given preretention injections of AVP showed no improvement in

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memory retrieval whether treated with the ineffective low or the amnesiainducing high doses of epinephrine. Taken together, these ﬁndings (1) indicate that the VP facilitation of memory consolidation occurs only in the presence of an adequate amount of peripherally circulating epinephrine and (2) suggest that the failure of AVP to inﬂuence retrieval among the epinephrine-treated ADX rats might have been because postlearning-administered epinephrine was no longer present in the circulation at the time of vasopressin treatment. It was also noted that the memory consolidation effect induced by AVP in ADX rats that received the normally amnesia-inducing dose of epinephrine is consistent with the antiamnesic property of vasopressin (e.g., Rigter et al., 1974; Chapter 2), and indicates the importance of peripherally circulating epinephrine for the expression of this VP function. In experiment 3, the authors tested the effect of AVP on memory retrieval in ADX or sham operates given preretention injections of epinephrine. That is, epinephrine (0.0025, 0.50, or 125 g/kg, subcutaneous) and AVP (1 g/rat, subcutaneous) were both injected 1 h before the 24-h retention test. The vehicle for AVP and epinephrine was used for control injections. The results indicated that (1) given alone, AVP failed to normalize memory retrieval in ADX rats; (2) on its own, only the middle dose of epinephrine (50 g/kg) markedly facilitated memory retrieval in ADX rats; and (3) AVP markedly improved PA memory retrieval in all ADX rats that received preretention epinephrine injections. These data were interpreted as indicating that AVP-induced facilitation of memory consolidation, as well as retrieval, requires an adequate level of peripherally circulating epinephrine at the time each phase of memory processing is active. The ﬁnding that the peptide was active in ADX rats that received the high dose of epinephrine also reinforces the earlier interpretation that AVP has an antiamnesic property as well as a facilitative inﬂuence on memory consolidation and retrieval. Experiment 4 tested the effect of posttraining centrally administered AVP (5 ng/rat, intracerebroventricular) on PA behavior in ADX and shamoperated rats, given an injection of vehicle control or a high dose of epinephrine (50 g/kg, subcutaneous) immediately after the training trial. On its own, this high dose of epinephrine has amnestic properties when injected into intact rats (Gold and Van Buskirk, 1975). It was observed that centrally administered AVP treatment was ineffective in the vehicle-treated ADX rats but signiﬁcantly improved PA retention in those ADX rats given the posttraining high dose of epinephrine. Because intracerebroventricularly injected AVP likely acted at central receptors, the effects of the peptide were probably not mediated by an initial inﬂuence on receptors in the adrenal medulla; instead, the peripherally circulating epinephrine was in some way necessary for mediating the central effects of AVP on PA retention. Experiment 5 investigated PA retention in HODI and HEDI Brattleboro rats given sham operations or ADX surgery 48 h before the PA learning trial.

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These subjects were peripherally injected with vehicle control or AVP (2 g/rat, subcutaneous) immediately after the learning trial. The results were as follows: (1) in accordance with previous observations (De Wied et al., 1975; see Chapter 3), sham-operated, vehicle-treated HODI rats were signiﬁcantly impaired in PA retention relative to vehicle-treated sham-operated HEDI rats. AVP treatment signiﬁcantly improved PA behavior in both groups of Brattleboro rats, relative to the vehicle-treated, sham-operated HEDI comparison group; (2) adrenalectomy signiﬁcantly impaired PA behavior of the HEDI rats, but did not further impair the already severely deﬁcient PA performance of the HODI rats; and (3) the AVP dose that improved PA performance in the sham-operated HODI and HEDI rats had no effect on the deteriorated PA performance of these subjects after adrenalectomy. These results again demonstrated the necessity of peripherally circulating epinephrine for expression of the facilitative inﬂuence of AVP on PA retention. Experiment 6 tested the effect of a low (0.05 g/kg, subcutaneous) or a high (50 g/kg, subcutaneous) dose of epinephrine on PA retention in ADX or sham-operated HODI or HEDI rats. Results indicated that the ADXproduced retention deﬁcit was signiﬁcantly improved in HEDI rats given the low but not the high dose of epinephrine. On the other hand, the same high dose of epinephrine improved PA retention in HEDI rats with intact adrenal glands. Different results emerged for HODI rats given epinephrine replacement therapy. Posttraining injections of either the low (0.05 g/kg) or high (50.0 g/kg) dose of epinephrine failed to inﬂuence the behavioral deﬁcit of HODI rats, whether they were given ADX or not. This suggests that in the absence of brain AVP, peripherally circulating epinephrine loses its behavioral effects, indicating multiple interactions between peripheral epinephrine and AVP in memory processing. In summary, these experimental ﬁndings indicate that whether injected intracerebroventricularly or subcutaneously, the inﬂuence of VP on PA memory consolidation and retrieval is not expressed in rats deprived of the peripherally circulating catecholamine, epinephrine. Furthermore, noting the importance of the ceruleo–telencephalic NA projection system for mediating the facilitative inﬂuence of the peptide on PA retention (Kovacs et al., 1979a,b; discussed earlier), it was concluded that the ﬁndings suggest a close interaction between vasopressin and central catecholaminergic systems in modulating memory processes, and a dependence on an intact hormonal epinephrine system for expression of this VP/CA-ergic interaction. The authors noted that the exact nature of the involvement of VP with peripherally circulating epinephrine needs to be clariﬁed. As noted in earlier discussion, McGaugh and colleagues (e.g., Williams et al., 1998) have shown that one aspect of the central effect of epinephrine on memory processing involves NA release in the basolateral amygdala initiated by activation by the hormone of the vagus nerve. It is possible

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that a similar hormonal action results in activation of the locus coeruleus via a branch of the NA projection to the amygdala that originates in the nucleus of the solitary tract. If so, this could account for the ﬁnding that the expression of VP inﬂuence on memory processing depends on an intact peripheral epinephrine system. This is so, because interaction of VP with the cerulean/NA-ergic projection pathway is an important mechanism by which the peptide inﬂuences memory processing (Kovacs et al., 1979a). However, support for this speculation relies, in part, on demonstration of the required anatomical substrate.

V. Theoretical Propositions of the ‘‘VP/OT Central Memory Theory’’: Continued

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The research studies described in this chapter provided evidence relevant to two additional propositions of the ‘‘VP/OT Central Memory Theory.’’ These propositions and their supportive evidence are discussed below.

A. Proposition 7: The Central Anatomical Substrate for the Memory-Modulating Effects of VP and OT Includes Brainstem and Forebrain Limbic System Structures That Are Implicated in Memory Processing Microinjection and lesion studies have provided evidence in support of proposition 7. Lesion studies have indicated that VP-induced prolonged maintenance of a conditioned avoidance response is partially or totally prevented by a bilateral lesion of the thalamic parafascicular nucleus (Van Wimersma Greidanus et al., 1974), the septal/nucleus accumbens region (Van Wimersma Greidanus et al., 1975b), the anterolateral hippocampus (Van Wimersma Greidanus and De Wied, 1976b), and the central and basolateral amygdala (Van Wimersma Greidanus et al., 1979b). Lesion studies have also suggested that the various forebrain limbic system structures act more or less independently of one another in mediating the longterm effect of vasopressin on retention, but act in concert in mediating the short-term, presumably arousal-inducing effects of ACTH-like peptides on memory processing (Van Wimersma Greidanus et al., 1979c). Microinjection studies have indicated that exogenous AVP facilitates memory consolidation when locally injected into the thalamic parafascicular nucleus (Van Wimersma Greidanus et al., 1973); dorsal septal nuclei, hippocampal dentate gyrus, and dorsal raphe nuclei (Kovacs et al., 1979a); and retrieval when injected into the central nucleus of the amygdala (Bohus et al., 1982). Microinjection studies with anti-VP serum have indicated that endogenous VP facilitates memory consolidation when released into the dorsal raphe nucleus (Kovacs et al., 1980a), dorsal hippocampal

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dentate gyrus (Kovacs et al., 1982b), dorsal and ventral hippocampi (Veldhuis et al., 1987), and memory retrieval when released into the dorsal and ventral hippocampi and the dorsolateral septal area (Veldhuis et al., 1987). Local microinjections of OT into midbrain–limbic brain sites have demonstrated that exogenous OT opposes the inﬂuence of VP and attenuates PA memory consolidation when microinjected into the dorsal raphe nucleus and hippocampal dentate gyrus, but mimicks the inﬂuence of VP and facilitates memory consolidation when injected into the dorsal septal area (Bohus et al., 1982; Kovacs et al., 1979a). The similarity of VP and OT actions when injected into the septal area may be related to brain regional differences in rate of metabolism of these peptides, and the consequent generation of OT metabolites having VP-like effects on avoidance behavior (Bohus et al., 1982). Moreover, microinjection of anti-OT serum into the dorsolateral septum as well as the ventral hippocampus facilitated PA memory consolidation and retrieval, suggesting that endogenous OT released into these brain sites opposes the action of VP and attenuates these phases of memory processing (Van Wimersma Greidanus and Baars, 1988).

B. Proposition 8: Neurohypophysial Peptides Interact with Central Catecholaminergic Neurotransmitters in Mediating Their Influence on Memory Processing, and the VP/NA-ergic Interactional Effect Appears to Be Dependent on an Intact Hormonal Epinephrine System for Its Expression According to proposition 8, centrally located VP-ergic and OT-ergic neuropeptide systems interact with central NA as well as DA projection systems in exerting their inﬂuence on memory consolidation or retrieval in avoidance tasks. This interaction involves VP- or OT-induced modulation of release of NA or DA at relevant brain sites, which in turn mediates the inﬂuence of the neuropeptides on memory processing. In addition, peripherally circulating epinephrine plays an essential role in modulation by vasopressin of these phases of memory processing. Although proposition 8 emphasizes catecholamine involvement in the neurohypophysial peptide inﬂuence on memory processing, it does not deny participation of the serotoninergic and cholinergic forebrain projection systems. Thus Kovacs and colleagues suggested that serotonin-containing ﬁbers originating from the dorsal raphe nucleus may play a secondary role in mediating VP memory effects, because vasopressin appears to interact with noradrenergic terminals that modulate neural activity in this serotonin pathway (Kovacs et al., 1979b, 1980a). Interaction between central CA-ergic projections and the neurohypophysial peptides in memory processing has been supported by both behavioral

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and biochemical paradigms. Behavioral paradigms test the ability of exogenous VP or OT to inﬂuence PA retention after manipulation of a given CA-ergic pathway. Three behavioral studies support this proposition. Kovacs et al. (1977) demonstrated that inhibition of CA synthesis in the brain prevented facilitation of PA retention induced by peripherally injected LVP. Kovacs et al. (1979b, 1980a) showed that VP interacts with NA-containing ﬁber systems in mediating its effect on PA retention and that this interaction occurs at the NA terminals and not in the cell body region. Thus, chemically lesioning the dorsal noradrenergic projection to the forebrain, which originates in the locus coeruleus, prevented the facilitation of PA memory consolidation induced by peripherally injected AVP (Kovacs et al., 1979b). It has been demonstrated that NA terminals innervating the dorsal raphe nucleus are responsible for the facilitated PA retention induced by endogenous VP released in this structure (Kovacs et al., 1980a) as well as by locally microinjected AVP (Kovacs et al., 1979b). The biochemical paradigm tests the ability of VP or OT to inﬂuence neurotransmission in CA-ergic pathways innervating forebrain structures implicated in memory processing. If VP facilitates memory storage and retrieval by activating these CA-ergic pathways, and OT attenuates memory processing by inhibiting them, intracerebroventricularly or locally administered VP should increase, and OT decrease, pathway activation (the rate of utilization or release of the catecholamine). Moreover, correspondingly opposite effects induced by administration of the antisera of these peptides would further support an interaction between endogenous VP and OT and these CA-ergic pathways. The following observations concerning vasopressin are consistent with these predictions: (1) locally microinjected AVP signiﬁcantly increased NA release in the hippocampal complex (Kovacs et al., 1979a), and locally microinjected anti-VP serum signiﬁcantly decreased it in the dorsal and ventral hippocampus (Veldhuis et al., 1987); (2) intracerebroventricular injection of AVP (Tanaka et al., 1977a) and anti-VP serum (Versteeg et al., 1979) increased and decreased NA release in the thalamic parafascicular nucleus, respectively; (3) Van Heuven-Nolsen and Versteeg (1985) observed that microinjected AVP increased DA release in the caudate nucleus, a structure innervated by the nigrostriatal DA pathway, and this ﬁnding was supported by a subsequent push–pull perfusion experiment. Moreover, intracerebroventricular injection of AVP increased (Tanaka et al., 1977a), and anti-VP serum decreased (Versteeg et al., 1979) DA utilization in the caudate nucleus; and (4) locally microinjected AVP and AVP-like fragments (cyclo[Lys-Gly] and the OT tripeptide LPG), which exert VP-like effects on memory processing (Walter et al., 1975), increased DA utilization (release) in the amygdala, an interaction believed to be involved in mediating the retrieval effects of the neurohypophysial peptides (Van Heuven-Nolsen et al., 1984b). However, a study by Winnicka (1996) obtained results

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that indicated that chemical lesioning, which prevented DA release in the central amygdala, did not inﬂuence the facilitated retrieval effect induced by intracerebroventricularly injected AVP. Discrepant ﬁndings have been obtained for the NA-ergic pathways innervating the dorsal raphe nucleus and the dorsal septal region: (1) whereas intracerebroventricularly injected AVP increased NA utilization in the dorsal raphe nucleus (Tanaka et al., 1977a), a microinjection of AVP in this structure did not inﬂuence NA utilization (Kovacs et al., 1979a); and (2) in accordance with expectations, intracerebroventricular injection of AVP (Tanaka et al., 1977a) and of anti-VP serum (Versteeg et al., 1979) signiﬁcantly increased and decreased NA utilization (release) in the dorsal septal nucleus, respectively. However, when microinjected into this brain site, the opposite pattern of effects was observed: when microinjected into the dorsal septal nucleus, AVP signiﬁcantly decreased (Kovacs et al., 1979a), and antiVP serum signiﬁcantly increased (Veldhuis et al., 1987), NA utilization in this structure. Although fewer studies have been carried out with oxytocin, these too have been interpreted as generally consistent with the prediction that OT inhibits CA innervation of structures implicated in memory processing: (1) an intracerebroventricular injection of OT decreased the rate of NA utilization in the lateral and medial septal nuclei (Van Heuven-Nolsen et al., 1984a); and (2) an intracerebroventricular injection of OT and/or DG-OT decreased DA utilization in the midbrain and septal area but unexpectedly increased it in the striatum. These effects were reversed by an intracerebroventricular injection of anti-OT toxin, except for the septal area, where the antitoxin mimicked the effects of the peptide (Kovacs and Telegdy, 1983). Borrell and colleagues have suggested an interaction between AVP and peripherally circulating catecholamines, especially epinephrine. These researchers demonstrated the importance of circulating epinephrine for the expression of AVP facilitation of PA retention (Borrell et al., 1983a,b). Speciﬁcally, their experiments showed that whether administered centrally (intracerebroventricular) or peripherally, AVP-induced facilitation of PA retention is not observed in rats deprived of the medulla of both adrenal glands and hence of the peripherally circulating catecholamines secreted by these structures. These data are in accord with the possibility that peripherally circulating catecholamines interact with central catecholaminergic pathways in mediating the inﬂuence of AVP on memory processing (De Wied et al., 1988).

Barbara B. McEwen

De Wied and Colleagues IV: Research into Mechanisms of Action by Which Vasopressin and Oxytocin Influence Memory Processing

I. Chapter Overview

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This chapter describes research by De Wied and colleagues on several ways vasopressin (VP) and oxytocin (OT) may act in the brain to inﬂuence memory processing. These studies have investigated (1) the possibility that VP and OT act as precursors of smaller peptides, oligopeptides, that have memory-processing functions; (2) the nature of the receptor sites that may mediate the memory-modulating effects of VP and OT; (3) the inﬂuence of VP on hippocampal theta activity, a putative neural correlate of memory consolidation, during paradoxical sleep (PS); and (4) the neuromodulator functions of VP and OT and their relation to the role of the peptides in memory processing in the septal–hippocampus system. As in earlier discussions, the speciﬁc propositions derived from these research studies, together with a restatement of ﬁndings speciﬁcally relevant to each, are formally stated at the end of the chapter. Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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II. VP and OT: Precursors of Metabolic Fragments That Exert Memory-Modulating Effects in the Brain?

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A. The Neuropeptide Concept The ‘‘neuropeptide concept’’ was initially formulated in 1969 (De Wied, 1969) as ﬁndings indicated the neurogenic effects of pituitary hormones such as adrenocorticotropic hormone (ACTH) and related peptides, and it was periodically expanded and updated with continued neuropeptide research (De Wied, 1987). Essentially, the neuropeptide concept appears to embody three major points. First, many peptides localized within peripheral organs and tissues have also been found within the central nervous system (i.e., ‘‘neuropeptides’’ are synthesized within peptidergic neurons and possess neurotransmitter, neuromodulator, and/or neurotropic functions). Second, many neuropeptides, such as ACTH and related peptides, are derived from a common ancestral molecule [e.g., ACTH, -melanocyte-stimulating hormone (-MSH), and -lipotropic hormone (-LPH) derived from pro-opiomelanocortin) either within the same or different neuronal tissue. Third, the individual structural/functional proﬁles of neuropeptides derived from a common ancestral molecule result from the unique pre-and/or postranslation chemical alterations each is subjected to before its ﬁnal formation. De Wied and associates recognized that vasopressin and oxytocin are also peptides that can act at both peripheral target sites and at neural structures within the brain. In both periphery and brain, metabolic alterations mediated by peptidase enzymes (proteolysis) convert the parent peptides to smaller peptides, which in some cases are biologically active fragments with novel properties. The neuropeptide concept speciﬁcally proposes that in the brain the small active fragments generated by VP and OT can function as neurotransmitters or neuromodulators at brain target sites. It also suggests that some of these fragments act at relevant brain sites to inﬂuence memory storage and retrieval.

B. Early Behavioral Evidence of a Role for VP and OT Metabolic Fragments in Memory Processing Studies carried out during the 1970s led to two generalizations relevant to the neuropeptide concept as it applies to VP and OT. First, a number of studies using metabolic fragments of these peptides found that whereas the entire molecule of VP and OT was necessary to produce the characteristic hormonal activities of each peptide, it was not required for their effects on memory consolidation and retrieval. Thus, desglycinamide-lysine vasopressin (DG-LVP), produced by tryptic digestion of the LVP molecule, which removed the terminal amino acid (glycine), lost the endocrine activity of the parent peptide but retained its ability to inhibit extinction of the learned pole-jump shock avoidance response (De Wied et al., 1972). Second, a

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number of studies that investigated structure–activity relationships of the parent peptides found that VP and OT contained more than one active site contributing to memory consolidation and retrieval effects in active and passive avoidance learning paradigms. Thus, although the covalent ring structure of the arginine vasopressin (AVP) molecule (pressinamide) was most potent for memory consolidation in a pole-jump avoidance paradigm, the C-terminal tripeptide linear chain [prolyl-arginyl-glycinamide (PAG)] was a second, less important site (De Wied, 1976). The primary structures of VP, OT, and related peptides are given in Table I. Walter et al. (1975) had shown that the linear terminal segment of the VP and OT molecules is highly active in attenuating puromycin-induced amnesia, whereas the ring structure is inactive in this retrograde amnesia paradigm. Because these researchers view this paradigm as a test for memory retrieval (see Chapter 2), the ﬁndings of these two studies were interpreted to suggest that the ring structure might be most active in memory consolidation, whereas the linear terminal segment is most active in memory retrieval. Studies such as these TABLE I Peptidesa

Amino Acid Sequences of Neurohypophysial Hormones and Related

Sequence

a

Source: De Wied et al., 1988 (Table 1, p. 102). Copyright ß 1988 by CRC Press, Inc. Adapted with permission.

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led to the speculation that VP and OT within the CNS may act as precursors of smaller neuropeptides involved in memory processing (Burbach and De Wied, 1981). Further, studies on the biotransformation of neurohypophysial peptides in the brain provided evidence of aminopeptidase enzyme cleaving action in AVP (Pliska et al., 1971a,b) as well as in OT (Marks et al., 1973) molecules, consistent with proteolysis as the mechanism of fragment generation (Burbach and De Wied, 1981; De Wied et al., 1988).

C. Biochemical Support for the Formation of Biologically Active VP and OT Neuropeptide Fragments in the Rat Brain This evidence was obtained from both in vitro and in vivo investigation (for review see Burbach, 1987; Burbach et al., 1998). 1. In Vitro Research In vitro studies were undertaken to identify bioactive VP and OT fragments generated during metabolism of these peptides within synaptic membranes obtained from brain sites where these peptides were expected to be stored or released (Burbach et al., 1998). The following experimental strategies were included in these in vitro studies: (1) VP or OT was incubated with the synaptosomal membrane preparation (SMP) derived from chemical treatment of limbic tissues dissected from rat brains; and SMP was observed for its ability to convert the parent peptide into active metabolic fragments; (2) radioimmunoassay (RIA) and chromatographic (e.g., high-pressure liquid chromatography, HPLC) techniques were used to isolate and chemically characterize the VP and OT fragments thus generated; and (3) a time course procedure with radioactively labeled VP and OT was used to monitor the sequence of enzymatic events and the substances formed during the generation of the synaptic membrane fragments (Burbach, 1987). a. Selected Studies i. Burbach and Lebouille (1983) Burbach and Lebouille (1983) performed in vitro experiments to investigate the proteolytic conversion of AVP and OT by rat brain synaptic membranes. Radioactively labeled AVP(1–9) and OT(1–9) were incubated for 3 h with SMPs derived from midbrain and forebrain tissue of male Wistar rats. In separate preparations AVP and OT were radioactively labeled at either the tyrosine amino acid residue located at position 2 or at the terminal glycinamide amino acid residue. The fragments formed during synaptic membrane-mediated digestion of AVP and OT were isolated by HPLC and chemically characterized. The major peptide fragments of AVP were [Cyt6]AVP(2–9), [Cyt6]AVP(3–9), [pGlu4, Cyt6]AVP(4–9) [a pyroglutamic acid residue spontaneously formed by cyclization of the transiently formed [Cyt6]AVP(4–9)], and a peptide having

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the AVP(4–8) sequence. The fragments formed by digestion of OT were [Cyt6]OT(2–9), [Cyt6]OT(3–9), [pGlu4,Cyt6]OT(4–9), and [Cyt6]OT(5–9). The Cys-1–Cys-6 disulﬁde bridge was preserved in each of these fragments. Time course experiments were carried out by using differentially labeled samples of both AVP(1–9) and OT(1–9), radioactively labeled at either the tyrosine or glycinamide position. The sequential rate at which the fragments emerged indicated that AVP (Fig. 1) and OT (Fig. 2) digestion

FIGURE 1 Time course of conversion of AVP-(1–9) by brain synaptic membranes and accumulation of fragments. Arginine-vasopressin including ([14C][Tyr2]-AVP 22.2 104 dpm, &) or ([14C]GlyNH29]-AVP 14.8 104 dpm), in a ﬁnal concentration of 2 105 M was incubated with brain synaptic membranes (2.5 mg of protein/ml) at pH 7.0, 37degrees C in a total volume of 1300 l. At time intervals, aliquots of 200 l were taken and subjected to HPLC. The characterized components AVP-(1–9) (&, &) [Cyt6]-AVP-(2–9) (n, m), [Cyt6]-AVP (3–9) (!), [
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FIGURE 2 Time course of conversion of OXT-(1–9) by brain synaptic membranes and accumulation of fragments. Oxytocin, in the presence of ([14C][Tyr2]-OXT 20.8 104 dpm, &) or ([14C]Gly NH29]-OXT 22.4 104 dpm, &, was incubated as described in the legend to Fig. 1. The characterized components of OXT-(1–9) (&, &), [Cyt6]-OXT-(2–9) (n, m), [Cyt6]OXT-(3–9) (!), [
occurred by proteolytic cleavage beginning at the N terminus, a characteristic feature of aminopeptidase-like enzymatic activity. The authors concluded that aminopeptidases predominate in the proteolytic mechanisms by which brain synaptic membranes convert AVP and OT. The sites of cleavage in the VP molecule and the pathway of proteolytic processing of the peptide are shown, respectively, in Fig. 3A and B. Although not presented diagrammatically, this same pathway operates in OT processing. ii. Burbach et al. (1983b) Burbach et al. (1983b) incubated OT with rat synaptic plasma membrane preparation and identiﬁed, by HPLC

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FIGURE 3 (A) Primary structure of vasopressin (VP) and sites of cleavage by proteolytic enzymes: 1, aminopeptidase activity; 2, postproline-cleaving enzymes; 3, carboxamidopeptidase or trypsinlike enzyme. Source: Burbach, 1987 (Fig. 1, p. 499). Copyright ß 1987 by Plenum Publishing Corporation. Reprinted with permission. (B) Pathway of proteolytic processing of vasopressin (VP) by rat brain preparations. Source: Burbach, 1987 (Fig. 4, p. 502). Copyright ß 1987 by Plenum Publishing Corporation. Reprinted with permission.

analysis, the same peptide fragments observed by Burbach and Lebouille (1983). These researchers treated the OT hexapeptide fragment, [Cyt6]OT(4–9) with pyroglutamic acid and demonstrated that this treatment tended to prevent further proteolytic breakdown of OT and resulted in the accumulation of this hexapeptide fragment. The studies cited above illustrate two features described by Burbach (1987) as characteristic of in vitro-generated VP fragments. First, the disulﬁde bridge linking Cys-1 to Cys-6 is preserved (not reduced), producing asymmetric disulﬁde fragments that are stable under in vitro conditions. Second, VP metabolism in vitro leads to the formation of the pyroglutamic acid residue [pGlu4,Cyt6]VP(4–9), which in turn acts as a block for most aminopeptidases and therefore promotes the accumulation of this fragment during prolonged exposure of VP to brain membranes. It was further noted that a number of biologically active peptides carry this

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FIGURE 4 HPLC proﬁles of immunoreactive AVP peptides in an extract of rat hippocampus. An extract was made from 10 rat hippocampi and prepared for HPLC. A number of C-terminal AVP fragments were separated from AVP(1–9) by application of reverse phase HPLC to the extract. The radioimmunoassay for C-terminal AVP fragments (A) employed antiserum WIA; antiserum W4E was used in the radioimmunoassay speciﬁc for the N-terminal region of AVP (B). In both radioimmunoassay systems [125I]AVP(1–9) was used as a tracer and synthetic AVP(1–9) was used as standard. Numbers refer to the structure of the peptides with intact disulﬁde bridge or with reduced disulﬁde bridge. Further procedural details are described in the original article. Source: Burbach et al., 1984 (Fig. 1, p. 385). Copyright ß 1984 by Elsevier. Figure reproduced and legend adapted by permission of the publisher.

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derivative at their N terminus and often their biological activity depends on its presence. 2. In Vivo Research Subsequent studies provided immunological and chromatographic evidence that C-terminal VP and OT metabolites, similar to those observed in vitro, are present in the brain and that aminopeptidase activity is involved in their formation (Burbach et al., 1984; Stark et al., 1989; Wang et al., 1986). a. Selected Studies i. Burbach et al. (1984) Burbach et al. (1984) carried out a preliminary in vivo study to learn whether the C-terminal VP fragments obtained in vitro were present in the brain and pituitary gland of male Wistar rats. After 5 days of handling, the subjects were killed, the pituitary gland was removed, and the hypothalamus and hippocampus were dissected from the brain. Extracts of these tissues were fractionated by reversed-phase HPLC and analyzed by RIA systems with different speciﬁcities. The RIA systems used two types of antisera (W1A and W4E), [125I]AVP(1–9) as tracer, and synthetic AVP(1–9) as a standard. Antiserum W1A showed high speciﬁcity for the C-terminal portion of AVP(1–9), and cross-reacted with [Cyt6]AVP(2–9), [Cyt6]AVP(3–9), [pGlu4Cyt6]AVP(4–9), and [Cyt6]AVP(5–9) components extracted from the dissected brain areas. This antiserum also recognized the reduced forms (dissolved disulﬁde bridge) of these peptides. The RIA system containing W4E antiserum was speciﬁc for the N-terminal region of the AVP molecule and cross-reacted only with AVP(1–9) and AVP(1–8). Figures 4 and 5 depict the peptides, isolated by reversed-phase HPLC, immunoreactive with antisera W1A (Figs. 4A and 5A) and W4E (Figs. 4B and 5B), for the hippocampus and hypothalamus, respectively. Figure 6 displays the HPLC proﬁle of peptides immunoreactive with antiserum W1A for the pituitary gland. Structural similarities between the immunoreactive components depicted in Figs. 4 and 5 and a number of the synthetic C-terminal peptides generated by SPMs in the in vitro studies of Burbach and Lebouille (1983) were observed during HPLC: (1) components II and III comigrated with synthetic [Cyt6]AVP(2–9) and [Cyt6]AVP(3–9), respectively; (2) component IV had a retention time similar to that of the reduced form of [pGlu4, Cyt6]AVP(4–9); (3) component V eluted at the position of [Cyt6]AVP(4–9) and the reduced form of [Cyt6]AVP(5–9); and (4) component VI eluted similar to [Cyt6]AVP(5–9). Although the brain levels of these immunoreactive C-terminal fragments were well below those of the parent peptides, they were relatively higher in the hippocampus [levels between 3.4 and 17% of that of AVP(1–9)] than in the hypothalamus [levels between 1.5 and 3.0% of that of AVP(1–9)]. However, in the pituitary gland virtually all the immunoreactivity represented intact AVP(4–9).

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FIGURE 5 Fractionation by reversed phase HPLC of peptides immunoreactive with the C-terminal AVP antiserum W1A (A) and the N-terminal AVP antiserum W4E (B) in an extract of ﬁve rat hypothalami. See legend to Fig. 4 and original article for additional details. Source: Burbach et al., 1984 (Fig. 2, p. 386). Copyright ß 1984 by Elsevier. Figure reproduced and legend adapted by permission of the publisher.

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FIGURE 6 Reversed phase HPLC fractionation of peptides immunoreactive with the C-terminal AVP antiserum W1A in an extract of rat pituitary gland. The quantities shown represent those of a single pituitary gland. Source: Burbach et al., 1984 (Fig. 3, p. 386). Copyright ß 1984 by Elsevier.

Taken together these ﬁndings led these researchers to conclude that a number of peptides, structurally similar to the C-terminal AVP fragments identiﬁed after in vitro proteolysis of AVP(1–9) by synaptosomal membrane preparations, were present in brain tissue but absent in the pituitary gland. To the extent that these immunoreactive components represent endogenous metabolites of AVP(1–9), their presence in the brain and absence in the pituitary gland are in accord with ﬁndings from behavioral studies indicating that C-terminal fragments of AVP(1–9) exert highly potent positive effects on memory processing but lack the peripheral hormonal effects of the parent peptide. ii. Stark et al. (1989) Stark et al. (1989) provided the ﬁrst in vivo demonstration that aminopeptidase conversion is a major pathway of degradation for AVP microinjected into the brain, and that transient metabolites similar to those observed by in vitro study are generated by this degradation pathway. These authors microinjected radioactively labeled AVP, that is, [3H][Phe3]VP(1–9), into limbic system structures (hippocampus and central amygdala) and showed that the peptide was degraded with a half-life of less than 1 min after its administration. During the degradation, C-terminal fragments [i.e., [Cyt6]VP(2–9), VP(2–9), [Cyt6]VP(3–9), and VP(3–9)] were generated. Two observations indicated that the major pathway for degradation of the injected vasopressin involved aminopeptidase

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conversion: (1) the proﬁle of the transient metabolites that were formed and (2) the fact that the aminopeptidase inhibitor, amastatin, partly inhibited the degradation of microinjected vasopressin. In this in vivo study, AVP fragments were present that showed a reduced Cys-1–Cys-6 disulﬁde bridge [AVP(2–9) and AVP(3–9)]. However, in vitro study had indicated that cleavage of AVP by brain synaptic membranes occurred without prior reduction of the disulﬁde bridge, generating the peptide [Cyt6]AVP(2–9). Although this nonreduced metabolite was detected in the present study, smaller fragments also occurred in the reduced form, such as VP(3–9). The authors concluded that in vivo conversion is initiated by cleavage of the nonreduced form of VP followed by rapid reduction of the metabolites.

D. Demonstration of the Behavioral Activities of VP and OT C-Terminal Peptides The C-terminal peptide fragments, generated from parent peptides AVP(1–9) and OT(1–9) when incubated with synaptic membrane preparations, were shown to modulate memory consolidation and retrieval in passive and active avoidance paradigms even more potently than did the parent peptides. Representative studies are described below. 1. Selected Studies a. Burbach et al. (1983a) Burbach et al. (1983a) compared AVP(1–9) with two peptide fragments, the hexapeptide [pGlu4,Cyt6]VP(4–9) and its desglycinamide derivative, [pGlu4,Cyt6]VP(4–8), on their central activity [effect on passive avoidance (PA) retention] and peripheral endocrine activity (pressor effect). For the behavioral task, vehicle (saline) or graded doses of AVP (1.0 to 10,000 pg/rat), the hexapeptide (0.01 to 1000 pg/rat), or its desglycinamide derivative (0.10 to 10.0 pg/rat) was given intracerebroventricularly immediately after the single footshock (FS) learning trial. Retention was tested at 24 and 48 h after the learning trial. For peripheral endocrine effects (i.e., pressor activity), the peptides were injected intravenously over a dose range of 10 to 500 ng. The parent peptide and the peptide fragments facilitated memory consolidation in both the 24- and 48-h retention tests, although the fragments were much more potent in their effects. Thus, the parent peptide facilitated PA memory consolidation at a dose level of 100 pg and higher, whereas the hexapeptide fragment did so at dose levels as low as 0.03 pg (24-h test) and 0.01 pg (48-h test), but failed to inﬂuence PA behavior above dose levels of 0.10 pg (24-h test) and 1.0 pg (48-h test). Whereas the PA memory effects of the hexapeptide were dose dependent, the desglycinamide derivative facilitated PA memory consolidation over its entire tested dose range. However, this tested dose range was not as extensive as that for the hexapeptide. No aversive effects of

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the peptide fragments were observed, because PA response of nonshocked rats treated with the 10-pg dose did not differ from that of their salinetreated counterparts. Whereas an injection of 10 ng of the parent peptide signiﬁcantly increased blood pressure (BP) by 26 mmHg, the fragments failed to exhibit pressor effects over the entire tested dose range. The results of this study are consistent with the proposal, based originally on structure activity studies, that AVP is a precursor of metabolites formed in the brain by aminopeptidase cleavage. b. Burbach et al. (1983b) Burbach et al. (1983b) carried out a parallel study to the one cited above, using OT(1–9), OT(5–9), OT(4–9), and OT(4–8). For the PA behavioral task, the rats were intracerebroventricularly injected immediately after the PA learning trial with saline, OT(1–9) (10, 100, 1000, or 10,000 pg), OT(5–9) (10, 100, or 300 pg), OT(4–9) (0.1, 1.0, 10, 100, or 1000 pg), or OT(4–8) (1.0, 10, 100, or 1000 pg). PA retention was tested 24 and 48 h later. For evaluation of their peripheral endocrinological activity, the peptide fragments were tested in an in vitro study with rat uterus. With the exception of OT(5–9), which failed to inﬂuence PA retention, the glycinamide-containing peptides induced an amnestic action at lower dose levels [i.e., OT(1–9) (100 and 1000 pg) and OT(4–9) (1.0 and 10 pg)], but not at the higher dose levels. The two highest dose levels of OT(4–9) tended to facilitate retention (not signiﬁcant). The desglycinamide fragment OT(4–8) attenuated retention at all dose levels higher than the ineffective 1-pg level. The endocrinological test results indicated that the lowest dose of the parent peptide [OT(1–9)] necessary to contract the uterus was 0.14 ng/ml. In contrast, the hexapeptide fragment in the dose range of 0.001 to 100 ng/ml displayed no uterotonic effect, indicating that this OT fragment exerts central effects but no peripheral endocrine activity. c. Kovacs et al. (1986) Kovacs et al. (1986) tested graded doses of AVP(1–9) and of [Cyt6]AVP(5–9) and [Cyt6]AVP(5–8), its behaviorally active fragments lacking the pyroglutamic acid residue (pGlu4), for their ability to facilitate PA retention. The peptides were injected peripherally (microgram dose range), intracerebroventricularly (nanogram dose range), or intracerebrally (picogram dose range) into one of three limbic system sites (dorsal raphe nucleus, dorsal hippocampus, or ventral hippocampus). Effects on retention were tested at both 24 and 48 h after the PA learning trial. A consolidation design was used to test the behavioral effects of subcutaneously administered AVP(1–9) and [Cyt6]VP(5–8), of intracerebroventricularly administered AVP(1–9) and [Cyt6]AVP(5–8), and of intracerebrally administered AVP(1–9), [Cyt6]AVP(5–8), and [Cyt6]AVP(5–9). A retrieval design was added to test the effects of these peptides when

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microinjected into the ventral hippocampus. The results were as follows: (l) whether administered peripherally or centrally (intracerebroventricularly), both AVP(1–9) and [Cyt6]AVP(5–8) dose dependently facilitated memory consolidation in both retention tests, and the VP fragment was more potent (i.e., required a much smaller dose level than the parent peptide to achieve this behavioral effect); (2) when microinjected into the dorsal hippocampus and tested for PA memory consolidation, AVP(1–9) and [Cyt6]AVP(5–8), but not [Cyt6]AVP(5–9), dose dependently facilitated PA retention; (3) when microinjected into the dorsal raphe nucleus and tested for memory consolidation, all three peptides induced a dosedependent positive effect. Again, both VP fragments were more potent than the parent peptide and [Cyt6]AVP(5–8) proved the most potent; (4) when injected into the ventral hippocampus, both C-terminal fragments, [Cyt6]AVP(5–8) and [Cyt6]AVP(5–9), dose dependently facilitated memory consolidation and retrieval in both retention tests at a much lower dose level than that required for AVP(1–9); (5) moreover, when microinjected into the ventral hippocampus, the two peptide fragments were differentially active in tests of consolidation versus retrieval. That is, in the consolidation design [Cyt6]AVP(5–8) was more effective (i.e., a lower amount was required for facilitative effect) than [Cyt6]AVP(5–9), whereas the reverse was true for the retrieval design. The following conclusions were drawn from these data: (1) the pyroglutamic acid residue (pGlu4) is not required for the highly potent effects of these fragments on PA memory consolidation and retrieval; the fragments were more potent than the parent peptide in their actions on PA memory, whether administered subcutaneously, intracerebroventricularly, or into selected brain sites; (2) limbic system structures implicated in the memoryprocessing effects of AVP (e.g., Kovacs et al., 1979a; Van Wimersma Greidanus and De Wied, 1976b; see Chapter 4) are highly sensitive to these behaviorally active VP fragments; and (3) in the ventral hippocampus the fragments appear to selectively inﬂuence different phases of memory processing. d. Gaffori and De Wied (1986) Gaffori and De Wied (1986) investigated critical time periods for the memory-modulating effect on PA retention of the parent peptide, AVP(1–9), and metabolic fragments possessing the pyroglutamic acid residue (pGlu4) [i.e., [pGlu4,Cyt6]AVP(4–9) and [pGlu4, Cyt6]AVP(4–8)], or lacking it [i.e., [Cyt6]AVP(5–9) and [Cyt6]AVP(5–8)]. In the main experiment, independent groups of male Wistar rats received a subcutaneous injection of a given peptide at a dose of 3.0 g/rat for AVP(1–9), and of 0.3 g/rat for the peptide fragments. A control group for the parent peptide and one for the peptide fragments received equal volumes of physiological saline. Injections were given at various times after the PA learning trial: 0, 6, 12, 15, 18, 21, and 23 h. In a separate

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experiment, placebo or [Cyt6]AVP(5–9) was given either immediately posttraining or after a 15-h delay, and retention was tested 24, 48, and 72 h after the learning trial. The results of the main experiment, which tested retention only 24 h after the learning trial, indicated time-dependent effects for all the peptides, in accord with earlier reports for the parent peptide (Bohus et al., 1978a). Although AVP signiﬁcantly facilitated PA behavior at all injection intervals, the injection given immediately after the learning trial was most effective. The peptide fragments, which were more potent than the parent peptide at all effective injection times, exhibited differential effectiveness depending on the time of treatment. For example, [pGlu4,Cyt6]AVP(4–9) was maximally effective when injected either immediately after the learning trial or 1 h before the retention test. [pGlu4,Cyt6]AVP(4–8) and [Cyt6]AVP(5–8) exhibited maximal inﬂuence when injected immediately posttraining, and [Cyt6]AVP(5–9) was most effective when injected 1 h preretention. They were all least effective at delays of 6 and 21 h. Both the parent peptide and the VP fragments facilitated retention when given 15 to 18 h after the learning trial, indicating a third sensitive time period. However, when this effect was evaluated over a longer retention interval with the fragment [Cyt6]AVP(5–9), treatment at the 15-h delay interval was effective only in the 24-h retention test, whereas treatment given immediately after the training trial was effective in the 24-, 48-, and 72-h retention tests. In summary, this study corroborated the ﬁndings of Kovacs et al. (1986) and demonstrated again that the pyroglutamic acid residue is not necessary for the potent effects of the AVP fragments on PA retention. The time-dependent effects of the fragments, as for the parent peptide, strengthen the argument that these fragments modulate memory consolidation and retrieval. The authors noted that this study revealed three critical injection periods for a vasopressin effect in the 24-h PA retention test: immediately after the PA trial, 23 h later, and 15–18 h later, and the last period was most pronounced for [Cyt6]AVP(5–9). The ﬁrst two critical periods are well known and have been inferred from the effect of the peptide on consolidation and retrieval, respectively. The third critical period did not result in a retention effect beyond 24 h, at least for [Cyt6]AVP(5–9), a ﬁnding suggesting that mechanisms other than those responsible for long-term memory storage may have produced the 24-h memory effect. Further study is needed to clarify the nature of this third critical period effect, and whether retention may last beyond 24 h when subjects are treated with the parent peptide or with peptide fragments other than those tested here. e. De Wied et al. (1987) De Wied et al. (1987) compared the effects of the parent peptides, VP(1–9) and OT(1–9), and various C-terminal VP and OT fragments on several tests of memory processing. Speciﬁcally, VP and/or

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related fragments were tested for their ability to facilitate PA memory consolidation and retrieval, to block pentylenetetrazole (PTZ)-induced retrograde amnesia, and to retard extinction of a conditioned pole-jump avoidance response. OT and related fragments were tested for their ability to impair PA memory consolidation and retrieval. The results indicated that (1) peripherally administered AVP(1–9) facilitated, and OT(1–9) attenuated, consolidation and retrieval on the PA task; (2) similar to AVP(1–9), peripherally administered C-terminal fragments of AVP dose dependently facilitated PA memory consolidation and retrieval, prolonged extinction on the pole-jump avoidance task, and reversed PTZ-induced retrograde amnesia in the PA task; moreover, the shorter fragments, especially [Cyt6]AVP(5–8) and [Cyt6]AVP(5–9), had the most potent effects on these behavioral tests; (3) similar to OT(1–9), peripherally administered C-terminal OT fragments dose dependently attenuated PA memory consolidation and retrieval, and the shorter OT fragments [e.g., [pGlu4 Cyt6]OT(4–9) and [pGlu4,Cyt6]OT(4–8)] had the most potent behavioral effects; and (4) selected C-terminal VP fragments were three to four times more potent in their facilitation of memory consolidation and retrieval when injected centrally (intracerebroventricularly) than when given peripherally.

E. Localizing Brain Receptor Sites for the Behaviorally Active VP and OT C-Terminal Fragments Several lines of evidence suggest the presence of speciﬁc binding sites mediating the behavioral effects of the neurohypophysial fragments, and that these sites may overlap but nevertheless differ from those for the parent peptides. First, there are studies indicating that, in producing behavioral effects, various VP fragments do not simply bind to putative central AVP receptors. For example, Constantini and Pearlmutter (1984) and De Kloet et al. (1985b), using in vitro studies, showed that the VP fragment [pGlu4, Cyt6]VP(4–9) has low afﬁnity for hippocampal VP receptors, because it is much less effective than the parent peptide [AVP(1–9)] in displacing tritiated AVP ([3H]VP) from binding sites in synaptic membranes of hippocampal brain slices. Second, Brinton et al. (1984, 1986) and De Kloet et al. (1985a) demonstrated that the distribution of 35 S-labeled binding sites for VP fragments is distinctively different from the distribution of putative receptors for the parent peptide. The highest density of [pGlu4,[35S]Cyt6] VP(4–9)-binding sites was found in the pineal gland, organum vasculosum lamina terminalis (OVLT), arcuate nucleus, and the nucleus of the solitary tract (NTS), none of which are the limbic system sites where VP is considered to exert its effect on retention.

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III. Characterizing the Brain Receptors That Mediate the Effects of VP and OT on Memory Processing

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A. Selected Study: De Wied et al. (1991) De Wied et al. (1991) studied the speciﬁc nature of the binding sites in the limbic system that mediate the memory-processing effects of these peptides. Earlier behavioral observations suggested that a large portion of these binding sites had a ligand speciﬁcity resembling that of the V1 type of AVP receptor found in peripheral tissues (Dorsa et al., 1984). However, more recent data have indicated involvement of additional neurohypophysial receptor types, including a V2 type of AVP receptor (Cheng and North, 1989) and an OT receptor that binds AVP and arginine vasotocin (AVT) as efﬁciently as OT (Audigier and Barberis, 1985; Elands et al., 1988a). Given these more recent data, and the evidence of potent effects on avoidance retention by the metabolic fragments of neurohypophysial peptides, this study was designed to investigate the interaction of AVP, OT, AVT, and the behaviorally active fragments AVP(4–8) and OT(4–8) with selected V1, V2 vasopressinergic and OT-ergic receptor antagonists on passive avoidance retention. The receptor antagonists used in this study were as follows: d(CH2)5[Tyr(Me)]AVP (V1 antagonist), d(CH2)5[d-Ile2,Ile4]AVP (V2 antagonist), and desGly-(NH2)9,d(CH2)5[Tyr(Me)2,Thr4,Orn8]vasotocin (OT antagonist). The antagonist or vehicle was injected immediately after the PA learning trial; the agonist or placebo was injected 30 min later. Graded doses of each of the antagonists and peptides were given intracerebroventricularly (nanogram amounts for antagonists and parent peptides; picogram amounts for the metabolic fragments). A 2-s FS of 0.25-mA intensity was given for all treatments except for that of OT and the OT(4–8) fragment, for which a higher FS level (0.5 mA, 2 s) was used. When these neurohypophysial peptides were intracerebroventricularly injected on their own immediately after the PA learning trial, they produced the following PA retention effects on the 24-h and/or 48-h retention tests: (1) a signiﬁcant facilitation of retention for AVP(1–9) at the tested dose level of 1 ng, and for AVP(4–8) at the higher (10 and 30 pg) but not the lower (1 and 3 pg) tested dose levels; (2) a signiﬁcant inhibition of retention for OT(1–9) at the tested dose levels of 1.0 and 3.0 ng, and for OT(4–8) at the tested dose levels of 3.0 and 10.0 pg; and (3) a dual effect on PA retention for AVT, with a VP-like memory facilitation at the high end (10 ng) and an OT-like memory impairment at the low end (0.1 and 0.3 ng) of the tested dose range. The complex interactive effects that occurred between the neurohypophysial neuropeptides and the various receptor antagonists used in this study were as follows: (1) interactions between the antagonists and AVP(1–9) indicated that the facilitation of PA retention induced by a 1-ng dose of

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AVP was signiﬁcantly blocked on either or both retention tests by a low (0.1 ng) and a high (1.0 ng) dose of the V1 antagonist, a middle (0.3 ng) and a high (1.0 ng) dose of the OT antagonist, and all three doses of the V2 antagonist; (2) interactions between the VP(4–8) fragment and the various antagonists indicated that the facilitation of PA retention by 10and 30-pg doses of the fragment were blocked by appropriate doses of the V2 and OT antagonists, but not by the V1 antagonist at the dose used. However, the V1 antagonist and the 1-ng dose of the OT antagonist did block the behavioral effect produced by a small dose (3 pg) of the AVP(4–8) fragment; (3) interactions between the antagonists and OT(1–9) indicated that the attenuation of PA retention induced by the 1-ng dose of OT(1–9) was signiﬁcantly blocked in either or both retention tests by a 0.3-ng dose of the V1 antagonist, or of the OT antagonist, but not by the V2 antagonist; (4) the interaction between OT(4–8) and the OT antagonist was the only one tested, and it was signiﬁcant. When a low (0.3 ng) dose of the antagonist was paired with a high dose (30 pg) of the fragment, the antagonist enhanced the attenuating effect of the fragment on PA behavior. However, when a high dose (1 ng) of the antagonist was paired with a middle dose (10 pg) of the fragment, the antagonist blocked the attenuating effect of the fragment; and (5) the interaction between AVT and the three receptor antagonists indicated that the facilitated effect on PA retention induced by the high dose (10 ng) of AVT was blocked by the 0.3-ng dose but not the 1-ng dose of the V1 and V2 receptor antagonists and by both doses of the OT receptor antagonist. The attenuation of PA retention produced by the 0.1-ng dose of AVT was blocked by the V1 and OT receptor antagonists, but not by the V2 receptor antagonist, at the one dose level (0.3 ng) of antagonist used for this experimental test. In their discussion of these results, the authors made the following points: (1) the results conﬁrmed earlier observations that posttraining intracerebroventricularly administered AVP(1–9) and AVP(4–8) facilitated PA retention (De Wied et al., 1984a; see Chapter 3). Moreover, the V1 antagonist that blocked these effects was expected from previous study of interactions between this receptor antagonist and AVP-related peptides [De Wied et al., 1984a; Koob et al., 1981; Le Moal et al., 1981 (Chapter 6)]; (2) however, the interaction between VP-related analogs and the V1 receptor antagonist was not a selective one, because the V2 and OT receptor antagonists blocked the facilitated retention produced by AVP(1–9) and AVP(4–8) as efﬁciently as did the V1 receptor antagonist; and (3) the interactions between these receptor antagonists and OT, and also AVT, the ancestral neurohypophysial peptide, were also nonspeciﬁc in nature. Thus, a 0.3-ng dose of both the V1 and OT receptor antagonists blocked the memory impairment effect produced by the 1-ng dose of OT and the low dose (1 ng) of AVT, whereas the memory facilitation effect of the high dose (10 ng) of AVT was antagonized by a 0.3-ng dose of all three antagonists.

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The authors concluded that the effects of the neurohypophysial peptides on avoidance retention are apparently mediated at nonselective receptors that have the same afﬁnity for V1 and OT receptor antagonists, and a somewhat lesser afﬁnity for the V2 receptor antagonist. Because studies have shown that AVP can bind to OT receptors, the authors questioned whether V1 receptors exclusively mediate the behavioral effects of vasopressin. Given that the ventral hippocampus, the amygdala, and the septal area all demonstrated the effect of the neurohypophysial peptides on PA retention (Kovacs et al., 1986) they proposed that a nonselective receptor complex in the limbic system contains separate receptive sites responsive to both VP and OT. AVP has its facilitative retention effect at this receptor complex by serving as an agonist, whereas OT has its amnesic effect by acting as an ‘‘inverse’’ agonist (i.e., a drug that attaches to a receptor-binding site and interferes with the action of the receptor without blocking the binding site for the principal ligand; Carlson, 1998). Understandably, the OT and VP receptor antagonists can block the actions of these peptides.

IV. VP, OT, and Hippocampal Theta Rhythm during Paradoxical Sleep

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A. Hippocampal Theta Activity: Characteristics, Genesis, and Behavioral Correlates The hippocampal theta rhythm, also known as rhythmic slow activity (RSA), refers to the electroencephalogram (EEG) activity pattern originally observed by Green and Arduini (1954), and consists of a synchronous train of sinusoidal waves of high amplitude, ranging in frequency from approximately 4 to 12 Hz (Stewart and Fox, 1990). It can be recorded from the granule cell layer in the dentate gyrus and also from the pyramidal cell layer in the CA1 ﬁeld of the hippocampus (Buzsaki, 1986; Vertes and Kocsis, 1997). This rhythm has been reliably demonstrated in a variety of nonprimate mammals (rats, cats, and rabbits). Early failure to observe hippocampal theta in primates led researchers to question the generalizability of relevant ﬁndings to humans (Robinson, 1980), but more recently Stewart and Fox (1990) successfully recorded theta activity in monkeys. Hippocampal theta is thought to be generated by the interaction of excitatory principal cells (granule cells and pyramidal cells) and inhibitory interneurons [-aminobutyric acid (GABA)-ergic cells] within these hippocampal regions (Vertes and Kocsis, 1997). Neuronal input from the neocortex (via the entorhinal cortex and hippocampal commissural and association projections) and from polysynaptic pathways originating in cholinergic and monoaminergic cell populations in the brainstem reticular formation is

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important to the initiation of hippocampal theta (Vertes and Kocsis, 1997). Populations of GABA-ergic and cholinergic rhythmic bursting cells in the medial septum and diagonal band (MS/DB) nuclei project to and ‘‘pace’’ activity of the hippocampal cells that generate the theta rhythm. Because the cholinergic rhythmic bursting cells in the MS/DB are excited by an ascending pathway from the brainstem reticular formation, the MS/DB comprises an important link in this ascending inﬂuence from the reticular formation (see Vertes and Kocsis, 1997, for a review). The hippocampal synchronized (theta) activity co-occurs with neocortical desynchronized (low voltage, fast activity) EEG activity during paradoxical sleep (PS; characterized by rapid eye movement and an ‘‘arousal’’ EEG pattern) and during the performance of certain behaviors during the waking state (Vertes and Kocsis, 1997; Winson, 1990). The dual hippocampal synchronized theta/neocortical desynchronized pattern has been suggested as a signal of active involvement of the neocortex and hippocampus in the psychological and behavioral processing occurring at the time (Vanderwolf, 1971; Vertes and Kocsis, 1997). Vanderwolf (1971) studied the relation between hippocampal RSA and motor behavior (movement and postures). Other researchers have attempted to relate hippocampal theta activity to psychological processes such as cognitive mapping of the spatial surround (O’Keefe and Nadel, 1978), arousal/attention (Berry and Swain, 1989), and memory storage (e.g., Vertes and Kocsis, 1997; Winson, 1985, 1990). Presumably its role in memory storage is initiated during waking time when the animal is actively responsive to the stimulus environment (the learning experience), and is reestablished during REM sleep, during which time the learning experience undergoes further consolidation, and is perhaps integrated with other relevant memories. See Winson (1985) for an example of a comprehensive proposal concerning the functional role of hippocampal theta activity in memory storage. Evidence supporting its role during PS in memory processing is summarized below.

B. VP, OT, Hippocampal Theta Activity, and Memory Consolidation during Paradoxical Sleep 1. Hippocampal Theta Activity during Paradoxical Sleep: A Neural Correlate of Memory Consolidation? Relevant to this discussion is the proposal that the hippocampal theta rhythm during PS is a neural correlate of memory consolidation (Vertes and Kocsis, 1997; Winson, 1990). There is direct experimental support for a relationship between PS and memory storage (Winson, 1990) and between theta and neuronal events underlying memory consolidation (Vertes, 1986). Three lines of evidence from animal and/or human research support a signiﬁcant relationship between PS and memory storage: (1) time spent

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in PS is increased after periods of intense learning (Smith and Rose, 1997); (2) selective deprivation of PS after a learning experience interferes with its subsequent long-term retention in both laboratory animals (e.g., Fishbein, 1971; Leconte et al., 1974) and humans (Empson and Clarke, 1970); and (3) the time spent in PS during a learning task is positively correlated with task acquisition and/or retention (Portel-Cortes et al., 1989). Several lines of research, using rats or rabbits as subjects, support the view that theta activity is a neural correlate for memory consolidation (for reviews see Vertes, 1986; Vertes and Kocsis, 1997). One line of evidence shows that removal of hippocampal theta (e.g., lesioning the medial septum) produces severe memory deﬁcits (Winson, 1978, and other studies cited in Vertes, 1986). A second line of evidence relates hippocampal theta and longterm potentiation (studies cited in Vertes, 1986; Vertes and Kocsis, 1997). Speciﬁcally, these studies have demonstrated that (1) electrophysiological stimuli mimicking the theta activity pattern (e.g., its temporal and frequency patterns) are optimal for induction of long-term potentiation (LTP) (Aria and Lynch, 1992; Larson et al., 1986) and (2) stimulation delivered only in the presence of hippocampal theta induces LTP, which is most pronounced when the stimulation is delivered in the positive phase of the theta rhythm (Pavlides et al., 1988). 2. Influence of VP and OT on Hippocampal Theta Activity during Paradoxical Sleep: Relevance for Memory Processing The studies reviewed below demonstrate that endogenous AVP and OT inﬂuence hippocampal theta activity during PS, and relate this inﬂuence to the role of the peptide in memory processing. a. Selected Studies i. Urban and De Wied (1975) Urban and De Wied (1975) compared Brattleboro homozygous diabetes insipidus (HODI) and heterozygous diabetes insipidus (HEDI) rats in terms of their hippocampal theta activity associated with PS. Because HODI rats are inferior to HEDI rats in memory consolidation (De Wied et al., 1975; see Chapter 3), it was predicted that if VP contributes to the functional status of midbrain/limbic activity related to hippocampal theta rhythm (Green and Arduini, 1954; Klemm, 1970), differences in theta activity between the two groups should occur. In this study two HODI and two HEDI male rats were chronically implanted with recording electrodes in the dorsal hippocampus for EEG recording, and in the neck muscles for electromyographic recording. Theta activity during PS was recorded in a sound-attenuating experimental cage during 5 habituation sessions followed by 10 experimental sessions of 3-h duration for each rat. During four of the experimental sessions each subject received physiological saline only; in the remaining sessions the rat was given desglycinamide-arginine vasopressin (DG-AVP; 1 and 2 g/100 g

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body weight, subcutaneous). The total PS time under each treatment was divided into 100-s analysis epochs. The frequency composition of theta activity, in the absence and presence of exogenous DG-AVP treatment, measured the functional changes of the neural network participating in theta rhythm generation during PS. A power spectral analysis was used to quantify brain electrical activity. Four parameters were calculated for each 100-s epoch to characterize the theta frequency spectrum: peak frequency (hertz), mean frequency (hertz), and spectrum width and frequencies at onehalf peak amplitude. To illustrate global difference in theta spectra for HODI and HEDI rats, the data in both groups were pooled and expressed as the mean percentage of power per hertz. The spectral parameters were similarly pooled with respect to animals or treatment and their means were statistically compared by Student t test. The observations provided by visual inspection of the EEG records are graphically presented in Figs. 7 and 8 and the results of the statistical analysis are summarized in Table II. The major difference in theta activity during PS between HODI and HEDI rats was conﬁned to the frequency band lying between 5.5 and 10 Hz. As Fig. 7 indicates, the width of the theta frequency band was similar for both groups of rats, but it was dominated by lower frequencies in HODI rats (i.e., the power–frequency curve of theta activity was shifted to the left for HODI rats). The results of the statistical analysis of the four theta spectrum parameters, summarized in Table II, bore out these results and expanded on the HODI/HEDI differences in hippocampal theta activity. As Table II indicates, there was no signiﬁcant difference between the two groups in the width of the theta spectrum band, but that of the HODI rats was characterized by a greater number of lower frequency components, and by a mean and peak frequency positioned at its lower end. The effect of DG-AVP on hippocampal theta during PS in HODI rats is

FIGURE 7 Differences in hippocampal rhythmic slow activity between heterozygous and homozygous, hereditary diabetes insipidus rats during paradoxical sleep. Source: Urban and De Wied, 1975 (Fig. 1, p. 363). Copyright ß 1975 by Elsevier Scientiﬁc Publishing Company.

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FIGURE 8 Effect of DG-AVP treatment on hippocampal rhythmic slow activity of homozygous diabetes insipidus rats during paradoxical sleep. Source: Urban and De Wied, 1975 (Fig. 2, p. 364). Copyright ß 1975 by Elsevier Scientiﬁc Publishing Company.

TABLE II Differences in Spectral Parameters of Rhythmic Slow Activity between Heterozygous and Homozygous Diabetes Insipidus Rats during Paradoxical Sleepa

Mean frequency (Hz) Peak frequency (Hz) Spectrum width at one-half peak amplitude Frequencies at one-half peak amplitude

DI homozygous (X SD, N ¼ 32)

DI heterozygous (X SD, N ¼ 30)

p level

7.6 0.1 6.8 0.5 0.7 0.2

8.1 0.04 7.8 0.4 0.9 0.3

p < 0.001 p < 0.001 NS

6.4 0.4–7.1 þ 0.5

7.0 0.3–7.9 0.4

p < 0.001

Abbreviations: DI, diabetes insipidus; NS, not signiﬁcant. a Source: Urban and De Wied, 1975 (Table 1, p. 364). Copyright ß by Elsevier Scientiﬁc Publishing Company.

depicted graphically in Fig. 8. As can be seen, with the exception of peak frequency that remained signiﬁcantly lower, a single peripheral injection of this peptide dose dependently normalized their theta frequencies in the direction of the HEDI rats. Because DG-AVP has no endocrinological effect,

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the restored electrophysiological activity could not be attributed to the restoration of a disturbed sodium or potassium balance associated with abnormal water balance, and these disturbances remained uncorrected by the treatment. ii. Urban and De Wied (1978) Urban and De Wied (1978) continued their investigations of a putative role for vasopressin on the mechanism underlying hippocampal theta rhythm during paradoxical sleep (PS). The subjects were male rats of the Brattleboro strain, homozygous for DI (HODI), heterozygous for DI (HEDI), and homozygous normal (HONO) at the genetic site encoding VP synthesis. Both PS and the frequency composition of hippocampal theta during PS were initially monitored under nontreatment conditions and subsequently under selected peptide treatment conditions. During treatment sessions, a peptide or placebo (saline) was randomly injected with two subsequent peptide treatments separated by a 48-h interval; each peptide was repeated at least four times per rat, with each rat serving as its own control. HODI and HEDI rats were tested in separate experiments with each of the following peptides: HODI rats received DGAVP (0.02 and 0.6 g/rat, intracerebroventricular) and ACTH(4–10) (0.06 g/rat, intracerebroventricular), and HEDI rats received DG-AVP (0.02 and 0.04 g/rat, intracerebroventricular), ACTH(4–10) (0.06 g/rat, intracerebroventricular), and [d-Phe7]ACTH(4–10) (0.06 g/rat, intracerebroventricular). HONO rats were tested under AVP antiserum (1 l, intracerebroventricular) and normal rabbit serum (1 l, intracerebroventricular) treatment conditions. The results with untreated rats indicated that (1) compared with HONO rats, HODI rats spent signiﬁcantly less time in slow wave sleep (SWS) and more time in a waking state, probably associated with the markedly increased water intake of these subjects. However, HONO and HODI rats did not signiﬁcantly differ in the average percentage of PS exhibited over the 8-h recording session, nor did any of the peptide treatments inﬂuence this measure; (2) visual inspection of the raw hippocampal records indicated that the rate of HODI theta synchronization was not as high as that of HEDI or HONO rats (Fig. 9); and (3) the power spectral analysis indicated that while the widths of the theta frequency band [4–10 cycles per second (cps)] were comparable for all three groups, HODI rats exhibited signiﬁcantly lower mean and peak frequencies in this band than did HEDI rats, and these differences were even more marked in comparisons with HONO rats. Results of the drug treatments were as follows: (1) either DG-AVP or ACTH(4–10) temporarily normalized the frequency composition of theta activity in the HODI rats, although three times more ACTH(4–10) was needed for equal effectiveness. A slight acceleration of theta frequencies began in the ﬁrst hour, peaked between 1 and 2 h, gradually decreased over several hours, and returned to pretreatment levels 24 h after injection;

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FIGURE 9 A segment of theta activity during paradoxical sleep in HEDI and HODI rats. HID, dorsal hippocampus; PTH, posterior thalamus; EMG, electromyogram. Source: Urban and De Wied, 1978 (Fig. 1, p. 53). Copyright ß 1978 by Ankho International Inc.

(2) ACTH(4–10) mildly increased the amount of the high-frequency component of the theta rhythm, whereas [d-Phe7]ACTH(4–10) suppressed this component and stimulated the appearance of low frequencies in HEDI rats, making the theta activity pattern similar to that observed in HODI rats. DG-AVP (0.02- and 0.06-g dose levels) accelerated theta activity in HEDI rats as it did in HODI rats; (3) HONO rats given VP antiserum exhibited a hippocampal theta frequency pattern equivalent to that of HODI rats. This effect began within 60 min, persisted 4 h or more, and had returned to the pretreatment level 24 h after injection; and (4) none of the peptides signiﬁcantly affected the amount of PS observed over the 8-h recording sessions. The following points were made in the discussion. First, it was suggested that vasopressin normally contributes to the production of hippocampal theta during paradoxical sleep. This was based on the marked decrease in theta frequency, but not in other EEG frequencies, observed during PS in rats severely deﬁcient in VP (HODI rats and VP antiserum-treated HONO rats), and on the ability of DG-AVP to normalize theta activity when injected subcutaneously or intracerebroventricularly in amounts that restore memory deﬁcits in these animals (De Wied et al., 1975; Urban and De Wied, 1975).

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Second, it was suggested that the contribution of DG-AVP and related peptides and ACTH(4–10) to hippocampal theta activity may occur by way of their ability to enhance neural activity within the mesodiencephalic limbic structures [cell group(s) in the midbrain reticular formation and ascending polysynaptic pathway(s)] that are important for the induction of the theta rhythm (see Section IV.A). Indirect support for this suggestion comes from the present ﬁndings and earlier studies showing that (1) ACTH(4–10) enhanced the high-frequency components of theta activity induced by electric stimulation of the midbrain reticular formation in freely moving rats (Urban and De Wied, 1976), and the effect of the peptide on this activity was mimicked by increasing stimulus intensity, which presumably activated more reticular neurons engaged in theta generation; and (2) both the mean and peak frequency of theta activity is positively correlated with neural activity within mesodiencephalic limbic structures (Klemm, 1970; Paiva et al., 1976). Given the observations noted in ﬁndings 1 and 2, it is possible that the peptide-induced alterations in the theta activity frequency pattern observed in this study reﬂected an increase [induced by ACTH(4–10) or DGAVP] or decrease (induced by [d-Phe7]ACTH(4–10) or AVP antiserum) in excitability of the midbrain limbic network. Third, the authors related the action of these neuropeptides on hippocampal theta activity to their roles in memory processing. They noted that the altered excitability of the midbrain limbic structures (measured by changes in frequency content of the hippocampal theta pattern) induced by these neuropeptides is correlated with their effects on retention in avoidance paradigms. Thus, ACTH(4–10) and DG-AVP, which enhanced this excitability, also improved retention (increased resistance to extinction) in active and passive avoidance tasks (Bohus et al., 1973; De Wied et al., 1975; see Chapter 3). In contrast, [d-Phe7]ACTH(4–10) or AVP antiserum, which depressed this excitability, impaired retention (facilitated extinction) of conditioned avoidance behavior (De Wied, 1969; Van Wimersma Greidanus et al., 1975a; see Chapter 3). iii. Bohus et al. (1978b) Bohus et al. (1978b; see Chapter 2) subsequently tested the effects of OT and OT antiserum on hippocampal theta rhythm during PS in male Wistar rats, as part of a larger study designed to compare the central effects of VP and OT (see Chapter 2). Their electrophysiological experiments focused on the inﬂuence of OT on this EEG activity because earlier studies had reported on the nature of the inﬂuence of AVP (De Wied et al., 1975; Urban and De Wied, 1975). OT (5 or 20 ng/rat, intracerebroventricular) was given to three of the ﬁve rats and anti-OT serum (1 l, undiluted, intracerebroventricular) was given to the remaining two subjects. Physiological saline and normal rabbit serum served as placebos for these experiments. Each treatment was replicated three times with a minimal interval of 48 h between any two

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consecutive injections. The power spectral analysis was used to quantify brain electrical activity as in Urban and De Wied (1978). Both doses of OT signiﬁcantly decreased the percentage of the higher, and increased the percentage of the lower, frequencies in the hippocampal theta frequency band. OT antiserum produced the reverse pattern. Neither OT nor OT antiserum affected the total amount of paradoxical sleep during the 8-h observation periods. The results of these electrophysiological experiments with OT, together with those from comparable studies on VP (De Wied et al., 1975; Urban and De Wied, 1975, 1978), indicated that the two peptides exert opposite effects on theta activity, with OT depressing and VP enhancing the high-frequency end of the hippocampal theta spectrum during PS episodes. Opponent actions of these neuropeptides on retention in active and passive avoidance paradigms have also been observed with OT impairing, and VP facilitating, retention in active and passive avoidance tasks (Bohus et al., 1978b). Previous discussion in this chapter (Section IV.B.1.) summarized supporting evidence that hippocampal theta during PS is an important neural correlate of memory consolidation. The ﬁnding that centrally administered OT, which induces an amnestic action in avoidance conditioning tests (Bohus et al., 1978b), depressed the high-frequency end of the hippocampal theta spectrum during PS episodes suggests that low-frequency activity in this band correlates with impaired retention. This suggestion is supported by reports of both impaired retention (Bohus et al., 1978b; De Wied et al., 1975) and a greater than normal representation of low-frequency relative to high-frequency components in the hippocampal theta band in rats experimentally depleted of central AVP by intracerebroventricularly injected VP antiserum, or in Brattleboro HODI rats (Urban and De Wied, 1975). The co-occurrence of amnestic and theta frequency-depressing actions by OT, and memory storage-facilitating and theta frequency-enhancing actions by VP, is consistent with the suggestion that these neuropeptides inﬂuence memory storage, at least in part, by a modulating effect on the theta-generating system itself, or on the brain state reﬂected in this electrophysiological activity. iv. Urban (1981) Urban (1981) tested the effects of AVP and OT on the function of the septum, the pacemaker for hippocampal theta rhythm. AVP, OT, or saline was microinjected into the septum of male Wistar rats during slow wave sleep (SWS) or paradoxical sleep (PS). The results indicated that (1) intraseptal injections of AVP and OT had no effect on the percentage of time the rats spent in either PS or SWS; (2) intraseptal injections of these peptides altered the hippocampal EEG during PS in similar fashion; the alterations were conﬁned to frequencies of the theta band (4.1 to 12.0 Hz) and consisted of an increase in peak and mean frequencies relative to saline controls; (3) the hippocampal EEG during SWS was not

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signiﬁcantly affected by these peptides; and (4) microinjections of AVP in the same amounts into the frontal cortex or into the subicular region of the dorsal hippocampus had little effect on hippocampal activity during either SWS or PS. The ﬁnding that intraseptally injected OT increased hippocampal theta frequencies is in sharp contrast to the slowing of this frequency observed for an intracerebroventricular injection of the peptide (Bohus et al., 1978b). This difference in results is paralleled by differences in the effects of OT on PA behavior, so that intracerebroventricularly administered OT attenuates PA retention, whereas intraseptally injected OT mimicks the VP facilitation of PA retention (Kovacs et al., 1979a; see Chapter 4). Electron microscopic studies with speciﬁc VP and OT antisera suggest that the material within the septal ﬁbers and their synaptoid terminals is almost exclusively AVP (Buijs and Swaab, 1979). It is therefore possible that OT metabolites that mimic VP activity [e.g., prolyl-leucyl-glycinamide (PLG)] are responsible for the similar behavioral and EEG effects of intraseptally administered OT. Taken together, these observations were interpreted as suggesting that VP-ergic innervation of the septum is one inﬂuence on the regulation of neural processes related to hippocampal theta rhythm generation.

V. Effects of VP and OT on Neuronal Activities in the Septal–Hippocampal System, and Memory Processing

___________________

A. Introductory Comments: Electrophysiological Study of the Neurotransmitter and Neuromodulatory Activities of VP and OT Beginning in the mid and latter part of the 1970s, VP- and OT-containing neurons and ﬁber pathways were localized in extrahypothalamic brain sites (structures outside the hypothalamic–neurohypophysial system). Immunocytochemical, electron microscopic, and electrophysiological methodologies were employed to determine whether these peptides participated in central neuronal as well as peripheral hormonal signaling activity (see Chapter 1; also Buijs, 1983, for review of earlier research in this area). Joels, Urban, De Wied, and other colleagues have used electrophysiological technology with in vivo and in vitro paradigms to investigate the putative neurotransmitter–neuromodulator functions of VP and OT. For in vivo study the animal is anesthetized and prepared by removing the tissue over the brain surface selected for study. Iontophoretic technology permits VP or OT to be applied to a single cell, or numerous cells, in the exposed brain site; the test peptide is ejected from the micropipette by an electric current, the strength of which controls the amount of peptide applied to the target structure. This technique simulates the release of VP from

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the presynaptic terminal. In vitro studies, carried out with brain slice preparations from the septal–hippocampal system, add VP or OT to the tissue solution that bathes the preparation. This procedure has also been used to examine the role of VP in long-term potentiation in septal neurons after tetanic stimulation of ﬁmbria–fornix ﬁbers terminating on these neurons. A number of studies described below have examined the effects of iontophoretically administered VP or OT on spontaneous and elicited activity in septal or hippocampal neurons. In these studies, evidence of VP- or OT-induced neurotransmitter action is indicated by an immediately appearing but short-lasting response [i.e., increased alteration (excitatory response) or decreased alteration (inhibitory response) in spontaneous activity of the neuron]. In contrast, neuromodulatory action is indicated by long-lasting change in its responsivity to its synaptic inputs (i.e., change in its elicited activity). The septal–hippocampal system has been the focal point of study in this research because (1) anatomical and electrophysiological evidence has suggested a neurotransmitter role for VP in the septal area (Buijs, 1982; Buijs and Swaab, 1979), and for VP and OT in the hippocampus (Buijs et al., 1980; Muhlethaler and Dreifuss, 1982); and (2) both limbic structures have been implicated in the memory-modulating effects of these peptides (see Chapter 4). For further discussion see Joels (1987) and Urban (1987). 1. Selected Studies a. Joels and Urban (1982) Joels and Urban (1982) investigated putative neurotransmitter and neuromodulatory actions of VP and OT on single cells in the lateral septal complex (LSC) and dorsal hippocampus (DH). Speciﬁcally, neurotransmitter actions of the peptides were indicated by their effects on spontaneous activity of the cell, and neuromodulator actions were indicated by their effects on response of the cell to glutamate-evoked excitation. Glutamate (Glu), a major excitatory neurotransmitter at many CNS synaptic sites, was applied by microiontophoresis, as were VP and OT. Given the similar effects of septally injected VP and OT on avoidance retention (Kovacs et al., 1979a; see Chapter 4) and on hippocampal theta activity during paradoxical sleep (Urban, 1981, described above), it was also of interest to test for similarity in their neurotransmitter and neuromodulator actions on these septal and hippocampal cells. Anesthetized male Wistar rat subjects were prepared for study by removal of neocortex and corpus callosum overlying the LSC and DH. Extracellular recordings monitored spontaneous activity of the cell as well as its evoked activity. In the LSC, lateral septal (LS) and medial septal (MS) neurons were differentiated on the basis of their responses to stimulation of the ﬁmbria–fornix (ﬁ–fx) pathway (interconnects the hippocampus and septal area). LS cells, which receive hippocampal input, responded with short-latency orthodromic responses, whereas MS cells, which innervate the hippocampus, were identiﬁed by antidromic invasions observed in

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repeated collision tests. Neurotransmitter effects of the iontophoretically applied peptides and Glu were indicated by changes in extracellularly recorded spontaneous activity (increase or decrease in number of action potentials per unit time, and/or in spike amplitude) in these LSC and DH neurons. AVP was applied to 50 septal and 27 hippocampal neurons and OT was applied to 45 septal and 28 hippocampal neurons. The major results of these tests were as follows: (1) during iontophoretic application of AVP, 48% of tested LSC neurons and 67% of tested DH neurons were excited (responded with at least a 50% increase in spontaneous activity); the remaining neurons either failed to show an excitatory response (spontaneous activity increased by less than 50%) or remained unchanged. There was no evidence of an inhibitory response (i.e., a reduction in baseline level of spontaneous activity); (2) OT application produced an excitatory effect in 58% of tested LSC cells, and in 61% of tested DH cells. The remaining neurons were unaffected; (3) the response characteristics in more than 90% of the neurons that were excited by the applied VP and OT (rapid response onset at the beginning of peptide application and rapid offset at the end of administration) were similar to those observed with Glu. However, unlike VP- and OT-induced responses, application of Glu frequently resulted in an inhibitory effect (i.e., a decrease in spike amplitude and in the ﬁring rate of target neurons). An example of the excitatory effect of Glu and AVP on the spontaneous activity of a LSC neuron is given in Fig. 10; and (4) there was a

FIGURE 10 The effect of iontophoretic application of glutamate (GLU, 20 nA), vasopressin (AVP, 100 nA), and 165 ml of NaCl (SAL, 100 nA) on the spontaneous activity of a lateral septal neuron. Horizontal bars show the duration of administration. The ordinate represents the number of spikes per second. Inset: Three superimposed oscilloscope sweeps displaying the orthodromic response of the neurons to stimulation of ﬁmbria–fornix ﬁbers. Source: Joels and Urban, 1982 (Fig. 1, p. 81). Copyright ß 1982 by Elsevier Scientiﬁc Publishers Ireland Ltd.

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difference between orthodromically driven LS cells and antidromically driven MS cells in neuronal responsivity to VP and OT. In contrast to LS cells, relatively few MS neurons were excited by VP (3 of 18 tested cells) or OT (6 of 23 tested cells). The neuromodulatory activity of VP and OT was tested by determining their ability to inﬂuence responsivity to Glu application, especially in those cells that failed to show a rapidly reversible excitatory response to VP and OT. The results indicated that (1) low levels of iontophoretically released AVP (i.e., AVP released with low ejecting currents) enhanced excitatory responsivity to Glu by at least 50% in 10 of 18 tested LSC neurons and 7 of 11 tested DH neurons, and low levels of OT enhanced this evoked activity in 8 of 10 tested LSC neurons and 4 of 5 tested DH neurons (see Fig. 11); (2) the responses of LSC and DH neurons to Glu was increased by VP and OT application in neurons that were not excited by the peptides when administered alone; this indicated that the response enhancement was due to a neuromodulatory effect of VP and OT and was not the result of a mere summation of excitatory effects of the peptide and the amino acid; and (3) as in the case of neurotransmitter action, neuromodulatory effects of VP and OT on MS cells were seldom observed. The authors made the following points about these neuropeptides and the septal–hippocampal system: (1) the short latency in onset and offset of these neuropeptide-elicited excitatory responses suggested a neurotransmitter action, although the mechanism responsible for the short latency offset was not clariﬁed by these experiments; (2) the AVP-induced excitation of LSC and DH neurons observed in this study was consistent with reports by others that iontophoretically applied AVP excites LSC

FIGURE 11 Left: The effect of vasopressin (AVP, 100 nA) on the responses of a hippocampal neuron to glutamate (GLU, 35 nA). Right: The effect of oxytocin (OXT, 100 nA) on the glutamate-induced excitation (GLU, 25 nA) in a lateral septal neuron. Source: Joels and Urban, 1982 (Fig. 2, p. 82). Copyright ß 1982 by Elsevier Scientiﬁc Publishers Ireland Ltd.

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neurons (Huwyler and Felix, 1980), hippocampal pyramidal neurons (Tiberiis et al., 1982), and neurons in the locus coeruleus (Olpe and Baltzer, 1981) (neurotransmitter action); (3) the similarity between the VP and OT inﬂuences on neuronal activity in LSC cells observed in this study was in accord with earlier reports that, when injected into the LSC, these peptides exert similar effects on avoidance behavior (Kovacs et al., 1979a; see Chapter 4) and hippocampal theta rhythm during PS (Urban, 1981; see Section IV.B.2.a.iv); and (4) the ability of VP and OT to enhance responsivity of Glu-induced activity in both the LSC and DH, especially in cells that failed to respond to these peptides when applied on their own at the same ejecting current, strongly suggests a neuromodulatory action of these peptides. b. Joels and Urban (1984a) Joels and Urban (1984a) carried out singlecell and ﬁeld potential experiments in a continued effort to explore the neuromodulatory inﬂuence of AVP on neuronal activity in the LSC. The tests were performed on anesthetized male Wistar rats prepared for brain stimulation and recorded after surgical removal of the cranium, neocortex, and corpus callosum overlying the septal complex. The single-cell experiments examined the ability of AVP to inﬂuence LSC neuronal responsivity to electrical stimulation of the ﬁ–fx pathway (contains the hippocampal ﬁbers that synapse on LSC neurons), and to iontophoretically applied glutamate (Glu), an excitatory amino acid neurotransmitter that may mediate this synaptic input (Walaas and Fonnum, 1980). The speciﬁcity of the inﬂuence of AVP on Glu-evoked activity was examined by testing the effect of the peptide on responsivity to other excitatory [aspartate (Asp), quisqualate (Quis), and N-methyl-d-aspartate (NMDA)], and inhibitory (GABA), amino acids reported to inﬂuence this neuronal activity (Joels and Urban, 1984b; Segal, 1974). The orthodromically driven LS cells selected for the ﬁ–fx stimulation tests were those that consistently responded to ﬁ–fx stimulation, exhibited an action potential at a stable latency (about 5 ms), and were able to follow stimulus frequencies up to 50 to 150 Hz. The test for the effect of AVP on these synaptic-induced excitatory responses included the delivery of 32 stimuli at threshold stimulus intensity before, during, and after iontophoretic application of AVP. The expelling current of AVP was increased stepwise until AVP evoked a change that did not exceed an increase in synaptic activity by more than 50% (maximal expelling current for AVP was 50 nA, delivered through two micropipette barrels simultaneously). Neurons selected for the amino acid tests were those that evidenced no change in spontaneous activity to AVP application alone. The amino acid was applied at regular intervals with ﬁxed current intensity until the response stabilized, after which the response evoked by the amino acid was tested with simultaneously applied AVP. The AVP expelling current was

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increased in a series of tests until the peptide produced a criterion effect (a 50% change in the response of the cell to the amino acid). Using this effective current, the researchers attempted to record two test runs of at least three responses to a given amino acid (i.e., before, during, and after delivery of AVP). The effect of AVP on excitatory amino acid-induced responses was studied in 89 test units using an AVP-expelling current ranging from 10 to 100 nA (with an average current of 41.1 nA). The results were as follows: (1) 30% of the tested LSC neurons were excited by iontophoretic release of high levels of AVP (i.e., AVP applied with high currents of 100–150 nA), and these neurons were excluded from further study; (2) iontophoretically applied AVP (ejecting current of 20 nA for 1-min duration) increased the LSC response rate (number of action potentials per second) to ﬁ–fx stimulation; (3) AVP increased the responses to Glu by 50% or more in approximately 75% of the neurons tested, and this effect was replicated in repeated tests for all but seven of the responsive neurons; (4) once AVP stimulation was terminated, the enhanced responsivity to Glu promptly returned to the pretreatment level in 60% of the responsive neurons, but remained elevated for up to 15 min (maximal testing interval) in the remaining 40%; (5) AVP enhanced the excitations in LSC neurons elicited by Asp, Quis, and NMDA as it had for Glu. Thus, in the majority of tested LS neurons, responses to each of the excitatory amino acids increased markedly during concomitant application of AVP, and about 50% of these neurons showed responses that remained elevated for 3–15 min after termination of AVP application; (6) GABA, iontophoretically administered to 34 LS cells with low (5–25 nA) currents, readily depressed spontaneous activity in all tested neurons. The ability of concomitantly administered bicuculline (GABA antagonist) to reverse the GABA-induced inhibition presumably indicated that the inhibition was mediated by a GABA-sensitive receptor on LSC cells; (7) AVP did not alter the GABA-evoked inhibitory responses in any of the 12 neurons tested, although in all of these AVP did enhance excitatory responses to Glu; and (8) the effect of AVP on transsynaptic excitations was studied in 18 orthodromically activated LS neurons. In 12 of these, iontophoretic administration of AVP with currents that increased responses to Glu led to an increase of at least 50% in the number of action potentials elicited by 32 ﬁ–fx stimuli. In the ﬁeld potential experiments, a comb consisting of four pairs of stimulating electrodes was inserted into the ﬁ–fx pathway. Before testing, the pair of electrodes that produced the largest ﬁeld potentials (FPs) was selected for testing, and stimulus intensity was adjusted to elicit an FP representing half the maximal amplitude. The results of these experiments indicated that (1) the FPs elicited by ﬁ–fx stimuli were each initially composed of a narrow negative wave of short duration that was then followed by a shorter, broader positive wave; the average latency to the peak of the negative wave did not differ signiﬁcantly from the mean latency of single LS cell action potentials

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in response to the same ﬁ–fx stimuli. These observations corroborate ﬁndings by De France et al. (1973a,b). They support the thesis that the FP negative wave represents the sum of excitatory, and the positive wave the sum of inhibitory, postsynaptic activations synchronously triggered in many LS cells by ﬁ–fx stimuli, the inhibitory activations being mediated by a negative feedback loop activated by excitation of the LS cells (De France et al., 1975); (2) the FP negative wave decreased during the course of the experiment (i.e., the negative wave recorded 3 h after the start of the experiment was on average 80% of the amplitude evoked by the initial series of stimuli). However, topical administration of AVP prevented this timerelated decrease in amplitude and increased it instead; (3) the positive wave of the FP did not show any consistent alteration in the presence of topically applied AVP; and (4) superfusion of the LS with Glu and GABA altered the negative and positive waves of the FP response to ﬁ–fx stimulation, with Glu decreasing both the negative and positive waves, and GABA producing a marked decrease of the positive wave and a small increase in the negative wave. Taken together, the results stated in ﬁndings 2, 3, and 4 indicate that topically applied AVP did not directly inﬂuence cell membrane conductivity the way it did for Glu or GABA, because the inﬂuence of AVP on the positive and negative waves of the FP response to ﬁ–fx stimulation differed from that induced by these excitatory and inhibitory amino acid transmitters. The ﬁndings of this study together with those of Joels and Urban (1982) support the idea of a neuromodulatory action of VP in the septal– hippocampal system. The authors made the following points: (1) the present ﬁndings, together with results obtained from biochemical (Walaas and Fonnum, 1980) and electrophysiological (Joels and Urban, 1984b,c) studies, support the interpretation that the hippocampus-originating afferents in the ﬁ–fx pathway to the LS contain Glu or a closely related excitatory amino acid (EAA), and that AVP normally modulates (increases) septal responsivity to this input; (2) the failure of AVP to inﬂuence the inhibitory response induced by GABA-ergic input to these cells also argues in favor of an AVPinduced modulation of neuronal responsivity to EAA input and against the interpretation that this observation could be attributed to a nonspeciﬁc effect on the postsynaptic membrane per se; and (3) the AVP-induced inﬂuence on the FP is also in accord with a proposed VP-induced modulation of LS neuronal responsivity to EAA transmitter input from the hippocampus. Thus, unlike Glu and GABA, topical application of AVP gave no evidence of direct alteration of cell membrane conductivity in producing its effect on the FP response to ﬁ–fx transsynaptic input. c. Joels and Urban (1985) Joels and Urban (1985) examined the inﬂuence of monoamines on spontaneously active LSC cells, and the ability of subsequently administered AVP to alter the monoaminergically induced effects. The monoamines tested, noradrenaline (NA), dopamine (DA), and

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serotonin (5HT), are normally found in the pathways projecting to the septum from cell groups originating in various levels of the brainstem. The major stimulus for this study was the evidence supporting a putative transmitter role for monoaminergic projections to the LSC (Biegon et al., 1982; Kohler et al., 1982; Lindvall and Stenevi, 1978), a putative VP modulation of NA-ergic neurotransmission in the LSC during memory processing (Kovacs et al., 1979a), and an AVP-induced modulation of cellular responsivity to EAAs in this brain site (Joels and Urban, 1984a). Extracellular recordings of LSC neuronal activity were carried out in anesthetized male Wistar rats, prepared for iontophoretic application of AVP and the test transmitters (Glu, NA, DA, and 5HT) as described in Joels and Urban (1984a). These LSC neurons were initially tested for their responsiveness to orthodromic stimulation of the ﬁ–fx pathway; those exhibiting an action potential within 5 ms and able to follow stimulus frequencies of 25–150 Hz were classiﬁed as responders. The monoamines were tested on all LSC neurons exhibiting spontaneous ﬁring rates of at least three spikes per second. The action of AVP alone on spontaneous activity was examined before testing its ability to inﬂuence the response of a cell to Glu or to the monoamines. Neurons that did not change their spontaneous activity in response to AVP, applied with ejecting currents of 200 nA or less, were selected to assess the effect of the peptide on neuronal responses to the test transmitters. These test agents were iontophoretically applied for 15–20 s at ﬁxed intervals (either 30, 60, or 90 s) and at ﬁxed current intensities. An excitatory/inhibitory response to the test transmitter was deﬁned as an increase/decrease in the rate of spontaneous activity, by at least 50%, in the tested neuron during iontophoretic application of the transmitter. The results indicated that (1) the majority of tested LSC cells were inhibited in the presence or NA, DA, or 5HT. Only 30% of tested LS neurons failed to be inﬂuenced by monoamine application; (2) at submaximal ejecting currents the monoamine-inhibitory effect, especially for 5HT and DA, had a latency of 5–10 s and lasted for 10–15 s after stimulus termination; (3) monoamine-responsive LSC neurons did not differ from nonresponsive neurons with respect to characteristics of their spontaneous activity (action potential amplitude and waveform) or percentage responding to ﬁ–fx stimulation (63 and 64%, respectively); (4) the two types of cells did differ in their localization within the LSC, that is, responsive cells were mainly in those parts of the LSC that receive ascending monoaminergic input (Kohler et al., 1982; Lindvall and Stenevi, 1978); (5) in the majority of tested neurons, AVP did not inﬂuence spontaneous discharge rate inhibition exerted by the monoamines or by the inhibitory amino acid GABA. However, in a minority of neurons, AVP ejected with moderately lowamplitude (50 nA) currents attenuated the inhibition induced by NA, DA, and 5HT; and (6) Glu-induced excitatory responses were markedly increased during AVP iontophoresis with low AVP-ejecting currents

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(20–50 nA), and this lasted for at least 5 min after ending AVP application in about half the responding cells. In the discussion the authors made the following points: (1) the speciﬁc location of LSC neurons that responded to the exogenous monoamines suggests that they are the normal recipients of ascending monoaminergic input, and their lower spontaneous activity may further indicate that they are tonically inhibited by this input; (2) the primarily inhibitory effect of the monoamines on lateral septal cells conforms with reports on this and other sites in the brain and spinal cord (e.g., Biscoe and Straughan, 1966; Krnjevic and Phillis, 1963; Phillis et al., 1968; Segal, 1975; Stefanis, 1964). However, occasional excitatory effects have also been reported (Jones, 1982; Kadzielawa, 1983; Parry and Roberts, 1980; Stefanis, 1964). Moreover, neurons that under light (urethane) anesthesia show no spontaneous activity and that prevail in the LS were not studied in this investigation, and may be affected differently by their monoamine inputs; (3) the failure of AVP to inﬂuence the inhibitory effects elicited by NA, DA, and 5HT and also by GABA, using ejecting currents in the range that was effective for inﬂuencing responses to Glu and other EAAs (e.g., Joels and Urban, 1984a), suggests that the neuromodulatory action of AVP on these cells was speciﬁc to EAA transmitter input from the hippocampus to the LS cells; and (4) the ﬁnding that AVP attenuated monoamine-induced inhibition in some neurons could not be attributed to a direct excitatory action of AVP, because spontaneous activity in these neurons was not elevated during AVP application with currents of up to 200 nA. This ﬁnding does provide some support for the proposal that AVP modulates neuronal responses to NA-ergic input to this brain site (Tanaka et al., 1977a). Nevertheless, according to the present results, the inﬂuence of AVP on EAA neurotransmission in the LSC appears to be far more prominent than its effect on monoaminergic transmission. d. Urban and De Wied (1986) Urban and De Wied (1986) examined the effect of VP and OT and their behaviorally active derivatives on ﬁeld potential (FP) responses to electrical stimulation of the ﬁ–fx pathway. The presence of synapses that can release AVP in the LSC (Buijs, 1983, 1985; Buijs and Van Heerikhuize, 1982; Landgraf et al., 1988), together with evidence that AVP released into brain synapses is biotransformed into highly active oligopeptides (Burbach et al., 1983a), suggest the possibility that these derivatives may mediate the AVP-induced modulation on EAA neurotransmission observed in the LSC (Joels and Urban, 1982, 1984a). The rationale for including OT and its derivatives in this study was not given by these researchers. However, it may have been due, in part, to earlier ﬁndings that intraseptal administration of OT and certain of its derivatives (e.g., PLG) has effects on avoidance behavior and EEG activity, similar to those observed for AVP (Kovacs et al., 1979a; Urban, 1981). The FP was used as a measure of the effects of the peptides on septal neuronal responsivity to stimulation of

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the ﬁmbria–fornix (ﬁ–fx) pathway. The VP and OT metabolic derivatives tested in this study were as follows: [pGlu4,Cyt6]VP(4–9), [pGlu4,Cyt6]VP(4–8), [Cyt6]VP(5–9), DG-AVP, 1-desamino-8-d-arginine vasopressin (DDAVP), [pGlu4,Cyt6]OT(4–9), and [pGlu4,Cyt6]OT(4–8). The results were as follows: (1) the FP evoked in the LS by ﬁ–fx stimuli consisted of a large negative wave, followed by a smaller positive wave; (2) the amplitude of the negative wave decreased over time in control rats [dorsal surface of the LS superfused with artiﬁcial cerebrospinal ﬂuid (ACSF)] but not in AVP-treated subjects. Instead, 15 min after topical application of AVP(1–9) an increase in this amplitude was initiated and continued, reaching a value 40% higher than that of control rats by 60 min after the initially observed FP; (3) AVP(1–9) affected neither the amplitude of the positive wave, nor the onset latency of the negative or the positive wave; (4) the AVP metabolites [pGlu4,Cyt6]AVP(4–9), [pGlu4,Cyt6]AVP(4–8), [Cyt6]AVP(5–9), DG-AVP, and DDAVP were much less potent than the parent peptide in their effect on the FP (i.e., required a concentration 1000 to 10,000 times greater than that used for the parent peptide to produce a comparable increase in amplitude of the negative wave); and (5) OT(1–9) increased the amplitude of the negative wave by about 35% and, at the one concentration level tested, the OT fragments [pGlu4,Cyt6]OT(4–8) and [pGlu4,Cyt6]OT(4–9) had no effect on this measure. The results were interpreted as follows: ﬁrst, the VP-induced increase in negative wave amplitude presumably represented the ability of the peptide to enhance the size and/or number of excitatory postsynaptic potentials and action potentials in LS neurons activated by ﬁ–fx input. Its lack of effect on positive waves presumably reﬂected its lack of inﬂuence on the inhibitory postsynaptic potentials produced by the inhibitory interneurons in the affected network (De France et al., 1973a,b). This interpretation is supported by previous ﬁndings showing that iontophoretically applied AVP increased the number of action potentials in LS cells responding to ﬁ–fx stimulation but failed to inﬂuence GABA induced inhibition (Joels and Urban, 1984a, cited earlier). Second, the ﬁnding that OT peptides produced effects similar to those produced by the VP peptides at equal potencies was considered to be of little physiological signiﬁcance given the few OT ﬁbers observed in the LS of rats (Buijs et al., 1978; Sofroniew, 1980). Third, the observation that the VP metabolites had to be at far higher concentrations than the parent peptide to produce similar effects renders it highly unlikely that these oligopeptides are responsible for the VP modulatory effect on EAA neurotransmission in this brain site. In addition, these ﬁndings suggest that the effect of VP on LSC neuronal responsivity to hippocampal input is not related to its role in avoidance retention. This is so because (1) intracerebroventricularly administered [pGlu4,Cyt6] VP(4–9), [pGlu4,Cyt6]VP(4–8) (Burbach et al., 1983b), and [Cyt6]VP(5–9)

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(Gaffori, personal communication cited by authors) are markedly more potent than the parent peptide in facilitating PA retention whereas the converse pattern of effects was observed in assessing the inﬂuence of the peptide on LS responsivity to EAA neurotransmission; and (2) DG-AVP (Kovacs et al., 1982) and DDAVP (Gaffori, personal communication cited by authors) have been shown to be comparable to the parent peptides in their effects on PA retention, but were 1000 to 10,000 times weaker than AVP(1–9) in their ability to increase the magnitude of the negative wave of the FP. Fourth, ﬁndings that the VP fragments had effects on neuronal responsivity only at high concentrations led to the speculation that ‘‘if the proposed N-terminus VP cleavage takes place in the LS, it presumably serves for termination of the VP action on LS neurons rather than for its initiation’’ (Urban and De Wied, 1986, p. 45). Although the authors cited evidence that aminopeptidase proteolysis does terminate some motor behavioral effects of intracerebroventricularly injected VP (Meisenberg and Simmons, 1984), it was noted that further study of the putative effects of other C-terminal VP fragments on LS responsivity to ﬁ–fx activation is clearly required. e. Van den Hooff et al. (1989) Van den Hooff et al. (1989) investigated a putative role for endogenous vasopressin in long-term potentiation (LTP) in the lateral septum. LTP refers to the long-lasting increase in neuronal responsiveness that has been observed in the hippocampus, lateral septum, and neocortex (Racine et al., 1983) after brief high-frequency stimulation of these neurons. It has been studied as a model of the type of increased synaptic efﬁcacy that may occur during memory formation, and glutamate is thought to be the transmitter involved in mediating LTP (Teyler and DiScenna, 1987). The possibility that the actions of VP on memory processes might stem from effects on neuronal processes such as LTP, as well as evidence that VP increased duration of LTP established in the hippocampal brain slice preparation (Chepkova and Skrebitski, 1982), were the major stimulants for this in vitro study. Brain slices composed of nearly the entire lateral septum (LS) and a long bundle of ﬁmbria ﬁbers were prepared from normal Wistar, normal LongEvans (LE), and Brattleboro HODI rats. Excitatory postsynaptic potentials (EPSPs) were recorded intracellularly, and ﬁeld potentials (FPs) were recorded extracellularly, before and after tetanic stimulation of the ﬁmbria ﬁbers innervating the LS neurons in these brain slice preparations. The same procedural protocol was used to test the presence of LTP in the intracellular recordings of EPSP and in the extracellular recordings of the ﬁeld potentials (FPs). The average amplitude of EPSPs, as well as the negative waves of the FPs before and after tetanic stimulation (a 2-s train of electrical stimulation of the ﬁmbria ﬁbers delivered at a frequency of 50 or 10 Hz), were calculated and expressed as a percentage of the amplitude before tetanic stimulation.

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An initial series of experiments tested for LTP in the EPSPs, and negative waves of the FPs, using brain slices from Wistar rats. The results were as follows: (1) mean amplitudes of EPSPs measured in six LS neurons were signiﬁcantly higher after tetanic stimulation of the ﬁmbrial pathway (2s train of 10-Hz pulses) than values obtained before stimulation, and this increased amplitude remained signiﬁcantly higher for 20 min after the offset of the tetanic stimulus; and (2) a signiﬁcant increase in the amplitude of the negative wave occurred in 9 of the 11 brain slices tested with a 2-s 50-Hz tetanic stimulus, and this increased amplitude persisted for 30 min after the tetanic stimulation. A similar negative wave increase after tetanic stimulation occurred in only 3 of 10 brain slices tested with a 2-s 10-Hz tetanic stimulus, indicating that the incidence of potentiation was markedly higher after a 50-Hz rather than a 10-Hz stimulus, and thus the former was used for all subsequent experiments. A second series of experiments studied the effect of tetanically stimulating the ﬁmbrial pathway (2-s 50-Hz electrical stimulation) on the FP negative wave in rat brain slices. Speciﬁcally, these experiments studied LTP induction and maintenance in the following brain slice preparations: (1) those obtained from nontreated HODI and LE rats; (2) those from HODI rats pretreated for 7 days with sufﬁcient AVP to normalize water metabolism, or slices receiving AVP that had been added to the superfusion medium 2 h before the recording period; (3) HODI slices that had been superperfused with the excitatory agent picrotoxin; (4) Wistar and LE slices to which the V1 antagonist d(CH2)5[Tyr(Me)]AVP had been added to the bath medium 30 min before tetanic stimulation and remained in the superfusion medium during the whole recording period; and (5) those from LE and HODI rats, tetanically stimulated 30 min after the NMDA antagonist D()-2-amino-5-phosphonovaleric acid (2-APV) had been added to the medium, and again at the end of a 60 min-long washout of the antagonist from the slices. The results of this series of experiments were as follows: (1) the brain slices of HODI rats showed normal induction of LTP to tetanic stimulation of ﬁmbria ﬁbers but failed to maintain it over an extended period of time. Although the posttetanic response amplitude of the negative wave was originally higher in the brain slices of HODI rats than in those of normal LE rats, this increase in response amplitude was not maintained during the posttetanic observation period, as it was for LE rats. Moreover, this failure in LTP maintenance in HODI rats was corrected by either superfusing the brain slices with AVP before the experiment or by implanting a device that released VP from subcutaneous implants over a 7-day period before sacriﬁce; (2) pretreatment of LS slices of HODI rats with the stimulant picrotoxin also corrected their failure to maintain their increased amplitude of the negative wave; (3) pretreatment of the LS slices of Wistar and LE rats with the V1 receptor antagonist d(CH2)5[Tyr(Me)]AVP did not prevent induction

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of LTP but did prevent its maintenance (i.e., the enhanced amplitude of the posttetanic negative wave of the FP diminished to a value signiﬁcantly below that of controls within 60 min); and (4) superfusion of the brain slices with 2-APV prevented the induction of LTP in brain slices obtained from both HODI and LE rats, indicating the necessity for NMDA receptors to establish LTP in this preparation. A second tetanic stimulus, applied after the NMDA receptor antagonist was washed out of the slices, induced LTP in both specimens, but the increase in amplitude of the negative wave was maintained only in slices from LE rats. In conclusion, the results of this study indicated the importance of endogenous AVP for long-lasting potentiated responses in LS neurons elicited by high-frequency stimulation of the ﬁ–fx pathway. These ﬁndings, together with those from earlier studies that showed that VP produces long-lasting enhancement of LS responses induced by iontophoretically applied EAAs (Glu, Asp, and NMDA) and by electrical stimulation of the ﬁ–fx pathway (Joels and Urban, 1984a, previously described), suggest that VP released in the LS by iontophoresis or by tetanic stimulation of ﬁmbria ﬁbers increases the efﬁcacy of excitatory transmission between ﬁmbria and LS neurons. f. Urban and Killian (1990) Urban and Killian (1990) provided evidence of both neurotransmitter and neuromodulatory actions of AVP on ventral hippocampal (VH) neurons in the rat. Previous research had indicated that AVP (Muhlethaler et al., 1982), glutamate (Glu) (Segal, 1976), and acetylcholine (ACh) (Bird and Aghajanian, 1975) excited VH neurons. This study reexamined the ability of AVP to directly excite VH neurons (its transmitter action), and also tested its ability to potentiate the response of VH neurons evoked by Glu and ACh stimulation (its neuromodulatory action). The ability of the V1 receptor antagonist d(CH2)5[Tyr(Me)]AVP to block the neurotransmitter and/or neuromodulator actions of VP was also tested. Finally, two EAA antagonists: 2-APV, an NMDA receptor antagonist, and glutamic acid diethyl ester (GDEE), a non-NMDA receptor antagonist, were tested for their effects on Glu-, AVP-, and ACh-induced excitations, as was the NMDA receptor antagonist for its effects on the putative neuromodulatory actions of AVP. The anesthetized rats were surgically prepared, the hippocampus was exposed, and VH cell testing was conducted with extracellular recording and iontophoretic procedures used in earlier described studies (e.g., Joels and Urban, 1984a). The recording electrode assembly was lowered stereotaxically through the subiculum or the CA1 ﬁeld of the VH until a spontaneously active neuron was encountered. Sensitivity to the drugs was tested and ejecting currents were adjusted to elicit a change of at least 30% in the ﬁring rate (spikes per second) of the neurons for subsequent tests. After testing for the neurotransmitter actions of Glu, ACh, and AVP, subsequent tests determined the neuromodulatory actions of AVP on responsivity to Glu

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and to ACh, and the inﬂuence of the receptor antagonists on these drugelicited responses. The effect of AVP on Glu and ACh excitations was examined in 61 neurons that failed to respond to AVP in the initial test. The average number of spikes for the direct responses of each neuron to Glu, ACh, and AVP, before testing for antagonistic and neuromodulatory VP actions with the antagonist, was set to 100%. The average number of spikes in the responses during these subsequent tests was expressed as a percentage of the controls. Comparisons between mean responses to the drugs before (control) and during release of the antagonists, or of AVP in tests of its neuromodulatory action, were statistically evaluated by the Wilcoxon test for repeated measurements. The results relevant to direct excitatory actions of AVP on VH neurons were as follows: (1) Glu and ACh excited nearly all the VH neurons tested, but AVP excited only about 30% of these neurons. Moreover, these were short-lasting effects (ACh- and AVP-induced excitations did not last more than several seconds after their application); (2) the V1 vasopressin receptor antagonist suppressed excitatory responses to VP and Glu, but had little effect on responses to ACh; Glu receptor antagonists 2-APV and GDEE also suppressed AVP and Glu excitations without affecting those to ACh. These actions make doubtful the speciﬁcity of these antagonists; and (3) the actions of the antagonists had disappeared, for the most part, within 5 min of their release. Observations relevant to the ability of AVP to enhance neuronal responsiveness to other neurotransmitters in the VH were as follows: (1) the 70% of tested neurons not directly excited by VP showed enhanced responsivity to Glu (i.e., resulted in an average 2-fold increase in the number of elicited responses), but not to ACh. The enhanced responsiveness to Glu appears to have been relatively long lasting because it was in evidence during the 15- to 20-min observation period after removal of VP application; and (2) the suppression of Glu-evoked activity by either V1 or NMDA receptor antagonists given alone was nulliﬁed when AVP was co-released with either antagonist. Thus neither receptor antagonist prevented the effect of AVP on neuronal responsivity to Glu stimulation. The following points were made in the investigators’ discussion: (1) the ﬁnding of a VP-induced direct excitatory effect (neurotransmitter action) on hippocampal neurons that can be blocked by the V1 receptor antagonist conﬁrms similar results obtained by in vitro methods (e.g., Muhlethaler et al., 1982); (2) the ability of the V1 receptor antagonist to block the direct excitatory action of the peptide on VH neurons suggests mediation by a V1 receptor. However, the speciﬁcity of this ligand–receptor interaction is questionable because this V1 receptor antagonist also blocked Glu-induced excitations; (3) the speciﬁcity of GDEE as an EAA antagonist was also challenged by these results, and by additional reports of its ability to block the excitatory actions of ACh (Watkins and Evans, 1981); (4) in contrast, the

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speciﬁcity of 2-APV as an EAA antagonist has not been challenged to date, and its ability to decrease the excitatory action of VP suggests that NMDA receptors are involved in this VP neurotransmitter action in the VH and probably in other brain sites as well; (5) in contrast to the small number of VH neurons that were directly excited by VP, most of the VH neurons tested showed a long-lasting enhancement of Glu-evoked activity in response to that peptide treatment; and (6) this VP-induced potentiation of Glu-evoked activity was not associated with noticeable changes in the spontaneous activity of the VH cells, and lasted for a relatively long time after peptide application. Moreover, the failure of VP and NMDA receptor antagonists to prevent the long-lasting VP increase in Glu-evoked activity suggests a VP action different from that responsible for direct and rapid neuronal excitations. g. Chepkova et al. (1995) Chepkova et al. (1995) carried out an in vitro study using brain slices to test for a neuromodulatory action of AVP and AVP(4–8) in the ventral hippocampus. As previously discussed, the observation that the brain metabolizes AVP into VP(4–8) and other behaviorally active peptide fragments has led to the suggestion that these metabolites themselves may mediate the effects of VP on memory (De Wied, 1991). Whereas picogram quantities of AVP or VP(4–8), injected into the ventral hippocampus of rats or mice, enhance memory processing in passive avoidance and discrimination tasks (Kovacs et al., 1979a; Metzger et al., 1993), much larger (micromolar) quantities of AVP are required for the fast-acting neurotransmitter effects of VP (e.g., Joels and Urban, 1982; Mizuno et al., 1984; Muhlethaler et al., 1982). Thus the mnemonic effects induced by picogram quantities of these peptides may occur by some other means, such as by the neuromodulatory effect of VP on glutamatergic neurotransmission in the septal–hippocampal system (Joels and Urban, 1982; Urban, 1987). The purpose of this study was to investigate the effects of picomolar amounts of VP and VP(4–8) on the excitability and glutamatergic transmission of neurons in the ventral hippocampus. The investigators made intracellular recordings from a brain slice preparation to test whether small concentrations (0.1 nM) of VP and VP(4–8), added to the superfusion medium, inﬂuenced glutamatergic excitatory postsynaptic potentials (EPSPs) and/or membrane responses to depolarization current in CA1–subiculum neurons in the ventral hippocampus. Glutamate (Glu)-mediated EPSPs were evoked in the CA1–subiculum neurons by electrical stimulation of stratum radiatum of the CA1 ﬁeld. The response measures recorded included (1) amplitude (in millivolts), slope (in nanovolts per millisecond), and latency (in milliseconds) of the evoked EPSPs, (2) voltage and time of onset of any action potentials (APs) superimposed on the EPSPs or the membrane responses to depolarization current; and (3) resting membrane potential (RMP) and input resistance (Rin) of the cells,

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which were monitored throughout the experiment and computed from the voltage response to hyperpolarizing current injections applied before the stimulus in each of the EPSP sweeps. Average EPSP amplitude, slope, and Rin values during and after peptide treatment were expressed as percentages of the average baseline of these parameters before peptide treatment. The signiﬁcance of the peptide treatment was assessed by statistical comparisons between the average baseline values and the average values obtained 60 min after peptide administration. Stimulus–response curves were graphed for certain neurons, relating EPSP responses to increasing stimulus intensity tested during the 15-min treatment interval. Two experiments tested the ability of these peptides to maintain their neuromodulatory effects on glutamatergic neurotransmission after pretreatments with antagonists that blocked either GABA receptor-mediated inhibition alone (picrotoxin) or GABA receptor inhibition plus Glu NMDA receptor-mediated excitation [picrotoxin and D(–)-2-amino-5-phosphonopentanoic acid (2-APV as per Van den Hooff et al., 1989)]. Picrotoxin (GABAA receptor antagonist) or picrotoxin plus 2-APV (Glu NMDA receptor antagonist) was added to the superfusion medium 30 min before these tests. One major ﬁnding of this study was the ability of a low concentration (0.1 nM) of both AVP and AVP(4–8) to produce a long-lasting enhancement of Glu-mediated neurotransmission in pyramidal neurons in the VH–subiculum. This was indicated by the following observations: (1) AVP induced an increase in EPSP amplitude and slope that peaked 30–45 min after peptide application and remained signiﬁcantly above baseline values for at least 60 of the 120 min of the washout period; (2) the AVP-induced increase in synaptic excitability was observed in the stimulus–response relationships obtained for EPSPs in neurons tested before and 60 min after AVP treatment; (3) the peptide facilitated the rising phase of the depolarization, a decreased threshold, and an increased number of action potentials in the membrane response to stimulation by depolarizing current injections; and (4) AVP(4–8) produced similar but even more pronounced effects because the EPSPs in the majority of neurons remained increased for the entire 60- to 120-min washout period. A second major ﬁnding was that AVP- and VP(4–8)-induced increases in EPSP amplitude and slope were not associated with any consistent changes in spontaneous activity, resting membrane potential, or input resistance in these VH–subicular neurons. This suggests a peptidergic effect different from the short-lasting reversible excitatory neuronal action associated with membrane depolarization or an increase in ﬁring rate or changes in input resistance, observed in in vitro studies using 103 higher concentrations of AVP (Charpak et al., 1989; Ma and Dun, 1985; Mizuno et al., 1984; Peters and Kreulen, 1985; Raggenbass et al., 1988). A third major ﬁnding was that pretreatment of brain slices with GABAA receptor antagonist alone, or in combination with NMDA receptor antagonist, did not inﬂuence the effects of VP or VP(4–8) on EPSP amplitude

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or threshold for action potentials. Thus, neither GABAA receptor-mediated inhibition nor the NMDA subtype of Glu receptor was necessary for the peptide-induced potentiation of Glu neurotransmission observed in this study. In their discussion, the authors noted that the mechanisms involved in the long-lasting enhancement of Glu neurotransmission induced by AVP and VP(4–8) await further clariﬁcation. However, it does appear that the NMDA receptor, a prerequisite for the long-lasting increase in Glu-mediated synaptic efﬁcacy associated with LTP (Collingridge and Bliss, 1987), was not necessary for the peptide-induced LTP-like effects observed here. More speciﬁcally, during LTP induction, the greater and longer lasting depolarization produced by tetanic stimulation removed the Mg2þ ions that block the NMDA channel, allowed it to open, and permitted the entry of Ca2þ ions that then triggered the sequence of events that resulted in enhanced synaptic efﬁcacy (Collingridge and Bliss, 1987). The authors noted, however, that an increase in intracellular stores of Ca2þ ions, resulting from stimulation of inositol phospholipid metabolism by VP and VP(4–8), might have mediated the enhancement of Glu neurotransmission observed in this study. As discussed in Chapter 1, during signal transduction involving the V1 receptor, the interaction of the receptor complex with a G protein in the plasma membrane leads to inositol phospholipid metabolism, and a subsequent mobilization of Ca2þ ions from intracellular storage depots. The increased intracellular Ca2þ ions, in turn, initiate a sequence of intracellular effects that produce the synaptic changes (e.g., increase glutamate release, or increased sensitivity in the postsynaptic receptor for glutamate) underlying the long-lasting enhancement of Glu synaptic transmission. If so, then a calcium-dependent mechanism mediated the VP and VP(4–8) neuromodulatory action observed in this study, as has been proposed for the synaptic changes responsible for LTP (Bliss and Collingridge, 1993). Relevant to this proposal, the authors cited ﬁndings that VP and VP(4–8) stimulate inositol phospholipid metabolism in neurons (Moratalla et al., 1989; Shewey and Dorsa, 1988), mobilize Ca2þ from internal stores in cultured hippocampal neurons as well as in the smooth muscle cells in blood vessels (Brinton et al., 1994; Van Renterghem et al., 1988), and increase transmitter release (Abdul-Ghani et al., 1990; Maegawa et al., 1993).

VI. Theoretical Propositions of the ‘‘VP/OT Central Memory Theory’’: Concluded

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The research discussed in this chapter has given rise to the last 3 of the 11 theoretical propositions characterizing the ‘‘VP/OT Central Memory Theory.’’ These propositions are discussed below, together with relevant research support.

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A. Proposition 9: VP(1–9) and OT(1–9) Are Precursors of Metabolic Fragments That Modulate Memory Processing in the Brain Proposition 9 suggests that both exogenous and endogenous AVP and OT are metabolized into smaller bioactive peptides that mediate the memory-processing functions of the parent peptide. Relevant to this proposition are in vitro (Burbach and Lebouille, 1983; Burbach et al., 1983b) and in vivo (Stark et al., 1989) biochemical studies demonstrating the presence of aminopeptidase enzymes in rat brain that convert the parent peptides into smaller C-terminal hexapeptide fragments. The fragments so formed were of two types: those with, and without, a pyroglutamic acid residue (pGlu4) that acts to preserve the fragment from further proteolytic conversion (Burbach et al., 1983a,b). Once these fragments were isolated and synthesized, behavioral studies assessed their effectiveness in modulating memory consolidation and retrieval, especially in avoidance memory paradigms. These studies showed the similarity of the fragments to the parent peptide in their long-term effects on memory. Whether administered peripherally (De Wied et al., 1987), centrally (intracerebroventricularly), or directly into limbic system structures associated with a VP inﬂuence on memory processing (Kovacs et al., 1986), these VP and OT fragments inﬂuenced memory consolidation and/or retrieval in a PA paradigm. VP fragments also prolonged extinction in pole-jump shock avoidance task and reversed pentylenetetrazole (PTZ)-induced retrograde amnesia in the single-trial PA task (De Wied et al., 1987). The VP fragments have time-dependent effects similar to those of the parent peptide (Gaffori and De Wied, 1986), and several ﬁndings have suggested that, depending on the speciﬁc fragment, it may play a greater role in one or another stage of memory processing (Gaffori and De Wied, 1986; Kovacs et al., 1986). Finally, these behavioral studies have indicated that both the VP (Burbach et al., 1983a) and OT (Burbach et al., 1983b) fragments, which lack the endocrine effects of the parent peptide, are nevertheless far more potent in their behavioral effects. The ﬁndings that these fragments are three to four orders more effective when injected centrally (intracerebroventricularly) than peripherally (subcutaneously) (De Wied et al., 1987) suggest that they act at central rather than peripheral receptor sites. Studies exploring the distribution of the peptide fragment-binding sites suggest that they are localized in brain areas that may overlap with, but differ from, those mediating the effects of the parent peptides (Brinton et al., 1986, 1984; De Kloet et al., 1985a). In several earlier studies (Constantini and Pearlmutter, 1984; De Kloet et al., 1985b), it was reported that receptorbinding sites for VP fragments were located mainly in endocrine/autonomic processing sites (e.g., sites in the pineal gland, the OVLT, the arcuate

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nucleus of the hypothalamus, and the nucleus of the solitary tract), whereas receptor sites for the parent peptide were localized in these areas plus limbic sites implicated in memory processing (see Chapter 1). Nevertheless, Du et al. (1998) cites evidence that VP(4–8) has tight and speciﬁc binding to synaptosomal membranes in the anterior cortex and hippocampus, and a wide range of regional distribution in the brain, especially in limbic system sites. Detailed analysis indicated a substantial difference in the exact locations that bind VP(1–9) and VP(4–8) in these brain areas (Du et al., 1994a,b). Although the evidence discussed is generally supportive of proposition 9, several observations appear to this author to raise difﬁculties for this proposition. First, there is evidence that the C-terminal hexapeptide fragment is less effective than the parent peptide in binding to receptor sites in the hippocampus (Brinton et al., 1984; De Kloet et al., 1985b), a brain structure strongly implicated in VP-associated memory processing by research cited in this chapter and in Chapter 4. Second, it has been observed that C-terminal VP and OT fragments are much less potent than the parent peptides in enhancing lateral septal neuronal responsivity to excitatory neurotransmission from ﬁmbria–fornix ﬁbers of hippocampal origin (Urban and De Wied, 1986). This ﬁnding was interpreted at the time as an indication that the effect of VP on lateral septal neuronal responsivity to hippocampal input is unrelated to the role of the peptide in avoidance retention. However, subsequent study indicated that the effect of VP on this neural activity was related to its ability to maintain LTP established in this brain structure, and LTP has been considered an experimental model for synaptic events underlying memory storage (Van den Hooff et al., 1989; Carlson, 1998). Although the septal data pose some difﬁculty for this proposition, the fragments do appear to be more potent than the parent peptide in inﬂuencing neuronal synaptic excitability in the hippocampal area (Chepkova et al., 1995). The reason for the disparity between the septal and hippocampal neuronal responsiveness to VP fragments needs further study but could be related to the fact that an NMDA receptor was involved in the maintenance by VP of LTP in the study by Van den Hooff et al. (1989), but not for the enhancement by VP of Glu synaptic transmission in the study by Chepkova et al. (1995). It is possible that the whole VP molecule as well as the VP fragments both inﬂuence memory processing but act in different ways on the structures involved in memory processes (De Wied, personal communication). It is hoped that future research will help resolve these difﬁculties, and permit us to fully characterize the receptors of these behaviorally active C-terminal metabolites, which appear to differ from those identiﬁed for the parent peptides (Burbach et al., 1998), and to more clearly understand their speciﬁc role in memory processing and how it relates to that observed for the parent peptides.

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B. Proposition 10: The Influence of Vasopressin on Hippocampal Theta Activity during Paradoxical Sleep Is Related to Its Role in Memory Consolidation Two important correlates of proposition 10 are as follows: (1) hippocampal theta activity during paradoxical sleep plays a crucial role in the long-term storage of information in the brain of those mammals in which this EEG pattern is present; and (2) endogenous vasopressin contributes to this process by means of its inﬂuence on this neuronal activity. Evidence supportive of the ﬁrst correlate was brieﬂy presented in an earlier discussion in this chapter (Section IV.B) and has received more extensive consideration in discussions by Winson (1985, 1990) and by Vertes and colleagues (Vertes, 1986; Vertes and Kocsis, 1997). Several studies by Urban, Joels, and De Wied reviewed in this chapter are relevant to the second correlate. First, Urban and De Wied (1975) observed the frequency–power curve for the hippocampal theta rhythm during paradoxical sleep for HODI and HEDI rats, and noted that the curve was shifted to the left for HODI rats, indicating lower frequencies at all parts of the curve. DG-LVP treatment dose dependently restored the theta frequencies toward HEDI levels without correcting the diabetes insipidus condition itself, indicating that the altered brain rhythm was not merely a secondary effect of the endocrine disorder. Second, Urban and De Wied (1978) demonstrated an even wider discrepancy in hippocampal theta frequencies between HODI and HONO subjects. Moreover, intracerebroventricularly injected DG-AVP temporarily normalized the theta frequency disturbance in HODI rats, and intracerebroventricularly injected VP antiserum shifted the theta frequencies of HONO rats toward those of HODI rats. Third, Bohus et al. (1978b) noted that when AVP and OT were intracerebroventricularly injected into normal Wistar rats, the two peptides produced opposing effects on hippocampal theta frequencies, consistent with their opposing effects on avoidance memory consolidation.

C. Proposition 11: Vasopressin Exerts Both a Neurotransmitter and a Neuromodulator Action on Neurons in the Septal–Hippocampal System; Its Neuromodulator Action at Glutamatergic Synaptic Sites Is Especially Important for Memory Consolidation Findings from in vivo and in vitro electrophysiological studies reviewed in this chapter, using iontophoretically or bath-applied AVP, support proposition 11. Evidence of the neurotransmitter action required higher iontophoretic ejecting currents, and higher concentrations of bath-applied VP, than did that of its neuromodulatory action (Joels and Urban, 1982; Urban and Killian, 1990). The neurotransmitter action of VP was characterized by an

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excitatory response (e.g., depolarized membrane associated with action potentials, or an increase in spontaneous activity) of rapid onset and relatively short duration once the applied peptide had been withdrawn; in contrast, its neuromodulating action involved a relatively long-lasting potentiation (enhancement) of the responsivity of the target neuron to its synaptic input (Joels and Urban, 1982; Urban and Killian, 1990). In the studies reviewed herein, neuromodulatory action was exhibited as a VPinduced long-lasting facilitation of glutamate-mediated excitatory neurotransmission in the lateral septum (Joels and Urban, 1982, 1984a; Urban and De Wied, 1986), dorsal hippocampus (Joels and Urban, 1982), and ventral hippocampus (Chepkova et al., 1995; Urban and Killian, 1990). These studies permit one to drawn two parallels between the characteristics of the neuromodulating and mnemonic actions of VP. First, the long-lasting nature of its neuromodulatory action (observed up to 90 min in ﬁeld potential studies; e.g., Joels and Urban, 1984a) is paralleled by the long-term effect of VP in avoidance conditioning, repeatedly demonstrated in the research studies described in this text. Second, as noted above, larger concentrations of VP are needed to demonstrate its neurotransmitter actions compared with its neuromodulatory actions. Chepkova et al. (1995) noted that concentrations in the picomolar range, which induce the longer-lasting neuromodulatory action of the peptide, parallel the picogram dose levels of centrally administered AVP that facilitate retention in learning tasks [Kovacs et al., 1979a (Chapter 4); Metzger et al., 1993]. The importance of endogenous VP to LTP maintenance in the lateral septal nucleus, observed by Van den Hooff et al. (1989), is also relevant to proposition 11. This in vitro study demonstrated that septal neurons in brain slices from Brattleboro HODI rats failed to exhibit long-lasting enhancement of neuronal responsivity normally observed after high-frequency stimulation of the ﬁmbria–fornix pathway. Moreover, VP pretreatment of the rats before their sacriﬁce, or of the brain slices before the LTP procedure, resulted in a long-term potentiated response, whereas adding a V1 vasopressin receptor antagonist to the bath solution prevented it. Many neuroscientists regard the LTP phenomenon as a valid model of the cellular mechanisms involved in the strengthening of synaptic connections postulated to occur during memory consolidation (e.g., Laroche et al., 1995). As previously noted, glutamate is the putative neurotransmitter involved in LTP, and both NMDA Glu and non-NMDA Glu types of receptors are important to this process (see Carlson, 1998, for a clear and updated introductory discussion). The long-lasting enhancement by VP of glutamatergic neurotransmission in the septal–hippocampal system (cited above) is in accord with the ﬁnding that this peptide is important to the long-lasting enhancement of LTP of septal responses to high-frequency stimulation of Glu-mediated synaptic input in the VH (Chepkova and Skrebitski, 1982) and LS (Van den Hooff et al., 1989).

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Findings from the in vitro study of Chepkova et al. (1995) further reinforced the earlier drawn distinction between the neurotransmitter and neuromodulatory actions of VP and related peptides. Low concentrations of VP and VP(4–8), judged to be too low to exert neurotransmitter effects in a brain slice preparation, produced a long-lasting enhancement of EPSPs evoked in ventral hippocampal–subiculum neurons by stimulation of presumably glutamatergic terminals in the stratum radiatum. Moreover, this response enhancement was unaccompanied by changes indicative of a VPinduced neurotransmitter effect. It was further found that the presence of the NMDA glutamate receptor antagonist in the superfusion medium did not inﬂuence the effects of VP(1–9) and VP(4–8) on stimulus-evoked EPSP responses. This indicated that Ca2þ inﬂux through NMDA receptors is not the likely mechanism by which these peptides enhance glutamatergic neurotransmission in the septal–hippocampal system. Instead, this enhanced neurotransmission may have resulted from pre-and/or postsynaptic changes that resulted from interaction of the peptides with intracellular phospholipid metabolism.

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Part III

Research Studies of Koob and Colleagues: The ‘‘Vasopressin Dual Action Theory’’

I. Overview

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By the early 1980s, numerous experimental studies by De Wied and collaborators had shown that posttraining peripherally administered vasopressin prolonged extinction in a multitrial active avoidance task (De Wied, 1971; see Chapter 2) and reentry latency in a single-trial inhibitory (passive) avoidance task (Ader and De Wied, 1972; see Chapter 2). Similar effects were observed if the injection was given just before the retention test trial in the inhibitory avoidance task (Ader and De Wied, 1972; see Chapter 2). Centrally (intracerebroventricularly) administered vasopressin produced identical results, but at much lower dose levels (Bohus et al., 1978b; see Chapter 2). These behavioral effects after posttraining and preretention treatment were interpreted as signifying, respectively, a vasopressin-induced facilitation of memory consolidation and memory retrieval. It was theorized that the effect of the peptide on memory storage and retrieval was Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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mediated by an action at vasopressin receptors localized within the brain (see Chapters 2–5). Several considerations, however, prevented wholesale acceptance of the ‘‘VP/OT Central Memory Theory.’’ First, there were reported failures to obtain a vasopressin-facilitated memory effect on avoidance behavior [e.g., Hostetter et al., 1980 (Chapter 9); Sahgal et al., 1982 (Chapter 7)] and the ﬁndings from the relatively few studies that had used appetitive paradigms were even less consistent than those of studies employing aversive stimuli (see Chapters 9–11). Second, it was difﬁcult to understand how peripherally administered vasopressin could directly inﬂuence vasopressin receptors in the brain because many researchers argued against the probability that the peptide could cross the blood–brain barrier (BBB) (Pardridge, 1983a; Reppert et al., 1981; Wood, 1982; Zaidi and Heller, 1974). Third, there were alternative explanations of how peripherally administered vasopressin might exert its observed effect on retention behavior. Two alternative suggestions soon crystallized: the ‘‘VP Dual Action Theory’’ formulated by Koob and associates, and the ‘‘VP Central Arousal Theory’’ of Sahgal and colleagues (see Chapter 7). This chapter describes the arginine vasopressin (AVP)/memory research studies of Koob and associates carried out between 1981 and 1986, and those propositions of the ‘‘VP Dual Action Theory’’ relevant to this research. With the exception of two physiological studies, this experimental research manipulated peripheral or central levels of vasopressin, primarily by injection of AVP and analogs (agonists and antagonists), and examined the effect on retention behavior in appetitive as well as aversive tasks. They employed the same multitrial pole-jump active avoidance and single-trial inhibitory avoidance paradigms used by De Wied and colleagues. These studies had three major objectives: (1) replication of the ﬁndings reported by De Wied and colleagues indicating that peripherally and centrally administered AVP facilitates retention performance in aversive conditioning tasks, (2) determination of whether vasopressin exerts a memory-facilitating effect on appetitive learning and memory tasks, and (3) clariﬁcation of the mechanism(s) by which behaviorally active doses of peripherally and centrally administered vasopressin contribute to memory processing. The experimental ﬁndings replicated the reported vasopressin-induced facilitation of long-term memory in aversive tasks, and demonstrated a similar outcome for appetitive tasks. Concerning the third objective, the research evidence led Koob and associates to reject the ‘‘VP/OT Central Memory Theory’’ proposal that vasopressin treatment facilitates retention by a direct action on central VP-ergic receptor sites involved in memory processing. Instead, these researchers formulated the ‘‘VP Dual Action Theory,’’ which proposes that the retention effect is secondary to an AVP-induced increase in behavioral arousal, achieved by independent mechanisms depending on whether the peptide is administered by peripheral or central injection.

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II. Memory Tasks Used in VP/Memory Research

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To assess the effects of peripherally or centrally administered vasopressin and analogs on memory storage, Koob and colleagues have used both single- and multiple-trial learning tasks with either aversive (footshock) or appetitive (food or water reward) learning incentives. Four of these learning/memory tasks are described below. The two avoidance tasks are essentially the same as those used by De Wied and colleagues (see Chapter 2, Section II) and therefore permit a direct comparison between results of studies of the two research teams. The two appetitive tasks permit evaluation of the effect of vasopressin on memory processing in a nonpunitive learning environment. A brief description of each of these behavioral tasks is given below. For all these tasks a ‘‘blind’’ protocol was used, with the experimenter remaining unaware of the subject’s experimental treatment (e.g., vasopressin or placebo).

A. Pole-Jump Footshock Avoidance Task The apparatus, training, and testing procedure for the pole-jump footshock avoidance task are described in Koob et al. (1981). Brieﬂy, the apparatus is a modiﬁed Skinner box, with a grid ﬂoor and a centrally located ink-blackened pole extending from ﬂoor to ceiling. A light bulb centered over the pole, just above the Plexiglas ceiling, serves as the conditioned stimulus. Light onset signals the beginning of each trial. Footshock, delivered through the electriﬁed grid ﬂoor, is avoided if a jump response onto the central pole is made within 5 s of light onset. Failure to make a successful avoidance response results in footshock delivery (0.3 to 0.7 mA) lasting up to 30 s or until the rat jumps onto the pole. During the ﬁrst three trials on the ﬁrst day of acquisition, rats failing to jump onto the pole within 5 to 10 s are gently placed on the pole by the experimenter. Rats failing to climb down from the pole within 30 s of jumping are gently turned around on the pole and thus forced to return to the grid ﬂoor. Each rat receives 10 acquisition trials/day with intertrial intervals of 40, 60, or 80 s, with an average of 60 s. Three days of acquisition training (10 trials/day) are followed by four blocks (10 trials/block, 1 block of trials every 2 h) of extinction test trials (no footshock) on day 4. Only those subjects that achieve the learning criterion (seven or more successful avoidance responses during the ﬁrst block of extinction trials) remain in the experiment for further study. Experimental treatment is given immediately after completion of the ﬁrst block of extinction trials. The effect of the treatment is assessed in the remaining blocks of extinction trials, given on day 4 at 2, 4, and 6 h after the experimental treatment. The underlying assumption is that the greater the number of avoidance responses during

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extinction (i.e., the slower the rate of extinction), the stronger the retention for the learned avoidance response.

B. Single-Trial Inhibitory (Passive) Avoidance Task The apparatus, training, and testing procedure for the single-trial inhibitory (passive) avoidance task are described in Lebrun et al. (1984). The animal is trained and tested in an apparatus consisting of a small illuminated white safe chamber separated from a larger darkened black chamber box by a guillotine door. Although this apparatus differs from the one used by De Wied and colleagues, the training/testing procedure is the same. On day 1, the subject is placed in the larger darkened chamber, where it remains for a 2-min interval to familiarize it with the enclosure. It is then removed from this chamber and placed in the adjoining white chamber for the ﬁrst pretraining trial. On the following day, two additional pretraining trials are given followed by the single acquisition trial. During pretraining, the rat has free access to both chambers and, because of its species-speciﬁc preference for dark over light, the rat normally enters the dark chamber within 15 s. During the single acquisition trial, entry into the preferred dark chamber results in delivery of a footshock (0.25 mA for 3 s) followed by brief conﬁnement (additional 5-s interval) in the chamber. Experimental treatment is administered immediately on removal of the rat from the shock chamber. The animal is returned to its home cage and tested for retention 24 h later by being placed in the white compartment and the latency to enter the dark chamber (reentry latency), up to a maximum of 300 s, is recorded. No footshock is given during the retention test. The reentry latency is assumed to reﬂect the strength of the memory for the aversive experience.

C. Single-Trial Water (Food)-Finding Task See Ettenberg et al. (1983a,b) for a complete description of the task apparatus and training procedure. For this task, a non-food- and non-waterdeprived rat is given a single 5-min exploratory trial in a square box housing a tube ﬁlled with water, placed in a single alcove located along one of its sides. Typically the nondeprived subject ingests little water at this time. The experimental treatment is given immediately on removal of the rat from the apparatus. The rat is then returned to its home cage and deprived of water for the next 48 h. At the end of this interval, the deprived rat is returned to the apparatus, which at this time contains a dry drinking tube in the alcove. The latency for the thirsty rat to locate the dry drinking tube is recorded. The underlying assumption is that the shorter the time required for the thirstmotivated rat to locate the drinking tube, the stronger the memory of the stimuli experienced during the exploratory (learning) trial. This behavioral task has also been adapted for use with a food (sucrose solution) reward.

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D. Radial Maze Task See Packard and Ettenberg (1985) for a description of the task apparatus and testing procedure. In this task, the food-deprived rat is placed in the center of an elevated eight-arm radial maze; food is available in a sunken food cup at the outer end of each arm. Initially, the animal is allowed to make 16 choices in each of the daily performance trials. However, once the learning criterion has been met (i.e., 7 different arms are selected in the ﬁrst 8 choices on 3 consecutive days) the subject is removed from the apparatus after the ﬁrst 8 choices (instead of after 16 choices). A single food-rewarded trial is given on each day for 18 consecutive days. Extinction training is carried out on day 19. During the initial extinction trial, a nonrewarded visit to each of the eight unbaited arms provides an opportunity to experience the changed reward contingency. After the animal has visited each of the eight arms, it is removed from the maze and transferred to an adjacent room where it receives the vasopressin treatment. Four additional extinction trials are successively given at 2-h intervals, in a manner similar to that used for assessing the rate of extinction in the pole-jump avoidance task. However, unlike the active avoidance study, a vasopressin-induced increase in the rate of extinction is interpreted as memory facilitation (i.e., facilitation of the retention of the changed contingency experienced in the initial extinction trial).

III. Studies with Pole-Jump Active Avoidance, and Single-Trial Passive Avoidance Paradigms

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A. Selected Studies 1. Le Moal et al. (1981) Le Moal et al. (1981) demonstrated a relationship between the pressor and retention effects of peripherally circulating vasopressin, using adult male Wistar rats in the pole-jump shock avoidance task. Independent experimental tests were given to determine (1) dose levels at which peripherally administered AVP induces a pressor response; (2) the dose ratio required for the peripherally injected V1 pressor antagonist 1-deaminopenicillamine-2(O-methyl) tyrosine-argine vasopressin (i.e., [dPTyr(Me)]AVP) to block this pressor effect; (3) reproducibility of the earlier observed AVP-induced extinction effect in this task (e.g., De Wied, 1971; see Chapter 2); and (4) the degree to which pretreatment with the peripherally injected V1 antagonist can block this retention effect, using the same dose ratio that blocked the peptide-induced pressor response. Blood pressure was monitored in instrumented, conscious, freely moving rats. Each rat was tested only once per day and placed in a soundattenuated experimental chamber for monitoring of heart rate and blood

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pressure. Blood pressure was monitored at various times after a peripheral injection of saline or various doses of AVP (1, 5, or 25 g/kg, subcutaneous). AVP led to prompt elevations of systolic blood pressure (SBP) at both the 5- and 25-g/kg dose levels, which peaked 10–20 min after injection and generally returned to normal levels within 1 h. Using the threshold pressor dose of AVP (5 g/kg), various doses of the antagonist were tested for the ability to block the AVP-induced pressor response. This pressor response was completely blocked by a 25-g/kg dose of the V1 antagonist, a dose that, by itself, did not cause hypotension. This antagonist-to-agonist dose ratio of 5:1 was then used for subsequent behavioral testing. For behavioral testing, rats were trained and tested in the pole-jump shock avoidance task according to the aforementioned procedures (Section II.A). After meeting the learning criterion during the ﬁrst 10-trial extinction session, the subjects received two successive injections, separated by a 2-min interval, of either saline/saline, saline/AVP, or [dPTyr(Me)]AVP/AVP. AVP was injected subcutaneously at a dose of 6 g/kg, and the V1 antagonist was injected subcutaneously at a dose of 30 g/kg. Three successive 10-trial extinction sessions were given at 2-h intervals (i.e., 2, 4, and 6 h after the injections). Saline/AVP-treated rats exhibited signiﬁcantly more conditioned avoidance responses than did saline/saline controls at both the 4- and 6-h time points, and pretreatment with the antagonist completely blocked this prolonged extinction effect. In summary, this study showed that peripherally administered AVP at a dose level that facilitated retention (i.e., prolonged extinction) also produced a pressor response. Moreover, peripherally injected V1 receptor antagonist blocked the AVP-induced retention effect at the same antagonist-to-agonist dose ratio (5:1) that effectively blocked the pressor activity of the peptide. The authors suggested two possible explanations for this latter ﬁnding. The ﬁrst explanation, consistent with the ‘‘VP/OT Central Memory Theory,’’ was that the antagonist inﬂuenced a type of receptor that is present in peripheral tissues as well as in the brain. Peripherally, the receptor mediates the pressor effect of hormonal VP; centrally, it mediates the behavioral (memory-facilitating) effect of the peptide. Therefore, the pressor and behavioral effects were unrelated to each other. The second possibility, consistent with the subsequently formulated ‘‘VP Dual Action Theory,’’ was that the pressor and retention effects produced by the peripherally administered AVP were related to each other. That is, the pressor response somehow mediates the retention effect, perhaps by an ‘‘altered perception of visceral status that is sufﬁcient to place the animal in a state of alerted surveillance’’ (Le Moal et al., 1981, p. 493). 2. Koob et al. (1981) Koob et al. (1981) attempted to reproduce and extend the vasopressin retention effects reported by De Wied and colleagues to conditioned polejump shock avoidance responding. To ensure replication success, Koob and

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associates carefully followed the procedures used by the former research team for training, experimental treatment, and subject selection. Experiments 1 and 2, respectively, tested the effects on extinction of the pole-jump response of peripherally (0 or 1 g/rat, subcutaneous) and centrally (0, 1, 10, or 50 ng/rat, intracerebroventricular) administered AVP. The results replicated the observations of De Wied and colleagues (e.g., Bohus et al., 1978b; De Wied, 1971; see Chapter 2) by demonstrating that AVP prolonged extinction of the avoidance response when injected peripherally at a 1-g dose level, and centrally at a 1-ng dose level. The two higher doses of centrally administered AVP (10 and 50 ng/rat) failed to prolong extinction and, at the highest dose level, produced more rapid extinction. The failure of these higher doses to facilitate retention may have been due to disruptive effects because Sahgal et al. (1982; see Chapter 7) subsequently reported seizure-like activity (barrel rotation) after intracerebroventricular injections of AVP at and beyond the 10-ng dose level. Experiments 3 and 4, respectively, tested the effect on response extinction of the pole-jump avoidance response of peripheral (0, 10, or 100 g/rat, subcutaneous) and central (0, 10 ng, 100 ng, 1 g, or 10 g/rat, intracerebroventricular) injections of the AVP V1 receptor antagonist [dPTyr (Me)]AVP. The footshock levels in experiments 3 and 4 were increased to produce greater resistance to extinction to better assess a possible facilitation of extinction exerted by the V1 antagonist. When peripherally injected, the antagonist dose dependently facilitated response extinction with a statistically signiﬁcant effect for the highest dose (100 g/rat, subcutaneous) in the last two blocks of extinction testing (6- and 8-h time points). When centrally injected, the antagonist did not inﬂuence extinction within the nanogram dose range as expected, but required a 10-g dose level to signiﬁcantly facilitate extinction, and this occurred for the last two blocks of extinction testing. The results of this study replicated ﬁndings by De Wied and associates indicating that both peripherally and centrally administered AVP exert a long-term effect on retention (prolong extinction of a conditioned avoidance response) and at a much smaller dose when centrally administered. Moreover, the ﬁndings that a VP antagonist that blocks VP receptors weakens retention (shortens the rate of extinction) when given on its own, indicates that endogenous vasopressin normally has a role in this behavior. The ﬁnding that the intracerebroventricularly injected V1 antagonist [dPTyr (Me)]AVP did not inﬂuence retention, except when injected at a much higher dose level than that effective for centrally administered AVP, was puzzling. The researchers suggested that part of the explanation may have ben related to the fact that because the antagonist is more lipophilic than AVP, it can cross the cerebrospinal ﬂuid (CSF)–blood barrier, in contrast to AVP. Hence, whereas intracerebroventricularly injected AVP remained in high concentration in the compartment into which it was injected, the antagonist, whether

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injected subcutaneously or intracerebroventricularly, was distributed into a larger volume because it diffused out of the compartment in which it was injected. 3. Lebrun et al. (1984) Lebrun et al. (1984) simultaneously conducted replicate experiments in the Netherlands and in the United States to achieve three major objectives. The ﬁrst objective was to replicate earlier reports by De Wied and colleagues that posttraining peripheral injections of AVP facilitate passive avoidance behavior (see Chapters 2–5). Replicability was desirable because of failures reported by several other laboratories (e.g., Hostetter et al., 1980; Sahgal et al., 1982). The second objective was to test for an AVP-induced bimodal effect on reentry latency. This effect had been reported by Sahgal and colleagues and composed a major source of support for the ‘‘VP Central Arousal Theory’’ that challenged the ‘‘VP/OT Central Memory Theory’’ (see Chapter 7). The third objective was to test the potential of the V1 pressor antagonist to block the effect of vasopressin on inhibitory (passive) avoidance behavior as it had been previously shown to do for active avoidance behavior (Le Moal et al., 1981). This outcome would further support the proposition that the retention effect induced by peripherally circulating (hormonal) AVP is an indirect result of the pressor action of the peptide. This study was jointly carried out in the laboratories of Koob and associates in the United States, and of Rigter in the Netherlands. The experiments in both laboratories employed adult male rats of the Wistar line and used essentially the same training/testing procedures, peptide analogs (i.e., AVP; V1 antagonist [dPTyr(Me)]AVP), antagonist-to-agonist dose ratio (i.e., 5:1), and peptide treatment schedules. The two laboratories differed with respect to the passive avoidance (PA) task apparatus, level of footshock (FS), and speciﬁc doses of peptides used for these replicate experiments. In addition, the Koob (U.S.) group carried out a second experimental test with an antagonist-to-agonist dose ratio of 25:1. Depending on the group, rats received posttraining peripheral injections of saline (controls), AVP alone, AVP antagonist alone, or AVP plus AVP antagonist. Subjects were returned to the home cage after treatment and were tested for retention 24 h after the PA learning trial. An analysis of variance (ANOVA) tested for group differences in average performance. For testing bimodal effects, each of the experimental and control subjects was subdivided into two subgroups based on their reentry latency scores (i.e., below 30 s or above 120 s). Results of the ANOVA on average performance were as follows: (1) posttraining AVP (1 g/rat, subcutaneous, United States; 3 g/rat, subcutaneous, the Netherlands) enhanced PA retention in both laboratories; (2) depending on the dose, V1 antagonist given alone had no effect (5 g/rat, subcutaneous, United States; 25 g/rat, subcutaneous, United States) or

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showed a VP-like effect (15 g/rat, subcutaneous, the Netherlands) on reentry latency; and (3) pretreatment with V1 antagonist at a 5:1 antagonist-to-agonist dose ratio exhibited a partial (the Netherlands) or no (U.S. experiment 1) ability to block the VP-induced facilitation of PA retention, whereas a dose ratio of 25:1 (U.S. experiment 2) completely blocked this retention effect. The results of the tests for a bimodal distribution of reentry latencies revealed two major ﬁndings: (1) in contrast to the vehicle-treated controls, the distribution of latency scores for the AVP-treated subjects was positively skewed with a higher number of animals failing to reenter within 120 s; and (2) despite this difference in average performance between AVP- and vehicletreated subjects, a signiﬁcant number of AVP-treated animals showed short reentry latencies. Taken together, the results of these replicate experiments suggested the following conclusions: ﬁrst, peripherally administered AVP does facilitate PA retention (i.e., prolong reentry latency) when average performance scores are used for analysis. Second, the retention-enhancing effect of AVP could be blocked by the pressor antagonist but only at an antagonist-to-agonist dose ratio of 25:1, which is higher than that required for a multitrial active avoidance task but the same as that observed for a single-trial appetitive task (Ettenberg et al., 1983b). The ﬁnding that an antagonist-to-agonist dose ratio of 5:1 blocked the pressor, but not the PA behavioral, response was interpreted as signifying that ‘‘factors other than a simple reversal of the pressor effects of AVP may be important’’ (Lebrun et al., 1984, p. 1511). Third, although it is true that VP shifted the distribution of the reentry latencies to the right (i.e., longer reentry latencies signifying better retention), the fact that a signiﬁcant number of VP-treated rats showed short latencies (below 30 s) is consistent with the ‘‘VP Central Arousal Theory’’ of Sahgal, which proposes that a behaviorally active dose of AVP increases the level of central arousal, which in turn affects retention performance in accordance with the proposed inverted U-shaped arousal–performance relationship (see Chapter 7 for further discussion). 4. Koob et al. (1985a) Koob et al. (1985a) studied the effect on active avoidance retention of increasing the rat’s level of endogenous vasopressin and the ability of a V1 pressor receptor antagonist to inﬂuence this behavioral effect. Retention was assessed by examining the rate of extinction of a conditioned pole-jump shock avoidance response. Endogenous vasopressin was increased by an osmotic challenge (i.e., treatment with hypertonic saline). The hypertonic saline was peripherally (intraperitoneally) injected at a dose range known to release vasopressin both centrally (Rodriquez et al., 1983) and peripherally (Dunn et al., 1973). Moreover, the 1 M NaCl dose of hypertonic saline used in this study had been shown to produce a plasma AVP level of 10 pg/ml1

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in rats of similar body weight (Dunn et al., 1973), and this plasma level was similar to that observed by Koob and associates which indicated ‘‘plasma levels of 13.1 2.2 to 28.4 3.5 pg ml1 20 min after s.c. injection of behaviourally effective doses of AVP (0.1–1.0 g per rat)’’ (Koob et al., 1985a, p. 751; and see Deyo et al., 1986). In experiment 1, the rat received a subcutaneous injection of either placebo (physiological saline) or AVP (1 g/rat, subcutaneous) or an intraperitoneal (intraperitoneal) injection of hypertonic saline (0.5, 1.0, 1.5, or 2.0 M NaCl, intraperitoneal) after the completion of the initial block of 10 extinction trials used to assess criterion learning. Water was available throughout testing to avoid dehydration effects on the experimental outcome. The results of this experiment showed that (1) as expected, the 1-g dose of peripherally administered AVP prolonged extinction of the avoidance response relative to the saline controls; (2) the hypertonic saline injections dose dependently prolonged extinction of the conditioned avoidance response, and was signiﬁcant for all dose levels except the lowest one (0.5 M NaCl); and (3) there were no indications that the behaviorally effective dose levels of hypertonic saline produced ill effects greater than those induced by the vasopressin treatment—an observation consistent with previous unpublished observations demonstrating that behaviorally active AVP (1 g/rat, subcutaneous) and hypertonic saline (1.5 to 2.0 M NaCl, intraperitoneal) produced similar taste aversions. In experiment 2, the same procedure was followed except that the experimental treatment consisted of two injections, a subcutaneous injection of placebo or the V1 pressor antagonist ([dPTyr(Me)]AVP; 5.0 or 10.0 g/rat, subcutaneous) followed by the osmotic stimulus (intraperitoneal injection of hypertonic saline at a dose of 1.0 or 1.5 M NaCl). The results showed that both dose levels of the V1 pressor antagonist effectively reversed the prolonged extinction induced by the osmotic challenge; this effect of the pressor antagonist may be dose dependent because pilot work showed that a smaller dose of the antagonist (2.5 g/rat) failed to inﬂuence the retention effect produced by the hypertonic saline treatment. In summary, the results of this study demonstrated that (1) an interoceptive osmotic challenge known to release both central and peripheral AVP mimicked the effects of exogenous AVP on learned behavior, and (2) this retention effect was reversed by a peripheral injection of the AVP V1 receptor antagonist. The authors drew two major conclusions from the results of this study. First, the effect on extinction behavior produced by the pressor antagonist was due to its interaction with the osmotically released vasopressin rather than to some unique action of its own, because previous study found no ability of the V1 antagonist to affect response extinction in this task, except at the high dose level of 100 g/rat (Koob et al., 1981). Thus,

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the endogenously released vasopressin was causally related to the behavioral change produced by the osmotic challenge. Second, it is possible that an increase in blood pressure, associated with the peripheral vasopressin released by the osmotic challenge, contributed to the observed behavioral effect. Consistent with this interpretation was the ﬁnding that a 5-g dose of the V1 antagonist reversed the retention effect induced by the osmotic challenge, and this dose level was the same as the one observed to reverse a retention effect induced by a 1-g dose of peripherally injected AVP in the same task as used in this study (Le Moal et al., 1981). In this author’s opinion the interpretation of the results given by these researchers would have been strengthened by additional experiments that (1) measured blood pressure in a group receiving the intraperitoneally injected hypertonic saline simultaneously with a control group receiving similarly injected physiological saline; and (2) provided pretreatment with an intracerebroventricular injection of VP antiserum in sufﬁcient dose to prevent a VP neuronally induced facilitation of retention. Given that the osmotic challenge simultaneously releases neuronal and hormonal VP, and that the peripherally injected V1 pressor antagonist can cross the blood– brain barrier and block V1 receptors in brain sites engaged in memory processing, these additional experiments would reinforce the interpretation that it was the VP hormonal effect on blood pressure that was responsible for the facilitated retention observed in this test paradigm. 5. Lebrun et al. (1985) Lebrun et al. (1985) used agonist–antagonist interactive effects to help answer the question of whether the retention effects induced by peripherally administered AVP occurred by direct action at central receptors or was mediated by pressor-associated effects of the peptide. Speciﬁcally, the centrally administered V1 antagonist ([dPTyr(Me)]AVP) was tested for its potential to block the pressor and the (behavioral) avoidance effect of peripherally injected AVP. It was predicted that if peripherally administered AVP achieved its effect on avoidance behavior by acting at peripheral vascular receptors—rather than directly at central receptors—an amount of the antagonist would be required that was sufﬁcient to diffuse from central injection sites to block the effect of the peptide on these peripheral sites. On the other hand, if the AVP behavioral effect was achieved by an action at central receptors, a far smaller amount of the centrally injected antagonist would be required. The subjects (adult male Wistar rats) were trained and tested in the polejump shock avoidance task according to training and selection procedures established by De Wied and colleagues (see description in Section II.A). Subjects that achieved the learning criterion were assigned to one of the following three treatment regimens: (1) saline intracerebroventricular/saline

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subcutaneous (controls); (2) saline intracerebroventricular/AVP (1 g/rat, subcutaneous); and (3) antagonist (0.2, 1.2, or 6.0 g/rat, intracerebroventricular)/AVP (1 g/rat, subcutaneous). The intracerebroventricular injection was given 2 min before the subcutaneous injection. In separate independent experiments, chronically instrumented freely moving rats were tested for blood pressure and heart rate. After recording these physiological parameters for 10 to 20 min, intracerebroventricular injections of physiological saline or the V1 antagonist (0.2, 1.2, or 6.0 g/rat) were given followed 2 min later by a subcutaneous injection of AVP (1 g/rat). The results showed that (1) consistent with previous ﬁndings in this task, peripherally injected AVP (1 g/rat, subcutaneous) signiﬁcantly prolonged response extinction relative to saline controls in the last two blocks of extinction testing (i.e., at 4- and 6-h time points); (2) the centrally injected antagonist prevented the AVP-induced behavioral effect only at the highest dose level (i.e., 6.0 g/rat, intracerebroventricular); and (3) peripherally administered AVP (1.0 g/rat, subcutaneous) signiﬁcantly increased blood pressure (BP) and decreased heart rate (HR) and these cardiovascular effects were also completely blocked only by the highest dose level of the centrally administered antagonist (i.e., 6.0 g/rat, intracerebroventricular). The results of this study were interpreted as support for the proposition that the (behavioral) avoidance effect observed for peripherally administered AVP is mediated by pressor-associated endocrinological effects of the peptide rather than by a direct action at central receptors. This interpretation was based on the most important ﬁnding of this study: centrally injected V1 pressor antagonist blocked the behavioral effect of peripherally injected AVP only at a dose level that also blocked the pressor effect of the peptide. This is noteworthy because antagonism of the pressor effect of systemic AVP can be mediated only by a peripheral action. 6. Koob et al. (1986) Koob et al. (1986) obtained further evidence relevant to the ‘‘VP Dual Action Theory.’’ First, this study tested the reproducibility of the earlier ﬁnding that a nanogram dose of centrally administered AVP would facilitate retention (i.e., prolong extinction) in a pole-jump shock avoidance task (Koob et al., 1981). Further experiments tested whether or not this memory effect is related to aversive and pressor properties of the centrally administered peptide, as is presumably the case for the peripherally injected peptide. Finally, the study examined the ability of the peripherally injected pressor antagonist to block the behavioral actions of centrally injected AVP. Adult male Wistar rats were trained and tested in the pole-jump shock avoidance task. Two behavioral experiments were conducted. The ﬁrst tested the effect on extinction of the avoidance response of various dose levels of centrally injected AVP. The second examined the ability of the peripherally injected V1 antagonist, [dPTyr(Me)]AVP, to block the behavioral effect

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produced by the 1-ng dose level of centrally injected AVP. In both experiments, the experimental treatment was given immediately after achievement of the learning criterion (i.e., after the ﬁrst block of extinction trials). In the ﬁrst experiment, four independent groups of rats received a single central injection of placebo or AVP (0.1, 1.0, or 10.0 ng/rat, intracerebroventricular). The results demonstrated that centrally injected AVP signiﬁcantly prolonged response extinction, relative to saline controls, for the two higher dose levels (1.0 and 10.0 ng) but not the lower dose level (0.1 ng). In the second experiment, each subject received two injections, one via a central, the other via a peripheral, route of administration. Depending on the group, the subject was assigned to one of the following treatment regimes: (1) saline intracerebroventricular/saline subcutaneous, (2) 1 ng of AVP intracerebroventricular/saline subcutaneous, or (3) 1 ng of AVP intracerebroventricular/30-g/kg [dPTyr(Me)]AVP subcutaneous. Once again, the 1-ng dose of centrally administered AVP prolonged response extinction (facilitated retention), and this effect was blocked by the peripherally injected V1 antagonist at both the 4- and 6-h time points, when response extinction had occurred for the saline controls. Taste aversion conditioning was carried out to test for potential aversive properties of centrally administered AVP. The one-bottle consumption test measured consumption of a sweetened milk solution after its pairing with a central injection of AVP (0, 0.1, 1.0, or 10.0 ng of AVP, intracerebroventricular). The results of this test demonstrated an AVP-induced conditioned taste aversion only at the 10-ng dose level, a dose level that also produced barrel-rolling in ﬁve of the six rats tested. The two-bottle preference test was used to determine whether centrally injected AVP (1 ng/rat, intracerebroventricular) would alter a previously established preference for saccharin over plain water after two pairings of AVP with saccharin ingestion. The peptide failed to alter the preestablished preference, providing additional proof that a behaviorally active dose of the centrally injected peptide lacks aversive properties. The inﬂuence of the peptide on BP was monitored in freely moving, chronically instrumented rats under two sets of conditions. In the ﬁrst condition, baseline BP was measured for a 15-min period before, and for 60 min after, either centrally (1.0 ng/rat, intracerebroventricular) or peripherally (5.0 g/kg, subcutaneous) injected AVP. In the second condition, BP was measured before and after centrally injected (1.0 ng/rat intracerebroventricular) AVP; in addition, BP monitoring that followed the AVP injection occurred for 15-min intervals every 2 h for 6 h to simulate the time regimen used to monitor extinction of the conditioned avoidance response. The test results indicated an increase in BP after treatment with a behaviorally active dose of AVP when it was administered peripherally, but not centrally. Altogether, the results of this study demonstrated that (1) when centrally administered, a behaviorally active dose of AVP fails to exhibit the pressor

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or aversive effects associated with its peripheral administration; and (2) when peripherally injected, the V1 receptor antagonist prevents the behavioral effect of centrally injected vasopressin. The behavioral interaction between the peripherally injected antagonist and the centrally administered agonist was explained as follows: the antagonist, being more lipophilic than the agonist, can readily cross the blood–brain barrier (BBB), whereas the agonist does not do so (Deyo et al., 1986, cited later in this chapter). The demonstration that intracerebroventricular administration of small doses of the antagonist can reverse behavioral effects of systemically injected AVP, but only at a dose that also reverses the pressor effects of the agonist (Lebrun et al., 1985, cited above), has also been interpreted as additional support for the ability of this antagonist to penetrate the BBB. The results of this study, together with previous ﬁndings demonstrating aversive (Ettenberg et al., 1983a) and pressor (Koob et al., 1981) properties of behaviorally active doses of the peripherally injected peptide, were interpreted as supporting a major proposition of the dual action theory: although central (neuronal) and peripheral (hormonal) AVP produce the same behavioral outcome (i.e., facilitated retention), they do so by different and independent mechanisms.

IV. Aversive Effects of Behaviorally Active Doses of Peripherally Administered VP

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Koob, Dantzer, and associates carried out several studies to test whether behaviorally active doses of AVP result in aversive afferent signals, which could in turn increase the arousal level (Fray et al., 1982) and secondarily affect learning and memory without exerting a direct action on central memory substrates. The conditioned taste aversion (CTA) paradigm (Garcia and Ervin, 1968) was the primary behavioral bioassay chosen to assess the aversiveness of peripherally or centrally administered AVP.

A. Selected Studies 1. Dantzer et al. (1982) Dantzer et al. (1982) designed several experiments to learn whether a behaviorally active dose of peripherally injected AVP is characterized by aversive stimulus properties. Having demonstrated a linkage between the pressor effects of peripherally injected AVP and its facilitative effect on retention (Le Moal et al., 1981), these researchers hypothesized that visceral afferent signals (aversive properties) associated with pressor effects would then place the animal in a state of altered arousal. An increase in the arousal level, within the optimum range, was considered the mechanism by which pharmacological doses of AVP improved retention. In this study, the ability

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of a behaviorally active dose of peripherally administered AVP to produce a conditioned taste aversion (CTA) was used as a demonstration of its aversive stimulus properties. Experiment 1 tested the ability of a behaviorally active dose of peripherally administered AVP (0 or 6 g/kg, subcutaneous) to establish a CTA after ﬁrst two, then four, saccharin–AVP pairings. CTA training began after 7 days of preliminary training during which the rats learned to ingest their daily ﬂuid allowance within a 20-min exposure session. During an initial 4-day training period, the subjects were exposed to two saccharin–AVP pairings: On days 1 and 3, 0.1% saccharin solution replaced water during the 20-min drinking session, after which half the rats were injected with AVP and half were injected with vehicle solution; on days 2 and 4, only water was available for drinking and no injection was given. Saccharin consumption (expressed as a percentage of total ﬂuid intake) was measured on day 5 in a two-bottle free-choice (preference) test given during the 20-min drinking session. Two additional saccharin–AVP (or vehicle) pairings were given on days 6 and 8 of the second training period (days 6–9) and a ﬁnal preference test (day 10) measured saccharin consumption relative to total ﬂuid intake after all four drug pairings. The results indicated that saccharin–vehicle pairings did not alter the rat’s preference for saccharin, but saccharin–AVP pairings greatly reduced subsequent saccharin consumption. Statistical comparisons indicated that saccharin intake of AVP-treated rats differed signiﬁcantly from that of the vehicle-treated rats after four saccharin–AVP pairings. The stressful experience of the injection itself was not the cause of the CTA because saccharin paired with an injection of placebo did not result in a CTA. Experiment 2 tested the generality of the ﬁndings obtained in experiment 1. This experiment used a one-bottle test and paired various doses of peripherally administered AVP (0, 0.6, 3, or 15 g/kg, subcutaneous) with sweetened milk. During an initial 5-day training period, subjects were trained to drink on cue in the home cage by having available a 20% sucrose solution for 15 min during each day of the 5-day training period. CTA training was carried out for an additional consecutive 5-day period during which sweetened milk was available for the 15-min drinking period, and was followed by a peripheral injection of physiological saline or one of the three dose levels of AVP. A ﬁnal session was run on day 6 without any injection of the peptide. CTA occurred for the highest (15 g/kg) dose of AVP, as indicated by a steady decline in milk consumption that began after the ﬁrst day of CTA conditioning and continued toward a minimal intake, which occurred on day 5. Although milk consumption increased after the ﬁrst CTA trial for the lowest (0.6 g/kg) and intermediate (3 g/kg) dose of AVP as it did for milk– saline pairings, the researchers suggested that these results did not necessarily imply that a dose of AVP lower than the 15-g/kg dose would not produce

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CTA; this is because the single-bottle test is less sensitive than the two-bottle free-choice test, especially because food and water deprivation may force the rat to overcome possible slight aversive consequence of taste–drug pairings. Experiment 3 was designed to test a possible interaction between footshock and AVP in this CTA paradigm. The rationale for experiment 3 was twofold: (1) a precedent for an interaction between an interoceptive (gustatory) and an exteroceptive (footshock, FS) unconditioned stimulus (UCS) had been established by the ﬁnding that electric shock facilitated a taste aversion to apomorphine (Lasiter and Braun, 1981); and (2) FS is often used as a UCS in avoidance tasks assessing behavioral effects of AVP and analogs. Neither food nor water deprivation was used in experiment 3. Peripherally administered AVP (0, 5, or 15 g/kg, subcutaneous) was paired with sweetened milk. Taste aversion conditioning was carried out according to the general procedure used for experiment 2 except for the absence of food deprivation and placement in the Skinner box after presentation of the milk solution and AVP injection. Each of the three injected groups was subdivided into two subgroups: a footshock (FS) and a nonfootshock (non-FS) subgroup. The major results of this experiment indicated that (1) after the initial taste aversion conditioning trial, milk consumption decreased in a dosedependent manner in both groups treated by AVP, but increased in rats injected with vehicle. Thus, under conditions in which the subjects were neither food nor water deprived, the lower dose of AVP (5 g/kg) produced a CTA; and (2) FS did not potentiate the taste aversion developed in response to AVP treatment; in fact, regardless of AVP or vehicle injection, all the subjects given inescapable footshock after each conditioning session tended to show an increase in milk consumption compared with their no-shock partners. The inability of FS to potentiate the CTA produced by AVP was interpreted as probably indicative of the fact that the interoceptive signals elicited by AVP differ from those of the emetic agents for which FS does potentiate the taste aversion effect. The authors noted that AVP along with a variety of other agents including emetics, toxins, and even self-administered drugs (e.g., morphine and amphetamine) are capable of inducing a CTA. The mechanisms underlying their ability to produce a CTA probably differ from each other. The authors speculated that the visceral effects associated with increased blood pressure as well as other effects [e.g., release of adrenocorticotropic hormone (ACTH)] by which a behaviorally active dose of AVP may induce an altered state of arousal and alertness may be sufﬁciently aversive to account for the CTA produced by AVP. 2. Bluthe et al. (1985a) Bluthe et al. (1985a) provided evidence supporting the view that the V1 AVP receptor is the peripheral receptor mediating the aversive properties of peripherally circulating AVP. As noted in Chapter 1, the two known types of peripheral AVP receptors have been categorized as V1 and V2 receptors.

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V1 receptors, located in liver cells and in the smooth muscles of blood vessels, mediate, respectively, the glycogenolytic and pressor effects of the peptide. V2 receptors, located in the cells of the kidney tubules, mediate the antidiuretic function of AVP. This study consisted of several experiments designed to establish whether one or both of these receptors are involved in mediating the aversive properties of peripherally administered AVP (e.g., Dantzer et al., 1982). Adult male Wistar rats served as subjects and conditioned taste aversion (CTA) was used to assess the aversive properties of the VP peptides tested in this study. CTA was assessed by either the one-bottle consumption technique or the two-bottle preference technique. The former was used to assess the aversiveness of AVP and the V2 agonist 1-desamino-8-d-arginine vasopressin (DDAVP), and the ability of the vasopressor antagonist [dPTyr(Me)]AVP to block an AVP-induced CTA. The two-bottle preference test was used to study the speciﬁcity of the pressor antagonist to block the AVP-induced CTA. The one-bottle technique for testing a CTA was used in experiments 1, 2, and 3. In all three experiments, aversion conditioning was carried out for a consecutive 5 days, during which the drug or vehicle was administered directly after a 15-min session of drinking sweetened milk, given at the same time every day. Fluid intake of a sucrose solution on each of 2 days before conditioning, and of the sweetened milk solution during the ﬁve conditioning days, was measured. The acquisition of a taste aversion is indicated by a progressive decline in milk consumption over successive days of conditioning. The two-bottle preference technique for testing a CTA was used in experiment 4 and involved the same procedure as used by Dantzer et al. (1982, described above). Brieﬂy, the drug or vehicle was injected directly after a 30-min drinking session with a 0.1% saccharin solution (day 1 or 3) and no injection was given on days 2 and 4; the preference test, given on day 5, indicated saccharin intake relative to total ﬂuid intake after the two conditioning trials. Experiment 1 was designed to verify the observation that peripherally injected AVP displays aversive properties (Dantzer et al., 1982, cited earlier) and establish a dose–effect relationship for this peptide. Depending on the subgroup, the subjects were injected with vehicle or AVP (0.6, 3, or 15 g/kg, subcutaneous) immediately after each daily 15-min drinking session. AVP produced a CTA at a dose level of 15.0 g/kg but not at a dose level of 3.0 or 0.6 g/kg. Experiment 2 was designed to determine whether the nonpressor V2 receptor agonist DDAVP can produce a CTA. Depending on the group, vehicle (physiological saline) or DDAVP (25 g/kg, subcutaneous) was injected after the daily 15-min drinking session with sweetened milk. This V2 agonist, which is devoid of pressor activity, did not induce a CTA even at a high dose level of 25 g/kg. Experiment 3 tested the ability of the V1 (pressor) antagonist [dPTyr (Me)]AVP, at an antagonist-to-agonist dose ratio of 5:1, to block the ability of

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AVP to produce a CTA. Speciﬁcally, four groups of rats each received aversion training with vehicle (physiological saline) or AVP (10 g/kg) preceded 5 min earlier by an injection of saline or [dPTyr(Me)]AVP (50 g/kg). Rats in the saline/AVP group drank signiﬁcantly less sweetened milk than those of the three other groups, and the [dPTyr(Me)]AVP/AVP group did not signiﬁcantly differ in their milk consumption from the groups given saline treatment. This result indicated that the V1 antagonist blocked the AVP-induced CTA at the same dose ratio at which it blocked the pressor effects of the peptide. A two-bottle preference test was used in experiment 4 to test the speciﬁcity of the ability of the V1 antagonist to block the CTA produced by AVP (i.e., inhibit the aversive properties of the peptide). This was done by comparing the ability of [dPTyr(Me)]AVP to block a CTA produced by AVP as well as by the emetic agent apomorphine (APO). Conditioning began after the rats were trained to drink their daily amount of water during a 30-min drinking session. A taste aversion to a 0.1% saccharin solution was conditioned by pairing saccharin intake with either a subcutaneous injection of AVP (10 g/kg, subcutaneous) or APO (0.5 mg/kg, subcutaneous) on days 1 and 3 of the 5-day training/testing period according to the procedure used by Dantzer et al. (1982, described above). The ability of the pressor antagonist [dPTyr(Me)]AVP (50 g/kg, subcutaneous) to block this aversive conditioning with AVP or APO was tested in two subgroups. Speciﬁcally, on conditioning days, the subjects received two subcutaneous injections, separated by a 5-min interval, directly after the 30-min drinking session. Depending on the subgroup, these two subcutaneous injections were either saline/saline, saline/AVP (10 g/kg), saline/APO, [dPTyr(Me)]AVP/AVP, or [dPTyr(Me)]AVP/APO. The two-bottle preference test was given on day 5. Both AVP and apomorphine produced a CTA. However, the V1 pressor antagonist blocked only the AVP-induced CTA, indicating the speciﬁcity of this V1 pressor antagonist for blocking the aversive effects of AVP. Two major conclusions were drawn from these results: (1) the observation that a V2 receptor agonist that lacks pressor effects is unable to produce a CTA indicates that the antidiuretic effect of AVP is not responsible for its aversive effects; and (2) the speciﬁcity with which the V1 receptor antagonist [dPTyr(Me)]AVP blocked the AVP-induced CTA, at a dose ratio of 5:1, which completely blocks the pressor response of AVP, suggests the possibility of a linkage between the pressor action and the aversive properties of AVP. 3. Bluthe et al. (1985b) Bluthe et al. (1985b) designed several experiments to provide further support for the relationship between the pressor and aversive properties of peripherally administered AVP, suggested by earlier ﬁndings (Bluthe et al., 1985a). The observation that repeated preexposure to an aversive drug treatment lessens the subsequent CTA induced by that drug (Cappell et al., 1975) provided the basis for the hypothesis tested in this study. That is, if the pressor

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effects are responsible for the aversive properties of AVP, then preexposure to this peptide or to another pressor agent, but not to a similarly noxious nonpressor agent, should lessen the effects of the subsequent treatment. This hypothesis was tested by means of several experiments that evaluated aversiveness by means of the one-bottle conditioned test for the CTA. The results of experiment 1 showed that prior exposure to AVP, but not the noxious nonhypertensive agent apomorphine, inhibited the CTA produced by peripherally administered AVP. This outcome was not due to the lack of aversive properties of apomorphine because, on its own, the dose of apomorphine used for pretreatment produced a more intense CTA than did AVP on its own. Moreover, previously reported observations indicated that pretreatment with apomorphine was able to attenuate its own conditioned taste aversion (Brookshire and Brackbill, 1971, as cited by Bluthe et al., 1985b). The results of experiment 2 indicated that peripheral injection of the hypertensive agent angiotensin II (Ang II), as well as AVP, had aversive effects because each of these treatments disrupted scheduled control operant behavior and produced a CTA. Experiment 3 demonstrated that Ang II and AVP were reciprocal in their ability to block the aversive properties of each other in pretreatment–treatment regimens. Thus, pretreatment with Ang II blocked the CTA produced by AVP treatment and vice versa. The results of experiment 4 did not support the argument that the aversive effects of Ang II were simply due to its ability to release endogenous AVP, because pretreatment with the AVP V1 pressor antagonist failed to block the CTA produced by Ang II. In summary, these ﬁndings demonstrated attenuation of the AVP-induced CTA after preexposure to AVP or to the hypertensive agent Ang II, but not to the nonpressor but equally noxious agent apomorphine. This fully reciprocal generalization between AVP and Ang II was interpreted as indicating that the hypertensive action of the two hormones contributed, directly or indirectly, to the interoceptive cues responsible for the CTA that they produced. These investigators thus suggested, ‘‘the interoceptive cues which are responsible for the conditioned taste aversion induced by vasopressin are related to the hypertensive action of this peptide’’ (Bluthe et al., 1985b, p. 31).

V. Research Studies with the Single-Trial Water (Food)-Finding Task

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A. Selected Studies 1. Ettenberg et al. (1983a) Ettenberg et al. (1983a) recognized the desirability of extending the study of the potential role of vasopressin in memory enhancement to an appetitive motivational paradigm, and selected the single-trial waterﬁnding task (task description described in Section II.C). Several additional

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experimental tests were conducted to help clarify the mechanism by which peripherally administered vasopressin may enhance retention performance in this task. Three theoretical explanations were suggested: a direct effect on memory-processing circuitry, an arousal reaction that indirectly aids such processing, and a pure behavioral effect totally independent of any memoryimproving property. Given our incomplete understanding of the precise cellular substrate for memory processing, and therefore an inability to directly test this alternative, the researchers reasoned that a direct effect of the peptide on central memory substrates could remain a viable hypothesis only if one could rule out a peptide-induced indirect effect on memory storage or a purely behavioral, nonmemory factor as a potential explanation for the experimental outcome. Experiments were performed to determine whether improved performance in this test (i.e., a reduced latency to locate the drinking tube when replaced in the water-ﬁnding box 48 h after the posttrial AVP injection) might be attributed to (1) the aversive properties of the AVP treatment producing an arousal-induced facilitation of memory stored during the exploratory trial or (2) a conditioned increase in activity level induced by pairing the arousal effects of the peptide with the cues present in the test environment. The ﬁrst explanation represents a peptideinduced indirect effect on memory storage; the second, a purely behavioral, nonmemorial effect of the peptide. To test the effects of AVP on task retention, the subjects (adult male Wistar rats) received a single posttraining injection of vehicle or AVP (1.2, 6.0, or 20 g/kg, subcutaneous; note that 6.0 g/kg is approximately equal to 1.0 g/rat). Other experiments tested the ability of the nonpressor vasopressin analog desglycinamide vasopressin (DG-AVP; 0, 1.2, 6.0, or 20.0 g/kg, subcutaneous) as well as the highly aversive agent lithium chloride (LiCl; 0 or 50 mg/kg, intraperitoneal) to inﬂuence retention under identical testing circumstances. The ﬁndings indicated that all three doses of AVP signiﬁcantly reduced the latency to relocate the water tube in the retention test. LiCl, the aversive nature of which has been demonstrated in numerous taste aversion studies, improved retention performance in this task to an even greater degree than AVP. On the other hand, none of the doses of DG-AVP affected performance. Peripherally injected AVP, as well as DG-AVP, were tested for their ability to produce a conditioned taste aversion (CTA) and a conditioned place aversion (CPA). For the CTA test involving AVP, the subject received an injection of vehicle or AVP (6 g/kg, subcutaneous) after ingestion of a 0.1% saccharin solution for a 20-min period. Preference testing occurred after two, and then after four daily AVP–saccharin pairings. For the CPA test involving AVP, the rat received a single injection of AVP (1.2 or 6.0 g/kg, subcutaneous, depending on the treatment group) followed by placement into one of two distinctive Plexiglas boxes for 30 min. On the next day, the rat received an injection of vehicle before placement in the other Plexiglas

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box. This procedure was continued for a consecutive 8 days, by which time the subject had received four AVP–place and four vehicle–place pairings. On day 9, the preference-testing day, the rat was offered a choice between the two boxes used during the conditioning phase. The comparable experimental tests employing DG-AVP used vehicle or a dose of 6.0 g/kg for the CTA and vehicle or a dose of 6.0 or 20.0 g/kg for the CPA test. The aversive effect of AVP was indicated by the ﬁnding that AVP (6 g/kg, subcutaneous) produced a CTA after four but not two AVP– saccharin pairings and a CPA at both AVP dose levels, although the higher dose (6.0 g/kg) was more effective than the lower dose (1.2 g/kg) in this respect. On the other hand, neither of the dose levels of DG-AVP produced either a CTA or a CPA. One nonmemory factor that could explain the improved retention performance in this water-ﬁnding task relates to an AVP-induced increase in activity level. That is, arousing or aversive properties of the posttraining injection of AVP could have become paired to environmental cues in the test situation so that when the AVP-treated subjects were returned to the learning environment 2 days later their conditioned heightened activity led them to locate the tube more rapidly than did the vehicle-treated control subjects. Experiments were carried out to determine (1) whether AVP or DG-AVP treatment increases spontaneous locomotor activity, and (2) whether locomotor activity does increase in an environment previously associated with a single injection of AVP. The acute effects on locomotor activity of AVP (0, 1.2, 6.0, and 20.0 g/kg, subcutaneous) and DG-AVP (0, 6.0, and 20.0 g/kg, subcutaneous) were tested in separate experiments with individual activity cages equipped with photocells. Locomotor activity was assessed every 10 min over a 90-min test session. The results were as follows: (1) compared with vehicle controls, AVP dose dependently decreased locomotor activity throughout the ﬁrst half of the 90-min test session; (2) the progressive decline observed in the locomotor activity of the control subjects over the course of the test session apparently contributed to the reduced difference between the vehicle- and peptidetreated rats that occurred during the second half of the test session (i.e., the low level of spontaneous activity in the vehicle-treated subjects made it difﬁcult to observe any further reductions in the AVP-treated groups); and (3) DG-AVP did not signiﬁcantly inﬂuence spontaneous locomotor activity relative to placebo controls. The effect of AVP on ‘‘conditioned locomotor activity’’ was assessed in two experimental tests. The ﬁrst test monitored locomotor activity in the activity cage. A single injection of vehicle or AVP (6.0 g/kg, subcutaneous) was given immediately after removal of the rat from the activity cage. The subject was returned to the home cage, deprived of water for 48 h, and then retested in the activity cage. The second test used the water-ﬁnding task apparatus. AVP (0 or 6.0 g/kg, subcutaneous) was injected on removal

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from the apparatus in which the rat had been placed for a 5-min exploratory period. Unlike the standard water-ﬁnding task paradigm, the water tube was absent during this exploratory period to prevent latent learning of its position. After AVP treatment, the rat was returned to its home cage, water deprived for 48 h, and then returned to the test apparatus, which now contained the water tube. Latency to locate the water tube was recorded. The results of this experimental test indicated no evidence of an AVP effect on conditioned locomotor activity in either the activity cage (i.e., no increased activity relative to controls when returned to the activity cage) or in the water-ﬁnding test apparatus (i.e., no signiﬁcant difference between AVP-treated rats and saline controls in the latency to locate the water tube). The overall results of this study may be summarized as follows: (1) peripherally administered AVP improved retention performance in a single-trial appetitive task; (2) this improved retention cannot be attributed to a nonmemory effect involving a conditioned locomotor activity effect; (3) at a behaviorally active dose, peripherally administered AVP appears to have aversive effects, as indicated by its ability to promote a conditioned taste and place aversion. The decrease in spontaneous locomotor activity that followed acute treatment with AVP was interpreted as being consistent with this interpretation. However, if the aversive effects of the peptide induced an increase in behavioral arousal, one might expect an increase rather than a decrease in spontaneous locomotor activity after injection of a behaviorally active dose of the peptide. On the other hand, behavioral depression occurring shortly after an acute injection of AVP has also been observed by other investigators using appetitive motivational paradigms (e.g., Van Haaren et al., 1986; discussed in Chapter 9); and (4) the aversive agent lithium chloride (LiCl) was even more potent than AVP in enhancing retention performance in this appetitive task. Moreover, the effect on retention induced by LiCl treatment may be related to an aversively produced arousal effect, because lithium treatment has been demonstrated to increase selective attention in rats (Cappeliez and White, 1982). On the other hand, the nonaversive agent DG-AVP failed to inﬂuence retention, at least in this appetitive task. 2. Ettenberg et al. (1983b) Ettenberg et al. (1983b) attempted to further clarify the mechanism by which peripherally administered AVP can enhance retention behavior in the single-trial appetitive water-ﬁnding task. Earlier study with this task established support for a correlation between the aversive properties and the retention performance effects of peripherally administered AVP (Ettenberg et al., 1983a; see above). The objective of this study, however, was to relate the pressor properties of peripherally acting AVP to the behavioral effects it produces in this appetitive task. Accordingly, the ability of a peripheral injection of the V1 receptor pressor antagonist [dPTyr(Me)]AVP to block

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the behavioral effects of this peptide in this water-ﬁnding task was tested. Earlier experimental ﬁndings by this research team had demonstrated the ability of this antagonist to block both the pressor and retention performance effects of the peptide in a multitrial avoidance task (Le Moal et al., 1981). This study conﬁrmed the observation that peripheral AVP [1 g/rat (i.e., roughly 6 g/kg), subcutaneous] facilitates retention in this task and further showed that pretreatment with a selective V1 antagonist, [dPTyr(Me)]AVP, completely blocked the AVP-induced retention effect at a dose of 25 g/kg but not 5.0 g/kg. Thus, for this single-trial learning task, effective prevention of the AVP-induced retention effect requires a much higher antagonistto-agonist dose ratio (25:1) than that for the multitrial pole-jump avoidance task, which required the same 5:1 antagonist-to-agonist ratio as that needed to prevent the pressor effect produced by a behaviorally active dose of the peptide. A similar antagonist-to-agonist ratio of 25:1 is necessary for effective prevention of the VP-induced retention effect in the single-trial passive avoidance task (Lebrun et al., 1984). 3. Ettenberg (1984) Ettenberg (1984) conducted three experiments using interactional effects between a centrally injected V1 antagonist and peripherally injected AVP to test the hypothesis that the retention-improving properties of peripherally injected AVP are related to its aversive and hence arousal-inducing actions. In the introduction section of the study, it was noted that the aversive effects of peripherally administered AVP are presumably due to the pressor-inducing action of the peptide, because prevention of the pressor response also removes the aversive effects (Bluthe et al., 1985a; Dantzer et al., 1982). In addition, the assumed linkage between the aversive properties of stimulation and increased arousal was explicitly expressed by the author’s statement that ‘‘aversive stimuli are inherently arousing by nature’’ (Ettenberg, 1984, p. 202). In the ﬁrst experiment the generality of the AVP inﬂuence on an appetitive task was tested by adapting the water-ﬁnding task for use with a food reward (sweetened milk). Substitution of food for the water reward avoids the problem that water deprivation itself can release endogenous AVP (Hays, 1980, as cited by Ettenberg, 1984). The degree to which the behavioral effect induced by peripherally injected AVP (6 g/kg, subcutaneous) could be reversed by pretreatment with the centrally (intracerebroventricularly) injected pressor antagonist [dPTyr(Me)]AVP (0, 1, 5, or 10 g/kg) was also examined in this experiment. The results indicated that AVP facilitated memory for the location of the feeding tube when the reward was food and that pretreatment with the pressor antagonist attenuated this behavioral effect at a dose of 5 g/ kg and completely blocked it at the 10-g/kg dose level. The second experiment was designed to replicate the observation that peripherally administered AVP (0 or 6 g/kg, subcutaneous) can establish a

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CTA (Ettenberg et al., 1983b), and to determine whether pretreatment with a centrally administered pressor antagonist, [dPTyr(Me)]AVP (0, 1, 5, or 10 g/kg, intracerebroventricular), could block this CTA. Four AVP–saccharin pairings resulted in a profound reduction in the amount of saccharin consumed during the saccharin-versus-water preference test. This AVP-induced CTA was prevented only in rats pretreated with the highest dose of the centrally administered V1 antagonist (10 g/kg, intracerebroventricular). The objective of the third experiment was similar to that of the second experiment except that the behavioral task was a conditioned place aversion (CPA). The procedure involved four pairings of peripherally injected AVP with placement in a distinctive environment (a black or white box). The AVP treatment resulted in a CPA that was dose dependently attenuated by pretreatment with the centrally administered pressor antagonist [dPTyr (Me)]AVP. Here, too, only the highest dose of the antagonist completely blocked the AVP-induced CPA. These investigators interpreted their results as supporting the proposition that the retention effect produced by peripherally administered AVP is attributable to the aversion-induced increase in arousal level that results from the pressor response evoked by the peptide. The aversive nature of peripherally injected AVP was demonstrated by the ability of the peptide to produce both a CTA and a CPA. That this aversive effect was responsible for the AVP-induced behavioral effect was indicated by the observation that only that dose of the antagonist that was able to block the aversive effects of AVP was also able to prevent the peptide-induced retention effect. In addition, the observation that full antagonism of the aversive and behavioral effects of the AVP treatment required an antagonist dose level in the microgram, rather than the nanogram, range was interpreted as support for the proposition that the antagonist interacted with a peripheral receptor site far removed from the lateral ventricle where the V1 antagonist was injected.

VI. Research Study with the Radial Maze Task

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A. Selected Study: Packard and Ettenberg (1985) Packard and Ettenberg (1985) employed an eight-arm radial maze to investigate the effect of peripherally administered AVP and the nonpressor analog DG-AVP on extinction of a spatially learned response reinforced by food reward. Unlike extinction testing in an avoidance task, where an opportunity to learn about the changed reinforcement contingency occurs only after an error, each response given during extinction testing in an appetitive task informs the subject that the originally learned response is no longer rewarded. Given this distinction it should be expected that, when a subject is treated with an agent that facilitates memory formation immediately after an

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extinction experience in an appetitive task, the agent should facilitate rather than prolong the subsequent extinction of the response. For this task, treatment with AVP and DG-AVP was given after an initial extinction trial during which the subject had the opportunity to experience the changed contingency (i.e., entry into each of the eight arms of the radial maze no longer resulted in a food reward). The procedure used for training and testing the subjects (male SpragueDawley rats) in this radial maze task is described in Section II.D. of this chapter. Depending on the group, the rat received a single injection of vehicle, AVP (1.0 or 5.0 g/kg, subcutaneous), or DG-AVP (1.0, 5.0, or 10.0 g/kg, subcutaneous) after completion of the initial extinction trial in the radial maze task. The animal was injected in a room adjacent to the test room and returned to its home cage. For each of these extinction trials, the rat was placed in the empty maze, and the number of arms entered during each of the 5-min extinction trials was recorded. The effect of these peptide treatments on spontaneous locomotor activity was tested 2 weeks after the completion of extinction testing in approximately half the subjects used in the radial maze task. Five instead of the original six treatment groups were formed at this time: saline, AVP (1.0 or 5.0 g/kg, subcutaneous), or DG-AVP (5.0 or 10.0 g/kg, subcutaneous). Each animal received the same treatment it had received during extinction training. At 2 h and again at 4 h postinjection, unconditioned locomotor activity was monitored over a 5-min period for each subject. Analysis of the data for the radial maze task indicated the following results: (1) AVP-treated rats did not differ from saline controls in the ﬁrst extinction trial given after peptide treatment, but did exhibit a reliably faster rate of extinction thereafter; this observation was interpreted as indicating that the postextinction AVP treatment potentiated the new learning that was occurring over the successive extinction trials; and (2) the rats that received DG-AVP treatment, at the higher dose levels of 5 and 10 g/kg, exhibited a higher rate of response in the maze relative to the saline controls. This disruption of the extinction process occurred at the ﬁrst postinjection extinction trial for both dose levels and at all subsequent extinction trials for the highest dose level (10 g/kg). Analysis of the data collected for the unconditioned locomotor activity test indicated that (1) the AVP-treated rats did not differ from the saline controls in tests of locomotor activity for either the 2- or 4-h posttreatment time points, indicating that nonspeciﬁc effects of AVP on locomotor activity did not contribute to extinction performance; and (2) DG-AVP at the dose levels of 5 and 10 g/kg produced a dose-dependent and statistically signiﬁcant increase in locomotor activity at both the 2- and 4-h posttreatment time points; it was suggested that the inﬂuence of this peptide on unconditioned locomotor activity may have contributed to the enhanced response that these animals exhibited during extinction testing in the radial maze task.

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Taken together, the results indicated that AVP and the nonpressor analog DG-AVP had opposing effects on extinction of a previously learned food approach response in the eight-arm radial maze. Interpreting a more rapid rate of extinction as indicating a faster rate of learning about the new response–outcome relationship, it was clear to the authors that AVP treatment given immediately after an initial extinction session potentiated the new learning that occurred during subsequent extinction trials. On the assumption that the postextinction trial injection of AVP reinforced the memory consolidation of the most recently learned information, this result was consistent with previous ﬁndings indicating an AVP-induced facilitation of retention. Moreover, a number of observations suggest that the reduced responding during extinction in the AVP-treated rats should not be attributed to an AVP-associated depression of general activity (e.g., see the results of the locomotor test) or to aversive effects of the peptide, because the postinjection extinction trials did not begin until 2 h after the injection, a time when endocrinologically associated aversive actions of AVP have long since subsided (Le Moal et al., 1981). The failure of DG-AVP to potentiate the new learning during extinction is consistent with a previously observed inability of this nonpressor vasopressin analog to enhance retention in the single-trial water-ﬁnding task (Ettenberg et al., 1983a). The observation that DG-AVP-treated rats actually made more arm entries during extinction than did saline controls may have been due to the effect of the peptide on the activity level of the subjects. The present ﬁndings suggesting a DG-AVP-induced increase in activity level is at variance with a previous report by this research team (Ettenberg et al., 1983a), which indicated no statistically signiﬁcant change in spontaneous locomotor activity measured over a 90-min observation period that began immediately after injection. Procedural factors may have contributed to discrepant ﬁndings of the two studies (e.g., interval between injection and locomotor activity assessment). However, there have been other observations suggesting that DG-AVP increases arousal level (Skopkova et al., 1987, 1991; see Chapter 3). If so, the arousal enhancement effect of the parent peptide would appear not to be completely dependent on its pressor effect. The inconsistency with which DG-AVP induced facilitation of retention has been reported in the literature [i.e., positive effects reported by Bohus, 1977 (see Chapter 2); De Wied et al., 1972; Gaffori and De Wied, 1985 (see Chapter 3); and failures reported by Ettenberg et al. (1983a), Packard and Ettenberg (1985; this chapter), and Sahgal (1987a; see Chapter 7)] could indicate an arousal-mediated effect on retention (Sahgal, 1984; see Chapter 7). Alternatively, the arousal effects induced by AVP and, perhaps, by DG-AVP could be independent of the inﬂuence of these peptides on memory storage per se (Gaffori and De Wied, 1985; Skopkova et al., 1991; see Chapter 3).

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The majority of studies designed by Koob and colleagues in connection with the role of vasopressin in memory processing have been behavioral pharmacological studies. However, the two studies discussed below have used physiological paradigms. Each study was designed to obtain evidence on the issue of whether peripheral or central receptor sites mediate the behavioral effects of peripherally administered vasopressin on learning and memory tasks. Thus, according to the ‘‘VP Dual Action Theory,’’ the retention effect induced by peripherally administered AVP is due to the sequelae that follow from activation of peripheral and not central V1 receptors.

A. Evidence Relevant to the Issue of Whether VP Crosses the Blood–Brain Barrier 1. Selected Study: Deyo et al. (1986) Deyo et al. (1986) designed experiments to determine whether an experimentally induced increase in peripherally circulating vasopressin might penetrate the blood–brain barrier (BBB) to reach central receptors in the brain. The level of plasma AVP was increased either by osmotic stimulation (water deprivation) or by a peripheral injection of AVP. The ability of the increased level of plasma AVP to penetrate the BBB was determined by radioimmunoassay (RIA) analysis of AVP concentration in the brain. Specifically, immunoreactive (ir) AVP concentration was measured both in the plasma and in various regions of the brain, some of which were protected by the BBB (thalamus–hypothalamus, amygdala–temporal cortex, hippocampus, and remaining cerebral cortex) whereas others were circumventricular organs, located outside the BBB (median eminence and area postrema). Adult male Wistar rats were either peripherally injected with AVP (0.0, 0.05, 0.50, or 5.0 g/kg, subcutaneous) or water deprived for either 24 or 48 h. After treatment, the animals were decapitated. Immediately thereafter, trunk blood was collected for plasma samples and the brain was dissected to assess the concentration of immunoreactive irAVP in the target regions selected for study. A brain perfusion procedure was performed only on those subjects that were injected with the 5-g/kg dose of the peptide to remove the blood-borne AVP (i.e., AVP lodged within brain capillaries). This control procedure permitted assessment of the degree to which the immunoreactive AVP of the assay indicated an accumulation of immunoreactive AVP in the microvasculature of the brain rather than in the brain tissue itself. The following results were obtained for experiments involving peripherally injected AVP and the brain perfusion control procedure: (1) AVP dose dependently increased the concentration of plasma immunoreactive AVP and this was statistically signiﬁcant for each dose level; (2) the plasma levels

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of irAVP peaked 5 min after the subcutaneous injection and declined in a biphasic manner over the next 115 min; (3) depending on the dose and brain region sampled, a subcutaneous injection of AVP elevated immunoreactive AVP to a level three to nine times greater than that which occurred in vehicle controls; (4) the concentration of AVP in brain tissue samples peaked 20 min after the subcutaneous injection of AVP, and the rate of decline was the same as that occurring in the plasma for brain regions protected by the BBB (the time course of decline differed for the brain regions lying outside the BBB); and (5) brain perfusion, which removed the blood-borne AVP, also reduced to normal control levels the concentration of AVP in the brain regions protected by the BBB (i.e., thalamus–hypothalamus, amygdala–temporal cortex, and hippocampus). The following results were obtained for the osmotically induced release of AVP: (1) plasma irAVP was signiﬁcantly increased, relative to nondeprived subjects, after 48 h of water deprivation; however, this increase in plasma irAVP was much less than that induced by peripherally injected peptide; and (2) both levels of water deprivation signiﬁcantly increased irAVP in the hypothalamus and the amygdala–temporal areas, but not in the other brain regions sampled. The authors drew the following conclusions from the results of this study: (1) the difference in the time course at which AVP reached peak accumulation in plasma versus the brain (i.e., 5 versus 20 min) represented the complex shift of AVP from circulating plasma to the blood vessels of the brain and in the regions lying outside the BBB to the brain cells themselves; (2) the increase in irAVP observed for the brain regions protected by the BBB resided chieﬂy within the microvasculature of these areas; this conclusion was based on the lack of differences in irAVP levels in the brains of AVPinjected and vehicle-injected controls when the brains of AVP-injected subjects were perfused of blood before dissection; and (3) the elevated AVP in brain regions after water deprivation differed from that produced by systemic injection of large doses of AVP and was considered to be a reﬂection of direct effects of dehydration on AVP-containing neurons within these brain regions. This latter conclusion is consistent with the observation that osmotic stimulation elevates endogenous AVP within the brain as well as within the plasma (Koob et al., 1985a).

B. Electroencephalographic Activational Effect Induced by Behaviorally Active Doses of Peripherally or Centrally Administered AVP 1. Selected Study: Ehlers et al. (1985) Ehlers et al. (1985) carried out a spectral analysis of electroencephalographic (EEG) activity in awake, freely moving male adult rats of the Wistar strain. The study had two objectives: (1) to compare the effects on EEG

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activity of behaviorally active doses of AVP given by peripheral and central routes of administration; and (2) to investigate EEG changes, given a report that multiple central injections of high doses of AVP can induce behavioral seizures (Kasting et al., 1980). Because only the ﬁrst objective has direct relevance for the ‘‘VP Dual Action Theory,’’ only those experimental ﬁndings relevant to it are described. EEG activity was tested in two groups of rats, each of which received either two central or two peripheral injections. The two peripheral injections were spaced 1 week apart, and the two central injections were 1 h apart. Half the rats in the peripherally injected group received two injections of saline, and half were given one injection of saline and one injection of AVP (6 g/kg, subcutaneous). The rats receiving central injections of AVP were subdivided into several subgroups and, depending on the subgroup, the subject received either two injections of saline, or one injection of saline and one intracerebroventricular injection of AVP at a dose level of 0.1, 0.5, or 1.0 ng/rat. For all treatment subgroups, the computerized EEG data were transformed and compressed into six frequency bands (1–2, 2–4, 4–6, 6–8, 8–16, and 16–32 cycles per second [cps]) and mean power density was calculated for each frequency band. The results indicated some similarity in the distribution of spectral power for various frequency bands of EEG activity between the groups receiving the behaviorally active doses of peripherally and centrally injected AVP. When peripherally injected, AVP produced a signiﬁcant decrease in spectral power in the low- and moderate-frequency ranges (2–4 and 8–16 cps). When centrally injected, peptide at the behaviorally active dose of 1.0 ng/rat produced a signiﬁcant decrease in all frequency bands except for the lowest (1–2 cps) frequency band. On the other hand, at a dose level of either 0.05 or 0.1 ng/rat, centrally injected AVP signiﬁcantly decreased spectral power only in the highest frequency range (16–32 cps), exhibiting no similarity to the EEG activity produced by peripherally injected peptide. The authors noted that Urban and De Wied (1975, 1978) and Urban (1981) had reported several research ﬁndings suggesting a link between the EEG theta rhythm and brain vasopressin. First, hippocampal theta rhythm was observed to be slower during paradoxical sleep for rats severely deﬁcient in brain AVP, and AVP replacement therapy helped to correct this abnormality (Urban and De Wied, 1975, 1978; see Chapter 5). Second, picogram amounts of vasopressin microinjected into the septal area of the brain of normal rats were observed to increase the average frequency of hippocampal theta activity during paradoxical sleep (Urban, 1981; see Chapter 5). In the present study, neither central nor peripheral AVP treatment produced a shift in theta activity, but these subjects were fully awake and the EEG recordings were made from the cortex and not from the hippocampus. However, the behaviorally active doses of AVP did decrease overall spectral power in selective frequency ranges, and the decreases in EEG power in the 2–4 and

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8–16 cps frequency bands were interpreted as suggesting that ‘‘the AVPtreated rats were less drowsy during the recording periods than rats given only saline injections’’ (Ehlers et al., 1985, p. 432). Although both peripheral and central injection of a behaviorally active dose of AVP produced EEG changes with some overlap, the authors noted that there are two reasons why it is unlikely that these EEG effects could be attributed to a common neural mechanism. First, it is highly unlikely that peripherally administered AVP, at the dose level used here, penetrated the BBB (Deyo et al., 1986, cited above). Second, although an increase in blood pressure may have been involved in mediating the inﬂuence on EEG activity resulting from AVP injected peripherally at the 6-g/kg dose level (Le Moal et al., 1981), this could not have occurred for the centrally injected 1-ng dose of the peptide (Lebrun et al., 1985). These considerations led the authors to conclude that the two routes of injection may have produced a ‘‘common brain state’’ as reﬂected in the EEG activity, but did so by different mechanisms.

VIII. The ‘‘VP Dual Action Theory’’

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A. Opposing Views Concerning the Means by Which Exogenous Vasopressin Influences Retention Behavior While the De Wied et al. and Koob et al. research teams both reported that centrally or peripherally administered vasopressin facilitated retention performance in aversive and appetitive learning paradigms, they disagreed about the mechanisms responsible for this memory effect. According to the De Wied et al. ‘‘VP/OT Central Memory Theory,’’ either peripherally or centrally administered VP acts directly on central AVP receptors. In contrast, the Koob et al. ‘‘VP Dual Action Theory’’ proposes that different mechanisms mediate the memory-facilitating effects produced by the two routes of administration. According to Koob and associates: ‘‘there are two AVP systems operating on behavior, one central and the other peripheral, although these systems may ultimately act in a similar behavioral substrate, they do so by different and independent mechanisms’’ (Koob et al., 1985b, pp. 196–197). The ‘‘behavioral substrate’’ in this quotation is understood to refer to the central arousal system (Dantzer, personal communication, 1998). Of crucial importance to these opposing theoretical viewpoints are their positions on whether the peptide can cross the blood–brain barrier. Adherents of the ‘‘VP/OT Central Memory Theory’’ assume that vasopressin, or one of its behaviorally active metabolites, is able to penetrate the blood–brain barrier (see Chapters 2–5). Proponents of the ‘‘VP Dual Action Theory’’ argue that this is highly improbable. Deyo et al. (1986) designed a study to help clarify this issue and found no evidence that a behaviorally active dose of peripherally injected AVP was able to pass the blood–brain barrier and enter CNS tissues. Because of its importance to this ﬁeld of study, experimental

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evidence relating to the interaction of vasopressin with the blood–brain barrier is more fully discussed in Chapter 14.

B. Propositions of the ‘‘VP Dual Action Theory’’ On the working assumption that peripherally administered vasopressin does not penetrate the blood–brain barrier, the ‘‘VP Dual Action Theory’’ proposed that behaviorally active peripheral doses of the peptide act on peripheral vascular receptors to increase blood pressure, resulting in a ‘‘visceral arousal’’ effect. However, when injected intraventricularly or directly into brain sites, its inﬂuence on retention is mediated by central VP receptors directly involved in the modulation of central arousal (Dantzer, 1998). While the ‘‘VP Dual Action Theory’’ has much in common with the Sahgal et al. ‘‘VP Central Arousal Theory’’ (see Chapter 7), the latter does not elaborate the means by which peripherally administered VP can bring about a central arousal effect. Moreover, the Sahgal et al. theory emphasizes the inverted U-shaped relation between arousal and performance in predicting the effects of the peptide on behavior. For discussion purposes the various proposals in the writings of Koob and associates relevant to the long-term memory research covered in this chapter are now formulated as a series of propositions, as was done for the ‘‘VP/OT Central Memory Theory’’ (see Chapters 2–5). 1. Proposition 1: The Posttraining Retention Effect Induced by Treatments That Increase Peripherally Circulating AVP Is Unlikely to Be Due to a ‘‘Nonmemorial’’ Performance Effect These investigators have attempted to control for nonmemorial performance factors by careful consideration of their testing procedures and by experiments speciﬁcally designed to test this possibility. Examples of these control procedures and tests are illustrated below. First, the probability of short-acting acute effects of peripherally administered AVP that could inﬂuence learning or retrieval was prevented by testing at times when these effects were not present. It has been observed that the pressor effects produced by behaviorally active doses (e.g., 5 and 15 g/kg, subcutaneous) of peripherally injected AVP dissipate 1 h after injection (Le Moal et al., 1981). Retention testing was thus initiated no earlier than 2 h after vasopressin treatment in the pole-jump shock avoidance task (e.g., Koob et al., 1981) and in the radial maze task (Packard and Ettenberg, 1985), and no earlier than 24 h after AVP treatment in the single-trial inhibitory avoidance task (e.g., Lebrun et al., 1984) and in the appetitive water-ﬁnding task (e.g., Ettenberg et al., 1983a,b). Second, several control experiments helped to rule out long-acting nonmemorial conditioned-performance effects. For example, Ettenberg and

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colleagues (1983a) eliminated the possibility that heightened locomotor activity, conditioned to cues in the water-ﬁnding task environment, was responsible for the speed with which the AVP-treated rats were able to locate the water tube in the retention test. 2. Proposition 2: Increased Blood Pressure Is an Intervening Causal Factor in the Learning/Memory Effects Induced by Treatments That Lead to High Levels of Peripherally Circulating VP Supportive evidence for proposition 2 comes from studies demonstrating that doses of peripherally injected AVP that facilitate retention also increase systolic blood pressure, and a vasopressin antagonist ([dPTyr (Me)]AVP) that inhibits the pressor action of the peptide also blocks this retention effect. Further, a nonpressor AVP agonist, DG-AVP, failed to enhance retention in two appetitive learning paradigms used in their studies. The following ﬁndings illustrate these points: (1) peripherally administered AVP at a dose level that facilitated retention (prolonged extinction) of a conditioned pole-jump avoidance response also increased blood pressure (Le Moal et al., 1981); (2) pretreatment with the peripherally injected VP antagonist [dPTyr(Me)]AVP blocked VP-facilitated retention in the multitrial pole-jump shock avoidance task at the same antagonist-to-agonist dose ratio (5:1) that blocked the pressor response (Le Moal et al., 1981); (3) pretreatment with this VP antagonist also blocked VP-facilitated retention in the singletrial passive avoidance (Lebrun et al., 1984) and appetitive water-ﬁnding (Ettenberg et al., 1983b) tasks, albeit at an antagonist-to-agonist dose ratio (25:1) considerably higher than that required to suppress the VP-induced pressor response; and (4) peripherally injected DG-AVP failed to facilitate retention in a single-trial water (food)-ﬁnding task (Ettenberg et al., 1983a) or in an eight-arm radial maze task (Packard and Ettenberg, 1985). 3. Proposition 3: An Experimental Treatment, Which Increases Plasma VP to a Sufficient Degree to Influence Learning and Memory, Results in Aversive Stimulus Properties Causally Linked to the VP-Induced Pressor Effect Proposition 3 is supported, in part, by experimental results demonstrating that a behaviorally active dose of peripherally administered AVP is an aversive stimulus. This aversive effect is illustrated in the following examples: (1) a peripheral injection of AVP induced a conditioned taste aversion (CTA) after being paired with saccharin and tested in a two-bottle preference test, or with sweetened milk and tested in a single bottle test (Dantzer et al., 1982); and (2) a behaviorally active dose of peripherally administered AVP that facilitated retention in the water (or food)-ﬁnding task also produced a CTA and a conditioned place aversion (CPA) (Ettenberg et al., 1983a). A number of other experimental observations by this research team, if not directly supportive, were at least consistent with the hypothesized causal

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linkage between the pressor and aversive effects of peripherally administered behaviorally active doses of AVP: (1) peripherally administered AVP at a dose level that facilitated retention also increased blood pressure and produced a CTA (Dantzer et al., 1982). Moreover, Ang II, another hypertensive agent, also produced a CTA (Bluthe et al., 1985b); (2) a V2 receptor agonist, which mediated the antidiuretic, but not the pressor, effects of VP, failed to produce a CTA (Bluthe et al., 1985a); (3) the V1 antagonist [dPTyr (Me)]AVP, which blocks the pressor response to AVP (Le Moal et al., 1981), also prevented its ability to produce a CTA (Bluthe et al., 1985a; Ettenberg, 1984) and a CPA (Ettenberg, 1984); (4) the attenuated VPinduced CTA that results from prior exposure to the peptide occurs after pretreatment with Ang II but not apomorphine (Bluthe et al., 1985b); and (5) the CTA produced by apomorphine (a nonpressor aversive drug) was insensitive to the V1 antagonist (Bluthe et al., 1985a), and footshock potentiated an apomorphine-produced, but not an AVP-produced CTA, suggesting that the aversive effects of the two treatments were qualitatively dissimilar (Dantzer et al., 1982). 4. Proposition 4: The Aversive/Pressor Accompaniments of High Levels of Peripherally Circulating VP Produce Arousal Effects That, in Turn, Mediate the Effects of the Peptide on Learning/ Memory in Avoidance and Appetitive Learning Tasks Two types of observations suggest that a dose of peripherally administered AVP, which has pressor- and retention-enhancing properties, increases the level of central arousal of subjects. First, Ehlers et al. (1985) observed that a peripheral injection of AVP (6.0 g/kg; i.e., approximately 1 g/rat) that increases blood pressure and improves retention on avoidance (Lebrun et al., 1984; Le Moal et al., 1981) and appetitive (Ettenberg, 1984) paradigms, produced an EEG proﬁle suggesting that ‘‘the rats given AVP were less drowsy during the recording periods than rats given only saline injections’’ (Ehlers et al., 1985, p. 432). Second, Lebrun et al. (1984) observed that a behaviorally active dose of peripherally administered AVP (1 or 3 g/rat, subcutaneous) produced a bimodal performance effect in the inhibitory avoidance task, consistent with the ‘‘VP Central Arousal Theory’’ formulated by Sahgal and associates (see Chapter 7). 5. Proposition 5: The Learning/Memory Effects Observed after Experimental Treatments That Increase Brain Levels of VP Are Due to a Direct Action on the Neurophysiological Substrates of the Arousal System Taken together, the following observations by these researchers support proposition 5: (1) a behaviorally active dose of centrally injected AVP (1.0 ng/rat, intracerebroventricular) facilitated retention on an avoidance (Koob et al., 1981) learning task, but without the concomitant pressor or

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aversive effects (Koob et al., 1986) that accompany a behaviorally active peripheral dose (6 g/kg, subcutaneous) (Ettenberg et al., 1983a,b; Le Moal et al., 1981); and (2) Ehlers et al. (1985) observed similar EEG proﬁles in rats injected peripherally and centrally with behaviorally active doses of AVP (i.e., 6 g/kg, subcutaneous and 1 ng/rat, intracerebroventricular). These data were interpreted as reﬂecting a common brain state (cortical activation, the neural substrate of behavioral arousal during waking behavior) directly activated by centrally applied VP, but indirectly activated by the pressor/aversive actions of peripherally administered AVP. An additional source of support for a VP-induced arousal effect is provided by evidence obtained by De Wied and colleagues. Urban and De Wied (1975, 1978; see Chapter 5) reported evidence that both endogenous and exogenous VP inﬂuence hippocampal theta activity during paradoxical sleep. This synchronized EEG activity pattern in old cortex (hippocampus) may reﬂect an activational state because it co-occurs with neocortical activation (EEG desynchronized wave form) present in the stage of activated sleep (i.e., paradoxical sleep when dreams are reported) and during alert waking behavior (Vertes and Kocsis, 1997; Winson, 1990). 6. Proposition 6: The Learning/Memory Effects That Occur after Experimental Treatments That Increase Peripheral Levels of VP Do Not Result from a Direct Action on Central VP Receptors Crucial to proposition 6 is the issue of whether peripherally circulating vasopressin or its behaviorally active metabolites can penetrate the blood– brain barrier. Each side of this issue has its proponents and supportive evidence, but it is still undecided (see Chapter 14). Koob and associates are skeptical about the ability of behaviorally active doses of VP to cross the blood–brain barrier and enter the brain substance. Deyo et al. (1986) tested for the passage of immunoreactive AVP from the plasma into the brain substance, including a control procedure for removing the contaminating effects of any AVP lodged within the capillaries of the brain before brain dissection and examination. They found no evidence that plasma AVP had entered the brain substance, whether its level had been experimentally increased by a behaviorally active dose of AVP (5 g/kg, subcutaneous) or by an osmotic challenge (48 h of water deprivation). A second line of relevant evidence was the ﬁnding by Lebrun et al. (1985) that intracerebroventricularly injected V1 receptor antagonist [dPTyr(Me)]AVP blocked the prolonged avoidance extinction effect induced by peripherally administered AVP, but only at a dose level that also inhibited its pressor effect. Because an intracerebroventricular injection of a behaviorally active dose of AVP has neither pressor nor aversive effects (Koob et al., 1986), it was concluded that the centrally injected lipophilic (able to cross the blood–brain barrier) antagonist interacted with AVP vascular receptors in the periphery.

The Vasopressin Dual Action Theory

IX. The Theoretical Controversy between the De Wied et al. and Koob et al. Research Groups

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The key aspect of this controversy revolves around the question of whether the ‘‘retention effect’’ of vasopressin results from a direct action on central memory substrates as claimed by De Wied et al. or is secondary to the effect of the peptide on the subject’s arousal system, which in turn mediates the retention effect. Koob and associates have commented on how peptide-induced arousal facilitates retention as follows: ‘‘a rat alerted and aroused either by LiCl or by AVP would be more likely to process information and consolidate various details and signals in the environment’’ (Le Moal et al., 1984, p. 337). The following discussion outlines evidence presented by each theoretical camp supporting its own position and challenging that of the other.

A. Two lines of Evidence Presented by De Wied and Associates One line of evidence obtained by De Wied and colleagues that challenges the ‘‘VP Dual Action Theory’’ involved dissociation of the pressorinduced arousal effect of AVP from its facilitation of memory consolidation: (1) the AVP metabolite DG-AVP lacks the pressor action of AVP, but is nevertheless able to facilitate retention [De Wied et al., 1972 and Bohus, 1977 (see Chapter 2); Gaffori and De Wied, 1985 (see Chapter 3)]; (2) aside from DG-AVP, other AVP metabolites such as [pGlu4,Cyt6]AVP(4–9), derived from proteolytic breakdown of AVP by brain synaptic membranes (Burbach and Lebouille, 1983; see Chapter 5), also lack the pressor action of systemically injected AVP but are much more potent than the parent peptide in facilitating memory consolidation in a passive avoidance task (Burbach et al., 1983a; see Chapter 5); and (3) conversely, Ang II, a more potent pressor agent than AVP, was unable to facilitate retention of a learned avoidance response (De Wied, 1971; see Chapter 2). However, this line of supportive evidence has its problems: ﬁrst, DGAVP does not always facilitate retention (Ettenberg et al., 1983b), and has been shown to inﬂuence behavioral arousal (Skopkova et al., 1991), although facilitation of memory consolidation by the peptide appears to be independent of its arousal effect (Skopkova et al., 1991; see Chapter 3). More importantly, DG-AVP does not bind to central AVP receptors (Dorsa et al., 1984), indicating the need for further investigation into the means by which this VP analog can inﬂuence retention. Second, De Wied (1971) tested Ang II at a dose level (1 g/rat) behaviorally active for AVP, but as noted by Koob (personal communication, 1997), this experimental study did not indicate whether Ang II increased blood pressure at this dose level. Third, the behaviorally potent peptide metabolites derived from synaptic

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membranes have such low afﬁnity for AVP-binding sites in the brain (Brinton et al., 1984, 1986; De Kloet et al., 1985b) that it is not clear that they bind to V1 receptors at all. If not, it is questionable whether their action is relevant for a proposal concerning the mechanism by which peripherally administered AVP inﬂuences behavior (Koob, personal communication, 1997). Notwithstanding the speciﬁc problems besetting this ﬁrst line of evidence, the main argument is valid: in those numerous learning tasks in which the peripherally injected VP metabolites facilitate retention, the result cannot be attributed to a pressor effect. A second line of evidence relates to the question of whether peripheral or central vasopressin receptors mediate the retention effects of peripherally administered AVP. The observation that intracerebroventricularly injected AVP at a 1000-fold lower dose than that used for peripheral administration achieves a comparable effect on retention (e.g., De Wied, 1976) is consistent with De Wied’s proposal that only a small fraction of the amount injected peripherally must cross the BBB to inﬂuence central receptors. More to the point, De Wied et al. (1984a; see Chapter 3) demonstrated that a peripheral injection of the V1 antagonist d(CH2)5[Tyr(Me)]AVP (3 g/rat, subcutaneous) blocked both the pressor and retention effects of peripherally administered AVP (0.3, 1.0, or 3.0 g/rat), whereas the intracerebroventricularly injected antagonist (3 ng/rat) blocked the behavioral but not the pressor effects of peripherally administered peptide. These ﬁndings were interpreted as indicating that when peripherally administered, the antagonist, which can cross the BBB, blocked the AVP pressor effect by interacting with V1 peripheral receptors, and the retention effect by interacting with central receptors. It was also thought that the small quantity of intracerebroventricularly administered antagonist was able to reach central V1 receptors that mediate the behavioral effects of peripherally administered AVP, but that this quantity was insufﬁcient to reach the peripheral V1 receptors that mediate the pressor effects of the peptide.

B. Two Lines of Evidence Offered by Koob and Colleagues First, many of their research ﬁndings suggest a causal association between the pressor/aversive properties and the retention effect induced by peripherally administered vasopressin. These include the ﬁndings that (l) peripherally (Le Moal et al., 1981) but not centrally (Koob et al., 1986) administered AVP results in pressor effects at dose levels that also facilitate retention; (2) peripheral pretreatment with the V1 antagonist [dPTyr (Me)]AVP, at an antagonist-to-agonist dose ratio level that prevents the pressor effect of the peptide, also prevents its behavioral effect in a multitrial conditioned avoidance task (Le Moal et al., 1981); (3) peripherally (Dantzer et al., 1982; Ettenberg et al., 1983a) but not centrally (Koob et al., 1986)

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administered AVP resulted in aversive effects at dose levels which facilitated retention on aversive and/or appetitive behavioral paradigms; and (4) peripherally administered DG-AVP, which has neither pressor nor aversive (e.g., CTA-inducing) effects (Ettenberg et al., 1983a), did not enhance retention in the water-ﬁnding task (Ettenberg et al., 1983a) or in a spatial learning radial maze task (Packard and Ettenberg, 1985). Second, these investigators cited ﬁndings from their studies on antagonist–agonist interactions to argue against the proposition that peripherally administered AVP directly inﬂuences central receptors to affect retention: (1) pretreatment with the intracerebroventricularly injected lipophilic (able to cross the blood–brain barrier) V1 pressor antagonist [dPTyr(Me)]AVP blocked the retention effect of systemically injected AVP only at a dose level (6 g/kg) that also blocked the VP pressor effect (Lebrun et al., 1985). This indicates the need for sufﬁcient quantities of the antagonist to reach the peripheral V1 receptors that mediate both the pressor and behavioral effects of the peptide; and (2) similar ﬁndings were obtained after pretreatment with an intracerebroventricular injection of the more potent V1 receptor antagonist, d(CH2)5[Tyr(Me)]AVP (reported in Koob et al., 1989). This latter observation was in direct contradiction to experimental results reported by De Wied and colleagues (1984a; see above), even though both research groups used the same dose levels of AVP and V1 antagonist, and tested retention in the same behavioral task paradigm.

X. Commentary Relevant to the Koob et al. Position

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Although experimental replication may help clarify some of the speciﬁc contradictory ﬁndings reported by the De Wied et al. and Koob et al. research groups, there appears to be no simple resolution of their longstanding controversy. One major problem is the question of whether vasopressin or its metabolites can cross the BBB. Ongoing research designed to relate the interaction of vasopressin with the BBB to the behavioral effect of the peptide is complex in nature (see Chapter 14). It is worth noting that vasopressin, as well as numerous other peripherally circulating peptides with behavioral effects, can inﬂuence the central nervous system by interacting with receptors in various circumventricular organ sites in the brain (see Chapters 1 and 14). These specialized areas of the brain lying outside the BBB may provide a means by which hormonal ﬂuctuations in the body periphery can inﬂuence neural signaling in the brain. Thus, a peripherally circulating neuropeptide may not have to penetrate the BBB to exert an inﬂuence on CNS-mediated physiology and behavior. For example, peripherally circulating Ang II inﬂuences drinking behavior by interacting with receptors localized at circumventricular organs associated with the third ventricle (Carlson, 1991). The possibility that peripherally circulating AVP

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may interact with blood–brain barrier mechanisms to inﬂuence nutrient transport into the brain, and its relevance for memory processing, provides another mechanism by which peripherally circulating AVP may contribute to memory processing; this possibility was brieﬂy presented in Chapter 1, and receives further discussion in Chapter 14. It seems clear from this discussion that our knowledge of the mechanism by which central and peripheral VP has retention effects is too sketchy for appreciable resolution of the opposing theories. The controversy, however, has helped to sharpen interest and extend knowledge about how peripherally circulating vasopressin can produce the retention effects consistently observed in the studies carried out by both of these research groups. These studies have been concerned mainly with clarifying how pharmacological doses of AVP produce retention effects in aversive and appetitive learning tasks rather than with evidence of a potential role for endogenous vasopressin. A few of these studies did measure the effects of blocking endogenous VP-ergic receptors on retention by peripheral or central injection of the V1 receptor antagonist alone. The fact that the antagonist used in these studies was lipophilic and could therefore cross the blood–brain barrier made it difﬁcult to interpret the results, because this passage could dilute the concentration of the dose in the injected compartment. Inconsistent ﬁndings occurred, depending on the task. In a pole-jump active avoidance task, blocking endogenous VP receptors by peripheral or central injections of the lipophilic V1 antagonist interfered with retention, but only at high dose levels of the antagonist (i.e., 100 g/rat, subcutaneous; 10 g/rat, intracerebroventricular) (Koob et al., 1981). Lebrun et al. (1984) reported no effects on passive avoidance retention when this V1 antagonist was peripherally injected on its own at a dose level of either 5 or 25 g/rat, but a facilitation of PA retention when given at a dose level of 15 g/rat. Another line of evidence supporting a role for endogenous VP in facilitating retention in an avoidance-learning task was reported by Koob et al. (1985a). An osmotic challenge (hypertonic saline) known to release VP both centrally (Rodriquez et al., 1983) and peripherally (Dunn et al., 1973) facilitated retention in a pole-jump avoidance task, as did a peripherally administered behaviorally active dose of AVP (1 g/rat, subcutaneous). Evidence indicated that this dose of hypertonic saline produced a plasma level of AVP similar to that observed after behaviorally effective doses of the peripherally injected peptide. This suggested that increased arousal induced by pressor effects associated with the high level of plasma VP resulting from the hypertonic saline injection may have mediated the retention effect. However, the potential contribution of endogenously released AVP in this situation was not assessed. The ability of the peripherally injected V1 antagonist to cross the blood–brain barrier and hence block central VP receptors also made it difﬁcult to interpret the inhibitory effect of this pretreatment on the retention effect induced by osmotic challenge.

Barbara B. McEwen

Contributions of Sahgal and Colleagues: The ‘‘Vasopression Central Arousal Theory’’

I. Chapter Overview

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This chapter describes the views and studies of Sahgal and associates concerning the inﬂuence of vasopressin on memory processing. Many of Sahgal’s writings have critically evaluated the ‘‘VP/OT Central Memory Theory’’ and the research practices on which it has been based (Sahgal, 1984, 1985). Rejecting the thesis that VP directly inﬂuences substrates underlying memory consolidation and retrieval, the Sahgal et al. viewpoint, the VP central arousal theory proposes instead that the behavioral effects of the peptide are best explained by its ability to increase the baseline arousal level. Sahgal and colleagues (see Sahgal, 1986) regard their theoretical position as essentially agreeing with that of Koob and associates. Speciﬁcally, both research groups (1) emphasize the role of VP (as opposed to OT) in memory processing in their research studies, and (2) attribute the effects of VP on learning and memory tasks to a primary effect on the individual’s arousal level (Sahgal, 1986). In Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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contrast to the extensive research carried out by De Wied and colleagues, and to lesser extent by Koob et al., in the development and support of their theoretical viewpoints, Sahgal’s research team has expended relatively little research effort relevant to the ‘‘VP Central Arousal Theory.’’ Nevertheless, the innovative and creative research design and measurement that characterizes their research protocols, together with their employment of a variety of learning tasks, have yielded results supporting the view that a VP-induced arousal effect is especially prominent in those types and stages of memory processing, especially dependent on focused and sustained attentional processing for a successful outcome. However, in this author’s opinion, their research ﬁndings do not effectively challenge the central tenets of the ‘‘VP/OT Central Memory Theory’’ regarding the role of VP in long-term memory storage and retrieval.

II. Behavioral Arousal and Central Arousal System Constructs, and Their Relevance for Cognitive Behavior

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A. Introductory Remarks Beyond frequently alluding to the theorized inverted-U relationship between arousal level and performance efﬁciency, these researchers have not engaged in lengthy discussion either (1) to deﬁne behavioral arousal and arousal system constructs or (2) to explicate their relationship to those of cognition [attention, learning, formation of (and retrieval from) short-term memory, long-term memory], emotion, and motivation. Nevertheless, the comments made by this research group (e.g., Sahgal, 1983, 1984) suggest essential agreement with (1) Hebb’s concepts of behavioral arousal and the arousal system (Hebb, 1955; Hebb and Donderi, 1987); (2) Eysenck’s modiﬁcation of the postulated inverted U-curve relationship between arousal and performance (Eysenck, 1982); and (3) the views of Broadbent (1971) and Robbins (1984), which postulate the presence of neural mechanisms in the arousal system operating to maintain a fairly constant level of arousal in the waking subject, supportive of effective behavioral performance despite the disruptive inﬂuences that may occur in stressful situations. These views are important for understanding the positions of Sahgal and colleagues concerning arousal-relevant constructs and their relation to memory processing. They are discussed below (Sections II.B through II.D).

B. Views on Behavioral Arousal and the Arousal System: The Hebbian Influence In agreement with Hebb (1966), Sahgal and colleagues view arousal as a motivational (energizing) construct and have deﬁned behavioral arousal as ‘‘the state of alertness or wakefulness of the animal’’ (Sahgal, 1984,

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p. 222). This deﬁnition and other comments in the writings of Sahgal and colleagues (e.g., Andrews et al., 1983; Sahgal 1983, 1984) led this author to infer that their views regarding these constructs are in accord with the following statements: (1) the central arousal system, the neurophysiological substrate of behavioral arousal, consists of a number of well-known neurochemically deﬁned cell systems. They include catecholaminergic, serotoninergic, and cholinergic cell groups localized within the reticular core of the brainstem or in the basal forebrain. The widespread ascending and descending projections of these neuronal groups inﬂuence tonal neuronal activity in the CNS sites in which they terminate; (2) ﬂuctuation in the level of activity in these component neurotransmitter cell groups over the sleep– wake cycle is responsible for shifts that occur in both behavioral arousal and cortical activation. The ﬂuctuations in neocortical activation are reﬂected in the distinctive electroencephalographic (EEG) patterns that characterize the relaxed (synchronous, moderate amplitude, 9- to 14-Hz alpha pattern) and alert (desynchronous, low-amplitude, 14.5- to 30.0-Hz beta pattern) waking states, as well as the slow-wave (dominated by synchronous, highamplitude, 1.5-4.0-Hz delta waveforms) and rapid eye movement (REM; desynchronous, low-voltage fast activity seen in the alert waking state) sleep states (for a highly readable and up-to-date overview of the neurochemical substrates of the sleep–wake cycle see Carlson, 1998); (3) cortical processing, dependent on both the cue function of direct sensory input and the drive function of input from the arousal system (Hebb, 1955, 1966; Hebb and Donderi, 1987), mediates cognitive (e.g., attention, memory processing, and ideation) and emotional/motivational activities (e.g., registering emotional experience, initiating and sustaining speciﬁc types of goal-directed activities), and hence the efﬁciency of behavioral performance; and (4) in accordance with the postulated Yerkes–Dodson principle, performance efﬁciency is functionally related to the level of arousal during the waking state (Yerkes and Dodson, 1908). More speciﬁcally, this principle asserts that an inverted U-shaped curve describes the relation between arousal level and performance efﬁciency in cognitive tasks (attention, learning, memory, etc.). As Fig. 1A shows, task performance improves as behavioral arousal increases from a low to a moderate level, and thereafter declines as arousal level proceeds toward a high level.

C. The Yerkes–Dodson Postulated Arousal/Performance Curve Revisited: Qualifying Comments by Hebb and Eysenck Sahgal (1984) subscribed to the qualifying statements of Hebb (1966) and Eysenck (1982) concerning the Yerkes–Dodson postulated arousal/performance curve. Hebb (1966) proposed that the exact shape of the invertedU curve depends on the nature of the task. For example, if the task is

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FIGURE 1 (A) An inverted-U performance-versus-arousal curve; the exact shape will depend on the task (see text). Both performance and arousal are shown in arbitrary units; S1 and S2 represent subjects in different states of arousal, but note that they yield identical performance (score 4). However, a unit increase in arousal will improve performance in S1 (up to score 8), but disrupt it in S2 (down to performance score 1). Source: Sahgal, 1984 (Fig. 3, p. 222).

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relatively uncomplicated but requires a high degree of mobilized effort (e.g., avoidance), it is predicted that the curve will be skewed (characterized by a shallow ascending, and a steeper descending limb) and optimal performance will occur at a high level of arousal (Fig. 1B). Eysenck (1982) added the anxiety concept to explain the disorganized behavior associated with high excitability (overarousal) indicated by the low level of performance represented on the far right side of the inverted U-curve (Fig. 1C). Speciﬁcally, Eysenck (1982) noted that arousal per se may be consistently beneﬁcial (or at least not detrimental), but that anxiety becomes increasingly more disruptive at higher levels. These two processes would sum together, resulting in an inverted U-function between performance and arousal/high anxiety.

D. Broadbent’s Two-Component Model of Behavioral Arousal and Its Relevance for the ‘‘VP Central Arousal Theory’’ Eysenck (1982) noted that the proposals of the Yerkes–Dodson principle rested on three underlying assumptions: (1) arousal was regarded as a one-dimensional construct (i.e., there is a single, one-dimensional arousal system); (2) performance efﬁciency could be assessed by a single overall measure; and (3) the general concept of task difﬁculty could be assessed by use of a speciﬁc example (e.g., degree of discrimination required for discerning differences along a brightness continuum). Broadbent (1971) rejected a unifying view of behavioral arousal, and replaced it with a two-component model that better accommodated his ﬁndings in tasks of attention (Fig. 2). According to this model, a lower arousal mechanism responds passively to ﬂuctuations of interoceptive and exteroceptive stimulus input and directly inﬂuences behavioral output. As arousal in this lower mechanism intensiﬁes and threatens to disrupt discriminative performance, the upper mechanism, which monitors activity of the lower one, exerts feedback modulatory control over it, maintaining some Copyright ß 1984 by Springer-Verlag. Reprinted with permission. (B) Possible differences in the curve relating arousal to cue function or effectiveness of response in three habits. In (a), a simple, long-practiced habit such as giving one’s name when asked, maximal efﬁciency is reached with low arousal and maintained over a wide range. In (b), a complex skill, the maximum appears only with a medium degree of arousal. In (c), a performance such as running a race, which is relatively uncomplicated but demands full mobilization of effort, the maximum appears with higher arousal. Source: Hebb, 1966 (Fig. 76, p. 238). Copyright ß 1966 by Saunders Company. (C) Cartoon illustration of the Yerkes–Dodson inverted-U performance-versus-arousal curve (Yerkes and Dodson, 1908). Both low and high arousal produce poor performance; note that arousal enhancement will improve performance in the drowsy subject, but will disrupt it in the anxious subject. The exact shape of the curve depends on task difﬁculty (Hebb, 1966), and some research suggests that increasing arousal produces anxiety, which is actually responsible for poor performance (Eysenck, 1982). Reprinted from Sahgal, 1985 (Fig. 4, p. 344) by courtesy of Marcel Dekker, Inc.

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FIGURE 2 Schematized diagram of the proposed ‘upper’ and ‘lower’ arousal mechanisms. Examples of the types of conditions inﬂuencing the lower mechanism are given on the ﬁgure. Stimulus input is hypothesized to have 2 main aspects: nonspeciﬁc and arousing, produced by intense, or salient stimuli; and speciﬁc, involving informational properties such as spatiotemporal patterning. Both aspects are available to the upper mechanism, but only the arousing aspects are available to the lower mechanism. The latter controls primarily speed and probability of responding rather than response selection itself. The DNAB mediates arousal to the upper mechanism whereas the central DA systems may contribute to the activation of performance by the lower mechanism. (1), non-speciﬁc arousal, drive (intensive aspects); (2), informational (discriminative aspects); (3), response selection or choice; and (4), speed and likelihood of response. Source: Robbins, 1984 (Fig. 1, p. 19). Copyright ß 1984 by Cambridge University Press. Reproduced with permission.

degree of constancy in arousal and thereby helping to prevent the disruptive effects of supraoptimal arousal on discrimination (see Broadbent, 1971, for further discussion of this model). Robbins (1984) proposed, on the basis of pharmacological manipulations, that a distinction might be made between the effects of central dopaminergic (DA-ergic) activity and cortical noradrenaline (NA) activity on task performance control. Whereas the cortical-projecting NA system helps to maintain accuracy of response when arousing distracters, such as white noise, threaten to disrupt discriminative performance, the mesolimbic DA system seems to be implicated in activating effects (increasing the probability and speed of responding, but not involved in its accuracy). Applying these ﬁndings to the Broadbent model, Robbins (1984) speculated that activity in the cortical-ascending NA ﬁber system (dorsal noradrenergic bundle, DNAB) might serve the upper arousal mechanism while the mesolimbic DA system innervates the lower arousal mechanism in the Broadbent (1971) model (Fig. 2).

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Sahgal and colleagues (Andrews et al., 1983) have agreed with Broadbent’s thesis that although the arousal level of the subject is responsive to changing external and internal stimulus input, certain neural mechanisms constantly monitor arousal level, prevent transient ﬂuctuations, and maintain arousal at a broadly stable level (Broadbent, 1971). Although the actual magnitude of the arousal level depends on the reactivity of the subject, in the absence of any marked change the arousal level of the subject would remain fairly steady, although decreasing slightly with increasing familiarity (habituation) (Andrews et al., 1983). Sahgal (1984), noting the evidence that VP modulates activity in noradrenergic pathways (e.g., Kovacs et al., 1979a,b, 1980c), further speculated that the primary inﬂuence of VP might be on the upper arousal mechanism in the Broadbent (1971) model. This also led to the speculation (Sahgal, 1984) that during stress the interaction of VP with the NA-ergic system may modulate the arousal level, by reducing the disruptive effects of anxiety that would interfere with memory processing. In support of this speculation, Buwalda et al. (1993) showed that a behaviorally active dose of arginine vasopressin (AVP, 6 g/kg; subcutaneous), injected under resting conditions, induced behavioral arousal (increased grooming behavior, with a concomitant decrease in resting time) and activated the pituitary–adrenocortical axis. This AVP-induced stress response was terminated 60 min after peptide application. At this time, the rats were subjected to a mild stressor (transportation to and placement in a novel environment). The behavioral activation that followed this stressor was more rapidly decreased in the AVPtreated rats than in the vehicle-treated controls. This led to the interpretation that AVP has arousal-inducing properties, associated with pressor effects produced by its peripheral application, but may facilitate dearousal mechanisms after stress-induced behavioral activation.

III. Relationship Between the ‘‘VP Central Arousal Theory’’ and the ‘‘VP Dual Action Theory’’

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Sahgal (1984, 1985) theorizes that both central and peripheral vasopressin increase the baseline arousal level and thereby inﬂuence cognitive behavior in accordance with the postulated inverted-U relationship between arousal level and performance efﬁciency. For example, when the peptide is peripherally or centrally injected immediately after a passive avoidance learning trial, the resulting increment in arousal level will be expected to enhance learning processes (memory consolidation) in underaroused subjects and impair it in overaroused subjects. Thus, this theory would predict bimodal effects on retention (in a group heterogeneous for baseline arousal) in contrast to the ‘‘VP/OT Central Memory Theory,’’ which would predict a unimodal peptide effect (i.e., memory enhancement).

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In early writings, Sahgal (1984, 1985) did not accept the basic distinction of the ‘‘VP Dual Action Theory’’ that peripheral and central VP utilize different mechanisms to produce a homologous effect (an arousal-mediated inﬂuence on learning/memory task performance) (see Chapter 6). Although professing little understanding of how VP speciﬁcally interacts with the arousal system, Sahgal (1984, 1985) did not follow Koob and associates in positing a pressor/aversive effect as a prerequisite for the inﬂuence of the peptide on arousal level. Instead, Sahgal (1985) suggested that aversiveness was more likely to follow than precede the inﬂuence of the peptide on arousal. Noting that increases in arousal level beyond optimal levels can produce aversive states of anxiety and agitation (Eysenck, 1982), Sahgal (1985) suggested that VP given in large doses, or to overaroused subjects, might be expected to produce aversive consequences. On the other hand, he has also observed that neither centrally injected nor low doses of peripherally administered VP, which improve retention, appear to produce ‘‘obvious discomfort.’’ Sahgal (1985) recommended the use of dose–response curves in taste- and place-aversive conditioning studies with vasopressin, presumably to provide a more speciﬁc view of how the retention and aversive effects of the peripherally administered peptide are related to one another. In later writings, however, Sahgal (1986) agreed with one of the major tenets of the ‘‘VP Dual Action Theory,’’ that the cardiovascular actions of peripherally circulating VP could generate signals that alter arousal, thereby attributing to VP an indirect role in mnemonic processing rather than a direct action at brain sites that process input, storage, and retrieval of information. Sahgal’s acceptance of this VP peripheral action component of the dual action theory was inﬂuenced, in part, by two lines of supportive evidence: (1) Sahgal’s observation that subcutaneously injected AVP, but not desglycinamide-arginine vasopressin (DG-AVP), retarded step-through reentry latency (enhanced retention) in a combined passive/active avoidancelearning task (Sahgal, 1986). This failure of DG-AVP, which has limited pressor activity, to inﬂuence retention in an avoidance learning task was in accord with observations of Ettenberg et al. (1983a; see Chapter 6) that unlike AVP, DG-AVP exerted little if any effect on retention of a variety of learned behaviors; and (2) reports published at the time specifying the unlikely event that VP and its analogs could cross the blood–brain barrier (Ang and Jenkins, 1982; Pardridge, 1983a).

IV. Sahgal’s Critique of the Research Practices of De Wied and Colleagues

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Sahgal (1984, 1985) has pointed out a number of research practices of De Wied and colleagues that pose problems for proper tests of the ‘‘VP/OT Central Memory Theory’’ and opposing theories. First, the subject selection

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procedure of this research team precludes the use of a heterogeneous subject population, important for comparison testing of the ‘‘VP/OT Central Memory Theory’’ and the ‘‘VP Central Arousal Theory.’’ De Wied and colleagues use an inbred strain of Wistar rat and a research procedure that selects only the ‘‘good learners’’ for testing the effects of the peptide on task performance retention. According to Sahgal, subject heterogeneity is necessary for normal variation in baseline arousal level, and thus the bimodal effects in task performance expected with a peptide treatment that inﬂuences memory by a direct effect on arousal level. Second, the almost exclusive reliance of De Wied and associates on avoidance conditioning paradigms, with extinction and reentry latency as the sole measures of memory, also poses problems for theoretical tests. According to Sahgal (1984, 1985) a variety of task paradigms is necessary to establish generality for any theory proposing a VP-induced inﬂuence on memory processing. At the time of this criticism there were relatively few studies that had used appetitive conditioning paradigms, and among these were numerous inconsistent ﬁndings. Whereas positive effects were reported by some authors [e.g., Bohus, 1977 (see Chapter 2); Ettenberg et al., 1983a (see Chapter 6); Sara et al., 1982 (see Chapter 10)] other researchers failed to observe a VP-induced facilitation of learning or memory in appetitive tasks [Alliot and Alexinsky, 1982; Andrews et al., 1983; Buresova and Skopkova, 1980, 1982 (see Chapter 10); Sahgal, 1983]. Third, Sahgal (1984, 1985) has questioned the validity of the Brattleboro rat model as an example of memory impairment associated with vasopressin deﬁciency. De Wied and colleagues (e.g., De Wied et al., 1975; see Chapter 3) had reported that these rats were profoundly impaired in memory processing and that exogenously administered AVP helped to correct the deﬁcit. Subsequent research, however, produced inconsistent ﬁndings concerning this putative memory deﬁcit and led to alternative interpretations of the deﬁcit when it was observed (see Chapter 3).

V. Research Practices and Objectives of Sahgal and Colleagues

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Sahgal and colleagues have attempted to correct two of these perceived objections to the research of De Wied and colleagues in their attempt to provide support for the ‘‘VP Central Arousal Theory.’’ These corrections included both the use of a heterogeneous subject sample and a variety of tasks and tests of retention. Depending on the study, Sahgal and associates have used outbred rats representative of various lines of both hooded and albino rats, and females as well as males. The tasks have included appetitive as well as avoidance paradigms, latency as well as choice tests for memory, and short-term as well as long-term memory assessment.

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The research strategy of Sahgal and associates has been to (1) demonstrate that factors other than memory enhancement may account for observed VP-induced performance effects (Sahgal, 1987a; Sahgal et al., 1990); (2) improve the validity of assessing the effect of the peptide on retention by including on occasion a second measure of retention [e.g., the Carew paradigm for simultaneously measuring reentry latency and choice to test retention of an unavoidable footshock (FS) experience (Sahgal, 1986; Sahgal and Wright, 1984)]; (3) demonstrate a putative VP effect on behavioral arousal through comparative study with drugs known to inﬂuence the arousal level of the subject (Sahgal and Wright, 1983); and (4) demonstrate that AVP treatment disrupts performance of subjects judged to have a relatively high baseline level of arousal (Sahgal, 1983).

VI. Task Paradigms and Research Findings

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A. Passive Avoidance Task 1. Task Description The passive avoidance task is similar to that used by De Wied and colleagues and is described in Chapter 2. 2. Studies Using the Passive Avoidance Task a. Sahgal et al. (1982) Sahgal et al. (1982) employed a passive avoidance (PA) conditioning task to test for an AVP-induced bimodal effect on memory consolidation and retrieval in adult male Wistar rats. Physiological saline or AVP (1 ng/rat, intracerebroventricular) was administered 0, 3, or 6 h after the learning trial or 1 h before the 24-h retention test. The study also assessed the effects on PA behavior of lower doses of centrally administered AVP (0.1, 0.01, and 0.001 ng/rat, intracerebroventricular). These lower doses were administered either immediately after the learning trial or 1 h before the 24-h retention test. Nonspeciﬁc AVP-induced motor effects were tested by running parallel groups in the test apparatus while omitting the footshock. The results were as follows: (1) the 1-ng dose of AVP, but not the lower doses, prolonged reentry latency in the shocked rats and the 1-ng dose was effective for all posttraining injection times; (2) the number of shocked rats failing to reenter the shock chamber within the 300-s time limit was greater for the VP-treated than for the saline-treated subjects; (3) a substantial number of the VP-treated rats exhibited reduced rather than prolonged reentry latencies after the FS trial, and this bimodal distribution of reentry latencies (Fig. 3), although observed for all the shocked animals, was much greater for the VP-treated rats; and (4) centrally injected AVP produced neither a nonspeciﬁc motor response nor an aversive effect on PA performance.

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FIGURE 3 Individual scatter diagram of latency scores. Data from the different injection time groups have been pooled, because they were not signiﬁcantly different. Scores over 300 s (‘‘failures’’) are shown above the dashed lines. Source: Sahgal et al., 1982 (Fig. 1, p. 90). Copyright ß 1982 by Elsevier/North-Holland Scientiﬁc Publishers Ltd.

The fact that this bimodal reentry latency was especially marked in the AVP-treated rats was interpreted as support for the view that vasopressin exerted a direct effect on the behavioral arousal level of the subject. That is, in animals with low baseline arousal levels, VP treatment would be expected to increase arousal toward the optimal level and so improve retention, accounting for the prolonged reentry latencies observed in these subjects. In contrast, for those animals whose baseline arousal level was at an optimal or higher level, a further increment in arousal disrupted memory and resulted in the short reentry latencies. b. Sahgal and Wright (1983) Sahgal and Wright (1983) compared the abilities of vasopressin (AVP), oxytocin (OT), d-amphetamine (AMPH), and chlordiazepoxide (CDP) to induce a bimodal effect on PA performance. Both AMPH and CDP are known to inﬂuence behavioral arousal; that is, AMPH produces dose-dependent behavioral excitement correlated with low-amplitude and fast EEG activity (Bradley and Elkes, 1957), and CDP, at the doses used here, causes sedation (Sahgal and Wright, 1983). The behavioral effects of -ﬂupenthixol (FLU), a dopamine antagonist that should block the effects of AMPH, was also tested. All animals received a peripheral injection of physiological saline or a given drug treatment immediately after the learning trial in the single-trial, step-through PA paradigm. Vasopressin or oxytocin was given at a dose level of 0.0125, 0.125, or 1.25 g/rat, subcutaneous; AMPH (1.0, 5.0, or

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10 mg/kg) and CDP (2 or 20 mg/rat) were administered intraperitoneally. Rats that received FLU before the learning trial were given an injection of AMPH (2.5 mg/kg, intraperitoneal) or saline. Reentry latencies were recorded in the retention test trial 24 h after the learning trial. A nonparametric analysis of group averages indicated no drug effects on PA performance; according to the authors this failure was not surprising because of the inappropriateness of testing differences between group averages on data that exhibit a bimodal type distribution, as occurred in this study. The results of the tests for bimodality (see original article for details) indicated (1) no bimodal effect for the unshocked rats, which exhibited short reentry latencies; (2) a bimodal distribution of reentry latencies for the shocked animals treated with AMPH, FLU, CDP, or AVP; and (3) a nonsigniﬁcant bimodal trend in the OT-treated group, which was considered meaningful because of the similarity between this distribution and that observed for the AVP group. The bimodal effect of AMPH was primarily dopaminergic because this effect was blocked by the neuroleptic agent FLU. The results were interpreted as consistent with the ‘‘VP Central Arousal Theory’’ because (1) AVP produced signiﬁcant, and OT nearly signiﬁcant, bimodal effects on PA behavior as predicted by this theory; and (2) AMPH and CDP, drugs known for their ability to modulate arousal level as previously noted, were also observed to produce signiﬁcant bimodal effects in this study.

B. Autoshaped Lever Touch Task 1. Task Description This appetitive autoshaped lever touch task (Sahgal, 1983) assesses acquisition, extinction, and subsequent reacquisition of a lever press response for food reward. The automated operant chamber has two retractable levers, a magazine tray (containing food pellets), a food dispenser, a house light, and a cue light located above the magazine tray. The levers are in the retracted position throughout the 3 days of pretraining, during which the animal is adapted to the noise of the activated food dispenser and required to eat food pellets placed in the magazine tray. Acquisition training begins on day 4. Illumination of the house light and emergence of one of the levers initiate the onset of a learning trial. The lever is automatically retracted after a 30-s delay, at which time food is delivered and the cue light is illuminated. A lever touch at any time within the 30-s delay interval of a given trial results in immediate retraction of the lever, cue light onset, and food delivery. The subject receives 50 acquisition trials per day [intertrial interval (ITI) of 10 s] until 5 consecutive responses are made or 150 trials have been given. The learning criterion is achieved once a prescribed number of deliberate, nonaccidental lever touch responses are made (i.e., accidental tripping of the lever can occur if the rat enters its path before the lever emerges). On the day

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after completion of acquisition training, task competency is tested with the animal given 50 food-rewarded test trials. The levers remain available for 60 s in each trial to verify a ﬁrmly established lever response. Extinction training is initiated on the following day and is identical to the acquisition schedule except that the food dispenser is now inactive. Response reacquisition is tested on the day after extinction. The food dispenser is again functional and the animals are monitored for the number of trials required to reattain the learning criterion. 2. Studies Using the Autoshaped Lever Touch Task a. Sahgal (1983) Sahgal (1983) tested the effect of AVP on appetitive learning in female Brattleboro rats homozygous for diabetes insipidus (HODI rats) and found further evidence supporting an AVP-induced arousal effect. That HODI rats may be hyperactive and anxious (i.e., relatively overaroused) was suggested in a study by Bohus et al. (1975; see Chapter 3) and by informal observations by Sahgal (1983). The effect of AVP (1 g/ rat, subcutaneous) on acquisition, retention, extinction, and subsequent reacquisition in the autoshaped lever-pressing task was tested in these subjects. In experiment 1, the rats were injected with saline or AVP (1 g/rat, subcutaneous) 1 to 5 min before daily acquisition training (50 trials/day). Veriﬁcation of successful responding (retention behavior) was tested on the day after completion of acquisition training. For this test, the levers remained available for 60 s/trial. Extinction training was carried out on the following day, using an extinction criterion of failure to respond over four consecutive trials after the ﬁrst extinction response. Reacquisition training began on the day after extinction. The animal continued receiving daily injections as before. AVP disrupted (prolonged) acquisition of the autoshaped lever-pressing response, but did not prevent attainment of the learning criterion (two successful consecutive lever presses) or efﬁcient retention, as indicated in the retention test given on the day after acquisition. There was no signiﬁcant difference between the peptide- and saline-treated groups in the rate of extinction, although there was a tendency for a more rapid rate of extinction among the VP-treated animals. Reacquisition was rapid for all animals and no differential AVP effect on this measure was observed. Informal observations indicated a brief period of hypoactivity in the AVP-treated rats. The immobile postures and staring eyes were interpreted as overarousal rather then sedation and although brief, this hypoactive state was considered a possible contributor to the prolonged acquisition observed in the lever-pressing task. This possibility was tested in experiment 2, which assessed the effect of AVP on locomotor activity, tested in activity cages equipped with photocells. Indirect indicators assessed nonlocomotor behaviors such as grooming and rearing. Each rat received two injections of each of two doses of AVP (1 and 2 g/rat, subcutaneous) and four control

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injections of saline. Three days were allowed between AVP doses. Injections were given 1 to 2 min before placement in the activity cages. The lower but not the higher dose of AVP signiﬁcantly enhanced locomotor activity. The failure of the higher dose of AVP to facilitate locomotor activity was interpreted as due to AVP-induced competing responses such as grooming, mild stereotypy, or ataxia. Although the 1-g dose increased locomotor activity over the course of the 90-min observation period, the animals showed a tendency toward suppression of behavior during the ﬁrst 20 min of the test period. The VP-induced temporary suppression of motor behavior was suggested as responsible for the slower acquisition of the autoshaped response observed in the AVP-treated subjects tested in experiment 1. The results of both experiments were interpreted within the framework of the purported inverted-U relationship between central arousal and behavioral performance. Sahgal proposed that AVP increased the arousal level in the already highly aroused HODI animals and resulted in overarousal, which in turn led to temporarily suppressed behavior and concomitant poor acquisition. Indeed, Sahgal suggested that ‘‘the alert, staring and immobile postures frequently seen after AVP administration are highly suggestive of an excessive, disorganizing or anxiety-provoking state of behavioral arousal’’ (Sahgal, 1983, p. 323). b. Andrews et al. (1983) Andrews et al. (1983) further explored the possibility that VP has its primary effect on behavioral arousal. Having observed that a behaviorally active dose of AVP (1 g/rat, subcutaneous) disrupts acquisition of the autoshaped lever-touch task in the moderately aroused Brattleboro rat (Sahgal, 1983), it was predicted that the higher the initial arousal level in a population of normal rats, the more disruptive the effect of the peptide on cognitive behavior. This study was designed to test this hypothesis in a heterogeneous sample of female Norway rats. Vasopressin was tested for its inﬂuence on acquisition of the autoshaped lever-pressing response and the data were assessed for a bimodal distribution of performance scores as evidence of an interaction between the peptide treatment and baseline arousal level. Baseline arousal level was assessed on the last day of habituation by calculating the number of magazine entries (entries into the food tray) made during a 15-min observation period on last (third) day of habituation. It was predicted that anxious animals would make fewer entries into the food tray, and be more disrupted by the putatively arousal-enhancing AVP. In experiment 1, saline or AVP (0.5 or 1.0 g/rat; subcutaneous) was given immediately before each daily training session in the autoshaping lever-pressing task. The learning criterion was reached once the animal had executed ﬁve lever presses. The retention test (50 additional acquisition trials) was given on the day after completion of acquisition training to verify

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FIGURE 4 Trials to acquisition in the lever-pressing task plotted against number of magazine entries during the session before training. Best-ﬁt lines were drawn by minimizing the square perpendicular distances to the line itself, that is, error variances were assumed to be equal for both variables (x and y axis). None of the correlation coefﬁcients were signiﬁcant. Therefore, scores along the x axis (magazine entries) can be collapsed, but are shown here for illustrative purposes. Source: Andrews et al., 1983 (Fig. 1, p. 20). Copyright ß 1983 by Churchill Livingstone.

successful performance. The saline and AVP injections were given before this test as they were during original acquisition. A scatter plot relating rate of acquisition (trials to criterion) to magazine entries for each of the three treatment groups is diagrammed in Fig. 4. None of the correlation coefﬁcients were statistically signiﬁcant, indicating that number of magazine entries failed to predict acquisition performance in either of the AVP-treated groups. Analysis of general performance level indicated differences between the three groups in acquisition scores that

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just missed reaching statistical signiﬁcance. Inspection of the individual data points (Fig. 4) suggested a tendency for the peptide-treated subjects (especially in the 1.0-g dose group) to require either few (less than 50) or many (greater than 100) trials to reach the performance criterion. When reanalyzed with the information statistic, which took into account extreme scores, AVP did signiﬁcantly slow the rate of acquisition. However, despite the greater tendency for extreme scores in the AVP-treated subjects, the test for bimodality failed to reach statistical signiﬁcance. Experiment 2, conducted after completion of experiment 1, used a sample of the previously tested subjects to determine the effect of the same AVP dose levels on locomotor activity. After 1 week (90 min/day) of habituation to the activity cages, each subject was tested twice at each AVP dose level (0.0, 0.5, 1.0, or 2.0 g/rat, subcutaneous) with 3 days between successive AVP treatments. The injection was given immediately before activity testing. The behaviorally active dose of the peptide (1 g/rat) did not inﬂuence locomotor activity, but the high dose (2 g/rat) suppressed it. The results were interpreted as consistent with the hypothesis that AVP inﬂuences learning by increasing the baseline level of arousal of the subject. Thus, there was no evidence of a VP-induced uniform enhancement or disruption of learning or memory processes. Instead, some AVP-treated animals learned quickly, and others relatively slowly or not at all. According to the interpretation that AVP increased baseline arousal, AVP would be expected to disrupt learning and memory in those subjects with an initially high arousal level and to improve it in those with an initially low arousal level. Unfortunately, a reliable assessment of initial arousal level is not available at this time and the one selected in this study did not yield signiﬁcant results.

C. Combined Active/Passive Avoidance Task 1. Task Description The combined active/passive avoidance task measures both passive (e.g., reentry latency) and active (chamber entry choice) avoidance behavior in testing memory for a single-trial footshock (FS) experience. It was originally developed and used by Carew (1970) to test for retrograde amnesia effects after electroconvulsive shock treatment. He found that, of the two, choice was the more sensitive measure for amnesia. Sahgal (1986) used the step-through version of this test to assess the effects of VP and OT on retention for an aversive (FS) experience. Brieﬂy, the apparatus consists of a Perspex box, divided by a central partition into two chambers attached to a Y-shaped Perspex runway. The arms of the runway end at the two guillotine doors of the Perspex box. Beyond the choice point of the runway, the right side of the apparatus (runway ﬂoor, doors, walls of

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the chamber, and droppings tray under the grid ﬂoor) is painted white; the left side, black. At the beginning of each free trial, the rat is placed on the runway facing away from the doors and allowed to enter either compartment. Two trials are given on day 1: a free-choice trial followed immediately by a forced choice trial permitting entry only to the chamber not chosen in the free-choice trial, ensuring that the rat experiences both sides of the chamber. A single free-choice trial is given on days 2, 3, and 4. A footshock, 3 s in duration and at an intensity dependent on the experimental design, is given immediately after entry into the chamber of choice on day 4. Immediately after the footshock trial the rat is removed from the apparatus and receives a single injection of physiological saline or the experimental drug. The retention test trial occurs 24 h after the injection and the rat is scored for both choice (active avoidance) and step-through reentry latency (passive avoidance). The tendency of the subject to alternate its choice from day 4 to 5 to a greater degree than that from day 3 to 4, a behavioral change indicative of memory for the FS experience, is assessed. It was argued that an agent that enhances memory storage should increase reentry latency and produce a greater tendency to alternate choice from day 4 (FS day) to day 5, relative to the choice alternation observed from day 3 to 4. 2. Studies Using the Combined Active/Passive Avoidance Task a. Sahgal and Wright (1984) Sahgal and Wright (1984) evaluated the effects of VP and OT on retention in male Wistar rats in the combined active/ passive avoidance task. After a few days of entering the black or the white compartment, a single learning trial was presented wherein the animal received a 3-s FS (0.4-mA intensity) on entering the chosen side. Immediately after the footshock trial, the rat received a single injection of either saline, VP (1 or 10 g/rat, subcutaneous), or OT (1 or 10 g/rat. subcutaneous). A nonFS peptide-treated (10-g dose of VP or OT) group was included to control for nonspeciﬁc peptide effects. The retention test was given 24 h after the learning trial; at this time the subject was returned to the test box for a single-choice trial. Both the choice of the chamber entered and the latency to enter this chamber were recorded. Latencies were categorized as short (<120 s), medium (120–600 s), and long (>600 s). Scores of 600 s (the cutoff limit) and beyond indicated that the animal failed to enter either test chamber. Analyses of the reentry latency data indicated that (1) for the nonshocked rats, neither peptide inﬂuenced reentry latency, which ruled out nonspeciﬁc drug effects in this measure; (2) for the shocked rats, VP signiﬁcantly prolonged reentry latencies at both dose levels, relative to saline controls, whereas OT had no effect at either dose level; and (3) bimodal scores were observed only in the group given the higher dose level of AVP.

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Choice behavior was affected by FS in all treatment groups, as indicated by the percentage of subjects that alternated their choice before FS (from day 3 to 4) compared with that after FS (from day 4 to 5). Thus, in the saline–FS group, only 22% of the tested subjects alternated choice of chamber entered from day 3 to 4, whereas this ﬁgure increased to 53% from day 4 to 5; for the nonshock–saline group, 29% of the subjects alternated from day 3 to 4 and again from day 4 to 5. Relative to saline controls, neither AVP nor OT signiﬁcantly inﬂuenced choice alternation in either the FS or non-FS condition. The saline–FS group was tested for a possible relationship between choice behavior and latency, but no signiﬁcant relationship between these two measures was found. That is, there was no tendency for subjects scoring above the median on reentry latency to alternate more or less than those scoring below it. The major ﬁnding with this paradigm was that latency behavior suggested that AVP improved memory, but choice behavior indicated that it did not. The possibility that choice behavior was the less sensitive of the two measures of memory was rejected because Carew (1970) found that choice but not latency scores indicated memory for an FS experience after electroconvulsive shock treatment. The lack of a signiﬁcant correlation between reentry latency and choice behavior meant that the subjects exhibiting longer latencies did not necessarily choose the safe compartment, a discrepancy interpreted as indicating that long latencies need not imply improved memory. The authors concluded that these data offered no support for either vasopressin- or oxytocininduced inﬂuence on retention. However, given the bimodal effect on latency scores, these data did not rule out a vasopressin inﬂuence on arousal level. The authors’ conclusion that the choice measure indicated that vasopressin did not inﬂuence memory on this task may not be totally warranted. First, it may be noted that in this paradigm there is no a priori reason why prolonged reentry latencies should be highly correlated with choice alternation when there is a good memory for the aversive FS experience. Good retention could be manifested by an effective active avoidance response (i.e., rapidly entering the safe chamber and avoiding the dangerous one) or by highly generalized passive avoidance resulting in strong reluctance to enter either compartment. Vasopressin treatment, especially the 1-g dose level, appears to have enhanced this passive avoidance tendency. The signiﬁcantly longer median latency in the AVP group (376 s) relative to the saline controls (i.e., 51 s), and the greater percentage of VP-treated subjects that failed to enter either compartment (47%) relative to the saline controls (18%), both support this conclusion. Second, the failure of AVP-treated subjects to exhibit signiﬁcantly greater choice alternation relative to the saline controls may have been due, in part, to a ceiling effect. That is, because all drug treatment groups showed robust choice alternation after the FS experience, it may not have been possible for vasopressin treatment to improve on this performance measure.

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This latter possibility was a consideration in the design of the study described below. b. Sahgal (1986) Sahgal (1986) assessed the retention effects of AVP, DG-AVP, and amphetamine (AMP) in adult male Wistar rats in the active/ passive avoidance task. Two levels of FS intensity (0.4 or 1.0 mA, scrambled AC for 3 s) were compared in their effects on choice behavior. Because an increase in FS intensity is expected to increase the tendency of the animal to switch its previous choice of the chamber where the FS occurred, the proper FS intensity is important to prevent ceiling effects from obscuring a treatment-induced facilitation of memory for the experience. DG-AVP was included to investigate whether peripherally administered vasopressin exerted its inﬂuence at peripheral or central receptors. If AVP affects behavior via its peripheral pressor effects, then DG-AVP, lacking these effects, should prove behaviorally inert in this task. Amphetamine was chosen for study because it has been shown to have effects similar to AVP on PA tasks (e.g., Kovacs and De Wied, 1978; Sahgal and Wright, 1983), and also increases reentry latencies at medium but not at low or high dose levels (Martinez et al., 1980). Each animal was given one forced choice trial and four free-choice trials on days 1 through 4 on this task. After the free-choice trial on day 4, the animal was exposed to either a low (0.4 mA, 3 s) or high (1.0 mA, 3 s) level of footshock. Within 10 s of removal from the chamber in which the FS was experienced, the animal received a single injection of either saline, AVP (5, 25, or 50 g/kg, subcutaneous), DG-AVP (5, 25, or 50 g/kg, subcutaneous), or d-amphetamine sulfate (AMP; 1, 3.2, or 10 mg/kg, subcutaneous). The following day, and at the same time, the rat was placed back in the apparatus and scored for reentry latency (up to a cutoff point of 600 s) and for choice alternation (selecting the alternate chamber from the one in which FS was administered). The data analyzed for each treatment group included a statistical comparison between (1) choice alternation occurring before FS (percentage of animals that alternated their choices between days 3 and 4) and after FS (percentage of animals that alternated their choices between days 4 and 5), and (2) median step-through latency on days 4 and 5. The results were as follows: (1) for the saline-treated subjects, median reentry latency after the FS experience was signiﬁcantly increased for both levels of FS. On the other hand, the tendency to avoid the shock chamber after the FS (percentage alternating choice on day 5 versus day 4) was statistically signiﬁcant for the 1.0-mA, but not the 0.4-mA, FS condition. Moreover, an association between choice alternation and reentry latency, tested only for the saline–FS groups, indicated that subjects scoring above the group median on reentry latency also tended to exhibit more choice alternations than those scoring below the median and vice versa, and this

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association was statistically signiﬁcant for the high-FS group; (2) because there was no dose-dependent effect for the AMP-treated subjects in either behavioral measure, the AMP data were pooled across the three dose levels. AMP signiﬁcantly increased reentry latencies and choice alternation relative to saline controls for the low-FS, but not the high-FS, condition. This latter lack of signiﬁcance was attributed to ceiling effects; (3) there was no AVPinduced dose-response effect on choice alternation. Neverthless, saline-AVP treatment comparisons were carried out for each AVP dose level as well as for the data pooled across all AVP dose levels, and indicated no signiﬁcant VP inﬂuence on day-5 alteration behavior; it should be noted, however, that the increase in average percent alternation from day 4 to day 5 for the AVP dose groups (i.e., from 26 to 47%) was similar to corresponding scores obtained for the AMP dose groups (i.e., from 20 to 50%). AVP treatment produced a dose-dependent effect on reentry latency in the low-shock condition (i.e., the middle dose of the peptide signiﬁcantly prolonged reentry latency relative to the saline controls); and (4) for the DG-AVP-treated subjects, no dose–response effect was observed for either dependent measure for the low- or high-shock condition, and pooling the data across dose levels resulted in no peptide inﬂuence on reentry latency or choice alternation relative to the saline controls in either FS condition. The results of this study were interpreted as follows: (1) reentry latency is signiﬁcantly correlated with choice alternation only after a sufﬁciently strong punishment (e.g., high versus low FS intensity); (2) strong punishment may act to obscure potential drug effects on retention because of ceiling effects. Thus, nearly optimal performance on latency and choice behavior measures of retention induced in saline controls after intense FS may have prevented further improvement by any of the drugs tested in this study; (3) AMP, which has been shown to inﬂuence both memory (Glick and Jarvik, 1969; Martinez et al., 1980) and arousal (Bradley and Elkes, 1957), exerted a highly potent effect on retention, enhancing both latency and choice measures of retention; (4) in contrast, the failure of AVP to inﬂuence choice behavior suggested that in this paradigm the peptide had only a weak effect on retention. Nevertheless, although failing to reach statistical signiﬁcance, the close correspondence between the pooled AVP and AMP data regarding postshock alternation behavior is noteworthy; and (5) the failure of DG-AVP to inﬂuence either choice behavior or reentry latency, consistent with ﬁndings by Ettenberg et al. (1983a), was given a similar interpretation: that is, when peripherally administered AVP enhances retention it is due to a pressor-associated arousal effect, rather than a direct effect of the peptide on mnemonic processing. Carew (1970) and Sahgal (1986) proposed that choice alternation complements reentry latency, and therefore both measures in a single study provide a more powerful test of the effect of a drug on memory. However, it may be noted that for the low-FS condition, in which ceiling effects

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are presumably not a problem, the close association between these two measures, expected if they both measure the same theoretical construct consistently, did not occur (Sahgal, 1986; Sahgal and Wright, 1984).

D. Delayed Matching and Nonmatching to Position Tasks 1. Task Description The tasks assess the ability of the animal to remember, after a designated delay interval, which of two retractable levers was presented as the sample for that trial. To obtain the reward, the rat must choose either the lever that matches the sample (delayed matching to position task; DMTP) or the alternate lever (delayed nonmatching to position task; DNMTP). The test apparatus (an automated operant box with two retractable levers) and task procedure were developed by Dunnett (1985) and is described in Sahgal (1987a) and Sahgal et al. (1990). Brieﬂy, each trial consists of a sample presentation followed immediately, or after a designated delay, by the two-lever choice presentation. During sample presentation the right or the left lever extends into the test chamber and the cue light above it is illuminated; the rat is required to respond to the lever within a 20-s hold period, at the end of which the lever is retracted and the magazine tray is illuminated. Between the sample and choice stages, the rat is required to approach and operate, by means of a nose poke, a central magazine ﬂap in order to proceed to the choice stage. This requirement was used by Dunnett (1985) to prevent the rat from maintaining a response orientation to the sample lever during the retention interval, After the nose poke response, both levers are extended into the test chamber and both cue lights are illuminated. The rat receives a food reward for pressing the correct lever within 20 s. An incorrect response or failure to respond within 20 s generates a ‘‘time out’’ interval during which all lights in the chamber are switched off. The rat is given 96 trials per day (ITI is 10 s). During early trials there is no delay between sample and choice lever presentations. Once the basic task is learned, the rat is then switched to the delay conditions. In a given trial a retention delay interval of 1, 2, 4, 8, 16, or 32 s is inserted between sample and lever choice presentations. Nose pokes are now ineffective until the delay has expired. Initially only short delays are used, but these are gradually increased. Within a few weeks the rats are exposed to an equal number of all delays, presented in a random order, across the 96 daily trials. In general, rats learn this task fairly quickly and unambiguous forgetting curves have been reported with delays ranging from 0 to 64 s (Pontecorvo, 1983, cited in Sahgal, 1987a). Subjects are scored for nose poke responses, latency to respond at the lever choice stage, correct responses, false positives (responding to the wrong lever), and misses (failures to respond to the

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correct lever). In addition to the percent correct responses, Sahgal and associates (Sahgal, 1987a,b; Sahgal et al., 1990) employ signal detection theory measures of accuracy and biased responding, based on models of Frey and Colliver (1973) and Grier (1971). These techniques enable assessment of the ability of a drug to inﬂuence the ability to detect stimuli and to produce perceptual (e.g., selective attention) or response (e.g., repetitive responding) bias. 2. Studies Using Delayed Matching and/or Nonmatching to Position Tasks a. Sahgal (1987a) Sahgal (1987a) compared the effects of AVP, DG-AVP, and d-amphetamine (AMP) in the delayed matching to position task in male Norwegian hooded rats. Training continued until performance stabilized; at this point drugs were introduced to determine their effect on memory during the retention delay interval (short-term memory assessment). On a given test day the subjects received an injection of saline or a designated dose of a selected drug. The doses used for AVP or DG-AVP were 1, 5, or 25 g/kg (intraperitoneal) and for d-AMP they were 0.05, 0.25, or 1.25 mg/kg (intraperitoneal). AVP or DG-AVP was administered immediately before the test and d-AMP was given 30 min before the test. A given subject received all doses of each of the three drugs in an order balanced for both dose levels and drugs: all doses of one drug were studied before the next drug was tested. At least 2 days elapsed between doses. Various measures were used to assess task performance at each delay interval. Aside from percent correct responses (a measure of accuracy), there were two other measures of accuracy and two measures of bias computed from ‘‘hit’’ and ‘‘false-positive’’ conditional probabilities based on two models of signal detection theory: (1) a sensitivity index (SI, accuracy measure) and a responsivity index (RI, bias measure) were based on a model of Frey and Colliver (1973) and (2) an accuracy assessment (A00 ) and bias assessment (B00 ) were based on a model of Grier (1971). RI was viewed mainly as a measure of response bias, and B00 as a measure of perceptual bias. Another measure, index Y (IY), developed by Sahgal (1987a) for memory tasks of this type, contrasted relative accuracy of the responses to the two levers. General responsivity was indicated by the average rate of magazine ﬂap responding (nose pokes) and by the total number of misses (whether at sample, nose poke, or choice stages). These measures were calculated for each delay interval. The results of the analyses of the measures of accuracy indicated (1) a decline in accuracy with progressively longer delays regardless of the treatment, indicating clear-cut forgetting curves on this task paradigm; (2) an AVP-induced dose-dependent improvement in accuracy across all delays, but only the highest dose (25 g/kg) having a signiﬁcant effect; (3) no DG-AVP treatment effect, at any dose level, on any measure of accuracy for any delay

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interval; and (4) an AMP-induced dose-dependent disruption in performance on all three measures of accuracy, signiﬁcant only at the highest dose level (1.25 mg/kg). The results of analyses carried out on the bias measures (IY, RI, and B00 ) indicated that (1) in general, IY, RI, but not B00 increased more or less monotonically with increasing delay; thus the signal detection theory measures suggested that response (RI) but not perceptual (B00 ) bias increased as the task became mnemonically more demanding; (2) AVP at a dose of 25 g/ kg markedly inﬂuenced IY (produced a shift in accuracy to one side); however, the lack of an effect on either RI or B00 suggested that no consistent response or perceptual bias was induced; (3) DG-AVP had no effect on any of the three bias indices; and (4) AMP shifted accuracy to one side in a manner similar to AVP (i.e., inﬂuenced IY), but unlike AVP this drug also affected RI but not B00 —suggesting that this dopaminergic agonist produced a response, but not a perceptual bias. Analyses of the rate of responding (nose pokes) and miss frequency indicated (1) no signiﬁcant effect of DG-AVP on either measure; (2) AVP at both the 5- and 25-g/kg dose levels reduced the overall rate of responding (nose pokes); the higher dose reliably increased misses (errors of omission), which occurred, for the most part, at the sample response stage of the trial sequence (i.e., inattention to sample lever on one side of the apparatus); and (3) AMP slightly increased the nose poke rate, but unlike AVP, had no effect on misses. In interpreting the experimental ﬁndings the authors noted that using the accuracy measure alone, the data suggest that memory in this task was facilitated by AVP (25-g/kg dose), disrupted by AMP, and not inﬂuenced by DG-AVP. However, a different interpretation emerges when one takes into account data from analyses of the bias indices, response rates, and responsivity index. In particular, the bias index (IY) indicated that AVP, like AMP but not DG-AVP, induced a tendency to attend to one or the other of the two levers—in the case of AMP this appears to have been due to a response bias (signiﬁcant RI, nonsigniﬁcant B00 ), whereas in the case of AVP a simple response or perceptual bias could not have been the reason because neither RI nor B00 was signiﬁcantly altered. Instead, the AVP-treated subjects tended to select a particular side, and then responded (at the sample stage) only if that lever emerged, resulting in few errors of commission (incorrect responses to the alternative lever). Although correct responding occurred when the sample was on the attended side it was at the cost of an increase in errors of omission to the alternative, ignored lever. This strategy simpliﬁes the task and has been observed in nontreated rats during early stages of DMTP learning. Thus, according to Sahgal, by heightening arousal level, AVP may have disrupted mnemonic processes and resulted in adoption of this more simpliﬁed problem-solving strategy. Because DG-AVP did not produce these biased effects, the adopted response strategy was ascribed to

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the pressor effects of AVP, supporting the Koob et al. viewpoint (e.g., Le Moal et al., 1981; see Chapter 6). The fact that AVP had different effects compared with AMP in this task could suggest that if both substances inﬂuence arousal mechanisms, as has been maintained, they must do so in different ways, acting at distinct anatomical and neuropharmacological sites. For example, in the arousal model favored by Sahgal (discussed in earlier section of this chapter), AVP may interact with noradrenaline to affect the higher mechanism of the twocomponent arousal system, whereas AMP may interact with dopamine at the lower mechanism. b. Sahgal et al. (1990) Sahgal et al. (1990) tested two independent groups of male PVG hooded rats in each of two variants of a spatial test for short-term memory: the delayed matching to position (MTP) task and the delayed nonmatching to position (NMTP) task. The hypothesis tested by this experimental design was as follows: if AVP increases a bias of attending and responding to one of the two levers, then the peptide will produce opposing results in the two tasks. Accuracy should be improved in the MTP task but impaired in the NMTP task, because correct performance in the latter requires attention to both levers during sample presentation for subsequent selection of the alternative lever. The delay between sample presentation and subject choice ranged between 0 and 32 s for each of the tasks. AVP was injected intraperitoneally at doses of 0 (physiological saline), 1, 5, and 25 g/kg immediately before each day’s behavioral test procedure. At least 2 days elapsed between injections, during which time the rats were not tested. Each rat received every dose in a random manner. Performance measures included many of the measures used by Sahgal (1987a; described above). These measures included percent correct, percent misses, index Y, and the responsivity index (RI). Index Y turned out to be a means of assessing a response tendency to attend to one side of the chamber at the expense of the other (i.e., a reduction in ﬂexibility in task responding). General responsiveness was assessed by monitoring latency to respond to the sample stimulus, and rate of responding to the magazine ﬂap (nose pokes). Analysis of variance (ANOVA) on overall performance accuracy (percent correct) indicated that all three main effects (task, delay, and dose of peptide) were signiﬁcant. These main effects showed that (1) the NMTP task was the easier task to acquire, especially in the early stages of acquisition; and (2) performance accuracy progressively declined (forgetting increased) as delay increased; only the highest AVP dose level affected performance. Both the group-by-dose and dose-by-delay interactions were signiﬁcant. Further analyses indicated that the high dose of AVP (25 g/kg) slightly, but signiﬁcantly, improved accuracy at the longest (32 s) delay interval in the MTP task while impairing accuracy at all delay levels in the NMTP task.

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Analyses of the responsivity index indicated signiﬁcant task and delay, but not dose, effects. Thus, greater response bias occurred in the NMTP task and increased monotonically with delay. None of the interactions were signiﬁcant, indicating that AVP did not inﬂuence response bias as measured by absolute RI. The ANOVA performed on the index Y measure, which compares response accuracy on the left and right lever positions, indicated that the nature of the task did not affect this index but both dose and delay interval did. Speciﬁcally, for both tasks the tendency to adopt the response strategy of attending and responding to one lever at the expense of the other increased as a function of increased delay interval and AVP dose level; a signiﬁcant increase in the use of this response strategy occurred at the highest VP dose level and at the three longest delay intervals. The ﬁndings pertaining to the number of misses, latency to respond to the sample stimulus, and the rate of response to the magazine ﬂap (nose pokes) indicated (1) although the majority (more than 90%) of missed opportunities (errors of omission) occurred at the sampling stage, the remaining 10% showed a dependence on the length of the delay and peptide dose level. An ANOVA on this measure indicated that the number of errors of omission was linearly related to delay (signiﬁcant at the 32-s delay) and curvilinearly related to AVP dose level (the 5- and 25-g/kg dose levels, but not the 1-g/kg dose level, increased the number of misses relative to control values); (2) AVP produced a dose-dependent increase in the latency to respond to the sample stimulus, a signiﬁcant effect for the two higher doses; and (3) the rate of response to the magazine ﬂap did not differ between the two tasks, but AVP treatment decreased responding, with signiﬁcant effects occurring for the two highest dose levels. Although the delay factor was signiﬁcant there was no consistent monotonic effect present: the fastest rate of response occurred for a 4-s delay, the slowest for a 32-s delay. Altogether, the results of this study were interpreted as conﬁrming the predictions of the tested hypothesis, that is, that AVP would improve performance in the MTP task, but disrupt it in the NMTP task. The AVP-induced improvement in accuracy in the MTP task was viewed not as a facilitative effect on memory per se, but rather as the outcome of biased responding in a memory-demanding task. More speciﬁcally, the drug was viewed as increasing arousal (anxiety), which in turn made performance of an already difﬁcult task even more so, especially at longer delays. In reacting to this situation, the animals adopted a simpliﬁed response strategy of attending to only one side of the chamber and ignoring the alternate lever during the sample phase. This response strategy was successful in the MTP task. When the lever, which was attended to during the sample phase, appeared during the choice phase, whatever the delay, the rat responded and this increased its ‘‘hits,’’ although at a cost of missed opportunities. Nevertheless, at the high delay

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level this strategy produced an improvement in performance compared with those relying on memory maintenance of the sample lever position. On the other hand, this same response strategy led to impaired performance in the NMTP task, which required a response to the lever that was not presented as the sample. The observation that with increasing difﬁculty, control rats tended to adopt this response strategy supports the argument that under ‘‘stressful’’ conditions, the animal will behave less ﬂexibly and adopt a more ﬁxed response strategy.

VII. Chapter Summary and Commentary on the ‘‘VP Central Arousal Theory’’

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The major objectives of the research carried out by Sahgal and colleagues have been to (1) support the ‘‘VP Central Arousal Theory’’ and (2) challenge the ‘‘VP/OT Central Memory Theory’’ and disprove the proposition that AVP exerts a direct effect on memory.

A. Propositions Derived from the ‘‘VP Central Arousal Theory’’ and Relevant Evidence The ‘‘VP Central Arousal Theory’’ can be basically characterized by two propositions. 1. Proposition 1: Increments in Peripheral or Central Levels of Vasopressin Raise the Baseline Behavioral Arousal Level, and by Implication Activity in Its Underlying Neurophysiological Substrate Sahgal’s own research studies have been limited to testing the effects of VP on the individual’s level of behavioral arousal. This testing is made difﬁcult by the lack of an unambiguous measure of behavioral arousal. Amount of locomotor activity is not in and of itself a useful measure because ‘‘frozen immobility’’ is as indicative of a high state of tension as ‘‘frenzied activity.’’ A disruption in performance is not a conclusive indicator of an overarousal effect because an agent may impair performance for reasons other than an effect on behavioral arousal (e.g., a sickness-inducing agent). The attempt by Andrews et al. (1983) to use the number of magazine entries made during habituation in an autoshaping task as a useful indicator of baseline arousal level did not yield signiﬁcant results. However, two lines of evidence consistent with a VP facilitation of arousal level were obtained from EEG studies reported earlier. Chapter 5 discusses ﬁndings obtained by Urban and De Wied (1975, 1978) indirectly supporting a VP contribution to EEG ‘‘activation’’ in old cortex [hippocampal synchronized EEG pattern (theta activity)]. Chapter 6 presents

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observations of Ehlers et al. (1985) supportive of a VP contribution to EEG activation in new cortex (neocortical desynchronized EEG pattern). 2. Proposition 2: The Behavioral Effects of VP on Learning and Memory Tasks Are Attributable to Increasing Baseline Arousal Level (Reflected in the Postulated U-Shaped Arousal–Performance Efficiency Curve) and Not to a Direct Facilitation of the Neural Processes Mediating Information Acquisition, Storage, and Retrieval Sahgal and colleagues have employed four types of paradigms in their attempt to refute the ‘‘VP/OT Central Memory Theory’’ and gain support for the ‘‘VP Central Arousal Theory’’. The results of ﬁndings obtained with these paradigms are relevant to proposition 2 and are discussed below. In one paradigm, a heterogeneous sample of male Wistar rats was tested for PA behavior (reentry latency) after a posttraining injection of central (Sahgal et al., 1982) or peripheral (Sahgal and Wright, 1983) AVP. The vasopressin-induced bimodal effects, observed in both studies, were interpreted as consistent with a VP/arousal but not a VP/mnemonic effect, because an increase in arousal level would be expected to enhance or impair PA retention depending on the baseline level of arousal. Moreover, the fact that the drugs d-amphetamine (stimulant) and chlordiazepoxide (minor tranquilizer), which are well known for their opposing effects on arousal, exerted bimodal effects on PA reentry latency in a manner similar to AVP was interpreted as additional support for the VP/arousal interpretation. The bimodal effect induced by posttraining peripherally injected OT was considered meaningful even though it failed to reach statistical signiﬁcance. This ﬁnding deserves replication and further study. A second paradigm tested the effect of presession injected VP on performance in an autoshaping appetitive lever-pressing task and in a locomotor activity test. One study (Sahgal, 1983) used ‘‘moderately aroused’’ female HODI rats, and the other (Andrews et al., 1983) used normal female hooded rats. The results of both studies were interpreted as being more consistent with the ‘‘VP Central Arousal Theory’’ than with the ‘‘VP/OT Central Memory Theory.’’ Sahgal (1983) did not observe either improved retention on the day after original acquisition nor prolonged extinction as would be predicted by the De Wied et al. theory. Instead, peptide treatment increased the baseline arousal level, which was expressed as an early period of reduced activity followed by increased activity. The suppressed behavior, expressed as reduced lever pressing, contributed to the prolonged acquisition observed in the AVP-treated subjects. The behavioral suppression was interpreted as an overarousal effect because of accompanying behavioral signs of tension (Sahgal, 1983). Andrews et al. (1983) cited the following evidence of a VP-induced arousal effect in their normal hooded female subjects: (1) the tendency (it

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failed to reach statistical signiﬁcance) in the VP-treated group to require either relatively few or many trials to reach the learning criterion suggested a bimodal effect; and (2) an AVP disruption of acquisition, especially when extreme scores were taken into account. However, neither magazine entries during habituation in the autoshaping task nor performance in the locomotor activity test were reliable independent measures of arousal effects associated with the behaviorally active 1.0-g dose of AVP. In a third paradigm, the effect of AVP on retention was evaluated by both a passive avoidance (reentry latency) and an active avoidance (choice alternation) measure of memory consolidation (Sahgal, 1986; Sahgal and Wright, 1984). Posttraining peripherally administered AVP signiﬁcantly prolonged reentry latency but did not increase choice alternation relative to saline controls in either study. Interpreting the choice alternation index as the more sensitive measure of memory, based on a previous study that assessed retrograde amnesia after electroconvulsive shock treatment (Carew, 1970), these authors concluded that this paradigm failed to demonstrate clear-cut support for a VP-induced mnemonic effect. However, as pointed out in an earlier discussion of the Sahgal and Wright (1984) study, a lack of one-to-one correspondence on these two measures in AVP-treated subjects does not necessarily imply that VP did not directly enhance memory consolidation, because other nonmnemonic effects could have been responsible for the discrepancy. For example, the peptide may have facilitated memory consolidation but acted to bias the type of coping strategy used to express the memory-driven fear response (i.e., by increasing a tendency to favor the use of a passive as opposed to an active mode of avoidance response) (Bohus et al., 1990; also see Chapter 1). Moreover, interpretation of the results of this paradigm is complicated by ceiling effects associated with choice alternation, especially under a higher FS level (Sahgal, 1986), and by a lack of correlation between choice alternation and reentry latency in the low-FS condition (Sahgal, 1986). The ﬁnal research paradigm that provided data interpreted as challenging a VP-induced mnemonic effect and favoring one based on arousal was the delayed matching (nonmatching) to position test of short-term memory (Sahgal, 1987a; Sahgal et al., 1990). Performance analysis using measures of accuracy and response bias based on signal detection theory indicated that, on closer analysis, the apparent AVP-enhanced retention effect resulted from biased responding produced by a VP-induced increase in arousal level added to the stress of increasingly longer retention intervals. In general, although these various paradigms have yielded results consistent with a vasopressin-induced inﬂuence on arousal level, they have been less effective in contradicting the De Wied et al. ‘‘VP/OT Central Memory Theory’’ for two major reasons. First, the evidence that VP increases baseline arousal, and therefore performance efﬁciency, is not in and of itself detrimental to the position of De Wied et al. because these investigators do not

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deny a vasopressin inﬂuence on arousal level, or that a VP-induced arousal effect can inﬂuence learning, only that the effect of the peptide on long-term memory storage and retrieval is not dependent on the VP-induced arousal effect (e.g., Skopkova et al., 1991; see Chapter 3). Second, the relevance of using VP’s failure to inﬂuence short-term memory [delayed matching (nonmatching) to position task] or learning (autoshaping task) to reject the ‘‘VP/OT Central Memory Theory’’ is questionable. This theory does not propose an important VP inﬂuence on the short-term memory processes involved in learning (see Chapters 2 and 3).

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Part IV

Role of Attentional Processing in Mediating the Influence of Vasopressin on Memory Processing

I. Introductory Remarks

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Muir (1996) has noted that attention is not a unitary construct, but rather has several theoretically useful forms, including vigilance (sustained attention or state of readiness to detect and respond to certain signals), selective attention (perceptual focus on certain stimuli while gating out others that are present in one’s immediate environment), and divided attention (switching focus between two or more stimuli at a given time). This chapter presents the work of several investigators who have examined possible effects of vasopressin on one or more of these forms of attention. Beckwith and colleagues designed several studies to examine the putative role of vasopressin in selective and/or divided attention, using both human and nonhuman animal subjects. The roles of the peptide in selective, and in divided, attention were investigated by Bunsey, Strupp, and colleagues, and by Meck, respectively; laboratory rats served as subjects in Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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each of these two studies. The human electrophysiological studies carried out by Fehm-Wolfsdorf and colleagues and by Timsit-Berthier and associates comprise the ﬁnal line of VP/attentional processing research presented in this chapter.

II. Beckwith and Colleagues

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Although not speciﬁcally stated by Beckwith and colleagues, it would seem that their interest in exploring the role of vasopressin in attention and memory came from two sources. First, there was the research by De Wied and colleagues comparing the inﬂuence on learning and memory induced by both anterior [melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH)] and posterior [arginine vasopressin (AVP) and oxytocin (OT)] pituitary hormones (see Chapters 2–5). Before 1982, Beckwith, in collaboration with Sandman, carried out a number of studies that had suggested a role for the anterior pituitary hormone MSH (a member of the family of neuropeptides that includes ACTH) on attentional processes (for a review see Beckwith and Sandman, 1982). Second, preliminary reports with human subjects, also stimulated by the vasopressin/memory research of De Wied and colleagues, suggested a vasopressin-induced improvement in learning and memory and in some cases attentional processes as well, in elderly cognitively normal volunteers (Legros et al., 1978), memory-impaired clinical volunteers (Le Boeuf et al., 1978; Oliveros et al., 1978; Weingartner et al., 1981), and cognitively normal college volunteers (Weingartner et al., 1981).

A. Research with Human Subjects 1. Overview The experimental protocols used by these researchers examined the effects of a single treatment with vasopressin on performance in a variety of task paradigms. These tasks included concept shift discriminations (Beckwith et al., 1982), reaction time required to determine whether a given probe was in an original set of memorized digits (Beckwith et al., 1983), and recall of sentences (Beckwith et al., 1984; Till and Beckwith, 1985) or prose (Beckwith et al., 1987a) requiring attention to relevant information. Their experimental ﬁndings are consistent with the thesis that this peptide inﬂuences selective attention at various stages of cognitive processing, which in turn contributes to memory processing. The subjects in these experiments were young adult college students, most male, 18 to 33 years of age. Initial interviewing and blood pressure monitoring were consistently carried out to screen for acute or chronic sickness and hypertension. Behavioral testing was initiated as soon as the vasopressin analog was judged to be absorbed into the body.

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Each of these studies used a 60-g dose level of the synthetic vasopressin analog 1-desamino-8-d-arginine vasopressin (DDAVP) administered intranasally, a route frequently used in human subjects. DDAVP was used in this research because of its relatively slow rate of metabolism and its lack of inﬂuence on blood pressure and heart rate (Couk and Beckwith, 1982). Beckwith et al., may have chosen the 60-g dose level because of the successful results obtained by Weingartner et al. (1981) with this analog, administered at an intranasal dose range of 30 to 60 g to college volunteers tested in a variety of verbal memory tasks. 2. The Studies a. Beckwith et al. (1982) Beckwith et al. (1982) explored the inﬂuence of DDAVP, when administered before learning, on selective attention using a concept shift discrimination learning task and on short-term memory (STM) using a visual memory task. The subjects, healthy male college students, between 18 and 25 years of age, were tested for blood pressure (BP) and heart rate (HR) and were administered the Spielberger State-Trait Anxiety Inventory (STAI) before peptide treatment and again after the completion of behavioral testing. This testing was done to monitor effects of the peptide on blood pressure and emotional arousal (anxiety). The subjects were also required to complete the vocabulary subtest of the Wechsler Adult Intelligence Scale (WAIS) and the test scores were used to ensure vocabulary skill equivalency among the peptide-treated and two control groups. The subjects received a single administration of either DDAVP (60 g/subject, intranasal), or placebo (physiological saline, intranasal) or were assigned to a no-treatment control condition, and behavioral testing began 20 min later. For the concept shift discrimination task, the subjects were consecutively trained in four discrimination problems requiring selective attention to the stimulus dimension of color or shape: the original discrimination, the discrimination reversal, the intradimensional shift, and the extradimensional shift. The stimulus material consisted of slides of geometric forms, distinctive in color and shape (e.g., red, black, blue, and white circles, squares, crosses, and triangles). For each discrimination problem the subject was shown a set of two slides and instructed to select the object he thought was correct. He was immediately informed if his choice was correct or not. After he reached the learning criterion on that problem (nine consecutive correct responses) he was presented with the next discrimination problem. For the original discrimination problem, color was relevant and form was irrelevant. For the discrimination reversal, the subject was required to choose the previously incorrect response of a two-item set (e.g., if red was the original correct response, the subject had to shift to black). For the intradimensional shift, a new set of stimuli was presented that differed in color and shape, but the original dimension (i.e., color) was still relevant. For the extradimensional shift, the heretofore irrelevant dimension of shape

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was now relevant. Efﬁcient performance depended on focusing attention on the relevant stimulus dimension. For the STM test, the subjects were shown 10 slides picturing geometric forms of varying complexity, one at a time, for 1 s. Thirty seconds after viewing each slide, the subject was asked to reproduce the ﬁgure with paper and pencil. The results were as follows: (1) the single dose of DDAVP improved performance (fewer trials to learning criterion) in each of the four discrimination problems (original learning, reversal, intradimensional shift, and extradimensional shift); (2) verbal ability, as assessed in the WAIS, did not inﬂuence any of the results of the four problems; (3) the single dose of DDAVP did not signiﬁcantly inﬂuence either the number of correct responses or the total number of errors made on the visual STM task; (4) there were no effects of the peptide on BP or HR; and (5) there were no differences among the three groups in general anxiety scores, nor did DDAVP affect anxiety state during the course of the experiment. The authors concluded that the rapid learning of the successive discrimination problems by the DDAVP-treated subjects suggested a vasopressin enhancement of selective attention. This is plausible to the extent that selective attention was an essential requirement for rapid solution of these discrimination problems. The failure of the peptide to inﬂuence STM is consistent with the theoretical views of De Wied and colleagues (Chapters 2–5) and also with ﬁndings in animal studies using the radial maze task (Buresova and Skopkova, 1980; Van Haaren et al., 1986; see Chapter 9). The observations that DDAVP did not inﬂuence BP, HR, or general anxiety during this study indicate that peripheral mechanisms (e.g., pressureassociated arousal effects) did not participate in the peptide-induced improvement in cognitive performance. b. Beckwith et al. (1983) Beckwith et al. (1983), noting that vasopressin improves human performance in tasks requiring attention, concentration, and memory (e.g., Legros et al., 1978; Weingartner et al., 1981), designed a study to separate these actions of vasopressin on behavior. The Sternberg item recognition task was selected for study because its results, according to Sternberg, theoretically allow attentional effects to be separated from those of memory (Sternberg, 1970, 1975). More speciﬁcally, Sternberg has used this reaction time (RT) task to study the cognitive operations involved in memory retrieval. Brieﬂy, the subject is presented with a set of digits (well within the subject’s memory span) to memorize (positive set). In the ﬁxed set procedure version of the task, used in this study, the positive set is presented once before a long series of test trials (e.g., 50 trials). Each test trial is initiated by a warning signal that announces the presentation of the test stimulus (a digit that may or may not have been in the positive set). The subject is required to execute a ‘‘yes’’ or ‘‘no’’ response (e.g., press the appropriate response key) and the task is designed to require as many

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‘‘yes’’ as ‘‘no’’ responses. The RT is the dependent measure. After completion of the ﬁrst series of the test, the procedure is repeated with a different positive set, varying in number of items (1, 2, 3, or 4 items) for a total of 4 sets of test trials (e.g., 50 trials/set). The data are graphed by plotting RT against set size (number of items per set). The results of numerous experiments with this task paradigm show a positive linear function relating RT to set size (a unit increase in RT with each additional item in set size). Sternberg has interpreted the slope of the linear function as indicating the time required for scanning the memory representation and comparing the target stimulus with each of the items in the positive set stored in active (shortterm) memory. The zero intercept is interpreted as the time required to engage in the one-time mental operations required for each trial: that is, encoding the test stimulus for representation and organizing and executing the response. Beckwith used this task to separate memory operations (i.e., scanning the representation in the retrieved memory) from attentional processes (deﬁned herein as encoding and response selection). The subjects were tested in two test sessions, separated by 1 week. Before behavioral testing, half the subjects were randomly assigned to either group A or B, which differed in the order of DDAVP (60 g/kg, intranasal) or placebo (physiological saline, intranasal) administration across the two test sessions. Group A received DDAVP in session 1 and placebo in session 2; this order of treatment was reversed for group B. In addition to behavioral testing, the subjects were also tested for peptide effects on BP and anxiety status (STAI). For each session, the subject was given 200 practice trials on the Sternberg item recognition task followed by administration of DDAVP or placebo while supine for 15 min, BP reassessment while sitting, and partial completion of the STAI. The subject then received the ﬁrst 200 test trials of the recognition task followed by a 5-min rest period, during which the STAI was completed, and then the ﬁnal 200 test trials of the recognition task, followed by a ﬁnal check of BP. Each session lasted about 1.5 to 2 h. Performance on the Sternberg item recognition task coincided with the estimated maximal drug-level time range based on biphasic DDAVP halflives of 7.8 and 75.5 min postadministration (Ferring Pharmaceuticals product information). Each of the 200 test trials was divided into 4 blocks of 50 test trials. Before each block of 50 test trials, the subject was visually presented with the target display to be memorized (a set of 4, 3, 2, or 1 digits) displayed on a cathode ray tube screen for 4 s. Fifty test trials followed, in which the subject was visually presented with a single digit and required to indicate whether the digit had been present in the target display by pressing a ‘‘Yes’’ or ‘‘No’’ response key. Reaction time (RT) was measured in milliseconds from stimulus onset (single digit) to response initiation by means of the Apple II-plus computer used for this task.

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DDAVP had no signiﬁcant effect on BP or self-reported anxiety, indicating that neither arousal induced by peripheral pressor effects nor emotional arousal was of inﬂuence in this study. The total mean error rate for all subjects was approximately 1.33%, with group A accounting for 0.93%, and group B 0.41%, of the total error rate. Group A made more errors than group B and also responded with slower reaction times, indicating that reaction time was inversely related to performance accuracy. Four-way analysis of variance (ANOVA; treatment session decision type memory set size) used to analyze the RT scores produced two signiﬁcant main effects (set size and decision type) and a signiﬁcant treatment session interaction. The signiﬁcant main effects indicated that reaction time slowed for each increase in set size, and that ‘‘no’’ responses were given more slowly than ‘‘yes’’ responses for each set size. The absence of main effects for session and treatment indicated that a practice effect (improvement from session 1 to session 2) was not a major inﬂuence in this task, and that there was no overall inﬂuence of DDAVP on reaction time performance, respectively. Subsequent analysis of the signiﬁcant treatment session interaction, however, indicated that during session 1, DDAVP-treated subjects responded slightly more slowly (not statistically signiﬁcant) than placebo controls, but that during session 2 DDAVP-treated subjects responded faster (statistically signiﬁcant) at each memory set size. Moreover, close inspection of the data indicated that when DDAVP did inﬂuence reaction time (session 2) it inﬂuenced the y intercept but not the slope of the function relating reaction time to memory set size. In accordance with the theoretical distinction formulated by Sternberg (1970, 1975) for this task, this ﬁnding indicated that attention, not memory per se, was inﬂuenced by the peptide treatment. Although a practice effect was not a major independent inﬂuence in this study, the authors suggested that slight practice and DDAVP effects could have combined, contributing to the improvement in reaction time in group B during session 2. That DDAVP did contribute to this improved performance was indicated by an analysis of covariance procedure that removed practice effects from second session performance, with the same results as the original ANOVA. Taken together, the results of the study were interpreted as indicating that the improvement in the Sternberg item recognition task, due to DDAVP, was complex in nature. The ﬁnding that treatment lowered the intercept only in the second testing period suggested that increased alertness, faster digit encoding, or speedier response selection occurred in subjects who had received both DDAVP treatment and prior experience in this task. An alternative possibility, not suggested by these authors, was that DDAVP might have exerted a slight enhancement of attention in both groups. When given to the less proﬁcient group A (session 1) its effects were masked by the performance of subjects in group B resulting from

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their superior baseline proﬁciency in this task. When administered to group B in session 2, the peptide-induced enhancement of attention magniﬁed the performance differences between the two groups. That group B was superior to group A in baseline task proﬁciency was evident by group B’s faster reaction times and fewer errors regardless of the treatment condition. c. Beckwith et al. (1984) Beckwith et al. (1984) assessed the effects of DDAVP on memory encoding and retrieval processes in male and female undergraduates, ranging in age from 18 to 33 years. The memory task used sentences (as opposed to digits or single words), worded so that certain implications could likely be drawn (e.g., ‘‘the pupil carefully positioned the thumbtack on the chair’’ implied performance of a prank). Encoding was manipulated by instructing the subjects to read the sentences for ‘‘comprehension’’ or for ‘‘memorization.’’ In the comprehension condition they were also asked to jot down a word implied by the sentence as soon as it was read (e.g., ‘‘prank,’’ or some equivalent, would be expected for the sentence cited above). Retrieval was manipulated by using either free or cued recall. For free recall, the subjects were allowed 5 min to write as many sentences as they could remember regardless of order. For cued recall, the subjects heard a tape-recorded list of 16 cue words (e.g., prank) and were told that each might remind them of a sentence heard earlier. The subjects recorded their responses to the cue words on separate pages of an answer book they were given. Before behavioral testing, the subjects were inventoried for health status to ensure that they were free of medication, illness, hypertension (blood pressure above 140/90 mmHg), and cardiovascular and renal disease. The vocabulary subtest of the WAIS was administered to assess verbal ability. The 64 male and 64 female subjects were randomly assigned to each of the treatment encoding retrieval conditions (n ¼ 8). Half the subjects received DDAVP (60 g /subject, intranasal), and half received placebo (physiological saline, intranasal) followed by behavioral testing 20 min later. Separate ANOVAs (treatment encoding retrieval) were conducted for males and females, in light of known sex differences in the performance of tasks requiring verbal skills (Macoby and Jacklin, 1974). For males, the ANOVA demonstrated a signiﬁcant main effect for treatment, with DDAVP enhancing recall across all conditions. There were marginal main effects (just short of statistical signiﬁcance) for encoding and retrieval conditions (recall tended to be better when instructed to comprehend and when tested under cued recall) with no signiﬁcant interactions. For the females, DDAVP treatment did not inﬂuence recall performance, but there was a main effect for retrieval (recall was enhanced under the cued-recall condition of retrieval). The authors suggested that the DDAVP-induced enhancement of memory in only the males might have been due, in part, to their consistently lower

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performance under all task conditions. That is, the peptide may have interacted with baseline proﬁciency level, enhancing performance only with low baseline proﬁciency levels. However, they recognized the need to further explore sex differences in vasopressin effects on both verbal and visuospatial memory tasks. d. Till and Beckwith (1985) Till and Beckwith (1985) used a cross-over design with a sentence memory task to accomplish four goals. First, the cross-over design was used to replicate the earlier reported DDAVPenhanced performance effect on a memory task for sentences with implications that favored easy comprehension such as those used by Beckwith et al. (1984). The subjects (male undergraduates, 18–33 years of age) were required to memorize and retrieve two orally presented lists of sentences, one of which was presented in session 1, and the other 1 week later in session 2. Two treatment groups were created to evaluate a cross-over interactional effect: the DDAVP (test session 1)–placebo (test session 2) group (V–Pl) and the placebo (test session 1)–DDAVP (test session 2) group (Pl–V). It was predicted that if DDAVP enhances immediate recall, the V–Pl group would perform better than the Pl–V group in session 1, and the reverse would be true for session 2. Immediate recall was tested in each session after presentation of the list of sentences. Second, an attempt was made to determine whether the effect of DDAVP on this memory task occurs at the encoding and/or the retrieval phase of memory processing. This was done by evaluating immediate versus delayed recall for session 1 material. Delayed recall for session 1 material was tested at the end of the immediate recall period in session 2. It was predicted that if DDAVP improved encoding, the V–Pl group would be superior to the Pl–V group at both immediate recall (session 1) and delayed recall (session 2). However, if the treatment effect was localized at the retrieval stage, then a cross-over interaction would be expected (i.e., the V–Pl group would do best for immediate recall, the Pl–V group for delayed recall). Third, because earlier study (Beckwith et al., 1984) suggested that DDAVP may preferentially improve task performance in low-verbal subjects, the inﬂuence of vocabulary proﬁciency on performance was assessed by splitting each group into high-verbal and low-verbal subgroups. The two subgroups were subsequently compared on immediate and delayed recall for treatment effects, and vocabulary treatment interactions were expected. Fourth, a practice effect on this task was evaluated in the absence of DDAVP treatment by adding a placebo group toward the end of the data collection period. Some degree of improvement from the ﬁrst to the second session was expected. Before assignment to the treatment conditions, health and biographical data were reviewed, BP was recorded, and the subjects were tested for verbal ability in the WAIS vocabulary subtest. Assignment to the V–Pl and Pl–V groups was arbitrary, although an effort was made to make them

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equivalent in verbal ability. For each of the two behavior sessions, the subjects were tested 20 min after intranasal administration of DDAVP (60 g/subject) or physiological saline. The test procedure was as follows. The subject heard the prerecorded sentence (one new sentence presented every 20 s) through binaural earphones from a cassette tape. Each sentence was read and immediately repeated to minimize perceptual errors. After presentation of the list, which the subjects were required to ‘‘memorize,’’ a 1-min counting task was given to minimize short-term memory or recency effects in free recall. During free recall, which immediately followed, the subject was given 5 min to write down, in any order, as many sentences as remembered, and encouraged to use as exact wording as possible. A second comparable list of sentences was used in behavioral session 2, held 1 week later. At the end of free recall in session 2, two additional recall tests were given. First, each subject received a list of cues for session 2 and was given 5 min to use the cues to help recall these sentences. The subject was allowed to write those words just given in free recall (but not allowed to look at the free recall sheet). The subject wrote the recall sentence alongside the cue that reminded him of the sentence. Second, the subject was given a delayed noncued recall test for the sentences presented in session 1 and had 5 min to recall them. The three treatment-order groups (V–Pl, Pl–V, and Pl–Pl) did not differ in age or vocabulary scores. There were three major effects of DDAVP on immediate and delayed recall: (1) the expected cross-over interaction effect for immediate memory was weak. That is, DDAVP signiﬁcantly improved recall in session 1, but it did not do so in session 2 (see Fig. 1); (2) both

FIGURE 1 Mean number of sentences recalled by subjects in the three treatment-order groups: vasopressin (V)–placebo (Pl), placebo–vasopressin, and placebo–placebo. Data are averaged across verbal ability and type of scoring. Source: Till and Beckwith, 1985 (Fig. 2, p. 400). Copyright ß 1985 by Ankho International Inc.

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FIGURE 2 Mean number of sentences recalled, during immediate and delayed tests of ﬁrstsession material, by subjects in the three treatment-order groups: vasopressin (V)–placebo (Pl), placebo–vasopressin, and placebo–placebo. Data are averaged across verbal ability and type of scoring. Source: Till and Beckwith, 1985 (Fig. 4, p. 401). Copyright ß 1985 by Ankho International Inc.

immediate and delayed recall of material presented in session 1 were better in the DDAVP-treated subjects than in the placebo-treated subjects (see Fig. 2), indicating a cross-over interaction for retrieval. Because immediate recall for session 1 material was superior in the V–Pl group, whereas delayed recall of this material was better in the Pl–V group, treatment at the time of retrieval was a decisive factor; and (3) subsequent analysis of the DDAVP vocabulary interaction indicated a signiﬁcant treatment effect for the lowvocabulary subgroup at immediate testing and for the high-vocabulary subgroup at delayed testing (Fig. 3). The peptide enhancement of immediate memory in the low-vocabulary group was indicated by the signiﬁcant increase in immediate recall from the ﬁrst to the second session, which occurred only for this subgroup (Fig. 4). In contrast, the high-vocabulary subgroups were quite similar on immediate recall but differed on delayed recall. It was suggested that these subjects, already highly proﬁcient in verbal learning, were helped by DDAVP only in the retrieval process. Taken together, the results of this study suggested that DDAVP improved memory processing during both encoding (enhanced immediate recall in the V–Pl group in session 1) and retrieval (cross-over effect for delayed recall of session 1 material), but the effect on retrieval was more consistent. It is not clear why DDAVP improved immediate memory for the material learned in session 1 but not for that learned in session 2. The possibilities that the V–Pl group was superior in baseline performance, or

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FIGURE 3 Mean number of sentences recalled by low-vocabulary and high-vocabulary subjects treated with vasopressin (V) and placebo (Pl) and tested for immediate and delayed recall of ﬁrst-session material. Data for all treatment groups are averaged across type of scoring. Source: Till and Beckwith, 1985 (Fig. 3, p. 401). Copyright ß 1985 by Ankho International Inc.

that DDAVP effects carried over to session 2, are ruled out by the results for delayed recall in session 2. There was no difference between the DDAVPtreated (Pl–V) and placebo-treated (V–Pl) groups for immediate memory in session 2, but the DDAVP-treated subjects were superior on delayed recall of material learned in session 1. This ﬁnding indicates that the peptide did inﬂuence retrieval of previously learned information. The authors concluded, however, that the locus of the effect of the peptide on encoding and/or retrieval stages of memory was not clearly delineated in this study and that future study should use successive tests of both immediate and delayed recall for further clariﬁcation of this issue. The ﬁnding that DDAVP differentially inﬂuenced performance in the low- and high-vocabulary groups suggests that vasopressin is more effective in subjects showing less proﬁciency in this task, at least for immediate recall. Moreover, this interactional effect may have contributed to the sex differences observed by Beckwith et al. (1984) because only males (consistently observed to have lower verbal proﬁciency than females; Macoby and Jacklin, 1974) beneﬁted from the peptide treatment.

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FIGURE 4 Mean number of sentences recalled by low-vocabulary and high-vocabulary subjects treated with vasopressin (V) and placebo (Pl) and tested for immediate recall during ﬁrst and second sessions. Data for all treatment-order groups are averaged across type of scoring. Source: Till and Beckwith, 1985 (Fig. 1, p. 400). Copyright ß 1985 by Ankho International Inc.

e. Beckwith et al. (1987a) Beckwith et al. (1987a) extended a test of vasopressin (DDAVP) effects to memory for narrative prose, which is more similar than digits, words, and sentences to the type of material normally learned, stored, and retrieved. It uses processes such as interpretation, inference, and abstraction to form themes, and so on, not required for recalling the simpler types of stimuli that may rely more on rote memorization. Healthy male undergraduates, ranging in age from 18 to 25 years, served as subjects in this study. On preliminary interview the subjects reported no chronic or acute illness and were free of medication. Blood pressure readings were 140/90 mmHg or less. Treatment assignment was conducted to ensure equivalency between the DDAVP and placebo treatment groups in verbal ability, assessed by WAIS vocabulary subscale scores. Behavioral testing occurred 20 min after a single intranasal administration of either saline or DDAVP (60 g/subject). After receiving instructions and 1 practice passage, the subjects heard, via audiotape, 6 experimental passages (200- to 250-word narrative) and were asked to recall as much of the passage as possible immediately after each was presented. Recall was typically ﬁnished within 3 min of hearing the audiotape. The passages were presented at three different speeds: two at slow, two at medium, and two at a rapid rate of presentation. Each story had been divided into idea units previously rated by independent groups of

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college students on a three-point scale for their importance to the theme of the story. The number of idea units at each level of importance was approximately equal. Recall for each passage was expressed as the proportion of idea units recalled under each level of importance. The proportion of idea units recalled was analyzed in a four-way mixed ANOVA (treatment WAIS-voc rate importance) with repeated measures on the last two factors. Several signiﬁcant main effects (treatment, verbal ability, rate, and level of importance) and a signiﬁcant interaction between treatment and level of importance were found: (1) DDAVP-treated subjects recalled a greater proportion of idea units than did placebotreated subjects; (2) subjects high in verbal ability recalled a greater proportion of idea units than did those low in verbal ability; and (3) proportion of recall was a function of rate of presentation (slow > medium > fast) and of level of importance (level 3 > level 2 > level 1). Further analysis of the signiﬁcant interaction showed that DDAVPtreated subjects recalled a greater proportion of idea units at both high and medium levels of importance compared with placebo-treated subjects. The peptide did not inﬂuence recall of low-importance idea units. The interaction of treatment and level of importance was interpreted as suggesting a DDAVP enhancement of selective attention to the most important idea units. The authors noted that (1) a DDAVP-induced enhancement of attention in this paradigm is consistent with their ﬁndings with human (e.g., Beckwith et al., 1982, 1983) and animal (Beckwith and Tinius, 1985) subjects; and (2) in addition to its enhancement of selective attention to the important idea units in this prose task, the peptide may have facilitated divided attention, which is involved in the comprehension of prose passages (Kintsch and Van Dijk, 1978). That is, the view that attentional processing capacity is limited necessitates its division between the continuous encoding of the text and maintenance in working memory of propositions abstracted from earlier parts of the text. Comprehension of the passage improves as interconnections among propositions become established. ‘‘Hence, treatment with DDAVP may have facilitated the divided attentional processes necessary to integrate text in working memory as evidenced by the increased attention to relevant as opposed to irrelevant details of the passages presented.’’ (Beckwith et al., 1987a, p. 431).

B. Research with Animal Subjects 1. Overview Beckwith and colleagues tested the role of vasopressin in attentional processing in laboratory animals with the aid of an analytic model developed by Mackintosh and Sutherland (Mackintosh, 1965; Sutherland and

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Mackintosh, 1971) to help explain the ability of certain manipulations (e.g., overtraining) during the original learning of a visual discrimination to enhance subsequent reversal learning. As discussed by Beckwith and Sandman (1982), this model proposes that two successive learning components are involved in mastering a discrimination problem: ﬁrst, learning to attend to the relevant stimulus cues (strengthening the appropriate sensory analyzers) and second, learning the choice response appropriate to the cue-reinforcement contingency that is in effect. In addition, the model postulates that analyzer strength accumulates at a different rate than does response strength and certain treatments such as overtraining and certain neuropeptides (e.g., MSH/ACTH analogs) increase the strength of the attention response to a greater degree than that of the choice response to that cue. In learning the reversed discrimination, it is only the choice response that needs to be extinguished because the attentional response remains relevant. Therefore treatment that strengthens the attentional component during original learning of the discrimination should facilitate learning the reversed discrimination. Thus, according to this model, if exogenously administered AVP facilitates attention processing, this treatment will be expected to enhance subsequent reversal learning compared with controls. More speciﬁcally, in white/black visual discrimination in a Y-maze, reversal requires that the animal relearn the new cue-reinforcement contingency that is rewarded (e.g., dislodge the black door) while selectively attending to the same stimulus cues (white- and black-painted doors). The treated subjects, presumably having a stronger attention response than the controls, will be less likely to extinguish this still relevant attentional response, whereas both groups will extinguish to an equal degree the earlier learned choice response. In animal experiments with vasopressin, this explanatory model has been tested with the use of the reversal discrimination task described below. 2. Description of the White/Black Reversal Discrimination Task The task apparatus and procedure are described in Couk and Beckwith (1982). This task typically involves testing original learning, reversal learning, and retention of reversal learning in a simultaneous visual discrimination task with black and white doors as the discriminative stimuli. The test apparatus is the Thompson–Bryant discrimination box, which is divided into three compartments: a start box, choice compartment, and goal box. A guillotine-type door separates the start box and choice compartments, and two solid doors (black and white) separate the choice compartment from the goal box. A grid ﬂoor enables delivery of footshock (5 mA, 5 s) in the aversive motivational version of the discrimination task. During pretraining the animal is trained to dislodge each of the doors that bar entrance to the goal box. Under reward conditions pretraining adapts the animal to eat in the test apparatus and shapes the response of

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dislodging the doors of the choice chamber to enter the goal box. In the aversive procedure, pretraining shapes the animal to leave the start box, cross the choice chamber, and enter the safe goal box by dislodging each door of the choice chamber. During original learning, only the solid white door (Sþ; a stimulus signaling availability of reinforcement) can be opened. The door positions are alternated according to a semirandom, that is, Gellerman series (Gellerman, 1933). The animal receives 10 trials/day until it achieves the learning criterion of 9 of 10 correct choices. The number of trials required to reach the learning criterion is the dependent variable. During discrimination reversal, which begins the day after completion of original learning, the black door is the Sþ. The procedure and learning criterion for reversal training is the same as that for original learning, as is the procedure and performance criterion for the reversal retention test, given after a 5- to 10-day retention interval. 3. Research Studies a. Couk and Beckwith (1982) Couk and Beckwith (1982) used the appetitive version of the white (Sþ)/black (S; stimulus not followed by reinforcement) discrimination reversal task in their investigation of the effect of vasopressin on the rate of original learning, reversal learning, and retention of the reversal-learned task in male Holtzman albino rats. This was the ﬁrst animal study to employ intranasally administered DDAVP, which these researchers were to use on a regular basis in VP/memory studies with human subjects. Two groups of subjects received an intranasal treatment of placebo or DDAVP (6 g/rat) peptide 15 min before the ﬁrst trial of each 20-trials/day test session throughout original and reversal learning. A third group, the nontreatment control group, received no intranasal solution. Training for each phase of learning continued until the subject reached the learning criterion (9 of 10 correct responses during 1 day’s test session). No treatment was administered during the retention test phase, which began 5 days after completion of the reversal learning phase. Comparisons of the peptide, placebo, and nontreatment groups indicated no signiﬁcant peptide effects on either original or reversal learning, or on retention of the reversed discrimination. However, the data suggested a trend toward facilitation of original learning in the DDAVP-treated animals, which was further studied by inspection of learning curves constructed for each phase of the study (original learning, reversal learning, and retention of the reversal problem). This inspection suggested that, relative to the placebo treatment and no-treatment control conditions, DDAVP treatment produced a trend toward facilitation of original learning, an initial learning impairment in the reversed discrimination, and had no effect on retention of the reversal discrimination. Separate ANOVAs performed on trials to criterion in original and reversal learning, and on savings scores

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calculated as a measure of retention of the reversal discrimination, indicated no overall treatment effects on either dependent variable and no signiﬁcant interactions. These results were interpreted as follows: (1) the clear-cut absence of a DDAVP-induced effect on retention of the discrimination reversal indicated that this nonpressor vasopressin analog did not inﬂuence long-term memory; (2) the tendency for facilitated original learning in the DDAVP-treated subjects suggested a peptide-induced facilitation of short-term memory processes; and (3) the initial impairment of reversal learning observed in the DDAVP-treated subjects reﬂected ‘‘a subtle perseveration of the originally learned response’’ (Couk and Beckwith, 1982, p. 525). The interpretation that this peptide had no inﬂuence on long-term retention may not be warranted by the design used to test this effect. Bunsey (personal communication, 1998) pointed out that because DDAVP was administered during both original and reversal learning, it is conceivable that the treatment enhanced retention of both discriminations and the absence of the subsequently observed retention effect on the reversal problem was simply the net outcome of interference from two types of opposing memories. The initial impairment of reversal learning among the peptide-treated rats is consistent with this interpretation. b. Beckwith and Tinius (1985) Beckwith and Tinius (1985) used the aversive Wþ/B discrimination task to test the effects of three vasopressin peptides, arginine vasopressin (AVP), arginine vasotocin (AVT), and pressinoic acid (PA), on original and reversal learning. In the AVP(1–9) molecule, six amino acids form the ring-shaped structure and three amino acids form the tail component. AVT, an ancestral form of vasopressin, is identical in structure to AVP with the exception of amino acid 3 (i.e., isoleucine instead of phenylalanine) appearing within the ring structure of the molecule. PA [AVP(1–6)] contains the ring structure of AVP but lacks the three C-terminal amino acids (amino acids 7, 8, and 9) that form the tail of the molecule. Comparisons among the three peptides in original and reversal learning permit assessment of the need for the integrity of the ring structure of AVP (AVP versus AVT) and the contribution of the tail structure (AVP versus PA) for the putative inﬂuence of this peptide in task performance. Depending on the treatment group, the subjects (young male adult Holtzman albino rats) received an intraperitoneal injection of vehicle, AVP (1 g/rat), AVT (1 g/rat), or PA (1 g/rat). These dose levels were chosen because a literature review indicated that the 1-g dose was the most frequently used behaviorally effective dose of AVP, and the researchers wanted to compare analogs at the same dose level. Injections were given 10 min before testing throughout both original and reversal learning. Several dependent variables (trials to criterion, percent correct responses per day, number of errors during learning) were each analyzed in separate one-way

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ANOVAs for original and reversal learning. In addition, savings scores (original learning minus reversal learning divided by original learning) were analyzed for the reversal learning phase of the study. Subsequent comparisons used the Newman–Keuls test. The results indicated that none of the peptides, at the dose level used in this study, signiﬁcantly inﬂuenced any of the dependent variables used to assess original discrimination learning (i.e., trials to criterion, percent correct responses per day, or number of errors). The results for reversal learning indicated that relative to placebo controls: (1) none of the peptides inﬂuenced trials to criterion; (2) AVP signiﬁcantly increased savings, increased percentage of correct responses per day, and reduced errors; (3) AVT increased the percentage of correct responses per day and reduced errors; and (4) PA increased the percentage of correct responses per day. Overall, these results indicated that chronic treatment with AVP and AVT during acquisition and/or reversal learning resulted in consistent enhancement or reversal learning (inﬂuenced at least two of the three measures of learning) whereas PA exerted a less consistent effect (inﬂuenced only one of the three). In this study, AVT and AVP produced similar effects on task behavior— neither inﬂuenced original learning and both enhanced reversal learning. Other studies have shown that AVT is less effective than AVP in retention enhancement. For example, it is ineffective against puromycin-induced amnesia (Walter et al., 1975) and has only 7% of the potency of AVP in prolonging extinction on a pole-jump avoidance task (Walter et al., 1978). Overall, these ﬁndings suggest that the integrity of the ring structure of AVP is not important for some behavioral actions but crucial for others. The speciﬁcs of this suggestion deserve further study. The ﬁnding that, unlike AVP and AVT, PA did not consistently enhance reversal learning was interpreted as indicating that the three C-terminal amino acids need to be present along with the ring structure for full potency of AVP-like peptides. This agrees with De Wied’s observation that whereas the ring structure of PA is essential for the behavioral action of the peptide, much higher doses of PA relative to the parent peptide are necessary to produce consistent results in conditioned avoidance paradigms (De Wied, 1976). The results of this study were interpreted as being consistent with the selective attention hypothesis. If the peptides had enhanced memory storage during original acquisition, the stronger memory should have interfered with reversal discrimination learning. This did not occur. Instead, the observed vasopressin enhancement of reversal learning conformed to the earlier described two-stage attentional model of discrimination learning espoused by Mackintosh (1965). c. Beckwith et al. (1987b) Beckwith et al. (1987b) conducted three experiments to obtain further information relevant to the putative ability

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of vasopressin to inﬂuence selective attention in the reversal learning discrimination task. The procedures and statistical analyses used in these experiments permitted (1) replication of the AVP-induced effect on reversal learning observed by Beckwith and Tinius (1985), (2) determination of whether this effect resulted from peptide treatment given during original and/or reversal learning (experiments 1 and 2), (3) further clariﬁcation of the mechanism by which the peptide inﬂuences reversal learning (experiment 3), and (4) determination of whether AVP exerted mnemonic effects on this task, independent of its effects on selective attention. The Sutherland–Mackintosh attentional model was used to guide the procedure and the interpretation of the results of these experiments. If AVP treatment acts like overtraining and enhances attention during original learning, the AVP-treated subjects should demonstrate more rapid reversal learning than do the placebo controls. Because overtraining occurs after completion of learning in the original problem (Sutherland and Mackintosh, 1971), AVP was given after daily training during original learning. The subjects, young adult male Holtzman albino rats, were trained and tested under aversive motivation (footshock) during original learning, reversal learning, and retention in the Wþ/B discrimination paradigm. In experiment 1, the subjects received an intraperitoneal injection of AVP (1 g/rat) or placebo (physiological saline) immediately after completion of each 10-trial/day session of original learning. No treatments were given during reversal learning. The subjects were tested for retention of the reversal (savings method) 10 days after achieving criterion for reversal learning. During retention testing, half of the subjects that received AVP, and half of those that received placebo during original learning were given AVP (1 g/rat), and the remaining animals received placebo. Injections were given 60 min before each 10-trial/day retention test session. The dependent measures (trials and errors to criterion) were separately analyzed in twofactor ANOVAs (treatment problem) with repeated measures for the last factor. Newman–Keuls procedures were used to assess interactions. Chronic AVP treatment during original learning did not affect acquisition of the original discrimination, or retention of the reversed discrimination. However, this treatment reliably facilitated reversal learning, as indicated by both fewer trials and errors to reach the learning criterion for the AVP-treated animals than for the placebo-treated animals. When given during retention testing, AVP signiﬁcantly impaired performance, an effect independent of the treatment given during original learning (see above). This pattern of results can be interpreted as indicating (1) a peptide-induced facilitation of selective attention to stimulus cues (represented as memory traces) present in the original discrimination, which in turn was responsible for enhanced reversal learning in accordance with the Sutherland–Mackintosh model; and (2) that this selective attention effect was independent of the inﬂuence of AVP on memory processing, because AVP inﬂuenced neither acquisition

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of the original discrimination nor retention of the reversed discrimination. Moreover, the peptide-induced impairment of retrieval was independent of initial treatment. Experiment 2 was designed to determine the effect of AVP on reversal learning when the peptide was given during reversal and to replicate the VPinduced retention effect observed in experiment 1. The procedure was the same as that in experiment 1 except that no treatment was given during original learning, and AVP (1 g /rat) or placebo was intraperitoneally injected 1 h before each 10-trials/day training session in the reversed discrimination. When given during reversal learning, AVP had no inﬂuence on trials or errors to the reversal learning criterion nor did it inﬂuence retention (savings) in the retention test given after the 10-day retention interval. Consistent with experiment 1, the daily AVP treatments given during retention testing impaired savings, as measured by both trials and errors to criterion, and this AVP-induced impairment of memory retrieval was independent of earlier treatment. Taken together, the results of experiments 1 and 2 localized the enhanced reversal learning effect observed in experiment 1 to the posttrial AVP treatment given during initial discrimination learning. The impaired retention of the reversed discrimination observed in experiments 1 and 2 could be localized to the pretrial AVP given during retention (i.e., a retrieval effect). Possible disruptive effects of the drug, due to sedation or overarousal, are unlikely causes of the impaired retrieval because pretrial AVP did not inﬂuence reversal learning. Experiment 3 was designed to help clarify the means by which AVP, given during original learning, is able to inﬂuence the rate of reversal learning. The authors noted that AVP has been shown to consistently enhance resistance to extinction in aversive conditioning tasks, interpreted as a retention effect by De Wied and colleagues (e.g., De Wied, 1971; see Chapter 2). Beckwith et al. (1987b) proposed an alternative explanation. Because overtraining results in greater resistance to extinction as well as enhanced reversal learning, AVP could prolong extinction of the originally learned discrimination by a process similar to that of overtraining. If so, the facilitated reversal learning may be a result of enhanced selective attention ‘‘caused by the same process used to explain the overtraining-reversal effect’’ (Beckwith et al., 1987b, p. 333). The ability of AVP to prolong extinction in the originally learned Wþ/B discrimination was tested in experiment 3. The subjects were trained to criterion learning, as in experiment 1, and then tested for extinction. AVP (1 g/rat) or placebo was intraperitoneally injected immediately after the last trial each day throughout learning. Footshock and injections were omitted during extinction training, which began the day after completion of original learning. The percentage of correct trials to the white door (Sþ) was assessed on each day of testing during the extinction phase. Posttrial AVP treatment during learning had no effect on learning or on the rate extinction on this task. That is, the ANOVA produced

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a main effect for day but no signiﬁcant treatment effect or treatment day interaction, indicating no AVP treatment effect on the rate of learning or extinction in this task. It was concluded that although both AVP and overtraining facilitate selective attention during original learning (i.e., enhance reversal learning), they do so by different mechanisms. The results of the various experiments taken together were interpreted as indicating that AVP facilitates reversal learning as a result of its enhancement of selective attention operating during original acquisition, in accordance with the attentional model presented by Sutherland and Mackintosh (1971). Moreover, in this task, the effect of AVP on selective attention appeared to be independent of its effects on memory processing because (1) neither posttraining AVP given throughout original learning, nor pretraining injections of AVP during reversal learning, inﬂuenced the rate of acquiring either discrimination; (2) neither of these treatments inﬂuenced memory consolidation of the information learned in the original, or in the reversed discrimination (i.e., no extinction effect in experiment 3 and no savings effect in experiment 2); and (3) the peptide-induced impairment of retrieval in the retention test was independent of selective attention effects in experiments 1 and 2. d. Tinius et al. (1989) Tinius et al. (1989) tested the effects of AVP on reversal learning with the aversive motivational version of the Wþ/B discrimination task, in male Holtzman albino and Long-Evans (LE) hooded rats. The purpose of this study was to determine the strain generality of the AVP-induced facilitation of reversal learning previously observed for Holtzman albino rats in this task (Beckwith and Tinius, 1985; Beckwith et al., 1987b). The research literature has indicated strain differences in responsiveness to other peptides that inﬂuence reversal learning on this task. Thus, -MSH facilitated brightness discrimination reversal learning in Holtzman albino rats (Sandman et al., 1972), but not in LE hooded rats (Sandman et al., 1973). For this study, the training procedure, footshock intensity level, AVP dose level, and so on were identical to those used by Beckwith et al. (1987b). Placebo or AVP (1 g/rat) was intraperitoneally injected immediately after each day of training in the original discrimination problem. Reversal learning, without AVP treatment, began the day after completion of original learning. Retention for the reversed discrimination was not tested in this study. The results indicated strain differences in performance efﬁciency during reversal learning as well as in responsivity to AVP-induced effects on reversal learning. The two strains of rat did not differ in the rate at which they learned the original discrimination. The LE hooded rats, however, were superior to the Holtzman albino rats during reversal learning, requiring fewer trials to reach the reversal learning criterion regardless of the treatments received during original learning. There were also strain differences in

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responsivity to the effects of AVP on reversal learning as tested in this task. Speciﬁcally, AVP did not affect original learning in either strain of rat. On the other hand, AVP treatment during original learning enhanced reversal learning in the Holtzman albino rats (AVP-treated rats required signiﬁcantly fewer trials to criterion than did placebo-treated rats), but not in the LE hooded rats (no signiﬁcant difference between the AVP- and placebo-treated rats in trials to criterion). However, given that the hooded rats were superior to the Holtzman albino strain in reversal learning, the peptide may have facilitated reversal learning had either a higher dose or a more difﬁcult discrimination been used in this study. Thus, a ceiling effect may have masked an AVP-induced facilitation of selective attention in the hooded rats. This suggestion was supported by the observation that AVP-treated hooded rats showed a tendency to learn the reversal faster than did their placebo-treated counterparts. Thus subject and task variables can interact with vasopressin to inﬂuence outcomes and such interactions may help explain discrepant ﬁndings in the vasopressin/memory research literature.

III. Vasopressin and Attentional Processing: Bunsey, Strupp, and Colleagues

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A. Introductory Remarks Bunsey, Strupp, and colleagues have also suggested a role for vasopressin in attentional processing (Bunsey et al., 1990; Strupp et al., 1984). They have proposed that in studies where the peptide has been injected before presentation of the stimulus material to be processed, memory effects that are observed may, at least partially, reﬂect the inﬂuence of the peptide on one or more forms of attention, as deﬁned earlier in this chapter. Before the vasopressin/attentional research conducted by these researchers, direct support for a vasopressin-induced facilitation of attentional processing, and in particular attentional selectivity, had been obtained for humans (e.g., Beckwith et al., 1982: this Chapter; Legros et al., 1978) as well as for laboratory animals (e.g., Beckwith and colleagues; this chapter). Bunsey et al. (1990) also noted two lines of indirect evidence consistent with a putative role for vasopressin in selective attentional processing. First, there is the evidence in the VP/memory research studies of De Wied and colleagues (see Chapter 4) that vasopressin interacts with the locus coeruleus–noradrenergic (LC–NA) projection system during cognitive processing. Moreover, this NA projection system has been studied for its potential role in attentional processes such as vigilance (maintaining an active state of surveillance of the environment) and selective attention (attentional focusing on certain stimuli while gating out others) (e.g., Aston-Jones, 1985;

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Foote and Morrison, 1987; Robbins et al., 1985). Of speciﬁc relevance is the observation that vasopressin microinjected into the noradrenergic cell bodies of the locus coeruleus augments the release of noradrenaline (NA) from that structure (Olpe and Baltzer, 1981) and NA release increases the signalto-noise ratio of neurochemical inputs in the target areas innervated by this NA pathway that originates in the locus coeruleus (LC) of the brainstem (for review see Foote and Morrison, 1987). The ‘‘signal-to-noise ratio’’ term is adopted from engineering terminology to refer to the differential effects that LC–NA axons have on two sets of target neurons: those neurons that are spontaneously active versus those currently activated by other neuronal inputs (e.g., neuronal activity in auditory cortical neurons elicited by species-speciﬁc vocalizations). That is, the LC–NA axons inhibit the spontaneously active neurons to a greater extent than do neurons excited by other neural inputs, thereby producing a relative enhancement of the evoked neuronal discharge. The increased signal-to-noise ratio is theorized to underlie selective attention (AstonJones, 1985; Foote and Morrison, 1987; Foote et al., 1983). This theoretical inference derives from electrophysiological data collected in single-cell experiments conducted with wakeful and active squirrel monkeys (Foote et al., 1975). A second line of indirect evidence of a VP-ergic contribution to selective attention is the linkage between vasopressin and CNS activation or arousal. Bunsey et al. (1990) cite evidence that treatments that increase the level of behavioral arousal (e.g., application of CNS stimulants, exposure to environmental stressors, experimental induction of a heightened emotional or motivation state) increase attentional selectivity (i.e., narrow the range of cues used by the subject). The study described below represents the contribution of Strupp, Bunsey, and colleagues to VP/attentional research; their studies, concerned with the role of VP in other aspects of cognitive behavior, are described in Chapter 9.

B. Research Demonstrating a Role for Vasopressin Fragment AVP(4–9) in Selective Attention in Rats 1. Selected Study: Bunsey et al. (1990) Bunsey et al. (1990) designed two experiments to test the hypothesis that vasopressin increases selective attention (focusing on relevant and ignoring nonrelevant information) during discrimination learning. In both experiments, the subjects (female Long-Evans hooded rats) were required to remove the lid from the correct of two differentially covered boxes, to obtain a desirable Froot Loop reward hidden within. To solve the discrimination problem, it was necessary to selectively attend to either the lid or the box, herein called the ‘‘relevant dimension,’’ depending on which had the speciﬁc

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cues (the distinctive coverings of the lids or the boxes) relevant to the reward. The reward was obtained once the subject chose the correct speciﬁc cue associated with the relevant dimension. An effort was made to minimize stress in the test environment to reduce the chance that increased arousal and, perhaps, stress-augmented release of endogenous vasopressin might narrow attention in vehicle-treated controls. To this end, subjects were tested in their home cages, and were neither food or water deprived nor subjected to aversive conditions. Moreover, the vasopressin metabolite used, AVP(4–9), although highly effective in behavioral studies (Burbach et al., 1983a; see Chapter 5), lacks the pressor/aversive effects of systemically administered AVP (LVP), which presumably increases arousal level, theorized by some to mediate the effects of the peptide on cognitive behavior (Ettenberg et al., 1983a; see Chapter 6). In experiment 1, two tasks were used: a multiple-cue task that contained both predictive and nonpredictive cues, and a single-cue task that contained only relevant cues (i.e., lid cues or box cues). The multiple-cue task required the animal to determine the predictive dimension (whether the lid or box cues were predictive of reward), ﬁlter out the irrelevant dimension (selectively attend to the predictive set of cues), and determine which speciﬁc lid or box cue within the predictive set was associated with the reward. The single-cue task was included to evaluate the extent to which nonpredictive cues impede learning. If, as predicted, vasopressin by itself does not alter learning, then the single-cue task can rule out certain alternatives to a selective attention interpretation of the inﬂuence of the peptide on the multiple cue-task performance (e.g., an effect on motivation, sensory acuity, and basic associational ability). The box cues were judged as perceptually more salient because of their larger size (i.e., attached to bases) but more importantly because, being lower in position than the lid cues, they seemed to fall more directly in the rat’s line of vision (and olfaction) during its investigative approach (Bunsey, personal communication, 1998). An initial training phase familiarized the subject with the procedure and enabled it to become skilled in removing the box lids and obtaining the rewards. Eighty subjects successfully completed this training and were assigned to one of four AVP(4–9) treatment groups (0, 1.0, 3.0, or 10.0 g/kg, subcutaneous). Each treatment group (n ¼ 20) was divided to form the multiple-cue and single-cue subgroups (n ¼ 10), each of which was then divided to form lid-relevant and box-relevant subgroups. Subgroups were matched for box-opening proﬁciency exhibited during initial training. For the multiple-cue condition, the subjects were presented with two boxes, each of which had distinctively covered lids (wire mesh or soft blue rubber) and distinctively covered boxes (white cloth or black rubber). For the single-cue condition, the two boxes had either distinctively covered lids (wire mesh or soft blue rubber) and plain boxes, or plain lids and

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distinctively covered boxes (white cloth or black rubber). The speciﬁc cues consistently predictive of the reward in the box- and lid-relevant subgroups were counterbalanced across all peptide treatment groups. Depending on the treatment group, each rat received a subcutaneous injection of vehicle or AVP(4–9) (1.0, 3.0, or 10.0 g/kg; average body weight of 195 g at start of experiment), 1 h before each 4-trial/day training session, 7 days/week until it reached the learning criterion. During training, the two boxes were placed on the ﬂoor of the home cage to the left or right of the subject, using a computer-generated placement procedure devised to prevent a position habit. Learning rate was assessed by an ANOVA with trials to criterion as the dependent variable. The results of this analysis were as follows: (1) a borderline effect (p ¼ 0.057) of task type (multiple versus single cue) suggested that the presence of irrelevant cues might have increased task difﬁculty; (2) in the single-cue task neither dose level, relevant dimension (i.e., whether boxes or lids were predictive), nor dose relevant dimension interaction inﬂuenced learning rate (Fig. 5); and (3) for the multiple-cue task, signiﬁcant interaction between peptide dose and relevant dimension indicated that, relative to vehicle controls, AVP(4–9) dose dependently facilitated learning when the more salient box cues were relevant, but hindered it when the less salient lid cues were relevant, an effect especially prominent at the 1.0- and 10.0-g/kg dose levels (Fig. 6). In addition, the box/lidrelevant subgroup comparisons indicated that the superior performance of the box-relevant subgroup was statistically signiﬁcant at the 10.0g/kg dose level. A response analysis was also employed in this study to determine additional ways in which the peptide may have inﬂuenced cognitive behavior in this task. For this analysis, the response given after either an error or a correct response in the trials preceding the criterion trials was categorized as adaptive (win-stay, lose-shift) or nonadaptive (win-shift, lose-stay) responding in accordance with learning set methodology. This response typology was used for both the side of the cage in which the correct object was placed as well as the object itself. For example, a peptide-induced bias on stay-side responding could indicate spatial perseveration, perhaps implicating hippocampal involvement, which, in turn, could serve as a subject for future research (Bunsey, personal communication, 1998). Altogether, there were eight types of responses: four for side and four for object. Only the four adaptive response types (win-stay: object; lose-shift: object; win-stay: side; lose-shift: side) were statistically analyzed. The dependent variable for this response analysis was the number of times a particular type of response was made divided by the total possible such responses, multiplied by 100 for a percentage. Finally, a t test was used to learn whether vasopressin (10-g/kg dose level) altered motivation (latency to begin eating, or time required to

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FIGURE 5 Mean number of trials to criterion for the box-relevant and lid-relevant versions of the single-cue task as a function of increasing dosage of AVP4–9 (n ¼ 5, for each subgroup deﬁned by dosage and relevant dimension). (The peptide treatment did not affect the learning rate in either version of the task.) Source: Bunsey et al., 1990 (Fig. 1, p. 280). Copyright ß 1990 by the American Psychological Association. Reprinted with permission.

retrieve and consume the four Froot Loops). This last test indicated no peptide effect on either motivational variable. The results of the response analyses revealed no signiﬁcant main effects or interactions for the side-choice responses (adaptive responding that pertained to the side of the cage in which the object was placed). However, signiﬁcant results were obtained in the analyses performed on the object-choice responding in both the single- and multiple-cue conditions. For the single-cue condition signiﬁcant results were obtained for both loseshift (Fig. 7, left) and win-stay (Fig. 8, left) adaptive response strategies, as follows: (1) a signiﬁcant dose relevant dimension interaction inﬂuenced the lose-shift response strategy; separate analyses for the lid- and box-relevant conditions indicated no dose effect in the box-relevant condition but a signiﬁcant linear effect in the lid-relevant condition, indicating a dose-dependent decline in the percentage of times this adaptive strategy occurred (i.e., a

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FIGURE 6 Mean number of trials to criterion for the box-relevant and lid-relevant versions of the multiple-cue task as a function of increasing dose of AVP(4–9) (n ¼ 5, for each subgroup deﬁned by dose and relevant dimension). (The interaction of dose and relevant dimension was signiﬁcant, reﬂecting the dosage-related facilitation of learning in the box-relevant task and the opposite trend in the lid-relevant task.) Source: Bunsey et al., 1990 (Fig. 2, p. 280). Copyright ß 1990 by the American Psychological Association. Reprinted with permission.

dose-dependent decrease in shifting object choice after an error) (Fig. 7, left); and (2) a signiﬁcant dose relevant dimension interaction was also a signiﬁcant predictor for the adaptive win-stay response strategy (Fig. 8, left). Separate analyses of the lid- and box-relevant conditions indicated no signiﬁcant peptide treatment effect on the win-stay response in the box-relevant condition but a signiﬁcant increase in the use of this adaptive response in the lid-relevant condition at the 3.0-g dose level, relative to placebo controls (i.e., increased tendency to repeat the choice of the correct object directly after performing a successful response). For the multiple-cue condition, signiﬁcant results for object-choice responding were obtained for the lose-shift (Fig. 7, right) but not the winstay (Fig. 8, right) adaptive response. Results of the analysis indicated the following signiﬁcant predictors for the lose-shift strategy: peptide dose, relevant dimension, and dose relevant dimension interaction. Separate analyses for the box- and lid-relevant conditions indicated that peptide dose had no effect on the frequency with which the lose-shift response was used in

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FIGURE 7 The mean percentage of trials after an incorrect response in which the rat appropriately shifted its object choice in the subsequent trial, termed lose-shift object responding. (This categorization was based on the animals’ choice of cue within the predictive dimension. In the single-cue task there was a dose-related decrease in lose-shift object responding for rats in the lid-relevant condition, but no dose-related effect in the box-relevant condition. In the box-relevant version of the multiple-cue task, there was an increase in loseshift responding at all doses of the peptide (relative to the vehicle controls), whereas there was no peptide effect in the lid-relevant version of this task. Source: Bunsey et al., 1990 (Fig. 3, p. 281). Copyright ß 1990 by the American Psychological Association. Reprinted with permission.

the lid-relevant condition, but increased the rate of its use in the box-relevant condition. That is, the tendency to shift object choice after an error was greater in all the peptide groups, relative to the vehicle controls in the box-relevant condition (Fig. 7, right). Taken together, the results of this experiment were interpreted as consistent with the hypothesis that vasopressin enhances attentional selectivity. First, the learning rate data for the multiple-cue task suggested that AVP(4–9) enhanced selective attention for the dominant (perceptually salient) cues in this task, increasing the extent to which learning was controlled by this dominant information. That is, under peptide treatment, learning was improved in the box-relevant condition because the animal was less subject to the inﬂuence of nondominant and nonpredictive lid cues, whereas learning was impaired in the lid-relevant condition because of an impaired ability to disregard the dominant but nonpredictive box cues. Second, the VP-selective attention hypothesis received some support from the lose-shift analysis for the multiple-cue condition. The increased tendency to shift box choice after an error occurred in the box-relevant condition for all three peptide groups relative to the placebo controls

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FIGURE 8 The mean percentage of trials immediately after a correct choice in which the animals appropriately chose the same cue that they had chosen on the previous trial, termed win-stay object responding. (The peptide treatment had little effect on this type of response, the only exception being the exceptionally high win-stay object responding in the 30-g/kg dosage group of the lid-relevant single-cue task.) Source: Bunsey et al., 1990 (Fig. 4, p. 281). Copyright ß 1990 by the American Psychological Association. Reprinted with permission.

and was interpreted as a reﬂection of greater control of attention by the dominant box cues. The expected counterpart, a peptide-induced reduction in this adaptive response for the lid-relevant condition, was not observed; this was attributed to the fact that the vehicle controls in the lid-relevant subgroup were already responding close to chance level. Interestingly, in the lid-relevant condition for the single-cue task, incremental doses of the peptide progressively reduced the tendency to shift lid choice after an error. The authors interpreted this result as further support for a vasopressin inﬂuence on selective attention, suggesting that the decline in this adaptive response may have been due to a peptide-related increased attentional focus on the boxes resulting in near chance performance, at the high dose level, with respect to lid choice. However, because the box cues were not present in the lid condition of the single-cue task it is unclear why the presence of the box itself should have been any more of a disruption to this form of cognitive behavior than its presence during the learning task. Bunsey (personal communication, 1998) offered the following post hoc explanation: even though experimentally manipulated irrelevant box cues were absent in the single-cue task, box odors and other uncontrolled box stimuli were undoubtedly present and served as salient distracters for the rat. Thus the difference between the single- and multiple-cue condition was merely one of degree. Granting that the multiple-cue task was more distracting (borderline effect on learning),

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it should not have been surprising that the effects on selective attention exerted in the single-cue task were more subtle, showing up only in one measure. In contrast to the hypothesis-conﬁrming results of the object analysis obtained for the lose-shift response, the comparable analyses done for the win-stay response were characterized by a relative absence of peptide effects. Although the reason for this discrepancy was not clear, subsequent analysis showed that the cognitive effects of vasopressin appear to be more inﬂuential after an error than after a correct response. It was suggested that under nondeprivation, loss of anticipated rewards might heighten negative emotions (e.g., frustration) to a greater degree than the emotional consequences of reward attainment. If so, then endogenous vasopressin released by the emotionality of a loss may have further increased the effectiveness of AVP(4–9) at the synapse in the time after a loss (Bunsey, personal communication, 1998). The fact that vasopressin-like peptides are present in the limbic system, a brain region implicated in emotional reactivity, is consistent with this suggestion, which, however, needs further clariﬁcation and more direct experimental support. Experiment 2 investigated whether the peptide-induced increased control of attention by dominant cues extends to situations in which cues have been rendered more salient by prior learning experience rather than perceptual characteristics. In this paradigm, which also included a choice between two boxes, discriminative lid cues predicted the reward both during the acquisition phase (only lid cues present) and during a subsequent redundant phase (box cues added as redundant predictors of reward). Failing to afﬁx them to the bases used in experiment 1 decreased perceptual saliency of the boxes. Learning with the redundant box cues was subsequently assessed in the reversal–nonreversal discrimination test phase (only redundant box cues present). See Fig. 9 for diagrams of the cues present during acquisition training, redundant training, and reversal–nonreversal test phases of this redundant learning paradigm. The authors predicted that AVP(4–9) given during the redundant learning phase would enhance selective attention to the lid cues, made salient on the basis of prior experience. As a result, peptide-treated subjects should learn less about the redundant box cues than do vehicle-treated controls and should exhibit this effect in the reversal–nonreversal discrimination test phase. The animals received four trials/day during each of the three phases of the experiment. In the acquisition phase, the subjects were ﬁrst trained to discriminate between two boxes characterized by only one set of discriminative lid cues [i.e., cork lids (Sþ) and carpet-covered lids (S)]. After mastery of this discrimination problem (12 of 15 correct consecutive choices), the animals were given a rest day. The 2-day redundancy training phase began on the following day, during which time the lid cues continued to predict the reward but a second set of equally predictive ‘‘redundant’’ box

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FIGURE 9 The three phases of the redundant learning paradigm. Source: Bunsey et al., 1990 (Fig. 5, p. 283). Copyright ß 1990 by the American Psychological Association. Reprinted with permission.

cues was added. The 2-day reversal–nonreversal test phase began on the day after the redundancy phase. Each subject was assigned to 1 of 12 conditions based on 3 parameters: (1) doses of AVP (0, 1.0, or 10.0 g/kg) received during the redundant phase, (2) which of 2 box types (white cloth or black rubber) was paired with the cork (Sþ) lid during the redundancy training phase, and (3) the test condition (reversal or nonreversal) to which the subject was assigned (see Fig. 9). Individual differences in original acquisition were counterbalanced across the 12 conditions. Vehicle or AVP(4–9) (1.0 or 10.0 g/kg, subcutaneous) was given on the rest day that followed the acquisition phase and 1 h before each of the two daily sessions of the redundant training phase. No treatment was given during the 2-day reversal–nonreversal test phase. At this time, half the subjects in each treatment group of the redundancy learning phase were assigned to the reversal task (reward located in the box that did not contain the reward during redundancy training), and the other half was assigned to the nonreversal task (reward located in the same box that had been correct during redundant training). It was expected that rats that had learned a great deal about the box cues during redundant training would commit few errors in the nonreversal test condition, but many errors in the reversal test condition. If the peptide enhanced selective attention to the experience-induced salient lid cues during redundancy training, then the peptide-treated groups should have learned less about the redundant cues than did the placebo controls. Accordingly, the peptide treatment groups should exhibit less

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difference in performance between the reversal–nonreversal test conditions than the placebo controls. The results were as follows: (1) AVP(4–9) did not inﬂuence performance in the redundant training phase (i.e., no differences in errors between the placebo controls and the peptide treatment groups during this phase of training); (2) overall, more errors were committed in the reversal than in the nonreversal subgroups during the test phase, indicating that redundant learning had taken place; and (3) the difference between reversal and nonreversal subgroups was signiﬁcantly reduced in subjects that had been treated with the 1.0-g/kg dose of peptide, relative to both the vehicle-treated subjects and the 10.0-g/kg peptide-treated subjects (see Fig. 10). The results of experiment 2 were interpreted as indicating a dosedependent peptide effect on selective attention. This interpretation was based on the pattern of results obtained during the reversal–nonreversal test phase (see Fig. 10). That is, in accordance with predictions from a VP/ attentional selectivity hypothesis, the 1.0-g/kg peptide treatment group

FIGURE 10 Number of correct trials (out of a total of eight) during the test phase of the redundant learning paradigm. (The difference between the reversal and the nonreversal subgroups within a dosage group provides an index of redundant learning for that group [the larger the difference, the greater the redundant learning].) Source: Bunsey et al., 1990 (Fig. 6, p. 284). Copyright ß 1990 by the American Psychological Association. Reprinted with permission.

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showed no evidence of redundant learning (i.e., no performance difference between the subgroups tested under reversal versus nonreversal conditions during the test phase). The authors suggested that the unexpected redundant learning effect exhibited by the high-peptide treatment group might have been due to attentional effects associated with the high level of arousal produced by this dose level. Heightened arousal has been inferred to increase both selectivity of attention (narrow attentional span) and lability of focus (Kahneman, 1973). If this occurred in this experiment, the resulting shifting of attention between the learning-salient lid cues and the redundant box cues, although having no differential effect on performance outcome during the redundant learning phase (both sets of cues predicted the reward), could have resulted in building up an association between the box cues and the reward (i.e., redundant learning). Taken together, the results of experiments 1 and 2 were interpreted as support for the proposal that AVP(4–9) increases the extent to which attention is controlled by dominant information. In experiment 1, the peptide increased preferential attention to the cues that were dominant on the basis of perceptual salience. In experiment 2, the cues were rendered dominant on the basis of previous learning experience. The authors also pointed out that these effects of AVP(4–9) on attention processing are similar to those resulting from a number of experimental treatments that are known to increase arousal level (e.g., incentives, stimulant drugs, and noise), that is, the aroused individual selectively focuses on the dominant sources of information in the environment. It was concluded that the emerging proﬁle of vasopressin effects on cognition (enhanced memory function and attentional selectivity) ﬁts involvement of the peptide in the body’s integrated response to stressful situations. This view is consistent with Kety’s suggestion that the increase in arousal level that occurs in emotionally/motivationally charged situations renders important experiences especially memorable (Kety, 1972). Thus, rather than producing an arousal-induced performance effect only, it is proposed that exogenous AVP interacts with the transmitter components of the ‘‘central arousal system’’ (e.g., the coeruleus–noradrenergic projection system) to directly inﬂuence cognitive processing, as proposed for the endogenous neuropeptide when it is released in certain learning environments.

IV. Other Lines of Evidence Supporting a Role for Vasopressin in Attentional Processing

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Additional support for a vasopressin inﬂuence on attentional processing is provided by a behavioral study on divided attention in laboratory rats, and by studies monitoring electrophysiological correlates of attentional processing in human volunteers.

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A. Animal Research: Divided Attention in Laboratory Rats 1. Meck (1987) Meck (1987) examined the effect of AVP(4–9) (0.3 g/kg, intraperitoneal) on divided attention in laboratory rats in a temporal discrimination task, with three modality-speciﬁc stimulus cues (tactile, visual, and auditory) asynchronously presented on each trial. Each cue signaled a different ﬁxed interval (FI) schedule of reinforcement [i.e., white noise (60-s FI), house light (30-s FI), and mild footshock (15-s FI)]. Although the onset of each cue occurred at a different time in each trial, all three were simultaneously present for a 5- to 15-s interval, and then were gradually terminated; the last cue indicated the reward interval in operation for the remainder of the trial. Because the procedure made it impossible to correctly predict which signal would continue to completion in an individual trial, optimum performance required divided attention among the three cues, and the timing of all three signals simultaneously, at least until all but one of the signals was turned off. The results indicated that the peptide treatment increased the probability that all three signals were attended to in each trial. It was concluded that the VP metabolite facilitated simultaneous temporal processing by increasing the speed of cognitive processing involved in memory storage and divided attention (see Meck, 1987, for further details).

B. Human Research: Electrophysiological Measures of Attentional Processing 1. Fehm-Wolfsdorf and Colleagues A number of scientists have investigated the putative role of VP in attentional processing using event-related potentials (ERPs) as the dependent measure. ERPs are those EEG waveforms that are time-locked to recurrent stimulus events present during performance of an attentional processing task. They are clearly revealed by averaging techniques that remove the spontaneous non-ERP activity from the EEG recording, and consist of a series of positive (downward deﬂections in graphic display of ERP data) and negative (upward deﬂections) waveform components. Depending on their poststimulus latency ranges, these components of the ERP have been categorized as ‘‘early’’ (e.g., P50, N1, and P2), ‘‘late’’ (N2 and P3), and ‘‘very late’’ (late positive component, LPC). It is theorized that (1) the early components are primarily sensitive to physical characteristics of the stimulus (e.g., pitch, loudness, and brightness), and reﬂect the attentional mechanisms in the early stages of stimulus processing; (2) the late components (especially P3) depend on the meaning the subject gives to the stimuli being processed (Timsit-Berthier et al., 1982) and are ‘‘associated with processing of attended stimuli requiring decision and response’’ (Dodt

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et al., 1994, p. M-183); and (3) the LPC reﬂects attentional mechanisms present during very late stages of stimulus processing, such as during encoding stimuli for memory storage (Naumann et al., 1991). In addition, a slow potential (contingent negative variation, CNV) is recorded in tasks containing a warning stimulus, and reﬂects cortical changes occurring in classic and operant conditioning paradigms. The CNV increases during heightened attention and decreases during habituation (Timsit-Berthier et al., 1982). In a selective attention task, a voluntary selective attention effect is indicated by higher amplitude in the waveforms correlated with the attended rather than with the unattended stimuli. On the other hand, automatic attentional processing (i.e., an attentional mechanism that monitors both types of stimuli) is indicated by amplitudes of waveforms that are higher than baseline values for both attended and unattended stimuli. Fehm-Wolfsdorf and colleagues examined the effects of VP on these ERP components reﬂecting attention mechanisms associated with different stages of stimulus processing. In a couple of their studies, behavioral tests of learning and memory were included. With one exception, VP (AVP, LVP, or DG-AVP) was applied intranasally. VP treatment had no effect on blood pressure or heart rate in those studies that monitored these parameters (Born et al., 1986; Fehm-Wolfsdorf et al., 1988). Their ﬁndings indicated that VP enhanced (1) attention during earlystage stimulus processing (increased the amplitude of the peak-to-peak difference between N1 and P2, vertex potential) in an auditory vigilance task (covert monitoring of tone pips), but exerted no effect on learning or retention tested with a 25-item word list. The authors concluded that the behavioral data indicated no VP effect on memory processing, but the EEG data indicated that VP facilitated ‘‘stimulus-related phasic cortical arousal’’ (Fehm-Wolfsdorf et al., 1988, p. 496) (Fig. 11A); (2) attentional mechanisms present in a late stage of stimulus processing (increased amplitude of P3 in the auditory oddball task after intranasally, but not intravenously, administered AVP (Pietrowsky et al., 1996; Fig. 11B); it was suggested that, unlike the intravenous route, the intranasal route may be a means by which peripherally administered VP can directly access the brain; (3) attentional mechanisms present in a late stage of stimulus processing (increased the amplitude of N2) in an auditory ‘‘oddball’’ task (requires responding to rare target tones interspersed among frequent standard tones) in both young and elderly subjects, but failed to improve ERP indicators of agerelated cognitive impairment (Dodt et al., 1994; Fig. 11C); (4) automatic attention in a late stage of stimulus processing (increased amplitude of N2) for target stimuli in a dichotic listening task [Born et al., 1986 (and see Fig. 12A and B); Pietrowsky et al., 1989]; and (5) very late-stage attentional processing (increased amplitude of LPC) during ‘‘structural’’ encoding (i.e., estimating whether each stimulus word has exactly six, fewer than six, or more than six letters) of emotional and neutral adjectives in an incidental

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FIGURE 11 (A) Auditory evoked responses to tone pips recorded from electrode locations along the midline (Fz, Cz, and Pz). VP-treated subjects (compared to control subjects) showed higher amplitudes of the vertex potential (range of 7–230 ms after tone onset). This effect was most pronounced after the longest interstimulus interval (ISI) of 3 s. Solid line, vasopressin; dotted line, placebo. Source: Fehm-Wolfsdorf et al., 1988 (Fig. 2, p. 498). Copyright ß 1988 by Springer-Verlag. Reprinted with permission. (B) Averaged auditory evoked potentials to target stimuli of an attended oddball task from a single subject. Responses are plotted separately for the three treatment conditions: placebo (thick solid line), intranasal administration of AVP, 20 IU (thin solid line), and intravenous administration of AVP (dashed line). Recordings were from Fz, Cz, and Pz (against linked mastoid references, vertex negative upward). N1, P2, and P3 components are marked for the electrode site of their respective maximum amplitudes. Source: Pietrowsky et al., 1996 (Fig. 1, p. 335). Copyright ß 1996 by the Society of Biological Psychiatry. Reprinted by courtesy of Elsevier Science, present publisher of this journal. (C) Examples of auditory evoked potentials of one young and one old male subject recorded from frontal (Fz), central (Cz), and parietal (Pz) leads of the EEG after intranasal administration of arginine vasopressin or placebo. The potentials were recorded while the subjects performed an attended ‘‘odd ball’’ task, ERPs are responses to the rare target tones of this task. Source: Dodt et al., 1994 (Fig. 1, p. M186). Copyright ß 1994 by the Gerontological Society of America. Reprinted with permission.

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FIGURE 12 (A) Auditory evoked potential (AEP) responses (obtained from Fz) to target stimuli from two pairs of twins. Under LVP, a negative shift of the AEP amplitude is apparent at about 200 ms poststimulus. Whereas in the upper waveforms the negative amplitude shift under LVP affects the P2 and N2 peaks about equally, in the lower panel the inﬂuence of LVP is limited to the N2 peak. (Negative is upward.) Source: Born et al., 1986 (Fig. 4, p. 192). Copyright ß 1986 by Ankho International Inc. (B) AEPs to standard and target pips after LVP and placebo recorded from frontal (Fz) and central (Cz) midline electrode locations. The average AEPs contain responses to unattended, as well as attended stimuli from the ﬁrst halves of the sequences. Under LVP, a negative shift of the AEP amplitude within 180–220 ms poststimulus is apparent. As the shift is especially prominent following the delivery of target pips deviating in pitch from standard pips, the LVP effect might represent an enhanced N2

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FIGURE 13 Grand means on adjectives during the structural encoding task in study 1 (ﬁrst row) and study 2 (second row) at each electrode location. Grand means for placebo (solid line) and AVP subjects (dashed line) are plotted together. (Positivity downward.) The vertical line indicates stimulus onset and gives the calibration in microvolts (V). Time axis: 100 ms before stimulus onset to 1400 ms poststimulus. Source: Naumann et al., 1991 (Fig. 3, p. 1383). Copyright ß by Pergamon Press plc.

learning task. Moreover, the AVP-induced facilitation of structural encoding, as well as of recall on the unexpected memory test, was more prominent for the emotional than for the neutral adjectives (Naumann et al., 1991) (Fig. 13). The results cited above indicate that, depending on the experimental paradigm used, AVP enhanced the amplitude of different ERP components. This inﬂuence at so many stages of stimulus processing led these researchers to conclude that the primary effect of VP is one of stimulating cortical arousal rather than exerting a direct and selective action on a speciﬁc stage or type of cognitive processing (Fehm-Wolfsdorf and Born, 1991; Dodt et al., 1994; Naumann et al., 1991). Moreover, Fehm-Wolfsdorf and Born (1991) suggested that this VP effect on the central arousal system could also alter affective aspects of stimulus processing, a suggestion consistent with results obtained by Dodt et al. (1994). 2. Timsit-Berthier et al. (1982) Timsit-Berthier et al. (1982) showed that, like other CNS stimulants, LVP given a relatively short time (6 h) before testing enhanced the CNV in an auditory reaction time task. Unlike other stimulants, however, LVP had a long delay of action (i.e., posttesting VP treatment in the ﬁrst session prevented the CNV habituation observed 1 week later in the placebo group component overlapping in this latency interval with the P2 component, rather than a change in the P2 component. This is also suggested by waveforms from single subjects [see (A)]. In the illustrated grand average waveforms (across subjects) the effect of LVP on N2 is probably smeared, due to the considerable variability of N2 latencies among subjects. (Negative is upward.) Source: Born et al., 1986 (Fig. 3, p. 191). Copyright ß 1986 by Ankho International Inc.

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FIGURE 14 Examples of CNV recorded under placebo and LVP conditions. The two top recordings (a and b) were obtained during the ﬁrst session. LVP or placebo was administered right after the session, and another recording (bottom, c) was performed after 1 week. Habituation of CNV persists after placebo (left), but is discontinued after LVP (right). Source: Timsit-Berthier et al., 1982 (Fig. 1, p. 254). Copyright ß 1982 by S. Karger. Reprinted with permission.

when tested in the same task paradigm) (see Fig. 14). This long-term effect of the peptide, which has been observed in behavioral learning tasks (De Wied, 1971; see Chapter 2), suggests that its effect on attentional processing may not be completely reducible to an action on the central arousal system.

V. Research Summaries: Beckwith and Colleagues, and Bunsey, Strupp, and Colleagues

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A. Beckwith and Colleagues The human and animal research ﬁndings of Beckwith and colleagues were consistent with the thesis that vasopressin has a primary role in attentional processing, which may help explain its ability to enhance both

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the memory-encoding (learning) and retrieval stages of memory processing. Their human research studies tested the effects of an intranasally administered nonpressor vasopressin analog, DDAVP, on performance in a variety of task paradigms. Two of their early studies, speciﬁcally designed to examine the role of vasopressin in attentional processing, showed that DDAVP (1) enhanced performance efﬁciency in a series of discriminations dependent on attention to the relevant dimension (color or shape), which shifted from problem to problem (Beckwith et al., 1982); and (2) enhanced attention as operationally deﬁned in the Sternberg item recognition task (Beckwith et al., 1983). Two studies that tested the effect of DDAVP on memory for implicational sentences used the same type of stimulus materials. The ﬁrst study (Beckwith et al., 1984), which used male and female subjects, found that DDAVP facilitated recall only in the males, and this occurred whether the sentences were encoded (learned) by rote or by comprehension of their implied meaning, and whether retrieval was tested using free or cued recall. The follow-up study (Till and Beckwith, 1985), which tested only males, found that the peptide facilitated both immediate and delayed recall, with the former effect greater in the low-vocabulary subgroup, and the latter greater in the high-vocabulary subgroup. Moreover, this peptide effect appeared to be on both the encoding and retrieval stages of memory processing. Subsequently, Beckwith et al. (1987a) observed that DDAVP facilitated recall of (1) aurally presented passages of narrative prose and (2) those idea units rated medium and high in importance to the theme of each passage. Their inference from these results was that DDAVP increased selective attention to meaningful idea units in the passage, and also facilitated the division of attention ‘‘between the continuous encoding of the text and maintaining the text propositions in working memory’’ (Beckwith et al., 1987a). Several of these human studies on attentional processing indicated that vasopressin might interact with baseline task proﬁciency. Thus, vasopressin proved more efﬁcacious in subjects on lower rather than higher baseline proﬁciency (Beckwith et al., 1984; Till and Beckwith, 1985) and this may have accounted for the observation by Beckwith et al. (1984) that vasopressin inﬂuenced sentence recall only in males, whose verbal proﬁciency is typically found to be below that of same-aged females (Macoby and Jacklin, 1974). In the animal studies, Beckwith and colleagues tested the Sutherland– Mackintosh attentional model developed in connection with discrimination reversal learning. According to this model, a treatment that enhances selective attention during acquisition of the original discrimination should increase the rate of learning the reversed discrimination. The subjects were mainly male adult Holtzman albino rats and the task was either an appetitive or an aversive white/black discrimination reversal. The predicted effect of vasopressin-induced enhancement of selective attention was not observed in the appetitive version of the discrimination task after treatment with

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intranasally administered DDAVP (Couk and Beckwith, 1982), but was obtained on the aversive discrimination task after intraperitoneally administered AVP (Beckwith and Tinius, 1985; Beckwith et al., 1987b). No single explanation for these discrepant ﬁndings was precluded because the two types of protocols differed with respect to the learning incentive, the peptide treatment, and the route by which it was administered. Because Bunsey et al. (1990) reported a vasopressin-induced facilitation of selective attention during appetitive learning after treatment with the nonpressor vasopressin metabolite AVP(4–9), the route of administration seems the more likely reason for the failure to obtain the expected vasopressin effect in the study by Couk and Beckwith (1982). The replication of the AVP-induced facilitation of reversal learning on the aversive version of the Wþ/B discrimination task (Beckwith et al., 1987b) demonstrates the usefulness of this task as a test for selective attention. It is herein suggested that the disparity between the inﬂuence of VP on selective attention and memory processing observed by Beckwith et al. with the Wþ/B discrimination tested in the modiﬁed T-maze was due, at least in part, to the possibility that the 1-g/rat dose of AVP facilitated selective attention to the relevant stimulus dimension during original learning, but was not sufﬁcient to enhance either acquisition or memory consolidation of the learned experience. This explanation is consistent with research ﬁndings presented in Chapter 10. Thus, Hostetter et al. (1977) found that a comparable dose of Pitressin, subcutaneously injected 20 min before each day’s training session, also failed to inﬂuence the rate of learning or retention for either the WþB or the BþW discrimination in the T-maze apparatus used in their appetitive motivational paradigm. In contrast, Sara et al. (1982) found that a 10-g dose of LVP, subcutaneously injected 90 min before the single day of training of a brightness discrimination in a modiﬁed Y-maze with food reward, facilitated learning of the more difﬁcult lightþ/dark discrimination but not the easier learned darkþ/light discrimination possibly due to a ceiling effect. The ﬁnding that AVP facilitated discrimination reversal learning in Holtzman albino rats but not in the LE hooded rats, suggested differential actions of AVP on memory processing in different strains of rat (Tinius et al., 1989). It is also possible that the greater proﬁciency of the latter strain in reversal learning created a ceiling effect, leaving little room for VP-produced improvement. If so, this would be another instance of a VP interaction with baseline proﬁciency in task performance.

B. Bunsey, Strupp, and Colleagues Bunsey et al. (1990) conducted two experiments in which they manipulated cue salience in discrimination tasks testing the ability of vasopressin to enhance attentional selectivity in laboratory rats. In the ﬁrst experiment, two

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discrimination tasks differing in the presence (multiple-cue test) or absence (single-cue test) of irrelevant distractor cues were used. For each test, reward-predictive cues were associated with either the lids (lid relevant) or the boxes (box relevant). If vasopressin increases attention selectivity (perceptual focus on relevant cues), AVP(4–9) treatment would be expected to enhance discrimination learning in the multiple-cue task, which contained irrelevant cues, but not in the single-cue task, which had none. As expected, there was no overall difference in learning rate between the peptide and the vehicle-treated subjects in the single-cue task. However, for the multiple-cue task, a dose relevant dimension interactional effect indicated only partial conﬁrmation of the prediction. AVP(4–9) dose dependently improved learning in the box-relevant condition whereas an opposite trend occurred for the lid-relevant condition. This pattern of effects led to the ad hoc hypothesis that vasopressin facilitated attentional focus on the dominant cues in the environment, in this case the perceptually more salient box cues. This hypothesis was subsequently tested in experiment 2, in which lid cue salience was increased by prior learning experience and box cue salience was reduced somewhat by removing their bases. In this experiment, the predicted vasopressin facilitation of selective attention to the learningsalient lid cues was observed for the subjects treated with the 1.0-g/kg dose of AVP(4–9). Subjects given the 10.0-g/kg dose of the peptide apparently focused sufﬁcient attention on the redundant box cues to learn their predictive value. One interpretation was that the heightened arousal level induced by the higher dose of the peptide increased lability of attentional focus, thereby periodically shifting attention to the box cues. The arousal/ attentional research literature (Kahneman, 1973) supports the conclusion that agents that increase arousal level increase attentional selectivity and also lability of attentional focus. In addition to the analyses of learning performance, a response analysis of win-stay and lose-shift adaptive responding was applied to side-choice and object-choice responding for both the multiple-cue and single-cue tasks in experiment 1. Side-choice responding was not inﬂuenced by the peptide, but object-choice responding, at least for the lose-shift response strategy, was affected. The peptideinduced increased tendency to shift object choice after an error in the boxrelevant multiple cue task was interpreted as a peptide-induced facilitation of attention to the dominant box cues in this task.

VI. Chapter Commentary: Vasopressin, Attention, and Memory Processing

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Taken together, ﬁndings from the human and animal studies of Beckwith and colleagues support the propositions that the inﬂuence of VP on attentional processing (1) occurs in both humans (Beckwith et al., 1982,

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1983, 1987a) and laboratory rats (Beckwith and Tinius, 1985; Beckwith et al., 1987b; Tinius et al., 1989); (2) can be dissociated from its pressorinduced arousal effects (Beckwith et al., 1982, 1983, 1984, 1987a; Till and Beckwith, 1985); (3) includes a number of forms of attention (e.g., selective attention, divided attention) occurring in various stages of cognitive processing, including those involved in memory encoding and retrieval (Beckwith et al., 1984, 1987a; Till and Beckwith, 1985); and (4) can occur independently of the inﬂuence of the peptide on memory processing (Beckwith and Tinius, 1985; Beckwith et al., 1987b), but where VP enhances both attention and memory processing in the same learning encounter, the two effects may be causally linked (Beckwith et al., 1987a). A vasopressin effect on selective attention received further support in a study by Bunsey et al. (1990), which examined the effects of AVP(4–9) on discrimination learning in laboratory rats, using stimuli judged to be of high or low salience depending on perceptual features or prior learning experience. The results of their behavioral analyses were interpreted as indicating a peptide-induced enhancement of selective attention to dominant sources of information in the environment. Meck (1987) obtained evidence that this VP metabolite also enhanced divided attention in laboratory rats tested in a temporal discrimination task. Finally, the ERP research of Fehm-Wolfsdorf and colleagues (Born et al., 1986; Dodt et al., 1994; Fehm-Wolfsdorf et al., 1988; Naumann et al., 1991; Pietrowsky et al., 1989, 1996) and of TimsitBerthier et al. (1982) supported the proposition that VP inﬂuences attentional mechanisms distributed throughout various stages of cognitive processing. Given the important role of VP in attentional processing, it is of interest to determine the degree to which the effect of VP on attention is related to its roles in arousal and in memory processing. Speciﬁcally, two questions may be raised. First, are the effects of VP on attention causally linked to the role of the peptide in memory processing? Second, is the inﬂuence of the peptide on behavioral arousal essential for its inﬂuence on both attention and memory processing? On the basis of their human (Beckwith et al., 1982, 1983, 1987a) and laboratory rat (Beckwith and Tinius, 1985; Beckwith et al., 1987b; Tinius et al., 1989) studies, as well as other studies with healthy and clinical human populations (e.g., Weingartner et al., 1981), Beckwith and colleagues arrived at a dual position regarding the ﬁrst question: (1) under conditions in which the inﬂuences of VP on attention and memory processing are congruent, the two effects may be causally related (i.e., the effect of VP on memory processing is secondary to its effect on attention) (e.g., Beckwith et al., 1987a); and (2) in a learning situation in which the effects of VP on attention and memory processing are disparate (e.g., Beckwith et al., 1987b), VP inﬂuences attentional processing but not memory processing. The writings of Strupp, Bunsey, and colleagues concerning the ﬁrst question appear compatible with the view expressed by Beckwith and

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colleagues, suggesting that a VP enhancement of attentional processing (e.g., selective attention and divided attention) during learning and of concentration during memory retrieval may be expected to contribute to these stages of memory processing (Bunsey et al., 1990; Strupp and Levitsky, 1985; Strupp et al., 1984). As for the arousal effects of VP on attention and memory processing, studies by Beckwith et al. indicate that a pressor-induced arousal action could not explain enhancement by the peptide of selective attention (Beckwith et al., 1982, 1983, 1987a) and memory (Beckwith et al., 1984, 1987a; Till and Beckwith, 1985) obtained in studies on human subjects treated with DDAVP. This treatment outcome may be due in part to the peptide analog itself. However, it may also be due to the intranasal route of application consistently used in their studies. If so, this treatment outcome is consistent with speculation offered by Pietrowsky et al. (1996) that an intranasal route of VP delivery of the drug may involve a nasal–brain pathway by which the peptide can directly access the brain independent of a blood-borne endocrine action. Nevertheless, the effects of the peptide on attention, and hence on memory-dependent attention, may result from interaction of the peptide with a central transmitter system, such as the LC–NA system, which inﬂuences both behavioral arousal as well as attentional processing (Kovacs et al., 1979b; Sara, 1985). Strupp, Bunsey, and colleagues acknowledge that VP-induced arousal contributes to the effect of VP on cognition, but unlike Fehm-Wolfsdorf et al. (Section IV.B.1.), do not attribute the effect of VP on memory processing solely to its arousal action (For additional discussion, see: Bunsey and Strupp, 1990; Strupp and Levitsky, 1985; Strupp, 1989; Strupp et al., 1990; and Chapter 15 [Section III.A.4.c.ii]).

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Part V

Expansion of Vasopressin/ Oxytocin Memory Research I: Peripheral Administration

I. Introductory Remarks

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Insofar as the researchers whose works are discussed in Chapters 9 and 10 are not committed to a speciﬁc theoretical viewpoint about the means by which vasopressin (VP) and oxytocin (OT) inﬂuence memory processing, they represent a more eclectic approach to this ﬁeld of study than those previously discussed in this book. The studies described in this chapter investigated the effects of VP and /or OT in laboratory rats and mice, and in young mentally healthy human subjects. In all instances these neurohypophysial hormones were peripherally administered and comprised either the parent peptide or their behaviorally active and highly potent metabolic fragments. The tasks were designed to assess one or more phases of longterm memory (acquisition, storage, and retrieval), and in some cases short-term memory. In keeping with the emphasis of this text on presenting ﬁndings from the animal research literature, only a few studies using human Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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subjects are discussed in this chapter. The animal studies, presented in Section II, examined the roles of the neurohypophysial hormones in learning and memory tested in a variety of aversive and apptitive tasks that, in turn, form the basis of the section’s organization. The several human studies included for discussion in this chapter are presented in Section III. These studies emphasize the role of OT in cognitive processing, and thereby complement the focus on VP that characterized the human research discussed in Chapter 8.

II. Animal Research Literature

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A. Aversive Paradigms 1. Conditioned Taste Aversion a. Selected Study: Vasopressin Vawter and Green (1980) investigated a vasopressin inﬂuence on resistance to extinction of a conditioned taste aversion (CTA) in which the unconditioned stimulus (US) is an internal cue (sickness) rather than an external cue (e.g., footshock). A CTA was established in male adult Wistar rats by several pairings of lithium chloride (LiCl) and saccharin-ﬂavored water. Desglycinamide-lysine vasopressin (DG-LVP; 0 or 1 g/rat, subcutaneous) was injected 1 h before saccharin ingestion during conditioning and extinction sessions in this CTA paradigm. After a 20-day adaptation to the drinking schedule (tap water available for 20 min/day), the tap water was replaced by saccharin solution on day 21, the ﬁrst day of the experiment. Only saccharin was available during the 3 days of aversive conditioning and the 8 days of extinction testing; tap water was available on days inserted between days of conditioning or extinction. On day 21 (the ‘‘neophobia’’ day), D and S groups were formed by subcutaneously injecting half the subjects with DG-LVP (1 g/rat, subcutaneous) and half with saline, 1 h before the 20-min drinking period. Before the conditioning period (days 22–30), D and S groups were subdivided to form four subgroups: D/L, D/ S, S/L, and S/S depending on whether they received LiCl [10 ml/kg (0.15 M), intraperitoneal] or saline during the conditioning days (days 22, 25, and 28). On conditioning days, DG-LVP or placebo was injected 1 h before the 20-min saccharin drinking period and 1 h later LiCl or physiological saline was injected. Before extinction, each of these four subgroups was matched for saccharin intake on the last conditioning day and subdivided to form eight subgroups: D/L–D, D/L–S, D/S–D, D/S–S, S/L–D, S/L–S, S/S–D, and S/S–S. One of each new pair of subgroups received DG-LVP (1 g/rat, subcutaneous) or placebo 1 h before saccharin presentation on each of the 8 days of extinction testing (i.e., every third day between days 31 and 52). The results were as follows: (1) on its own, DG-LVP did not inﬂuence saccharin intake (i.e., there were no signiﬁcant differences in saccharin intake between the subjects receiving DG-LVP and placebo treatment on

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the neophobia test day or during the three conditioning days for the subjects receiving intraperitoneal saline); this indicates that the peptide did not produce an aversive effect in this study; (2) DG-LVP treatment did not inﬂuence acquisition of the CTA because consumption of the saccharin solution was similarly decreased in both the peptide- and placebo-treated animals during each of the 3 days of aversion conditioning; and (3) DG-LVP prolonged extinction of the CTA whether it was administered during the conditioning or extinction phase of the experiment. The authors concluded that DG-LVP facilitated memory formation when administered during CTA conditioning, and memory retrieval when given during extinction testing. Because DG-LVP is almost devoid of endocrinological effects, the prolonged extinction could not be attributed to pressor-induced arousal effects. There was no evidence of aversive effects of the peptide because DG-LVP, on its own, did not inﬂuence saccharin intake whereas the aversive stimulus, and LiCl, did. b. Selected Study: Oxytocin Verbalis et al. (1986) conducted two experiments to learn whether a high plasma level of OT is able to induce the formation of a CTA (ﬁrst experiment) and, conversely, whether immunoneutralization of hormonal OT can alter the CTA induced by an intraperitoneal injection of LiCl (second experiment). The rationale for these experiments was the earlier ﬁndings in this study that, relative to injected vehicle, intraperitoneal injections of lithium chloride (LiCl), copper sulfate (CuSO4), and apomorphine dose dependently and signiﬁcantly increased plasma levels of OT and, to a lesser degree, VP in male Sprague-Dawley rats. Given that these sickness-eliciting agents produce CTAs when administered on their own (Coil et al., 1978; Garcia and Koelling, 1967; Nachman, 1963), it was of interest to learn whether their stimulated release of OT from the posterior pituitary lobe is causally involved in the learned aversions induced by these sickness-producing agents. Before experimental testing, the rats were adapted to drinking tap water for 30 min/day for days 1–7. On day 8, tap water was replaced by a 0.1% saccharin solution that the rats were allowed to drink during the scheduled 30-min drinking period. At this point, the procedures used in the two experiments diverged. In the ﬁrst experiment, immediately after consumption of the saccharin solution on day 8, the rats were injected intraperitoneally with OT (0.5 g/kg) or with 0.15 M NaCl (vehicle solution). On day 9, a CTA to the saccharin solution was assessed with the standard two-bottle preference test (compared consumption of tap water versus saccharin solution in the OT-treated rats with that of the saline-injected controls). At a later time, all the rats in this experiment were given another 30-min period to consume the saccharin solution and then were intraperitoneally injected with a 10-fold higher dose of OT (5.0 g/ kg) and retested as before. The results indicated that, relative to saline treatment, neither the low nor the high dose of intraperitoneally injected OT produced a CTA to saccharin. This was so, even though 20 min after these

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injections, the plasma OT levels (171 and 1897 U/ml, respectively) were signiﬁcantly higher than those obtained after an injection of LiCl or CuSO4. In the second experiment, half the subjects received 1.0 ml of nonimmune rabbit serum (NRS) and half received an equivalent volume of speciﬁc OT antiserum. This antiserum was estimated as possessing a high binding capacity for endogenous OT in accordance with results obtained from an in vivo study by Dunning et al. (1985). The NRS and OT antiserum were administered via preimplanted venous catheters 10 min before the ﬁrst exposure to a 0.1% saccharin solution on day 8. After the animals were allowed to drink the saccharin solution for 30 min, LiCl (5 mEq/kg, intraperitoneal) was administered to produce the CTA. On day 9, the rats were given the two-bottle preference test to determine whether the OT antiserum inﬂuenced the CTA to the saccharin solution that was paired with the noxious LiCl treatment. The results demonstrated that the course of development of the LiCl-induced CTA to the saccharin solution after pretreatment with the OT antiserum was identical to that after pretreatment with NRS. Taken together, the results of these two experiments suggest that hormonal OT is not causally involved in the production of a learned taste aversion, despite the evidence that OT levels in the plasma are substantially elevated in response to chemical agents that induce visceral illness and produce CTAs (see Verbalis et al., 1986, for further details). It was further noted, however, that these results do not rule out the possibility that OT-containing neurons projecting to other areas of the brain and/or to the anterior pituitary lobe contribute to the CTAs that develop in response to these noxious chemical agents. Nor do these ﬁndings indicate that the elevated levels of plasma OT, produced by their experimental administration or by ingestion of naturally occurring toxins, are without a physiological function. On the contrary, these authors pointed out that peripheral OT and arginine vasopressin (AVP) are potent stimulators of smooth muscle contraction (Bisset, 1974; Nakano, 1974) and, therefore, ‘‘when present in high concentrations in plasma, they may enhance gastrointestinal motility and thereby increase the rate at which ingested toxins are excreted’’ (Verbalis et al., 1986, p. 474). 2. Conditioned Response Suppression a. Selected Study: Vasopressin Hagan (1983) studied the effect of vasopressin on conditioned suppression of lever pressing for food reward in adult male CFHB Wistar rats. The subjects were initially trained to lever press for food reward on a 60-s variable-interval (VI60) reinforcement schedule (lever response rewarded on average once every min.). Responding to this schedule was stabilized for 15 days with one 30-min session run at approximately the same time each day. Shuttlebox avoidance training began on day 16 to a learning criterion of 10 correct consecutive avoidance responses. A correct

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avoidance response was deﬁned as shuttling between the compartments during the presentation of the conditioned stimulus (CS) alone. This CS, a compound light/tone signal, was presented alone for 10 s and continued for the subsequent 10 s accompanied by pulsed footshock. Animals failing to reach the learning criterion within the two consecutive days (50 trials/day) of avoidance training were discarded from the experiment. Immediately after reaching the shuttlebox avoidance learning criterion, the subjects were returned to the home cage and 30 min later received an injection of saline or LVP (1 g/rat, subcutaneous). Twenty-four hours later, the animals were returned to the Skinner box and tested for suppression of the lever response during presentation of the light/tone CS used in the avoidance task. After an initial 10-min warmup period, the CS was presented for 20 s on ﬁve separate occasions during the subsequent 10-min period. The number of lever presses given during period A (20 s immediately preceding presentation of the CS) and period B (20 s during which the CS was present) was used to calculate the suppression ratio, B/(A þ B). The lower the ratio, the greater the behavioral suppression in the presence of the CS. The results indicated that the peptide-treated rats exhibited signiﬁcantly more suppression of the operant response than the saline controls. Hagan discussed three possible behavioral explanations for the experimental outcome relevant to the theoretical views presented in Chapters 2–5 of how vasopressin inﬂuences retention behavior. First, posttraining LVP may have enhanced consolidation of the learned fear that, in turn, acted as a behavioral substrate for the long-term effects of the peptide (King and De Wied, 1974; see Chapter 2). Second, the aversiveness of this behaviorally active dose of LVP (e.g., Ettenberg et al., 1983a; see Chapter 6) became conditioned to the salient avoidance CS, thereby increasing its aversive properties. Third, the aversive properties of the peptide increased the subject’s arousal level, which then indirectly enhanced the conditioning of the fear response. The view that a vasopressin-induced increment in baseline arousal level was responsible for the experimental outcome is consistent with the theoretical views of both the Koob and Sahgal research teams. The author concluded that only further study would allow a choice among these three alternatives. 3. Shuttlebox Footshock Avoidance Conditioning a. Selected Studies: Vasopressin i. Hagan (1982) Hagan (1982) tested the effect of LVP on extinction of a shuttlebox avoidance response when the peptide was given after a series of response prevention (forced extinction) trials interpolated between avoidance learning and subsequent extinction testing. During these trials, the conditioned stimulus (CS) was repeatedly presented without the aversive unconditioned stimulus (US) while a barrier prevented performance of the conditioned avoidance response. This procedure has been repeatedly observed to facilitate subsequent extinction of the avoidance response (Baum, 1969, 1970; Bersh and Paynter,

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1972; King and De Wied, 1974), presumably because of the newly acquired information that the conditioned stimulus is no longer followed by the punisher (Seligman and Johnston, 1973). King and De Wied (1974; see Chapter 2) had previously used this paradigm to test the ability of vasopressin to inﬂuence the learning assumed to occur during the response prevention trials. Their study demonstrated that LVP (1 g/rat, subcutaneous), given 6 h after avoidance training and 1 h before the response prevention trials, prolonged rather than accelerated avoidance extinction. They interpreted their results as evidence that vasopressin does not inﬂuence the short-term memorial processes involved in learning. Hagan (1982) reasoned that because LVP was injected before the response prevention trials, it may have ‘‘altered aspects of motivation or attention during response prevention, reducing their efﬁciency through these mechanisms rather than through an interaction with consolidation processes occurring after prevention trials’’ (Hagan, 1982, p. 205). Accordingly, he replicated the study and injected the peptide after, rather than before, the response prevention trials. In this study, Wistar male rats were trained to a criterion of 10 consecutive shuttlebox avoidance responses within a maximum of two 50-trial training sessions held on consecutive days. After reaching the learning criterion, half the subjects received 30 response prevention trials, and half were returned to the home cage. Immediately, 30 or 60 min after the end of the response prevention procedure, the rats were injected with placebo or LVP (1 g/rat, subcutaneous) and returned to the home cage. Those subjects returned to the home cage without response prevention training were similarly injected at these times. Ten extinction trials were given 24 h after the injection. Because an analysis of variance (ANOVA) indicated no signiﬁcant effect of injection interval on extinction responding, the data were collapsed across the three injection intervals for subsequent statistical analysis. The results indicated that (1) the response prevention trials hastened extinction of the shuttlebox response for the placebo-treated subjects; (2) the subjects that received LVP after avoidance training and were not given response prevention trials exhibited fewer avoidance responses during extinction than the placebo controls; and (3) the subjects given LVP after the response prevention trials increased avoidance responding during extinction relative to the placebo-treated controls. The ﬁnding that the response prevention procedure hastened extinction of the avoidance response conﬁrms previous ﬁndings (Baum, 1969; Bersh and Paynter, 1972; King and De Wied, 1974) and supports the interpretation that response prevention trials reduce fear by informing the subject that the conditioned stimulus is no longer followed by the aversive reinforcer. Why did vasopressin treatment given to subjects that had no response prevention training hasten rather than prolong extinction of the avoidance response? The author pointed out that posttraining vasopressin may decrease

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subsequent extinction responding under certain conditions. This was seen in a study by Hagan et al. (1982; see Chapter 2), which demonstrated that the effects of posttraining vasopressin vary as an inverted U-shaped function of dose, and that the effects of a single dose may vary depending on the training–injection interval. However, the dose levels of LVP used for the 60-min training–injection interval by Hagan et al. (1982) did not include the 1-g dose level of LVP used in the present study. On the other hand, De Wied (1971; see Chapter 2) found that a 1-g dose level of LVP, when injected 60 min after avoidance training, did inhibit extinction of the conditioned avoidance response although less effectively than after zero delay. The failure of VP to hasten extinction in the subjects given the response prevention trials, although contrary to prediction, replicated the earlier reported ﬁndings of King and De Wied (1974). Hagan (1982) interpreted his ﬁndings, in conformity with King and De Wied (1974), that ‘‘shortterm memories are not invariably facilitated by the peptide’’ (Hagan, 1982, p. 209). However, this interpretation seems to imply that the procedure used in this study did not assess a vasopressin effect on consolidation. If so, it is unclear why the paradigm used in the present study is any less a demonstration of the inﬂuence of vasopressin on consolidation of long-term memory than is the paradigm that uses a single-trial inhibitory avoidance task. In both, vasopressin treatment follows an opportunity to learn information available in a limited period of time and retention of this information is tested 24 h later. Presumably some degree of learning occurs during the response prevention trials because, as noted earlier, they have consistently resulted in reduced avoidance responding during subsequent extinction testing (Baum, 1969; Bersh and Paynter, 1972; Hagan, 1982; King and De Wied, 1974). An alternative explanation for VP prolonging extinction of an originally learned avoidance response, whether administered before (King and De Wied, 1974) or after (Hagan, 1982) a series of response prevention trials, is that the peptide facilitated retrieval of the originally learned information (Bunsey, personal communication, 1998). This explanation has plausibility given the fact that the response prevention trials provide an opportunity for learning about a changed CS–US contingency as well as stimuli that can serve as retrieval cues for the originally learned information. However, it is still unclear, especially in the Hagan (1982) study, why the peptide would act to reinforce retrieval of the earlier learning rather than consolidation of the newly acquired information provided during the response prevention trials. ii. Hamburger-Bar et al. (1985) Hamburger-Bar et al. (1985) studied the effects of both acute and chronic peripheral administration of the synthetic vasopressin analog 1-desamino-8-d-arginine vasopressin (DDAVP) on

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acquisition and extinction of a shuttlebox response in intact and braindamaged rats. DDAVP is a V2 receptor agonist, more slowly metabolized and lacking the pressor effects of AVP. The brain damage, produced at 5 days of age by intracisternal 6-hydroxydopamine (6-OHDA) injections, reduced dopaminergic neurotransmission whereas noradrenergic neurons were spared by intraperitoneally injected desipramine. This damage produces transient hyperactivity and more permanent learning impairment (Hamburger-Bar et al., 1984) and is considered to be an animal model of minimal brain dysfunction in humans (Shaywitz et al., 1976). The normal intact and learning-impaired, brain-damaged subjects were tested in two separate experiments. For each experiment, the subjects were tested 3 days/week (10 trials/day) for acquisition (weeks 1–4) and extinction (weeks 5–6) in the shuttlebox avoidance task. For each experiment, they were randomly assigned to one of three treatment groups: DDAVP-chronic, DDAVP-acute, or saline control. The groups that received physiological saline or chronic treatment with DDAVP (20 g/rat, subcutaneous) were treated 1 h before each day’s test session throughout acquisition and extinction. The acute-DDAVP treatment group received 20 g/rat 1 h before the day’s test session throughout the ﬁrst week of acquisition and on the ﬁrst day of extinction. The two experiments were not done simultaneously and therefore cannot be directly compared. Statistical comparisons among the three treatment groups were carried out separately for the normal and brain-damaged subjects. In the intact rats, chronic treatment was not signiﬁcantly different from acute treatment in its effect on acquisition, but was somewhat superior to acute treatment in its inﬂuence on retention (prolonged extinction effect). Speciﬁcally, for acquisition, subjects given chronic injections of DDAVP performed slightly but signiﬁcantly better than saline controls whereas those given acute treatment showed a nonsigniﬁcant trend for improvement relative to controls. For extinction, there was a highly signiﬁcant difference in performance between the subjects receiving acute versus chronic treatment (i.e., chronic but not acute treatment prolonged extinction relative to saline treatment). In the brain-damaged rats, chronic treatment proved superior to acute treatment for both acquisition (nearly signiﬁcant) and retention (highly signiﬁcant) testing. Relative to the saline controls, both chronic and acute treatment regimens signiﬁcantly improved learning, whereas chronic but not acute treatment prolonged extinction. The authors concluded that both acute and chronic DDAVP treatment helped to normalize response acquisition in the learning-impaired, dopamine (DA)-depleted rats. On the other hand, chronic was clearly superior to acute vasopressin treatment in facilitating retention (maintenance of the conditioned response) in both the intact and brain-damaged animals. The demonstration that only chronic application of DDAVP signiﬁcantly improved retention in the braindamaged animals suggests the importance of chronic treatment with this

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peptide for clinical trials with human subjects. The superiority of chronic DDAVP treatment for normally functioning animals may also be applicable to experimental research with other vasopressin analogs and is deserving of further comparative study. However, future comparative studies with acute versus chronic treatment effects on retention (extinction) may provide more clear-cut information if the comparisons are limited to treatments given during only acquisition or extinction (i.e., in the present study the comparison between acute and chronic treatment effects on extinction included carryover effects from treatments given during acquisition). iii. Hamburger et al. (1985) Hamburger et al. (1985) compared six inbred strains of mice for learning and extinction in a shuttlebox avoidance task under vasopressin treatment. Depending on the treatment group, male mice of six inbred strains (A, C57BL, CBA/Lac, C3H, AKR/J, and BALB/c) received saline, AVP (1 g/mouse, subcutaneous), or no treatment 1 h before each day’s session (15 trials/session) of avoidance learning (days 1, 2, and 3), and extinction, (days 6, 24, and 78; i.e., 3, 21, and 75 days after learning). Two inbred strains, C3H and AKR/J, were given an initial learning session without any treatment (‘‘day 0’’). Each animal received three or four daily sessions. Extinction was studied 3, 21, and 75 days after the last active learning session. AVP was given 1 h before each extinction session. There were two major sets of results. First, there were signiﬁcant strain differences in acquisition and extinction of the avoidance response in nontreated animals. Thus, three of the strains (C57BL, C3H, and AKR/J) showed rapid learning compared with the three other strains (A, CBA/Lac, and BALB/c). Two strains (CBA/Lac and C57BL) showed little or no extinction for 2.5 months, two strains (C3H and A) exhibited rapid extinction, and the remaining two strains (AKR/J and BALB/c) were intermediate in their extinction rate. Second, strain differences were found in AVP inﬂuence on response acquisition and extinction. AVP had no signiﬁcant effect on acquisition or extinction in four of the six strains. However, in strain A, the peptide facilitated both learning and retention (retarded extinction) whereas it retarded learning for strain BALB/c. The authors suggested that differences between the mouse strains in endogenous vasopressin levels might have contributed to their different reactivity to exogenous vasopressin. The results also suggest that vasopressin may interact with the genetics of the strains and/or colonies of rats used by different research laboratories, thus contributing to some of the inconsistent results obtained by different research groups in this line of study. b. Selected Study: Oxytocin Uvnas-Moberg et al. (2000) investigated the ability of OT to improve avoidance learning in a low-performing, highemotional stock of Sprague-Dawley (S-D) male rats. Relatively low plasma

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levels of OT also characterize these rats and might be causally related to their emotional reactivity and avoidance learning deﬁcit (Uvnas-Moberg et al., 1999). Moreover, two lines of evidence provided additional reasons for this study. First, high emotional arousal induced by intense environmental stress or associated with long-term temperamental characteristics affect cognitive functions (Barden et al., 1995; Heinrichs, 1999; McEwen, 1998). Second, peripherally administered OT has antistress effects on behavior as indicated by the observations that (1) acute OT treatment transiently increased the plasma level of the stress hormone corticosterone (Gibbs, 1986b; Legros et al., 1988), which was followed a few hours later by a decrease in this level (Petersson et al., 1999); and (2) repetitive OT treatment produced longterm anxiolytic effects, which included suppression of the secretion of corticosterone (Petersson et al., 1999), reduction in blood pressure (BP), and an increase in latency to produce a tail-ﬂick withdrawal response to a nociceptive stimulus (Petersson et al., 1996a,b). The subjects of this study were rats obtained from two ‘‘behaviorally and hormonally distinct’’ colonies, herein referred to as stock A and stock B (Uvnas-Moberg et al., 2000, p. 28). Only the rats in stock B were characterized as highly emotional, and this was based on elevated plasma corticosterone, lowered plasma OT, and a decreased reaction time together with increased startle amplitude to an acoustic stimulus. The two stocks were compared on the rate at which they acquired a shuttlebox conditioned avoidance response (CAR) after a 5-day regimen in which the rats in both stocks received a daily injection of OT (1 mg/kg1, subcutaneous) or physiological saline (vehicle) before CAR training. This training began on day 6 and lasted for four consecutive days (20 trials/15-min daily training session). The rats were given 15 min of adaptation to the apparatus before the start of the ﬁrst training session, and thereafter a 3-min adaptation before the start of the daily training session. The results were as follows: (1) under nontreatment conditions, stock B rats showed no evidence of learning over the 4 days of training, whereas stock A rats displayed a normal learning curve with signiﬁcant improvement in performance in the last 2 days of training; (2) OT and vehicle pretreatment did not differentially inﬂuence CAR learning in stock A rats (i.e., both types of pretreatment produced similar learning curves and signiﬁcantly improved CAR acquisition over the 4-day training period); and (3) in contrast, stock B rats, pretreated with OT, signiﬁcantly improved learning performance over the 4-day training period, while those pretreated with vehicle showed no evidence of a learning curve. The authors interpreted the ﬁndings of this study as indicating that an antistress property of OT facilitated avoidance learning in a line of rats characterized by heightened emotional reactivity and cognitive impairment in stressful learning situations. On the basis of evidence from this study, together with other cited observations and experimental ﬁndings, these

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researchers further proposed that cognitive enhancement associated with this antistress property of the peptide signiﬁes a chronic and not a transient action of the peptide, and that this effect pertains only to individuals that are cognitively impaired in stressful learning situations. It was further suggested that the behavioral effect of peripherally injected OT was mediated by an action at central, rather than peripheral, OT receptor sites. This speculation was consistent with the ﬁnding that much lower doses of intracerebroventricularly injected OT produced antistress behavioral effects in an open ﬁeld test (Uvnas-Moberg et al., 1994). However, given the issue of the ability of these neurohypophysial hormones, or their metabolites, to enter the brain via the blood–brain barrier (Chapter 14), it is not clear how the peripherally administered peptide exerts its central effects, nor whether supraphysiological doses of OT represent the physiological role of the peptide in this action. In this connection, it is entirely possible that the stressor-induced release of OT from the posterior pituitary is paralleled by the activation of OT-ergic projections to limbic brain sites, which in turn exert the cognitive and behavioral effects of OT. The studies of Uvnas-Moberg et al. (1994) and of Windle et al. (1997) have provided evidence that centrally administered OT exerts antistress and antianxiety effects that are expressed in behavioral and neuroendocrine performance. This evidence, together with the present ﬁndings, warrant the use of stock B rats in future studies that examine the physiological role of OT in connection with its ability to inﬂuence cognitive performance via its putative antistress and anxiolytic properties. 4. Single-Trial Inhibitory (Passive) Avoidance Conditioning a. Selected Studies: Vasopressin i. Hostetter et al. (1980) Hostetter et al. (1980) failed to ﬁnd a vasopressin inﬂuence on passive avoidance retention in the laboratory rat. In a retrieval design, a single injection of LVP (0, 0.03, 0.06, 0.12, or 0.3 IU, where 0.3 IU ¼ 1 g) was administered 60 min before the 24-h retention test. Various footshock (FS) levels were used (0, 0.05, 0.10, or 0.25 mA) for the single passive avoidance (PA) learning trial. Attempts were made to replicate design features employed by De Wied and colleagues [Bohus et al., 1972 (see Chapter 2); Thompson and De Wied, 1973 (see Chapter 3); Wang, 1972]. Reentry latencies were measured 24 and 48 h after the FS. Although there was a signiﬁcant effect of FS intensity on reentry latencies, there was no VP treatment effect at any dose level tested. ii. Rigter (1982) Rigter (1982), noting the absence of extensive pretraining experience in the experimental design of Hostetter et al. (1980), designed a study to investigate the effect variation in passive avoidance pretraining experience might have on the outcome of vasopressin treatment. He pointed out that in the task procedure used by De Wied and colleagues

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(e.g., Ader and De Wied, 1972; Bohus et al., 1978a,b; see Chapter 2), the subjects were given both the opportunity to become familiar with the apparatus (F) and to practice the step-through response (ST) during pretraining. Three experiments tested the effect of posttraining AVP on PA retention in male Wistar rats given full experience (F and ST), partial experience (either F or ST), or no pretraining experience in a PA apparatus identical to that used by De Wied and colleagues. A single injection of AVP (0, 0.1, or 1.0 g/rat, subcutaneous) was given immediately after the acquisition trial. An additional experiment tested the effect of posttraining AVP given either immediately or 90 min after the single-FS trial. The results showed that (1) only the 1-g dose of AVP signiﬁcantly facilitated memory consolidation (prolonged reentry latencies) in rats given full pretraining experience (ST plus F components), but not in rats denied pretraining experience (neither ST nor F component); (2) the 1-g dose of AVP signiﬁcantly facilitated memory consolidation in fully pretrained rats when injected immediately but not 90 min after the FS trial; and (3) neither dose of the peptide signiﬁcantly inﬂuenced retention in rats given partial (either ST or F) pretraining experience. Given these results, it was concluded that the failure to observe a VP inﬂuence on PA retention by Hostetter et al. (1980) may have been due, in part, to inadequate pretraining experience. However, it should be noted that the Rigter (1982) study tested for VP effects on memory consolidation, whereas the former study used a retrieval design. It is not known why Rigter (1982) employed a different design. iii. Alescio-Lautier and Soumireu-Mourat (1990) Alescio-Lautier and Soumireu-Mourat (1990) obtained equivocal results concerning a vasopressin effect on memory consolidation and retrieval for a PA response in BALB/c mice. A subcutaneous injection of saline or AVP (0.5 or 1 g/mouse, equivalent to 25 and 50 g/kg, respectively) was administered either immediately after the learning trial (consolidation design) or 20 min before the 24-h retention test (retrieval design) in a single-trial step-through inhibitory avoidance task. Neither dose of the posttraining-injected peptide inﬂuenced reentry latencies, suggesting that the peptide had no effect on memory consolidation; however, because the majority of the controls showed reentry latencies well under 100 s, the FS level may not have aroused sufﬁcient fear to produce an effectively learned inhibitory response in any of the subjects. The high and low doses differentially inﬂuenced PA behavior in the retrieval design. None of the high-dose subjects reentered the FS chamber within 300 s, whereas all the low-dose subjects took far less than 100 s to reenter the chamber. The saline controls showed intermediate reentry latencies. The data seemed to suggest that a high dose of the peptide facilitated retrieval whereas the low dose impaired it. However, a subsequent test of activity

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level showed that a presession injection of the high dose of vasopressin signiﬁcantly depressed locomotor behavior. Thus, the prolonged reentry latencies observed in the high-dose subjects may have been due to the peptideinduced hypoactive state. Failure of the posttraining injection of the high dose of peptide to inﬂuence reentry latency does not contradict this argument, because the hypoactive state would have dissipated 24 h after the injection. iv. Faiman et al. (1987, 1988) Faiman and colleagues (1987, 1988, 1991) and Baratti and colleagues (1989) used reentry latency in a 48-h retention test of the single-trial, step-through inhibitory (passive) avoidance task as the measure of retention in their studies of memory consolidation and retrieval in adult male Swiss mice. Their protocols assessed the effect of both exogenous and endogenous vasopressin on memory processing. The V1 receptor antagonist [1-(-mercapto-,-cyclopentamethylenepropionic acid)-2-(O-methyl)-tyrosine]AVP is subsequently referred to as AAVP. In their training procedure, the subject is placed on a lighted platform outside a hole leading to a dark compartment and receives a footshock (0.8 mA, 50 cycles per second [cps], 1 s) on entering the dark compartment. On the retention test 48 h later, the subject is again placed on the lighted platform, and the reentry latency (cutoff time, 300 s) is recorded. Because these studies were also designed to evaluate a putative interaction between vasopressin and cholinergic mechanisms in memory processing they are described in more detail in Chapter 10. Faiman et al. (1987) demonstrated that the enhancement of memory consolidation produced by a single posttraining injection of LVP (0.03 g/kg, subcutaneous) was mediated by a V1 receptor, because this effect was prevented by a simultaneous injection of the V1 antagonist AAVP (0.01 or 0.03 g/kg, subcutaneous). Faiman et al. (1988) showed that a single injection of LVP (0.03 g/kg, subcutaneous) given 20 min before the 48-h retention test facilitated memory retrieval, which was also prevented by simultaneous administration of AAVP (0.01 g/kg, subcutaneous). v. Baratti et al. (1989) Baratti et al. (1989) replicated the earlier ﬁnding that exogenously applied LVP (0.03 g/kg, subcutaneous) facilitated memory consolidation of a passive avoidance response (Faiman et al., 1987), and further demonstrated that endogenous AVP, released by a posttraining injection of hypertonic saline, also facilitated memory consolidation in this task. Moreover, the facilitated retention effects of both endogenous and exogenous vasopressin were blocked by a posttraining injection of AAVP (0.01 g/kg, subcutaneous) given before the injection of LVP or experimentally induced release of endogenous vasopressin. vi. Faiman et al. (1991) Faiman et al. (1991) demonstrated that LVP facilitated, and AAVP impaired, PA retention in a dose-dependent manner. LVP (0.003, 0.01, 0.03, 0.1, 0.3, or 1.0 g/kg, subcutaneous) and AAVP

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(0.01, 0.03, 0.1, and 0.3 g/kg, subcutaneous) were administered immediately after the inhibitory avoidance training trial to independent groups of mice to obtain dose–response curves for shocked and unshocked mice. Speciﬁcally, the results of LVP treatment showed that (1) LVP dose dependently enhanced retention in accordance with an inverted U-shaped dose–response curve (signiﬁcant effects occurred at 0.01- and 0.03-g/kg dose levels) in the shocked mice; (2) given alone, the antagonist dose dependently decreased retention performance in shocked mice; the U-shaped response curve was a mirror image of that produced by LVP; this dosedependent impairment of PA behavior supports the thesis that endogenous vasopressin normally contributes to memory processing in this task; and (3) neither LVP nor the V1 antagonist inﬂuenced reentry latency in the nonshocked mice. Peripherally injected AAVP, at a dose level that by itself had no effect on PA behavior, antagonized the effects of exogenous vasopressin on behavior, as indicated by a shift to the right of the dose–response curve for LVP. Thus, when combined with AAVP, higher doses of LVP (0.3 and 1.0 g/kg) were required to produce the same retention effects observed for lower doses of LVP (0.01 or 0.03 g/kg) when injected alone. These mouse studies of Faiman and colleagues, using an inhibitory avoidance task, are consistent with the numerous rat studies carried out by the De Wied and/or Koob research teams. They have demonstrated a vasopressin facilitation of memory consolidation and/or retrieval [e.g., Ader and De Wied, 1972; Bohus et al., 1972 (see Chapter 2); Lebrun et al., 1984 (see Chapter 6)], a V1 receptor mediation of these effects [e.g., De Wied et al., 1984a (see Chapter 3); Lebrun et al., 1984], and memory enhancement induced by an osmotically stimulated release of endogenous vasopressin [Koob et al., 1985a (see Chapter 6); Lebrun et al., 1987]. b. Selected Study: Oxytocin Boccia et al. (1998) investigated the effects of OT and of a vasotocin analog that is also a selective OT receptor antagonist (AOT), and their interactive effects on memory storage in male Swiss mice tested in a single-trial step-through PA task. Additional experiments evaluated the speciﬁcity of the interaction between OT and its receptors. The general procedure used for all groups of shocked mice in all the experiments was as follows: the mouse was placed on a small lighted platform outside a hole leading to a dark compartment and received a footshock (0.8 mA, 50 Hz, 1-s duration) as it stepped into the dark compartment. Retention testing occurred 48 h after the learning trial, when the mouse was placed on the platform and reentry latency was recorded up to a maximum of 300 s. In experiment 1, the researchers tested dose-dependent effects of posttraining administered OT and AOT on PA behavior in mice assigned to either the shocked or nonshocked training condition. For both training conditions, independent groups of mice were subcutaneously injected with saline (control vehicle) or with a speciﬁc dose of OT (0.01, 0.03, 0.10, 0.30,

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or 1.0 g/kg) or AOT (d(CH2)5[Tyr(Me)2,Thr4,Tyr(NH2)9]OVT; 0.03, 0.10, 0.30, or 1.0 g/kg) immediately after the training trial. The results for shocked mice receiving either OT or AOT showed that (1) relative to saline controls, OT dose dependently impaired memory storage (shortened reentry latency), and AOT dose dependently facilitated it (lengthened reentry latency); (2) OT and AOT treatments produced mirror-image U-shaped response curves, with OT signiﬁcantly impairing PA retention at dose levels of 0.1 and 0.3 g/kg, and AOT signiﬁcantly facilitating it at a dose level of 0.3 g/kg; (3) neither peptide inﬂuenced reentry latency in the nonshocked mice, indicating that results of these peptide treatments could not be explained as a nonspeciﬁc effect on PA behavior. Experiment 2 was conducted to examine time-dependent effects of posttraining administered OT and AOTon PA retention. Independent groups of mice received OT (0.10 g/kg, subcutaneous) or AOT (0.30 g/kg, subcutaneous) 0, 30 or 180 min after training. The effect on PA retention of both OT and AOT was time dependent. The results indicated that OT and AOT, respectively, impaired (decreased), and enhanced PA retention (increased reentry latencies) when administered within 30 min after the training trial, but had no effect on retention when given 3 h after this trial. In experiment 3, the researchers investigated a putative OT–AOT interactional effect on PA retention. Four independent groups of mice received a posttraining injection of saline or AOT (0.03 g/kg, subcutaneous) immediately after the training trial and, 10 min later, were subcutaneously injected with either saline or OT (0.10 g/kg, subcutaneous). The results showed that pretreatment with a dose of AOT, which on its own had no effect on PA retention, prevented the OT-induced impairment of PA retention observed when OT was given 10 min after the training trial. Experiment 4 tested for cross-reactivity between OT and AVP receptor antagonist d(CH2)5[Tyr(Me)2]AVP (i.e., AAVP), and between AVP and the AOT used in previous experiments. Eight groups of newly trained mice received a posttraining injection of saline, AOT (0.03 g/kg, subcutaneous), or AAVP (0.01 g/kg, subcutaneous), and 10 min later either saline or OT (0.10 g/kg, subcutaneous). Eight other groups of mice received a posttraining injection of saline, AAVP (0.01 g/kg, subcutaneous), or AOT (0.03 g/kg, subcutaneous), and 10 min later a subcutaneous injection of saline or AVP (0.03 g/kg). The results demonstrated that (1) at the dose level used in these experiments, neither AOT nor AAVP affected PA retention on their own; (2) unlike AOT, neither saline nor AAVP pretreatment prevented the impaired PA retention induced by the posttraining OT treatment; and (3) unlike AAVP, pretreatment with AOT failed to prevent the enhanced PA retention effect induced by posttraining administered AVP. The following points were made in the course of discussing these results: (1) the ﬁndings that posttraining AVP facilitates, and OT attenuates, PA memory consolidation in mice (experiment 4) is consistent with ﬁndings

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from studies with laboratory rats (Bohus et al., 1978a,b); (2) like vasopressin (see Chapter 2), the vasotocin analog d(CH2)5[Tyr(Me)2,Thr4,Tyr(NH2)9] OVT (AOT) used in this study dose dependently facilitated memory consolidation and produced an inverted U-shaped dose–response curve that was the mirror image of that produced by comparable doses of OT (experiment 1); (3) the time-dependent effects on PA retention observed for posttraining OT and AOT (experiment 2) have also been reported for OT and VP in studies with laboratory rats (Bohus et al., 1978a; see Chapter 2). The authors of this study further noted these time-dependent effects suggest a peptide action ‘‘on a neural or neurohumoral process underlying the storage of recently acquired information’’ (Boccia et al., 1998, p. 142; McGaugh, 1989); and (4) the results of the cross-reactivity experiment (experiment 4) suggested that the OT receptor mediating the amnestic effect observed in this study can interact with the vasotocin analog (AOT) shown to be a highly potent OT receptor antagonist (Elands et al., 1988b; Manning and Sawyer, 1993), but not with d(CH2)5[Tyr(Me)2]AVP, one of the most potent and selective V1a antagonists tested to date (Elands et al., 1988b; Manning and Sawyer, 1993). Taken together, the results of experiment 4 were interpreted as suggesting that, in mice, the posttraining OT-induced PA memory impairment was due to an OT interaction with speciﬁc receptors. The ﬁndings of experiment 4 are not consistent with observations reported by De Wied et al. (1991). This latter study showed that the V1a receptor antagonist used in this study, and a different AOT (another vasotocin analog), nonselectively interacted VP and OT, effectively blocking both the VP facilitation and OT attenuation of PA behavior. These ﬁndings led De Wied et al. (1991) to propose a central receptor complex at which VP acted as an agonist, and OT as an inverse agonist. Given that, in contrast to the central route of administration used by De Wied et al. (1991), the authors of the present study peripherally administered these peptides, and given the question of whether these neurohypophysial hormones can cross the blood–brain barrier (BBB) and reach central receptor sites, it would be of interest to reexamine the VP and OT interactive effects with these receptor antagonists, using central (intracerebroventricular or local) injection of these peptides. The proposed study should be of particular value for its relevance to the receptor complex model proposed by De Wied et al. (1991; see Chapter 5). 5. Ethologically Relevant Avoidance Behavior in Mice a. Selected Studies: Vasopressin i. Leshner and Roche (1977) Leshner and Roche (1977) compared the effects of a single injection of adrenocorticotropic hormone (ACTH) with lysine vasopressin (LVP) on avoidance of attack in 7-week-old male albino CD-1 mice. This experimental paradigm employed an ethologically relevant learned passive avoidance (PA) response that involved learning to inhibit entry from a start chamber into an attack chamber where the mouse

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was subjected to repeated attacks for 5 s by a trained ﬁghter mouse. The PA learning criterion was the number of trials required to learn to remain in the start chamber for 300 s. Retention tests were conducted 24, 48, and 240 h after acquisition of the PA response. The subjects were not attacked during the retention sessions because the ﬁghter mouse was restrained within the attack chamber. Two measures of avoidance responding were used: the number of trials to reach the PA criterion and the latencies to enter the attack chamber during each of the three retention sessions. The effect of a single injection of ACTH on PA responding was tested in part A of the experiment. Each subject received three injections: the ﬁrst injection was given 1 h before PA training, the second immediately after the last training trial, and the third 1 h before the 24-h retention test. The mice were randomly assigned to one of four groups [the control group (P-P-P) received placebo at each injection time; the three treatment groups received two injections of placebo and one of ACTH (2 IU, subcutaneous). Depending on the group, ACTH was given before training (A-P-P), immediately after training (P-A-P), or 1 h before the ﬁrst retention test (P-P-A)]. The design of part B was identical to that of part A except that the peptide-treated mice received a single injection of LVP (0.08 IU, subcutaneous) at each injection time. The data for control groups of parts A and B were combined for analysis because the two groups did not differ signiﬁcantly in any measure of learning or retention. The data were analyzed in a two-way factorial design. One factor was the hormone injected; the other, the time of injection (i.e., never, before training, immediately after acquisition, or before the ﬁrst retention test). Neither peptide inﬂuenced the rate of learning (the number of trials required to reach the PA learning criterion). Both ACTH and LVP treatment inﬂuenced retention of the PA response, but did so in quite different ways. The ACTH treatment facilitated retention relative to placebo controls in the 24- and 48-h, but not the 240-h, retention test, and this effect occurred whether ACTH was injected before or after learning, or before the retention test. However, ACTH proved most effective when injected 1 h before the 24-h retention test (i.e., there was a signiﬁcant difference at this time between P-P-A and the other two ACTH treatment groups), indicating that the peptide produced a relatively short-term effect on retention. Regardless of when it was given, LVP treatment did not inﬂuence retention at 24 h but did so in the 48- and 240-h tests. Relative to the placebo-treated subjects, posttraining LVP facilitated retention (signiﬁcantly longer mean retention latency) in both the 48- and 240-h retention tests, and preretention LVP did so for the 240-h test. Although an injection of LVP 1 h before the 24-h retention test did not prolong reentry latency on the day of treatment, reentry latency for this group lengthened on the following day (48-h test) and matched the latency effect exhibited by the posttraining LVP treatment group in the 240-h retention test.

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In summary, this study demonstrated that whereas ACTH hormonal treatment was effective whenever it was administered, and produced relatively short-term effects on retention, LVP facilitated retention only when given immediately after learning or before the retention test and produced a relatively long-term effect. The authors noted the similarity between the results of this ethologically relevant passive avoidance paradigm and those of the typical inhibitory avoidance paradigm, which uses footshock as the punisher. In both, vasopressin facilitates long-term memory, whether given posttraining (consolidation effect) or preretention (retrieval effect), and its effect on retention is longer lasting than that of ACTH (e.g., De Wied, 1965, 1971; see Chapter 2). ii. Roche and Leshner (1979) Roche and Leshner (1979) investigated the effects of a single injection of ACTH (experiment 1) and LVP (experiment 2) on postdefeat submissive behavior in 7-week-old male albino CD-1 mice. Treatment (ACTH or LVP) was given immediately after defeat of the subject in an encounter with an aggressive male mouse. With the exception of the speciﬁc peptide injected, the design was identical for both experiments. Submissive behavior was induced by pairing each subject with a mouse, made highly aggressive by having been housed in prior isolation for 4–6 weeks. The subject and the aggressive mouse were allowed to interact in an arena until the subject submitted (displayed the upright submissive posture and failed to ﬁght back when subsequently attacked by the aggressive opponent). The subjects in each experiment were randomly assigned to one of three experimental and one of three control groups. The control groups received placebo (physiological saline, subcutaneous), and the experimental groups received the peptide hormone (2.0 IU of ACTH, subcutaneous, in experiment 1 and 0.08 IU of LVP, subcutaneous, in experiment 2). The subjects in each group were tested for submissive behavior either 24, 48, or 168 h after the original defeat by being placed in the same arena with the same aggressive opponent. The criteria employed for deﬁning submissive behavior in the original encounter were again used for this test. Two measures of submissive behavior were scored: the duration of the interval between the opponent’s initial attack and subsequent submission by the subject (latency to submit minus latency to attack), and the number of aggressions required to induce submission in the subject. The shorter the duration interval and the fewer attacks needed to produce submission, the more submissive the subject was judged to be. Relative to the placebo controls, the ACTH-treated mice exhibited signiﬁcantly greater submissive behavior (both measures of submission) at the 24- and 48-h, but not the 168-h, postdefeat test times. The LVP-treated mice exhibited signiﬁcantly greater submissive behavior than the placebo controls in both measures at the 48- and 168-h, but not the 24-h, postdefeat test times. Observation of the data points for the 24-h test indicated excessive

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submissive behavior in placebo controls in experiment 2, which may have created a ‘‘ceiling effect’’ obscuring the inﬂuence of vasopressin at this time. On the assumption that submissive behavior is a naturally occurring avoidance reaction, it was concluded that these results were similar to those reported by De Wied (1971; see Chapter 2), which demonstrated that ACTH has relatively short-term effects on maintenance of avoidance behavior whereas that of vasopressin lasts much longer. The authors further concluded that because both ACTH and vasopressin levels increase after exposure to stressful stimuli (Mason, 1968; Thompson and De Wied, 1973; see Chapter 3), the procedure used here resulted in a functional exaggeration of the normal reaction to certain stressors. Thus, the authors interpreted their results as supporting the proposal (Gold and Van Buskirk, 1976a,b; Levine, 1968) that these hormonal responses may in some way facilitate the memory of the stressful experiences that activate their release.

B. Appetitive Paradigms 1. Autoshaped Lever Touch Response Conditioning a. Selected Studies: Vasopressin i. Messing and Sparber (1983) Messing and Sparber (1983) assessed the effect of desglycinamide-arginine vasopressin (DG-AVP) on acquisition and extinction of an autoshaped lever touch response for food reward in Holtzman albino rats. ‘‘Autoshaping’’ refers to the assumption that the animal ‘‘teaches itself’’ to lever press for food reward. In each trial, the lever is extended into the operant chamber and is retracted after a 15-s interval, at which time a food pellet is automatically delivered into the chamber. However, if the subject touches the extended lever with suitable force at any time during the 15-s interval the food reward is immediately delivered. In addition to the autoshaped lever touch responses, nonspeciﬁc unconditioned activity (e.g., rearing activity) was scored to control for the possibility that a peptide-induced increase in general activity level may inadvertently facilitate performance on this task. Autoshaping training began the day after adaptation to the test environment. Twelve autoshaping trials were given on day 1 of training and 3 sessions (12 trials/session) were given 2 days later on day 3. Pilot work showed no indication of learning during the initial 12 trials. Asymptotic performance was tested on day 6, and 2 days later the subjects received 2 days of extinction (days 8 and 10) with 3 blocks (12 trials/block) of extinction testing per day. DG-AVP (5 or 10 g/kg, subcutaneous) or saline was injected 1 h before behavioral testing on each day of acquisition (days 1 and 3) and extinction (days 8 and 10). No injections were given on the day of the asymptotic performance test. Before extinction testing, the rats were reassigned to drug treatment groups to equalize for peptide treatments

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received during the earlier autoshaping training sessions and, as far as possible, to match the groups for asymptotic test performance. The results, graphed in Figs. 1 and 2, indicated that (1) the 5-g dose, but not the 10-g dose, of DG-AVP signiﬁcantly facilitated learning on blocks 2 and 3 (acquisition training day 3); see Fig. 1A; (2) there was no signiﬁcant peptide effect on basal responding (block 1) or on unconditioned rearing activity (strip touch behavior), indicating that the peptide-induced learning effect was not due to an inﬂuence on general activity level as measured in this study (Fig. 1B); and (3) during extinction, treatment with both peptide dose levels produced signiﬁcantly greater response in the second block of extinction trials given on the second day of extinction (day 10; Fig. 2A); again there was no peptide effect on unconditioned rearing activity (Fig. 2B). The authors interpreted these results as indicating that (1) DG-AVP facilitated learning on this appetitive task and this effect was uncontaminated by a concomitant inﬂuence on nonspeciﬁc activity; (2) the DG-AVPinduced retardation of extinction, coupled with its facilitation of original learning, indicated that the extinction effect on this task resulted from a peptide-induced strengthening of original memory rather than a deleterious

FIGURE 1 (A) Acquisition of the lever touch response. Rats were injected subcutaneously with saline or DG-AVP 1 h before each experimental session. They were subject to one block of trials in the ﬁrst session, and to three blocks 48 h later; n ¼ 8 or 9 per group. *p < 0.025, signiﬁcantly different from 5 g/kg by Dunnett’s t test. Source: Messing and Sparber, 1983 (Fig. 1, p. 46). Copyright ß 1983 by Elsevier Biomedical Press. (B) Strip touch behavior during the two acquisition sessions. Rats were treated as described in (A). Data are expressed as responses per minute of session length for a block of 12 trials. Source: Messing and Sparber, 1983 (Fig. 2, p. 47). Copyright ß 1983 by Elsevier Biomedical Press.

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FIGURE 2 (A) Extinction of the lever touch response. Rats were injected subcutaneously with saline or DG-AVP 1 h before each 36-trial extinction session. The interval between the two extinction sessions was 48 h; n ¼ 7 or 8 per group. *p < 0.005, signiﬁcantly different from 10 g/kg, and p < 0.01, signiﬁcantly different from 5 g/kg by Dunnett’s t test. Source: Messing and Sparber, 1983 (Fig. 3, p. 48). Copyright ß 1983 by Elsevier Biomedical Press. (B) Strip touch behavior during two extinction sessions. Rats were treated as described in (A). Data are expressed as responses per minute of session length for each block of 12 trials. Source: Messing and Sparber, 1983 (Fig. 4, p. 49). Copyright ß 1983 by Elsevier Biomedical Press.

effect on learning the new reinforcement contingency presented during extinction training; and (3) the extinction effect also suggested a facilitation of retrieval. The authors also considered that DG-AVP might have had a beneﬁcial effect on consolidation. That is, although the peptide given on day 1 of acquisition training showed no evidence of an effect on learning, it may have contributed to the improved learning observed on day 3. However, the treatment given on day 3 prevented a ﬁrm conclusion on this point. Moreover, the treatment reassignment procedure ruled out a conclusive test of a consolidation inﬂuence during the extinction test. ii. Messing and Sparber (1985) Messing and Sparber (1985) studied dose-dependent effects of DG-AVP on acquisition /retention of simple and difﬁcult versions of the autoshaping task in Long-Evans hooded rats. The animals received injections of placebo or one of three dose levels of DG-AVP (1.5, 5, or 15 g/kg, subcutaneous). Two experiments were conducted. Experiment 1 used the same simple version of the task as in the previous study (Messing and Sparber, 1983), with a slight variation in task procedure.

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Acquisition of the autoshaping response was carried out in 3 days (days 1, 3, and 5) with trial blocks (12 trials/block) 1 and 2 given on days 1 and 3, and blocks 3, 4, and 5 on day 5. Saline or DG-AVP treatment was given 1 h before daily testing on days 1 and 3 but not on day 5. Because asymptotic performance had not been attained by the second block of 12 trials, improvement could be observed during day 5 of acquisition training. The results, graphed in Fig. 3, indicated that the 15-g/kg dose signiﬁcantly improved performance on acquisition trial blocks 2 and 4 (acquisition days 3 and 5). This peptide effect was interpreted as selective for learning and not attributable to increased nonspeciﬁc activity because there was no peptide effect on basal response rates (block 1) or concomitantly tested nonspeciﬁc rearing activity. The improved learning observed in the 15-g/kg treatment group on day 5 of autoshaping acquisition was interpreted as a facilitation of memory consolidation induced by the drug given 2 days earlier and not due to state-dependent or other transient effects on performance. That a higher dose of the peptide was required to facilitate performance in this study in comparison with their earlier study (Messing and Sparber, 1983) was attributed to possible differences between Holtzman and Long-Evans rats in their susceptibility to this peptide.

FIGURE 3 Effects of DGAVP on acquisition of autoshaped lever-touch responses and interim nose-poke responses in the experiment with no delay interposed between lever retraction and delivery of food pellets. (Rats [n ¼ 4-5/ group] were injected s.c. with saline [1 ml/kg) or DGAVP 1 hr before Sessions 1 and 2. Values depicted are group means. DGAVP ¼ des-glycinamide arginine vasopressin). Source: Messing & Sparber, 1985 (Figure 1, p. 1116). Copyright ß 1985 by the American Psychological Association. Reprinted with permission.

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In experiment 2, the autoshaping lever touch response task was made more difﬁcult by insertion of a 6-s delay between lever retraction and the food reward. Previous work (Messing and Sparber, 1985) indicated that control subjects do not learn the task under these conditions. Each rat received 8 days of acquisition training (1 block of 12 trials/day). A 2-day weekend separated the ﬁrst 4 days from the last 4 days of acquisition training. Saline or DG-AVP (15 g/kg, subcutaneous) was injected 1 h before each daily testing session for the ﬁrst 6 days of training. No treatment was given for the last two training sessions. Autoshaped lever responding in the peptide treatment groups was signiﬁcantly higher than in saline controls on the last 2 days of acquisition (Fig. 4). Because treatment had been discontinued at this time, the improved performance was attributed to a peptide effect on learning and memory consolidation rather than to a transient-arousal enhancing effect on performance. This interpretation was supported by the lack of a DG-AVP effect on basal responding or on concomitant rearing behavior. iii. Mundy and Iwamoto (1987) Mundy and Iwamoto (1987) attempted to replicate the behavioral effects of DG-AVP treatment observed by Messing and Sparber (1983, 1985) for the autoshaped lever touch task, using Sprague-Dawley rats as subjects. Depending on the experiment, the simple or difﬁcult version of the task was used. In the simple version (no delay between lever retraction and reward), the animals were treated with placebo or a single dose of DG-AVP (10 g/kg, subcutaneous). For the difﬁcult version (an 8-s delay between lever retraction and reward), the dose of the peptide was varied (0, 10, 20, or 30 g/kg, subcutaneous). Several experiments were conducted using the simple version of the task. Experiments A through D used the same task procedure as that of Messing and Sparber (1983). Two experiments (A and B) were replicates of one another, and used a treatment schedule and reassignment before extinction identical to that used by Messing and Sparber (1983). A third experiment (C) omitted the treatment reassignment procedure before extinction; that is, each subject received the same treatment during extinction as it had during autoshaping training. Experiment C was designed to determine whether treatments given during training would inﬂuence extinction behavior. A fourth experiment (D) tested the usefulness of extinction as a measure of retention for this autoshaped task by testing each subject in two cycles of the autoshaping training/extinction procedure. Peptide treatment was omitted during experiment D. It was predicted that if extinction was a useful measure of retention, then extinction should be prolonged on the second cycle. The results of these experiments indicated (1) no signiﬁcant effect of the 10-g/kg dose of DG-AVP on either acquisition or extinction in experiments A and B (Fig. 5); (2) no signiﬁcant vasopressin effect on acquisition and extinction in experiment C (Fig. 6); and (3) no evidence of prolonged

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FIGURE 4 Effect of DGAVP (15 g/kg) on acquisition of autoshaped lever-touch responses and interim nose-poke responses during inter-trial intervals and 6-s delays of reinforcement. Rats were injected s.c. with saline (1 ml/kg) or DGAVP 1 hr before Sessions 1–6. Values depicted are group means. DGAVP ¼ des-glycinamide-arginine vasopressin). Source: Messing & Sparber, 1985 (Figure 2, p. 1117). Copyright ß 1985 by the American Psychological Association. Reprinted with permission.

extinction in the second cycle of experiment D (Fig. 6). The discrepancy between the ﬁndings of this study and those of Messing and Sparber (1983) may be due to differences in dose level of the peptide, strain or age of the rat, and /or to different interactional effects between dose level and subject variables. A second procedure, designed to assess the effects of DG-AVP on learning, but not extinction, was used in two ﬁnal experiments. Ten autoshaping trials were given each day until the subject reached a criterion of 10 of 10 correct responses in 1 day or until it had completed 10 daily sessions. One experiment compared heterozygous diabetes insipidus (HEDI) and

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FIGURE 5 Mean number of lever touches during acquisition and extinction of the autoshaped lever touch response, using procedure 1. Groups of six animals received saline (dashed line) or DG-AVP at 10 g/kg (solid line) 1 h before experimental sessions. During acquisition animals were exposed to 1 block of 12 trials in the ﬁrst session, and to 3 blocks 48 h later. Three days after the last acquisition session, animals were reassigned (see text) and subjected to two extinction sessions of 36 trials, separated by 48 h. Source: Mundy and Iwamoto, 1987 (Fig. 1, p. 309). Copyright ß 1987 by Pergamon Journals Ltd.

homozygous diabetes insipidus (HODI) variants of Brattleboro rats in task acquisition and found no signiﬁcant differences between vasopressin-deﬁcient HO and the normal HE Brattleboro rats. The other experiment tested the effect of the peptide (0.0, 10.0, 20.0, or 30.0 g/kg, subcutaneous) on response acquisition in male Sprague-Dawley rats, using the task made more difﬁcult by an 8-s delay between lever retraction and food reward. Saline or peptide was peripherally administered 1 h before daily autoshaping sessions. The results indicated no signiﬁcant effect of DG-AVP, at any of the dose levels used, on acquisition of this more difﬁcult version of the autoshaping task. This study thus failed to conﬁrm the DG-AVP-induced facilitation of acquisition and retention in this appetitive autoshaping task reported by Messing and Sparber (1983, 1985), despite attempts to follow behavioral and peptide treatment procedures used in the earlier studies. Moreover, the observation by Messing and Sparber (1985) that the facilitative effects of the peptide on acquisition and retention were even more robust in the difﬁcult version of the task was not conﬁrmed by this study. Mundy and Iwamoto (1987) did allow that the use of subjects from a different strain of rat (Sprague-Dawley versus Long-Evans hooded and Holtzman albino rats), of a lower average weight (i.e., average body weight of 305 versus 625 g) and of a younger age group (inferred from body weight data), may have

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FIGURE 6 Mean number of lever touches during acquisition and extinction of the autoshaped lever touch response. In experiment C, groups of six animals received saline (dashed line) or DG-AVP at 10 g/kg (solid line) 1 h before experimental sessions. During acquisition animals were exposed to 1 block of 12 trials followed by 3 blocks 48 h later. Three days after the last acquisition session animals were subjected to two extinction sessions of 36 trials, separated by 48 h. Animals were not reassigned before extinction sessions. In experiment D, 1 group of 12 animals received saline 1 h before acquisition and extinction sessions (dashed line). Two days after the last extinction session, the same animals were resubjected to the acquisition and extinction sessions for a second time (solid line). Source: Mundy and Iwamoto, 1987 (Fig. 3, p. 310). Copyright ß 1987 by Pergamon Journals Ltd.

contributed to the discrepancy between the two sets of ﬁndings. That is, the strain and age differences in learning ability or vasopressin sensitivity may have rendered the animals in this study less susceptible to the behavioral effects of the peptide. The authors of this study also concluded that, for this autoshaping paradigm, the rate of extinction proved not to be a good measure of retention. Finally, this study observed no evidence of a learning impairment in the VP-deﬁcient HODI Brattleboro rats in this appetitive task, a ﬁnding in accord with other reports that have examined learning and memory in positively reinforced operant tasks performed by Brattleboro rats (e.g., Laycock and Gartside, 1985; Sahgal, 1983; see Chapter 7). 2. Visual Discrimination Learning a. Selected Studies: Vasopressin i. Hostetter et al. (1977) Hostetter et al. (1977) examined the effects of vasopressin on both acquisition and extinction of a food rewarded black / white discrimination T-maze task in male Long-Evans hooded rats.

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The vasopressin analog used was Pitressin tannate in oil, a rather crude extract of vasopressin prepared from the posterior and intermediate lobes of the pituitary (De Wied, 1965; see Chapter 2). Placebo or Pitressin [0.4 IU/rat, subcutaneous (i.e., 0.3 IU is approximately equal to 1 g; Sahgal, 1984)] was injected 20 min before the day’s testing session. Speciﬁcally, three treatment groups were formed: the V–S group (received Pitressin during acquisition, saline during extinction); the S–V group (received saline during acquisition, Pitressin during extinction), and the S–S group (received saline during both acquisition and extinction). Ten massed training trials were given each day until the animals reached the learning criterion (10 of 10 correct choices in 2 consecutive days). On the day after the completion of extinction half the animals in each treatment group received saline or Pitressin 20 min before testing in the open ﬁeld, with number of squares crossed as the measure of general activity level. The three treatment groups were compared both for trials required to learn and extinguish the discrimination, and for running speed in the T-maze during acquisition. The results were as follows: (1) Pitressin did not signiﬁcantly inﬂuence running speed during acquisition training or activity level (number of squares crossed) in the open ﬁeld test; (2) Pitressin did not inﬂuence the rate of learning the discrimination to the white or black color (i.e., trials to acquisition did not differ among the S–V, V–S, and S–S treatment groups whether the black or the white goal arm was the positive discriminative stimulus); (3) the discrimination was easier when the black goal arm was the positive discriminative stimulus (i.e., regardless of treatment, the subjects required signiﬁcantly fewer trials to learn to choose the black as opposed to the white goal arm); (4) Pitressin given during acquisition only did not improve retention for either the learned black or white positive discrimination (i.e., no signiﬁcant difference between the V–S and S–S treatment groups for either the black or white positive discrimination); (5) Pitressin given during extinction training facilitated retention (i.e., prolonged maintenance of the learned discrimination) in those subjects trained to choose the black goal arm (i.e., for the subjects trained to choose the black goal arm, the S–V groups required signiﬁcantly more trials to extinction than did the V–S or S–S groups). Moreover, the peptide’s maintenance of discriminative responding during extinction could not be attributed to an effect on general activity because of the open ﬁeld data reported above; and (6) in contrast to the subjects trained to choose the dark goal arm, there were no differences in extinction performance among the three treatment groups trained to approach the white goal arm. The authors were not able to explain the failure of the peptide to retard extinction in the subjects trained to approach the white goal arm. The authors noted that result 5 contrasts with the failure of vasopressin, given during extinction, to maintain performance of an appetitively motivated

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runway response reported by Garrud et al. (1974; see Chapter 2). Because their study found no effect of Pitressin on speed to traverse the T-maze during acquisition of the discrimination (result 4), Hostetter et al. (1977) suggested that the failure to obtain a vasopressin effect on extinction of the runway response by Garrud et al. (1974) may have been due to their selection of runway speed as the dependent variable. ii. Sara et al. (1982) Sara et al. (1982) tested the effects of vasopressin on learning, long-term memory, retrieval, and subsequent reversal learning in a brightness discrimination task in two experiments using adult male Sprague-Dawley albino rats as subjects and LVP (10 g /rat, subcutaneous) as the vasopressin analog. The animals were trained and tested in a semiautomated Y-maze; entry into the correct goal arm at the point of the hinged-ﬂoor area activated the food dispenser, which delivered the food reward. In experiment 1, brightness was the positive discriminative stimulus and the peptide was tested for its effect on acquisition and retention of the brightness discrimination and then on learning the reversed discrimination. The subjects were trained to approach the brighter goal arm to a learning criterion of 9 of 10 consecutive correct choices. After 5 min of adaptation to the maze on day 1, 40 massed discrimination trials over a maximum 60-min period were given on day 2 to provide preliminary training experience in this difﬁcult discrimination. On day 3, the subjects were trained to a learning criterion of 9 of 10 correct responses or until 90 min had elapsed. Only subjects that attained the learning criterion within the 90-min period were tested for retention 19 days later, at which time they were trained to the original learning criterion and scored for the trials required to reach this criterion. Several days after the retention test, the subjects were rerun to a baseline of 9 of 10 correct and 24 h later trained in the reversed discrimination (dark goal arm positive). To test the effects of LVP on acquisition of the original discrimination, half the rats received placebo (saline) and half received LVP (10 g/rat, subcutaneous) 90 min before the testing session on day 3; to test the independent effects of the peptide on memory storage and retrieval, four treatment subgroups were formed 90 min before the 19-day retention test. Subjects that received LVP during acquisition were subdivided and received LVP or saline before retention testing, and those given saline during acquisition were similarly subdivided. To test the effect of the peptide on reversal learning, 90 min before the reversal test half the subjects that received saline injections for the retention test were injected with saline (Sal), the other half with LVP (10 g/rat, subcutaneous). The treatment groups thus formed permitted independent tests of the effect of the peptide on original learning (LVP versus Sal), memory consolidation (LVP/Sal versus Sal/Sal), retrieval (Sal/LVP versus Sal/Sal), and reversal learning (groups given LVP or saline

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before reversal but equivalent to each other in their preexperience with LVP and saline during original acquisition). Subjects that did not achieve the learning criterion during original acquisition were not included in the results reported below. The results showed that (1) LVP treatment on day 3 facilitated brightness discrimination acquisition (signiﬁcant difference between LVP- and placebo-treated subjects in the number of trials required to reach the learning criterion); (2) signiﬁcant forgetting occurred over the 19-day retention interval because, regardless of the treatment received during acquisition, the subjects required more than 30 trials to relearn the brightness discrimination; (3) LVP given before acquisition training did not inﬂuence long-term memory (no signiﬁcant difference in retention test performance between the LVP–Sal and Sal–Sal subgroups), nor was retrieval inﬂuenced by LVP injected before the retention test (no signiﬁcant difference in retention test performance between Sal–LVP and Sal–Sal subgroups); and (4) LVP impaired reversal learning (subgroups treated with LVP before reversal learning required signiﬁcantly more trials to reach the learning criterion than the saline controls). In experiment 2, naive subjects were trained in brightness discrimination to a learning criterion of 9 of 10 correct choices, with either the illuminated or the darkened goal arm as the positive discriminative stimulus. For each contingency, half the rats received LVP (10 g/rat, subcutaneous) and half received placebo (saline) 90 min before the single day (40 trials) of training (because many animals trained to the dark learn in less than 40 trials, the preliminary training procedure was not used in this experiment). Three weeks later, the rats trained to run to the darkened goal arm were tested for retention by retraining them to the same learning criterion, without peptide treatment. On the day after retention testing these subjects were reversal trained to approach the illuminated goal arm, and were given either saline or LVP (10 g/rat, subcutaneous) 90 min before the reversal training session. The results of experiment 2 were as follows: (1) presession LVP treatment replicated the ﬁnding in experiment 1 of facilitated learning when light was the positive discriminative stimulus but not when darkness was the positive cue. This latter failure may have been due to a ceiling effect, because both LVP- and saline-treated subjects exhibited peak performance in this easy discrimination; (2) LVP had no effect on memory consolidation for the easier discrimination (no difference in retention test performance between the groups receiving LVP versus saline before being trained to run to the dark compartment); and (3) presession LVP impaired reversal learning when the discrimination was switched from darkness to light as it had done for the switch from light to darkness in experiment 1. Altogether, the results of this study indicated that (1) pretraining LVP facilitated brightness discrimination learning provided the discrimination

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was sufﬁciently difﬁcult (i.e., ﬁndings for experiment 1 versus experiment 2); (2) LVP treatment inﬂuenced neither memory consolidation (assessed in experiments 1 and 2) nor memory retrieval (assessed in experiment 1 but not 2) in this discrimination task; and (3) LVP given before training on the reversed discrimination produced negative transfer effects (i.e., retarded reversal learning) whether the reversal involved a switch from a bright to a dark goal arm (experiment 1) or vice versa (experiment 2). It was argued that the LVP-induced negative transfer observed in reversal learning could have reﬂected a retrieval effect (enhanced retrieval of the original learned discrimination which interfered with reversal learning) or a perseveration effect (impairment of the underlying inhibitory processes that mediate extinction of the previously learned response). Because the peptide did not enhance retrieval in the 19-day retention test, it was hypothesized that the negative transfer reﬂected perseveration of the original learned response. Moreover, data reported by Bohus (1977; see Chapter 2), a VP retention effect reported for a learned sexually motivated T-maze discrimination, was reinterpreted by Sara et al. (1982) as indicative of a peptideinduced perseveration. In the Bohus (1977) study, ‘‘retention’’ behavior did not differ between the VP- and saline-treated controls for the initial trials of the retention test. However, differences were observed after the third retention trial when the VP-treated subjects continued to approach the sexual side of the T-maze as the saline controls began to avoid it. Sara et al. (1982) attributed this avoidance response to a ‘‘refractory’’ period in sexual motivation, and the continued sexual responsiveness in the VP-treated subjects was attributed to response perseveration. iii. Mulvey et al. (1988) Mulvey et al. (1988) also studied the effect of LVP on memory consolidation and retrieval for a brightness discrimination in adult male Sprague-Dawley rats. The animals were trained and tested for extinction and then for retention, using a savings method, in a modiﬁed Y-maze. Each subject was given 20 acquisition trials/day to a criterion of 8 consecutive entries into the illuminated goal arm. A single posttraining injection of either saline or LVP (1.8 g/rat, subcutaneous) was given immediately after the completion of acquisition training. Extinction training (20 trials/day) began 72 h after learning and continued until the response was extinguished (5 or fewer correct responses in 10 trials). No peptide treatment was given during extinction. The rate of extinction was used as one measure of retention (consolidation design). Forty-ﬁve days after the completion of extinction, the subjects were tested for savings of the originally learned discrimination by reacquisition training in the brightness discrimination. LVP (1.8 g/rat, subcutaneous) or saline was given 1 h before reacquisition training. Of the subjects that received posttraining vasopressin during the original discrimination, half received the peptide and half received saline during reacquisition training. A similar treatment reassignment was

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used for the subjects that received posttraining saline during the original discrimination. Treatment subgroup comparisons permitted evaluation of a peptide effect on consolidation (V–S versus S–S) and retrieval (S–V versus S–S), as measured by the savings method. The results indicated that LVP facilitated memory consolidation but not retrieval. The observation that posttraining vasopressin prolonged extinction as well as facilitated savings suggests that extinction was a useful measure of retention for this task. iv. Alescio-Lautier and Soumireu-Mourat (1990) Alescio-Lautier and Soumireu-Mourat (1990) used BALB/c inbred mice in a study designed to test the effect of high [1.0 g/mouse, subcutaneous (i.e., 50 g/kg)] and low [0.5 g/mouse, subcutaneous (i.e., 25 g/kg)] doses of AVP on different stages of memory processing in a go/no-go black/white (B/ W) discrimination task. At the end of discrimination testing, the effect of the same dose levels of AVP on spontaneous locomotor activity was assessed in a circular runway over a 24-h period. The apparatus for the discrimination task consisted of two side-by-side Plexiglas runways, one white and the other black. Each runway was divided into a start box, alley, and goal box, which contained the food reward. A guillotine door separated the start box and alley. The rats were given 1 daily training session (12 trials per session) for 3 days. One-half of each of the day’s test trials was spent in the rewarded runway (Sþ trials), the other half in the nonrewarded runway (S trials), in a randomized sequence. A food reward was provided in the cup of the goal box on Sþ (go) trials but not on S (no-go) trials. Depending on the group, the Sþ runway was either white or black in color. Learning corresponded to a decrease in Sþ running times and to an increase in S running times over successive trials. Retention testing consisted of an additional session, under the same conditions, given 24 days after the third learning session to ensure signiﬁcant but not complete forgetting. Depending on the treatment groups, the mice received either placebo or AVP at one of the following times: 20 min before or immediately after the ﬁrst learning session, 20 min before the second learning session, or 20 min before the retention test. Because performance was similar for the black and white Sþ stimuli groups, their data were combined for presentation of the results. Although the peptide exerted a dose-dependent effect on acquisition of this simultaneous (go/no-go) visual discrimination task, neither dose of AVP signiﬁcantly inﬂuenced the rate of learning relative to the saline controls. Speciﬁcally, the results for acquisition of the discrimination were as follows: (1) when given 20 min before the ﬁrst learning session, both doses of AVP depressed running speed (increased running times) in both runways, an effect that dissipated by the second learning session. Learning failed to occur in all three sessions for the high-peptide group but did occur for the low-peptide group which, however, did not differ from saline controls (i.e.,

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signiﬁcant differences between Sþ and S running times for sessions 2 and 3 occurred for the low-dose peptide and saline control groups but not for the high-dose peptide group); (2) when given immediately after the ﬁrst learning session, the high-dose AVP group exhibited improved learning performance relative to saline controls in the subsequent two learning sessions; on the other hand, the low-dose AVP group was delayed in learning, relative to the saline control group, during the second learning session (i.e., compared with the controls, they ran slower on Sþ trials, and faster on S trials), but not in the third learning session; and (3) when given 20 min before the second learning session, each dose level of AVP produced a bimodal effect on session 2 performance, improving learning, relative to saline controls, in some subjects (‘‘good learners’’) and impairing it in others (‘‘poor learners’’). In session 2, the peptides given to good learners improved learning performance (increased running time in S trials relative to Sþ trials) to a far greater degree than occurred for the saline controls. The peptide-treated poor learners exhibited slow running times equally for both Sþ and S trials. This bimodal effect did not persist to the third learning session because at this time there was no longer a signiﬁcant difference in learning performance between the peptide- and saline-treated subjects. AVP also exerted a dose-dependent effect on retention (memory retrieval) for the learned discrimination problem. Speciﬁcally, when administered 20 min before the 24-day postlearning retention test, the high dose of AVP improved retention relative to the saline controls, an effect that was maintained 24 h later. On the other hand, the low dose of AVP impaired retrieval relative to saline controls (i.e., there was no signiﬁcant difference in running time between Sþ and S trials for the low-dose peptide groups but there was for the saline controls). Assessment of the effect of the peptide on locomotor behavior indicated that AVP injected 20 min before this testing dose dependently inﬂuenced activity level. That is, the high but not the low dose of the peptide signiﬁcantly reduced locomotor activity relative to the controls, and this effect was observed only during the ﬁrst 4 h after treatment. In discussing these results the authors noted that the high dose of the peptide produced its main effects on behavioral performance in this task. This was probably because this dose level exhibited the greatest aversive effect, as indicated by the results of the locomotor activity test. AVP treatment appears to have produced two disparate consequences: an aversive effect (depressed locomotor activity and impaired learning) and a memoryenhancing effect (facilitated consolidation and retrieval). These two effects on performance interacted with the timing of the treatment (whether AVP was given before or after the day’s task session) and the degree of previous learning experience in the task. The aversive effect was most prominent when AVP was injected before the daily learning session, and when given to learning-naive subjects. The memory-enhancing effect of AVP dominated

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when the peptide was given after completion of the day’s learning session, and in subjects with some degree of learning experience. Moreover, once task learning had been completed, even the aversive effects of presession AVP treatment were insufﬁcient to outweigh the memory-enhancing (retrieval) effects of the peptide. The prolonged impairment in learning that occurred in the high-dose AVP group after treatment given before the ﬁrst learning session may have reﬂected an association between the aversive effects of the peptide and the experimental setting, that is, a result of an AVP-induced aversive place conditioning similar to that inferred by Ettenberg et al. (1983a) (see Chapter 6). The bimodal effect produced by AVP when given before the second learning session needs further clariﬁcation because there was no difference in session one performance between the good and poor learners. 3. The Radial Maze Task a. Task Description The radial maze apparatus used in the vasopressin/ memory research of Buresova and Skopkova (i.e., 1980, 1982) contains a circular choice platform, raised 5 cm above the ﬂoor and accessible through a central opening; it provides access to a variable number of regularly spaced tubular channels (radial arms). The arms have a transparent perforated ceiling permitting food odors to saturate the maze to prevent the inﬂuence of olfactory cues on choice behavior. A recessed feeder is located at the far end of each arm; an entrance and exit door within each arm channels the rat’s passage. After entering a given arm from the choice platform, the rat is required to descend to the main ﬂoor, return below the choice platform, climb onto it via the central opening, and make another choice. A 50-cm-high Plexiglas circular wall surrounds the entire maze. A typical procedure used to test short-term memory involves placing a food-deprived rat on the central platform with all the doors closed and all the arms baited. In a given trial, the doors are raised after a preselected interval (e.g., 15 s). Once the subject enters a given arm, the doors to the other arms are closed. After the subject returns to the central platform, the door of the arm last entered is also closed. After a 15-s interval, all the doors are raised and the process is repeated until the subject has completed a total of all available choices or, in the absence of this, until some selected time period has elapsed (e.g., 10 min). An error is recorded for entry into an arm of the maze previously visited during that trial. The radial maze task, a test of spatial memory, has been used to test both short-term (working) and long-term (reference) memory. For this task, short-term memory is operationally deﬁned as memory for those features of the task that remain constant within a trial but vary from trial to trial (memory for the arms visited within a trial, which enables efﬁcient foraging on the task). In contrast, long-term memory is operationally deﬁned as memory for those features of the task that are durable across many trials

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(e.g., memory for the general procedure learned during an extensive training period that permits the rat to forage efﬁciently and to consistently avoid entering the never-baited arms when only a speciﬁc number of arms are baited). b. Selected Studies: Vasopressin i. Buresova and Skopkova (1980) Buresova and Skopkova (1980) noted that numerous studies had obtained evidence supporting a role for vasopressin in acquisition and retrieval of long-term memory (LTM), but that comparable information was not available for short-term memory (STM). Thus, this study tested the effects of a number of vasopressin analogs on the decay of STM in a 12-arm radial maze. Adult male hooded rats of the Druckray strain served as subjects. l- and d-isomers of vasopressin analogs were used in this study, because previous reports had indicated that substituting the d-isomer for the natural l-isomer of some ACTH peptides reverses its behavioral effects. A total of ﬁve vasopressin analogs were tested. These were three natural l-isomers: 8-l-arginine vasopressin (AVP); a desglycinamide derivative of the natural isomer for LVP, desglycinamide-8-l-lysine vasopressin (desgly-NH2-VP); and the desamino l-isomer of AVP, desamino-8-l-arginine vasopressin (dAVP); and two d-isomers: 8-d-arginine vasopressin (DAVP) and 1-desamino-8-d-arginine vasopressin (DDAVP). Forgetting (decay of the STM trace) was induced by a 20-min interruption between the ﬁrst and last six choices in the daily test trial. The animals were removed from the maze and placed in a waiting cage during the 20-min retention interval. To test possible differential effects of the peptide on acquisition versus retention processes involved in STM, placebo (physiological saline, subcutaneous) or peptide (1 g/rat, subcutaneous) was administered either 40 min before the ﬁrst choice or immediately after completion of the ﬁrst six choices (immediately before the 20-min interruption). The peptides were tested at 3- to 4-day intervals, with each rat receiving all treatments. Because no errors occurred during the ﬁrst six choices only errors made after the 20-min interruption were statistically evaluated. The results were as follows: (l) although the 20-min interruption slightly increased the average error rate in choices 7 through 12 under control conditions, spatial STM nevertheless persisted over this interval because the performance level was signiﬁcantly better than predicted random choice behavior; (2) relative to the saline condition, none of the peptide analogs prolonged the duration of STM; (3) time of peptide application was of no inﬂuence on STM performance; and (4) multiple comparison tests of the treatments showed a signiﬁcant difference in errors committed during the last six choices between the l-isomer treatment (dAVP; fewest errors) and the d-isomer treatment (DDAVP; greatest number of errors), and the difference between the control and DDAVP conditions was almost signiﬁcant.

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In discussing these results the authors made the following comments. First, the absence of a difference between the effects of the peptides applied 40 min before the ﬁrst choice and immediately after the sixth choice indicates no differential inﬂuence of the peptides on acquisition versus retention and retrieval processes. Second, results of this study indicated that, as in the case of some ACTH peptides, substitution of the d-isomer for the natural l-isomer of vasopressin produces opposite behavioral effects. Thus, errors were fewest after treatment with the l-isomer dAVP, and greatest after treatment with the d-isomer DDAVP, whether the peptide was administered 40 min before initial testing or at the onset of the 20-min interruption. Third, the failure of these peptides to prolong short-term memory effects may have been due to an inadequate dose level used in the present study. Alternatively, the possibility was noted that duration of STM on this task cannot be inﬂuenced whatever the psychoactive agent. Moreover, prolonged duration of STM would seem disadvantageous for efﬁcient working memory, which requires prompt forgetting once the trial is completed lest the memory trace interferes with remembering the stimulus presented on the next trial. ii. Buresova and Skopkova (1982) Buresova and Skopkova (1982) tested the effect of three VP analogs on short-term memory capacity (number of items that can be efﬁciently stored in short-term memory) under conditions approaching the limits of the working memory capacity in rats. The subjects, male hooded rats of the Druckray strain, were young adult (6 months old) and middle-aged (9 months old) at the time of testing. Before testing, the subjects had been overtrained in the 24-arm radial maze, in which they were tested for spatial STM. Three VP analogs were tested: an l-isomer (dAVP), a d-isomer (DDAVP), and the desglycinamide derivative of the d-isomer (DG-DDAVP). For each day’s testing trial the rat was allowed to make 24 choices. Saline or one of the VP analogs (1 g/rat, subcutaneous) was given 40 min before the ﬁrst choice. Each subject received each treatment and the three analogs were tested at 5- to 7-day intervals. The number of errors (repeated visits to the same arms) was compared with mathematically predicted random choice behavior. The results were as follows: (l) the incidence of errors started to approach predicted random choice behavior only in the last six choices; (2) although the older rats made more errors than the younger ones in the latter part of the trial, this difference was not statistically signiﬁcant; and (3) although all three analogs tended to improve performance, only the desglycinamide d-isomer analog signiﬁcantly reduced errors compared with control conditions, and this occurred for both age groups. However, 2 weeks later a second application of this peptide was not effective. The authors suggested that the peptide may have improved performance by means of a general arousal effect and that the subsequent failure may have been due to a rapid habituation to the arousing effects of the drug.

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iii. Van Haaren et al. (1986) Van Haaren et al. (1986) tested the effects of AVP on short-term memory (STM) and long-term memory (LTM) in adult male Wistar rats, using an eight-arm radial maze. Separate groups of rats were tested in each of the two experiments that were conducted. For each experiment, vehicle or AVP (1.25, 3.75, or 6.25 g/rat, subcutaneous) injections were given 5 min before the start of the daily trial. Each rat was tested under all treatment conditions. AVP injections were given on Tuesday and Friday of each week; vehicle injections were continued on all other days. Experiment 1 tested STM effects (memory for the arms visited within a given trial) and all eight maze arms were baited. The seven subjects had previously been trained to retrieve food pellets from each of the arms. The authors scored the total time per trial that was spent in the maze (600-s maximum), total choices completed, the time per choice, and the number of STM errors per trial. Experiment 2 tested both STM (memory for the baited arms that were visited in a given trial) and LTM (memory for the four arms that were consistently baited). In this experiment, four of the eight arms were baited; the four arms chosen were consistent for a given subject but varied among the subjects. The seven subjects in this experiment had received previous training in the procedure of retrieving food from only half the maze arms. In addition to the measures scored in experiment 1, two additional measures were scored for each trial: the LTM errors and the total choices not completed (the baited arms not visited during the trial). There was no signiﬁcant difference between placebo and AVP treatment conditions in STM errors in either experiment, or for LTM errors in experiment 2. The peptide retarded locomotor activity in this task as indicated by a signiﬁcant dose-dependent increase in the total time spent in the maze and a signiﬁcant decrease in the number of choices completed in the maze for both experiments. The authors concluded that the AVP-induced behavioral inhibition was due to aversive endocrinological effects of the peptide that, in this setting, caused a deterioration in maze performance without, however, impairing STM or LTM per se. They also noted that the results of this study offered no evidence of a direct effect of the peptide on central processing involving either STM or LTM. iv. Strupp (1989) Strupp (1989) examined the effects of the vasopressin metabolite AVP(4–9) on spatial memory in the radial maze. The main objective of the study was to determine whether this nonpressor, nonaversive (Ettenberg, 1985, as cited in Strupp, 1989), but behaviorally potent (Burbach et al., 1983b; see Chapter 5) vasopressin metabolite of the parent peptide might improve long-term memory in an appetitively motivated task. This study used minimal food restriction because the stress of severe food deprivation might release vasopressin in control subjects [e.g., De Wied, 1984

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(review); Gibbs, 1986b; Laczi et al., 1984 (Chapter 3)] and thereby mask a potential effect of the peptide treatment. Task motivation was maintained by employing an appetizing food reward. Three experiments were conducted with radial mazes differing in number of arms, varying dose levels and time of injection, and varying durations of the retention intervals (RIs) interposed between the forced choice runs (during which information was stored) and the free choice runs [during which short-term memory (STM) and long-term memory (LTM) were tested]. Because this study evaluated only LTM errors, there is no further discussion of STM. Independent groups of subjects were tested in each experiment, but all had sufﬁcient training in the test maze to permit proﬁcient foraging performance. In all three experiments each day’s test session consisted of an initial forced run (guillotine doors raised, one at a time, to allow entry into half the baited arms of the maze), followed by a free choice run (free access to all the maze arms) after a designated RI. Each subject was tested under all peptide dose levels (includes the 0.0-level dose) twice with a minimum of two noninjection days intervening between injection days to avoid carryover effects of peptide treatment. Retention for information learned during the forced choice run was evaluated by scoring the LTM errors made during the free choice run. These errors consisted of reentering arms visited during the forced choice run. LTM errors comprised a major dependent measure in this study and were analyzed to determine (1) whether signiﬁcant forgetting occurred for the RIs chosen in experiments 2 and 3 and (2) whether the peptide exerted an inﬂuence on retention in all three experiments. A second dependent measure was the number of errors made by each subject during the ﬁrst set of choices that corresponded to number of arms not visited in the forced arm (e.g., choices 5–8 in experiment 1). This measure was used in statistical tests designed to determine whether retention under the control condition exceeded chance performance. In experiment 1, an eight-arm radial maze was used to test the effect of the peptide on memory after a 14-h RI. A pilot study had indicated that signiﬁcant forgetting occurred after this RI. Depending on the injection day, placebo (0.0 dose, i.e., vehicle solution) or AVP(4–9) (0.2, 0.67, 2.0, or 6.67 g/kg, subcutaneous) was injected 30 min before the forced choice run. The results indicated that (1) under nontreatment conditions, retention was signiﬁcantly above chance level; (2) vasopressin improved retention in this task as indicated by a signiﬁcant reduction in total errors, whether the comparison was between vasopressin treatment (all four dose levels combined) and the placebo condition or between each peptide dose level and the placebo condition; and (3) there was also signiﬁcant improvement over the successive test trials and signiﬁcant individual differences in the degree of retention exhibited. Because AVP(4–9) was injected before the forced choice run and 14.5 h before the retention test, it was clear that the effect of the

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peptide on memory processing occurred before the stage of retrieval; but it was not clear whether the retention effect was the result of an inﬂuence during an early (attention, perception, association processing) or later (consolidation) stage of memory processing. Experiment 2 was designed to test the inﬂuence of the peptide on memory consolidation. Depending on the injection day, vehicle (0.0 dose level) or AVP(4–9) (0.02, 0.2, or 2.0 g/kg, subcutaneous) was injected immediately after completion of the forced choice run in a 12-arm radial maze. A 9-h RI separated the forced choice and free choice test run. Each rat was tested under all treatment levels as in experiment 1, except that a 1.5-h RI was used for two test days in order to verify that signiﬁcant forgetting occurred over the 9-h delay. The results were as follows: (1) signiﬁcant forgetting occurred over the 9-h RI (i.e., signiﬁcantly fewer errors on days involving the 1.5-h RI than on test days involving the 9-h RI); (2) retention on injection days, which always involved a 9-h RI, signiﬁcantly exceeded chance level; and (3) none of the peptide treatments signiﬁcantly inﬂuenced total errors made during the retention test. These results seemed to suggest that the peptide retention effect in experiment 1 was due to a peptide inﬂuence on a stage of memory processing before memory consolidation. However, inspection of the raw data suggested that the failure of a vasopressin/memory effect in this experiment may have been due to either or both of two possible subject–peptide interactions. First, the peptide treatment may not have been effective for the highly proﬁcient subjects (i.e., subjects in which retention was good under control conditions) and inclusion of their data in the analyses may have masked any facilitatory effect of the peptide on the performance of the less proﬁcient subjects. Second, although there was no overall facilitatory effect for any dose of the peptide, observation suggested that subjects differed in the dose level optimal for their behavioral performance. These possibilities were tested in experiment 3. In experiment 3 each subject was tested with the dose that best facilitated its task performance in experiment 2. The procedure was the same as that for experiment 2 except that each subject received a single dose of the peptide, was trained in a 16-arm maze with a 7.5-h RI, and was allowed a maximum of 14 errors during the test session. In addition to the dependent measures already described, each subject was assessed for (1) baseline proﬁciency in retention (average LTM errors made on those noninjection days throughout the study that were preceded by a minimum of two drug-free days; the lower the error rate, the higher the baseline proﬁciency) and (2) the degree of improvement induced by AVP(4–9) treatment (average LTM errors made under peptide condition minus average LTM errors made under vehicle condition). The results indicated that (1) retention under control conditions was signiﬁcantly above chance level; (2) signiﬁcant forgetting occurred after the

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7.5-h RI (performance signiﬁcantly superior after the 1.5-h compared with the 7.5-h RI); (3) retention was slightly but signiﬁcantly improved by the effective dose levels used for posttrial AVP(4–9) treatment; (4) the subjects markedly differed in their baseline proﬁciency levels; (5) the memory facilitation effect of the peptide was inversely related to baseline proﬁciency (i.e., the less proﬁcient the subject under nontreatment conditions, the greater the peptide induced improvement); and (6) posttrial AVP(4–9) increased retention in the less, but not the more, proﬁcient subgroups. The author hypothesized that proﬁciency of the subject under nontreatment conditions may reﬂect, in part, the amount of endogenous vasopressin released in the test environment. If so, this suggests that the AVP released in the less proﬁcient performers at this time is suboptimal and retention is improved by vasopressin treatment. In contrast, the relatively high levels of endogenous AVP in highly proﬁcient performers is already optimal for retention and an increase by peptide treatment should result in no improvement and even a performance impairment, in accord with an inverted U-shaped dose–response function. 4. Socially Transmitted Information Regarding Food Preferences a. Task Description Strupp, Bunsey, and colleagues introduced a new type of social learning task for the study of vasopressin inﬂuences on memory in rats. This paradigm relies on olfactory-mediated social transmission of information from a conspeciﬁc regarding a recently consumed novel food. In the natural world this type of social transmission can be used to inform a conspeciﬁc that a novel food is safe to eat (Bunsey, personal communication, 1998). Thus, by snifﬁng the exhaled breath of a conspeciﬁc that has recently eaten a novel food, the observer’s memory of the inhaled odor can be used to determine the safety of eating a subsequently encountered food with that distinctive odor (see Galef and Stein, 1985). The essential features of the task paradigm are as follows: (l) a young female (the observer) is placed in the home cage of an older female (the demonstrator), where they are allowed to interact socially for 1 week; (2) then the demonstrator is removed from the home cage, placed in a single cage, and food deprived for 24 h, after which it is allowed to consume a distinctively ﬂavored diet for 30 min before being returned to the home cage for a 15-min social encounter with the observer; (3) after the 15-min interaction, the demonstrator is returned to the single cage with ad libitum access to food and water; (4) the observer’s food pellets are removed and it is given a choice between two novel diets, one of which was previously consumed by the rat demonstrator; the diets are equated for palatability, and the amounts of each diet consumed over a 24-h period are measured; (5) the choice test is given 1 day after the social encounter and again after a speciﬁed retention interval; the amounts consumed from each diet are compared between the two choice test days to estimate the degree of retention over the retention

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interval; and (6) vasopressin, administered before the retention test, can be used to assess the inﬂuence of the peptide on memory retrieval for the olfactory-based memory. b. Selected Studies: Vasopressin i. Strupp et al. (1990) Strupp et al. (1990) used the social learning paradigm to test the effect of the nonpressor, nonaversive peptide metabolite AVP(4–9) on memory retrieval in female Long-Evans hooded rats. A major objective was to expand the study of a vasopressin inﬂuence on memory retrieval to an appetitive paradigm to ‘‘dispel the concern that many previous reports of improved retrieval in avoidance tasks might not be generalizable’’ (Strupp et al., 1990). A second objective was to test the effects of vasopressin on memory retrieval under conditions that were assumed to reduce the possibility of ‘‘cueing’’ mediating the retrieval effect of the peptide. This is a distinct possibility in avoidance paradigms because the footshock (FS) stress activates the hypothalamic–pituitary–adrenocortical (HPA) axis, as does the parent peptide (AVP, LVP) when given before the retention test. The peptide may reinstate the same internal state that was present during learning, thus cueing the retrieval. In this study HPA activation was minimized during training by the absence of stressors such as FS or severe food deprivation, and during retention by use of a vasopressin metabolite that, unlike the parent peptide, does not stimulate the HPA axis. However, M. Bunsey (personal communication, 1998) has noted that cueing effects probably cannot be entirely ruled out in this study because evidence has indicated that vasopressin is released during conspeciﬁc social interactions similar to those occurring in this protocol (e.g., Garritano et al., 1996). With certain exceptions, the task procedure was the same for all three experiments. The observer was permitted a 15-min social interaction with the demonstrator, who had previously partaken of a diet of rat chow ﬂavored with a palatable spice, the odors of which could be detected by olfactory-mediated intercommunication. The choice test was given 1 day after the interactive communication and again after a speciﬁed retention interval. AVP(4–9) at one of several dose levels was subcutaneously injected 1 h before the retention test given after the speciﬁed retention interval (RI). Depending on the experiment, three to ﬁve pairs of diets were used in the test. Each subject was tested for each dietary pair, and under all treatment conditions, in the retention tests given on day 1 and after the speciﬁed retention interval. For data analysis, one of the two diets within each ﬂavor pair was randomly designated as food A. For each retention test, the percentage of the subjects’ total intake that comprised food A (the food consumed by the demonstrator rat) was calculated. For some subgroups food A was correct (the food ﬂavor consumed by the demonstrator), for other subgroups food

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A was incorrect (the demonstrator consumed the other dietary ﬂavor of the pair). The difference in the percentage of food A consumed by the two subgroups reﬂects the recall for the correct diet. Statistical analyses of these difference scores were carried out to determine whether the social interaction inﬂuenced choice behavior (i.e., evidence of olfactory memory) and to determine whether signiﬁcant forgetting occurred after the various retention intervals and whether vasopressin, at the dose levels used in this study, modulated (facilitated or impaired) retention over the selected retention interval. In experiment 1, the RI was 8 days and the peptide dose levels were 0.0 (vehicle), 0.02, 0.2, and 2.0 g/kg, subcutaneous Each observer/demonstrator was tested ﬁve times, so that each observer was tested under each of the four dose levels at the 8-day RI and with vehicle at the 1-day RI. The social interaction with the demonstrator inﬂuenced choice behavior of the observer after both the 1-day and 8-day RIs. The peptide did not facilitate but tended to impair recall relative to vehicle injection, and this may have been due to the 8-day RI not being of sufﬁcient duration to have allowed signiﬁcant forgetting to occur. This was supported by the ﬁnding that the social learning effect in vehicle-treated rats after the 8-day RI did not signiﬁcantly differ from that obtained in the 1-day retention test. The possibility that a vasopressin facilitative effect on retrieval would be demonstrated only if sufﬁcient forgetting occurred suggested the need for a longer RI. Accordingly, in experiment 2, the RI was lengthened to 10 days and peptide doses now included 0.0, 0.02, 0.2, and 6.67 g/kg, subcutaneous. Again, there was no signiﬁcant effect of AVP(4–9) on memory retrieval. Comparisons between day 1 and day 10 for the magnitude of the social learning effect once again indicated that signiﬁcant forgetting had not occurred for the 10-day RI. In experiment 3, the RI was lengthened to 14 days and the peptide doses were the same as in experiment 2. Comparisons for retrieval performance after an RI of 1 day versus 14 days indicated that signiﬁcant forgetting had occurred after the 14-day RI. Under these conditions, the AVP(4–9) peptide showed a dose-dependent facilitation of memory retrieval, with a signiﬁcant effect occurring for the highest dose level. In interpreting their data, the authors proposed that AVP(4–9) interacted with the degree of memory accessible at the time of treatment, impairing memory retrieval when memory accessibility was high but improving it when memory accessibility was low. ii. Bunsey and Strupp (1990) Bunsey and Strupp (1990) tested the hypothesis that vasopressin interacts with memory strength (accessibility) in its inﬂuence on retrieval of information concerning food preferences established by means of olfactory-based social encounters. This study used the same vasopressin peptide, task paradigm, and strain and age of female as

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used in the study by Strupp et al. (1990) cited above. Vehicle or AVP(4–9) (3.0 g/kg, subcutaneous) was injected 1 h before the choice test given either 1, 6, 11, or 16 days after the observer–demonstrator social encounter. Each subject was tested only once with a choice between two novel diets, cumin- and thyme-ﬂavored ground chow. As in the former study, the percentage of the observer’s total intake that was composed of the socially transmitted ﬂavored diet served as the index of memory retrieval. The peptide metabolite markedly improved memory retrieval when administered after the longest retention interval (the 16-day RI), when signiﬁcant forgetting had occurred under vehicle control conditions. On the other hand, AVP(4–9) impaired memory retrieval in the 6-day retention test, when controls showed excellent memory. There was no detectable peptide effect at those retention intervals (days 1 and 11) when memory retrieval was intermediate in nature. The results of this study, together with those obtained by Strupp et al. (1990), were interpreted as support for the hypothesis that vasopressin interacts with memory strength in mediating its inﬂuence on memory retrieval. In their discussion, the authors noted the following points: (1) the results of this study are consistent with those of other investigators who have reported that vasopressin facilitates memory retrieval under conditions in which forgetting has occurred in the controls [e.g., Alescio-Lautier et al., 1987; Bohus et al., 1972, 1978a (see Chapter 2); Till and Beckwith, 1985 (see Chapter 8)], but may impair it when recall is still evident in the controls (Beckwith et al., 1987b; see Chapter 8); (2) a bidirectional interaction between retention interval and pretest treatment with a putative memory modulator, similar to the present ﬁndings, has been reported for the cholinergic system by Deutsch (1971); and (3) the retrieval effects induced by vasopressin treatment suggest an interaction between endogenous biochemical changes that correspond to accessibility of the memory and the biochemical changes representing the intrinsic memory system (e.g., vasopressin, the extrinsic modulator of this system).

III. Human Research Literature

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A. Influence of VP and/or OT on Human Cognitive Processing 1. Effect on Memory Processing of OT Treatment Given for the Therapeutic Induction of Second-Trimester Abortion a. Selected Studies i. Ferrier et al. (1980) Ferrier et al. (1980) examined performance in several tests of learning and memory in consenting hospitalized females receiving OT treatment for the induction of therapeutic second-trimester abortion.

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This study was inspired in part by the results of animal research, reported by De Wied and associates, that suggested reciprocal actions on memory processing by AVP and OT, with VP facilitating and OT impairing memory processing (Bohus et al., 1978b; Van Ree et al., 1978). Given the extensive use of OT in obstetrical practice, these authors considered it of clinical as well as fundamental interest to determine whether the amnestic effect of OT observed in animals was also applicable to humans. The test protocol included testing ‘‘immediate recall’’ and ‘‘later recall’’ in a paired associate learning task, and memory recognition of photographs of faces in a picture recognition and matching task. The entire protocol was completed over a 40-min period, and given to each patient before and after 4 and 8 h of intravenous infusion of the OT synthetic, Syntocinon (200 mU/ min). The protocol was pretested on normal volunteers during the course of a working day. The results indicated that task performance remained stable or slightly improved on retesting after each of two 4-h intervals. The paired associate task consisted of 2 lists, each of which contained 10 word pairs. Learning ability was assessed by a test of immediate recall for each list. Speciﬁcally, the subject was given list 1, allowed 5 min to memorize the 10 word pairs, and then handed a list containing a word from each pair and asked to complete the pairs. This procedure was then repeated for list 2. Later recall for list 1 was tested after completion of the two tests for immediate recall, when the subject was shown a sheet containing single words from list 1 and asked to complete the word pairs. Once the subject completed the picture recognition and matching task, the same procedure used to test for later recall of the word pairs on list 1 was used to test later recall for list 2. The second task tested for recognition and matching pictures of faces presented in sets of two, four, or six in random order and counterbalanced across patients. Speciﬁcally, each patient was shown pairs of pictures of faces (1 male and 1 female), and then asked to select the matching pictures from a group of 25, presented with 1 of each pair of faces. Performance on tests of immediate and later recall of word pairs on each list was scored for number of words correctly recalled and the errors (word intrusions from previous lists, and new words not present on these lists) committed during this recall for each list. The dependent measures for the picture recognition and matching task included the number of correct matches, the number of errors made for each set size (two, four, and six), and the number of times patients changed their initial selections. The results of this study indicated that, compared with pretreatment test performance, 4 and 8 h of OT treatment (1) did not inﬂuence learning (immediate recall) on the paired associate learning task (i.e., no signiﬁcant effect of OT treatment on either the percentage of correctly recalled words or the percentage of errors committed during recall of either list of word pairs); (2) impaired later recall on the paired associate task [i.e., OT treatment

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reduced the percent correctly recalled word pairs on each list (statistically signiﬁcant) as well as increased percentage of errors committed when recalling these words pairs (short of statistical signiﬁcance)]; (3) did not signiﬁcantly interfere with the ability of patients to correctly select and match pictures, but increased the number of times the patients changed their initial selections (i.e., led to greater indecisions during cognitive performance); and (4) produced a subjective feeling of impaired ability in task performance on these cognitive tasks (i.e., the patients frequently commented on the fact that subjectively they found the tasks more difﬁcult to perform during treatment with OT, which did not occur at the outset). In conclusion, in spite of the absence of an external control group, the performance differences observed in the patients before and during OT treatment, together with the lack of performance deﬁcits in the pretested, nontreated volunteers, indicated that OT impaired cognitive performance. This cognitive impairment was expressed as (1) an amnestic action at a postlearning stage of memory processing that was assessed on the word associate task and (2) an interference with decisive, self-conﬁdent performance of the tasks used in this study. ii. Kennett et al. (1982) Kennett et al. (1982) validated the ﬁndings described above in their study of learning and memory in a group of women patients who were also hospitalized and treated for induction of therapeutic second trimester abortion. This group of patients provided an external control group for the patients studied by Ferrier et al. (1980), because they were treated the same with the exception that they were not given OT. Because this group was tested before and 4 h, but not 8 h, after the onset of their intravenously infused medication, test performance comparisons between these controls and the OT-treated patients pertained to these two points of time. As in the former study, the scores calculated for the paired associate and picture recognition and matching tasks were, respectively: (1) the number of words pairs correctly matched and the number correctly recalled although not correctly matched when tested for immediate and later recall of the memorized word pairs on each list of the paired associate task; and (2) the number of faces correctly matched as well as the number correctly recalled although not necessarily correctly matched after viewing photographs, each containing the faces of one male and one female, in sets of two, four, and six pairs. Six women from the experimental and control groups were selectively matched on the basis of the test scores they obtained in this protocol under pretreatment (baseline) conditions. The results were calculated as the percent change in test scores obtained under the pretreatment (baseline) condition compared with those obtained after 4 h of treatment. Statistical comparisons between the OT treatment and control groups regarding percent change in performance tested 4 h after treatment indicated (1) no signiﬁcant difference between the OT-treated and control

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groups for immediate recall (learning) of the word pairs on each list of the paired associate learning task; (2) in contrast to the controls, the OT-treated subjects were impaired in their later recall when tested after 4 h of OT treatment. Moreover, signiﬁcant differences between the two groups occurred for both the number of words correctly matched and the number of words correctly recalled but not matched; and (3) signiﬁcant differences between the OT-treated subjects and the controls occurred in the task involving immediate recall of faces whether originally viewed in sets of two, four, or six pairs. These differences were observed whether the results were expressed as correctly matched faces, or as faces correctly recalled although not necessarily correctly matched. The results of the data analysis performed in this study reinforce the preand posttreatment comparisons obtained in the study by Ferrier et al. (1980). Taken together, the results of the two studies were interpreted as strengthening the conclusion that the ‘‘impairment in performance in memory tasks after several hours of infusion of high doses of oxytocin is due to the hormone itself and not to any other treatment-associated factor’’ (Kennett et al., 1982, p. 275). These researchers further noted that these results are consistent with the following interpretations: (1) OT treatment exerts amnestic effects in humans as it has been shown to do in laboratory animals studies (Bohus et al., 1978b; see Chapter 2); (2) it is possible that a tiny amount of the high dose of intravenously infused OT used in the treatment procedure in the Ferrier et al. (1980) study reached CNS receptor sites, as proposed by De Wied and colleagues to explain the behavioral effects of peripherally administered neurohypophysial hormones in animal studies (see Chapters 2–5); and (3) the fact that high levels of hormonal OT, like AVP, exert a pressor effect, although a less potent one, suggests that the opponent effects of these hormones on memory processing is highly unlikely to be the result only of a systemic action of OT (Ferrier et al., 1980; Kennett et al., 1982). One matter of concern to this author is the degree to which the interpretations of the results obtained with the experimental protocols used by Ferrier et al. (1980) and Kennett et al. (1982) are generalizable to those obtained from VP/OT and memory-processing studies with animal subjects. Speciﬁcally, in these human studies the time interval between learning and retention that deﬁned LTM was less than 1 h, whereas the comparable interval typically used for the animal studies reported in this book ranged from several hours to many days (e.g., Chapters 2–5). 2. Intranasal Administration of VP and OT in Young Healthy Male and/or Female Human Subjects a. Selected Studies i. Fehm-Wolfsdorf et al. (1984) Fehm-Wolfsdorf et al. (1984) examined the effects of VP and OT on immediate and delayed recall tested in a verbal memory task. Of primary interest to this discussion was the objective

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of the researchers to test the De Wied et al. proposal that VP and OT exert opponent effects on memory processing (e.g., Bohus et al., 1978b; De Wied, 1979; De Wied and Versteeg, 1979) in a study with human subjects. A second objective was to determine whether the retrieval strategy used by a subject in the recall of a list of aurally presented words might inﬂuence the ‘‘recency effect’’ (deﬁned below), typically observed when tested under free recall conditions. This objective was relevant to ﬁndings reported by Fehm-Wolfsdorf et al. (1983) that VP-treated subjects exhibited better recall for words appearing in the earlier part of the list (primacy effect), whereas the controls showed better recall for the later appearing items (recency effect). In accordance with the theory that human memory contains STM and LTM components differing in storage capacities and other features (Wickelgren, 1979, 1981), the primacy effect has been assumed to reﬂect LTM, and the recency effect, STM (Baddeley, 1976; Craik, 1968). However, newer views concerning the recency effect have resulted in a proposal by Baddeley and Hitch (1977) that this effect results from a retrieval strategy, applicable to any available store, LTM or STM. The subjects in this study, male medical student volunteers, were subdivided into three groups and received an intranasal application of placebo, LVP, or OT (peptide dose of 10 IU/treatment) on three occasions (end of test session 1, and 24 and 1 h before session 2). During session 1, the subjects were ﬁrst pretrained with ﬁve lists, during which time instructions were given to manipulate retrieval strategies (i.e., form associations with words in the early part of the list, and begin recall with the last few words). After this pretraining experience, each subject was tested for immediate recall of a list of 15 common monosyllabic words presented orally at a rate of 1 word/ 2 s. At the end of the presentation of the ﬁrst list, the subject was given 1 min to write down as many words (any order) as he remembered. A tone signaled the initiation of the next list and the procedure was repeated for all 10 lists presented in session 1. In session 2, 1 week later, the subjects were presented with the same 10 lists used in session 1 and then given 10 new lists. At the end of this test session, the subjects were unexpectedly asked to write down within 6 min as many words as they could remember from all the lists. In a posttest interview, the subject was asked about his retrieval strategy, any subjective treatment effects he may have experienced, and to guess the treatment group to which he had been assigned in this double-blind study. Several months later each subject was retested for recall of the previously learned lists under a free recall condition (i.e., asked to write down all the words that he could recall). The results and their interpretations pertaining to the ﬁrst objective are listed below. First, statistical testing (ANOVAs) compared performance differences in immediate recall between sessions 1 and 2. A main drug effect (p ¼ 0.08) did not attain statistical signiﬁcance because of the extreme within-group variability. Further inspection of this drug effect indicated improvement in

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immediate recall in session 2 for the subjects treated with LVP and saline, but a relative decline (fewer words recalled) for the OT-treated subjects. The drug effect on task performance was due to the marked difference between the two peptide groups, a difference that, according to the authors, would have been signiﬁcant at the 0.05 level of signiﬁcance in a two-group comparison test. Second, the marked degree of improvement shown by the saline group, which although less than that of the LVP group was not signiﬁcantly different, was attributed to a prominent placebo effect. The placebo effect was attributed to the information that accompanied the treatment procedure (i.e., subjects were told that the intranasal treatment should affect attention, learning, and memory). Third, neither peptide inﬂuenced performance on the delayed recall tests given at the end of session 2, and several months later. This failure, together with the weak drug effect observed for immediate recall, could have been related to the dose levels of the peptides used in this study. According to the authors, these VP and OT dose levels were lower than those typically reported for other human studies. Fourth, responses on the posttest rating scale provided some indirect support for the proposed arousal effect of VP (Le Moal et al., 1981; see Chapter 6), and the amnestic action for OT (Bohus et al., 1978b; see Chapter 2). Whereas in most cases the posttest interview ratings suggested no differential subjective reactions to the various treatments received, several responses did differ between the two peptide treatment groups. The OT-treated group reported less learning and memory ability, but greater concentration, and the VP-treated group indicated more feeling of arousal than expressed by the other two groups. The results and interpretation relevant to the second objective in this study were as follows: (1) separate serial position curves, based on data averaged over all subjects, were constructed for the session 1 list, and for the old and new lists in session 2; each curve showed the typical shape characterized by a mild primacy effect and a strong recency effect; and (2) there was no signiﬁcant peptide interaction with serial position factors in this study as had been previously reported for vasopressin by Fehm-Wolfsdorf et al. (1983). The manipulation of subject recall strategies during pretraining in session 1 was interpreted as contributing, at least in part, to this result. Moreover, this interpretation was consistent with the aforementioned recency effect proposed by Baddeley and Hitch (1977). In conclusion, it is herein suggested that the results of this study provided some support for the proposed amnestic action of OT on memory, at least as it pertains to immediate memory, if not delayed memory as tested in this experimental protocol. The absence of a signiﬁcant VP-induced facilitating effect on immediate memory could have been related to one or all three of the following factors: a pronounced placebo action, an

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inadequate dose level, or the result of the manipulation of a behavioral variable (subject retrieval strategy) that acts to modulate peptide effects on memory processing. An inadequate dose level may have also contributed to the failure of these peptides to inﬂuence delayed recall tested in this study. ii. Bruins et al. (1992) Bruins et al. (1992) explored the effects of a single dose of intranasally administered DG-AVP (2 mg) or OT (20 IU) on mood status, attention, and memory processing assessed by performance on various tests within a neuropsychological test battery in young healthy volunteers. In this placebo-controlled trial, the males received placebo or OT, and the females received placebo or DG-AVP. The 2-h behavioral test session occurred either in the morning (9–12 a.m.) or afternoon (2–5 p.m.), with each subject tested in the same time period on test days 1 and 2, which were separated by a 1-week interval. Placebo or peptide treatments were given 10 min before the onset of behavioral testing. All subjects received placebo before baseline testing on day 1. On day 2, the male and female control groups received placebo, and their experimental counterparts received OT and DG-AVP, respectively. Neither hormone inﬂuenced blood pressure, which was measured immediately before and 1 h after placebo or peptide treatment on each test day. The neuropsychological test battery included a questionnaire assessing mood status, and various tasks of cognitive processing [learning/memory of abstract words (Buschke restrictive reminding method), vigilance (Bruins visual vigilance test); attention (Stroop color–word test), visual memory (facial recognition test), and a paper and pencil test version of the Sternberg task]. Aside from an OT-induced reduction in the vigor measure on the mood status questionnaire, and a DG-AVP-induced inﬂuence on the y intercept component of the Sternberg task (interpreted as an enhanced rate of perception and motor response), the only other peptide effects on this test battery were those found for certain of the dependent measures on the learning/ memory test of Buschke (Buschke restrictive reminding test). The procedure and dependent measures evaluated for this test are described below, followed by a discussion of the peptide effects on these measures. In this test of verbal memory processing, 15 abstract words are aurally presented by tape recorder (1 word/2 s) and the subjects are instructed to remember as many words as possible. After recall, only the words not recalled are presented again. Ten trials are given in which the words are to be recalled. The dependent measures on this task are as follows: (1) initial storage (number of items recalled after the ﬁrst presentation), (2) rate of storage (learning; i.e., number of trials or presentations needed until each item is recalled at least once); (3) short-term storage (items recalled only once either during the ﬁrst or any other trial); and (4) long-term storage with consistent retrieval (repeated recall without failures from either the

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initial list of learning, or any subsequent list of learning), or inconsistent retrieval (retrieval with failures). In addition, approximately 75 min later, the subject was tested for delayed recall (instructed to verbally recall the words again), immediately followed by a test of delayed recognition (replied yes or no to each of 30 verbally presented words containing an intermixture of 15 previously learned words and 15 distractor words). After a 1-week ‘‘washout period,’’ the subjects were mailed a list of words, some of which had been selected from previously presented lists whereas others served as distractors. The accompanying instructions required the subject to select the words recognized from previously learned lists. With the exception of three females (one from the DG-AVP treatment group) and four males (three from the OT treatment group) all of these mail-outs were returned to the institute. The test data for the males and females were analyzed separately, using ANOVAs with repeated measures and testing for drug (placebo or DG-AVP/ placebo or OT) and day (test day) factors. A post hoc analysis, using ANOVAs with repeated measures on the peptide-treated subjects, was done to directly compare performance scores between the DG-AVP and OT treatment groups. The results of these analyses indicated that most of the dependent measures assessed in the verbal learning/memory test were not signiﬁcantly inﬂuenced by treatment factors whether comparisons were made between peptide and placebo, or between OT and DG-AVP. The statistically signiﬁcant treatment effects were as follows. First, OT versus DG-AVP comparisons indicated statistically signiﬁcant opponent effects on two of the dependent measures scored in this task: (1) initial storage, which was reduced after OT treatment and enhanced after DGAVP treatment; and (2) the rate of storage, in which OT-treated subjects required more, and DG-AVP-treated subjects required fewer, trials to recall all words at least once. Second, the results of peptide/placebo comparisons indicated that 1 week after treatment with these peptides was discontinued the DG-AVP-treated females recognized more words than did their placebotreated counterparts. The following points were made in the course of discussing the interpretations of these results. 1. The ﬁnding that DG-AVP-treated subjects compared with placebo controls recognized more words in the Buschke test when tested in the recognition test 1 week, but not 75 min, after learning is puzzling. These researchers suggested that this ﬁnding, that a certain time period had to elapse before the effect of the peptide is manifested, may indicate an effect on retrieval of information. However, this interpretation is puzzling to this author, given that the inﬂuence of a drug on memory retrieval is typically tested by administering the drug shortly before the retention test, which was

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not the case in the present study, because 1 week had elapsed since treatment had been discontinued. 2. The absence of an OT effect on either measure of delayed recall or recognition was inconsistent with the observed OT-induced deterioration of memory in the paired associate task used in the studies of Ferrier and colleagues (Ferrier et al., 1980; Kennett et al., 1982; see above). The authors of this study suggested that the use of different test procedures, and the fact that the former studies used cued recall techniques and the present study used free recall techniques, may have contributed to the discrepancy. It may also be noted that the former studies used chronic treatment with a high dose of OT in contrast to the single injection of a much lower dose of OT used in this study. 3. The marginal treatment effects of the two peptides on learning processes did suggest opponent effects of these peptides even during an early stage of memory processing consistent with a VP-induced facilitation and an OT-induced attenuation of this processing. However, gender interactions with the VP analog may have acted as a confounding factor in these results, given that Beckwith and colleagues have reported the presence of such interactions in their verbal learning studies with a similarly administered VP analog (Beckwith et al., 1984; Till and Beckwith, 1985; see Chapter 8). 4. Whereas neither peptide inﬂuenced memory scanning in the Sternberg test, DG-AVP did improve the rate of perception and/or motor response with digits as the memory set. These DG-AVP effects on this task supported earlier ﬁndings reported by Beckwith et al. (1983; see Chapter 8) with a different AVP analog, which were interpreted as indicating that this hormone enhances attentional but not memory processing in this test (see Chapter 8). Several other studies discussed in Chapter 8 have obtained support for a VP-induced enhancement of attentional processing. However, neither peptide inﬂuenced selective attention or sustained attention as measured by the Stroop test and vigilance test, respectively. 5. The blood pressure (BP) monitoring undertaken in this study indicates that the peptide effects on learning and memory that did occur in this study could not be interpreted as secondary to peptide-induced modulation of BP, because neither DG-AVP nor OT inﬂuenced this physiological measure. On the other hand, an OT-induced decrease in vigor could have contributed to the presence as well as to the absence of the effects of this peptide on the various aspects of cognitive processing tested in this study. Chapter 11 summarizes the research ﬁndings presented in this chapter, comments on their contribution to the ﬁeld of vasopressin and oxytocin memory research, and relates them to the theoretical views of De Wied and colleagues and, where relevant, to the alternative viewpoints described in earlier chapters of this book.

Barbara B. McEwen

Expansion of Vasopressin/ Oxytocin Memory Research II: Brain Structures and Transmitter Systems Involved in the Influence of Vasopressin and Oxytocin on Memory Processing

I. Chapter Overview

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This chapter continues the discussion of the eclectic approach to vasopressin/oxytocin (‘‘VP/OT’’) memory-processing research. In contrast with research presented in Chapter 9, the studies described herein directly manipulated central levels of these neuropeptides. Taken together, these investigators used a variety of aversive and appetitive paradigms in studies designed primarily to identify the brain structures in which VP and/or OT acts (Alescio-Lautier et al., 1989; Engelmann et al., 1992a,b; Everts and Koolhaas, 1999; Herman et al., 1991; Ibragimov, 1990; Metzger et al., 1989, 1993), and the neurotransmitters with which it interacts (Baratti et al., 1989; Boccia and Baratti, 2000; Faiman et al., 1987, 1988, 1991; Hamburger-Bar et al., 1984) in its contribution to memory processing. Several of these investigators (Engelmann et al., 1992a; Ermisch et al.,

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1986; Ferguson et al., 2000) also examined the putative role of endogenous VP and/or OT in memory processing.

II. Central Neural Structures Involved in VP and/or OT Effects on Memory Processing

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A. The Hippocampus De Wied and colleagues provided several lines of evidence indicating that a number of limbic system structures are involved in mediating the inﬂuence of vasopressin and oxytocin on memory processing in aversive motivational tasks (see Chapter 4). Ibragimov (1990) obtained further information along this line in a study of the effects of chronic intrahippocampal injections of these peptides on memory processing in rats tested in an active avoidance paradigm. Alescio-Lautier, Metzger and colleagues (Alescio-Lautier et al., 1989; Metzger et al., 1989, 1993) have extended this research by studying the role of dorsal or ventral hippocampal VP in memory retrieval, using an appetitive go/no-go brightness discrimination task and an inbred strain of mouse. These studies are described below. 1. Effects of Lysine Vasopressin and OT and Metabolites on Memory Processing in an Aversive Paradigm a. Selected Study: Ibragimov (1990) Ibragimov (1990) compared the effects of chronic intrahippocampal application of lysine vasopressin (LVP) and OT, and of the vasopressin and oxytocin fragments desglycinamidearginine vasopressin (DG-AVP) and prolyl-leucyl-glycinamide (PLG), on acquisition and extinction of a shuttlebox footshock active avoidance (AA) response. Sixty minutes before daily test trials, independent groups of adult male CFY rats were treated with physiological saline or a 0.5-, 2.0-, or 4.0-ng dose of LVP or OT, or a 4.0-ng dose of DG-AVP or PLG, throughout the 6 days of acquisition and extinction testing in the AA task. With one exception (LVP was intraventricularly administered during extinction testing), all treatments were microinjected into the ventral hippocampus via a preimplanted cannula. The results indicated that relative to saline controls: (1) intrahippocampally applied LVP tended to facilitate acquisition, an effect that was statistically signiﬁcant for the lowest dose level, and intracerebroventricularly administered LVP severely retarded response extinction, an effect that was signiﬁcant at all dose levels; (2) intrahippocampally injected OT signiﬁcantly retarded the formation of the conditioned avoidance response at all dose levels, and showed a nonsigniﬁcant acceleration of extinction at the highest (4.0 ng) dose level; and (3) intrahippocampally injected DG-AVP tended to facilitate acquisition and retard extinction of the AA response, whereas

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similarly applied PLG produced the opposite effects. These opposing inﬂuences did not attain statistical signiﬁcance for response acquisition, but did so for response extinction. Taken together, these results suggest that chronic pretesting application of LVP and of the desglycinamide VP analog (DG-AVP) exert similar effects on AA behavior, producing a relatively weak facilitated learning effect but a stronger enhanced retention effect (i.e., greater resistance to extinction). These ﬁndings also suggest that both OT(1–9) and its C-terminal peptide PLG act in opposition to the VP family of peptides in their inﬂuence on AA behavior, retarding the rate of learning and attenuating retention in this paradigm. The ﬁnding that PLG produced an OT-like rather than the VP-like action on memory processing, observed by Gaffori and De Wied (1988), is another example of the puzzling inconsistent effects obtained regarding the role of OT in memory processing. These discrepant ﬁndings for OT and memory processing receive further commentary in Chapter 15. 2. Effect of VP on Memory Processing in an Appetitive Paradigm a. Selected Studies i. Alescio-Lautier et al. (1989) Alescio-Lautier et al. (1989) used the go/no-go appetitive visual discrimination task with two major objectives: ﬁrst, to determine whether hippocampal VP circuitry is involved in mediating retention in this behavioral paradigm; and second, to learn whether this same circuitry contributes to retention effects induced by central administration of exogenous AVP. Mice of the inbred BALB/c strain served as subjects and three experimental tests were conducted, each of which used a retrieval design. The apparatus and behavioral training/testing procedure for this successive go (Sþ), no/go (S) black/white (B/W) discrimination task are described in Chapter 9. Retention performance was evaluated by a discrimination ratio (i.e., the sum of the running times for Sþ divided by the sum of the running times for both Sþ and S trials) calculated for each subject for the retention test session given 24 days after original learning. The smaller the discrimination ratio, the better the retention. The role of endogenous dorsal hippocampal VP in mediating retention of the learned discrimination was tested in four groups of mice. For the two experimental groups, endogenous VP was neutralized by a bilateral dorsal hippocampal injection of anti-VP serum, diluted with artiﬁcial cerebrospinal ﬂuid (aCSF) at a ratio of either 1:50 or 1:10. The two control groups received a similarly placed injection of normal rabbit serum diluted with aCSF at the same ratio levels. Two experimental tests were conducted for the second objective, using this same task. First, two independent groups of mice were tested for retention after intracerebroventricular administration of either AVP (2 ng/kg) or

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physiological saline. Second, four independent groups of mice received an intracerebroventricular injection of AVP (2 ng/kg) in addition to either intrahippocampally injected anti-VP serum or normal rabbit serum diluted with aCSF at either a 1:50 or 1:10 ratio. Treatments were administered either 10 min (AVP or saline, intracerebroventricular) or 20 min (anti-VP or normal rabbit serum) before the retention test. The results of this study indicated (1) no impairment in memory retrieval after immunoneutralization of dorsal hippocampal VP whether diluted with a CSF at the 1:50 or 1:10 level; (2) enhanced memory retrieval induced by intracerebroventricularly administered AVP (i.e., mean discrimination ratio in the retention session was signiﬁcantly smaller for the AVP recipients than for the saline-treated controls); and (3) an impairment of the AVPinduced retrieval effect after treatment with the less diluted (1:10) but not the more diluted (1:50) anti-VP serum solution. The authors noted that the failure of dorsal hippocampal immunoneutralization of vasopressin to impair retention in this task was in sharp contrast to data obtained by Kovacs et al. (1982a; see Chapter 4) and by Veldhuis et al. (1987; see Chapter 4). These latter researchers observed that intrahippocampally injected VP antiserum, at even a weak dilution (1:50), impaired memory consolidation (Kovacs et al., 1982a) and retrieval (Veldhuis et al., 1987) in a step-through passive avoidance task in rats. However, the fact that the earlier studies used footshock stress, absent in this appetitive visual discrimination task, may have contributed to the discrepant ﬁndings, because release of hippocampal VP may not have occurred in this study in a degree sufﬁcient to mediate memory retrieval of the learned discrimination. Further, histochemical observation in the present study indicated that the anti-VP serum remained localized in the dorsal hippocampus, whereas Kovacs et al. (1982a) found that the serum had spread from the dorsal hippocampus into the lateral septum and ventral hippocampus. Thus it is possible that the retention impairment after vasopressin immunoneutralization observed by Kovacs et al. (1982a) involved the ventral, as opposed to, or in addition to, the dorsal hippocampus. The ﬁnding that memory retrieval was improved by AVP, injected intracerebroventricularly, was consistent with the observation that subcutaneously injected AVP also improved memory retrieval in this task (Alescio-Lautier and Soumireu-Mourat, 1990; see Chapter 9). The fact that intracerebroventricular administration of a 2-ng dose of AVP had the same behavioral effect as a subcutaneous injection at the 1-g dose level indicates that the peptide was 500 times more effective when administered centrally (intracerebroventricularly) than peripherally (subcutaneously). The increased effectiveness of the peptide when centrally administered has also been reported for rats tested in an aversive motivational task (e.g., De Wied, 1976; and see Chapters 2–5), and has been cited by De Wied and colleagues as support for the ‘‘VP/OT Central Memory Theory.’’

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The observation that intrahippocampal injection of less diluted anti-VP serum (1:10 ratio) blocked the enhanced retrieval produced by intracerebroventricularly injected AVP indicated involvement of dorsal hippocampal VP receptor sites in the retention effect induced by exogenous AVP. This ﬁnding is consistent with the demonstration that dorsal hippocampal lesions impair the retention improvement induced by intracerebroventricularly administered AVP in mice (Alescio-Lautier and Soumireu-Mourat, 1986) and rats (Van Wimersma Greidanus and De Wied, 1976b; see Chapter 4). In conclusion, whereas endogenous hippocampal AVP was not necessary for memory retrieval in this task, AVP binding to dorsal hippocampal sites seems to be required for the behavioral effect of intracerebroventricular AVP treatment. That is, the dorsal hippocampus is involved in mediating the effects of exogenous AVP on retention in this appetitive task. ii. Metzger et al. (1989) Metzger et al. (1989) assessed the effect of AVP microinjected into the dorsal or ventral hippocampus on memory retrieval for the successive, go/no-go B/W discrimination task in BALB/c mice. This study used the same task, training and testing procedure, and measure of retention (discrimination ratio) as employed by Alescio-Lautier et al. (1989). Saline or AVP (25 pg/mouse) was bilaterally microinjected into either the dorsal or ventral hippocampus 10 min before the retention test. A putative AVP effect on general activity level was evaluated after completion of retention testing by injecting saline or AVP (25 pg/mouse) into the dorsal or ventral hippocampus 10 min before placing the subject in a circular runway, where locomotor activity was recorded every 10 min for 3 h. Partial forgetting in the retention test occurred in both the VP- and saline-treated subjects, as has been previously observed in this task (e.g., Alescio-Lautier and Soumireu-Mourat, 1986). AVP injections into either the dorsal or ventral hippocampus improved memory retrieval relative to the saline controls. However, peptide placement in the ventral hippocampus led to signiﬁcantly greater improvement compared with placement in the dorsal hippocampus. In addition, locomotor activity was signiﬁcantly depressed, relative to saline controls, by AVP microinjected into the ventral, but not the dorsal, hippocampus. These authors noted that the greater effectiveness of AVP when delivered to the ventral rather than the dorsal hippocampus of rats has also been reported by Kovacs et al. (1986) in a passive avoidance paradigm. It was suggested that this may be because AVP-binding sites are more abundant in the ventral hippocampus than in the dorsal hippocampus (Van Leeuwen and Wolters, 1983). This difference in number of VP-binding sites may account for the greater sensitivity (depression) of locomotor behavior to the effects of VP microinjected into the ventral hippocampus as compared with the dorsal hippocampus. The VP-induced alteration in locomotor activity can

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be dissociated from the effect of the peptide on memory retrieval because the improved memory retrieval was exhibited simultaneously as a decrease in S (stimulus not followed by reinforcement) and an increase in Sþ (stimulus signaling availability of reinforcement) running speed. iii. Metzger et al. (1993) Metzger et al. (1993) designed three experiments to study the putative role of ventral hippocampal AVP in the retrieval and relearning of successive (go/no-go) B/W discrimination in BALB/c mice. In experiment 1, either AVP or anti-VP serum was microinjected into the ventral hippocampus (VH) to increase or neutralize, respectively, endogenous VP levels within this brain structure. The effects of these treatments on retrieval and relearning of the visual discrimination were tested shortly thereafter. The remaining two experiments were concerned with the degree to which involvement of the VH vasopressin system in these memoryprocessing activities depends on the integrity of the medial amygdaloid nucleus (AME). In experiment 2 immunohistochemical (IHC) techniques were used to localize the cell bodies in the AME that give rise to the VP-ergic ﬁbers that terminate in the VH. The rationale for this experiment is the evidence that AVP innervation of the VH comes from the AME in rats (Sofroniew, 1985a). In experiment 3, this pathway was lesioned before a microinjection of AVP into the VH, and the effect of the lesion on the ability of the microinjected AVP to inﬂuence retrieval and relearning of the B/W discrimination was evaluated. In experiment 1, the procedure used during initial training in this discrimination task was identical to that described by Alescio Lautier et al. (1989). Performance was expressed as the average running time for 6 go (Sþ) and 6 no-go (S) trials for each group in a given session (12 trials). Learning consisted of a decrease in running times for Sþ trials, and an increase for S trials. Retention testing occurred 24 days after the 3 daily sessions (12 trials/session) of acquisition training and consisted of an additional session of 12 trials under the original learning conditions. Surgical implantation of the cannula assembly into the ventral hippocampus (VH) for bilateral injection of the treatment solution was carried out after the completion of original learning or preliminary locomotor activity practice. Ten minutes before the retention test, each of four independent groups of mice received a bilateral intrahippocampal injection of VP (25 pg/rat), saline (Sal), anti-VP serum (1:10 dilution), or normal rabbit serum (NRS). Performance in trial 1 of the retention test was identiﬁed as a retrieval effect, and that on subsequent test trials as indicative of the rate of relearning. The learning data (running times for the go and no-go trials) were analyzed by a repeated measures multivariate analysis of variance (MANOVA) that computed the main effect of time (daily session) and its interaction effect with group and reinforcement. Then 4 (groups) 2 (reinforcement or not) ANOVAs for each daily session were computed. These analyses

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assessed the similarity of initial learning across the treatment groups. The retention test data were analyzed by a repeated measures MANOVA to determine the main effect of time (chronological series of trials) and its interaction effect with group and reinforcement. The 4 (groups) 2 (reinforcement or not) ANOVAS for each trial were computed. To test treatment effects on activity level, separate groups of animals were food deprived, placed in the same apparatus (each runway was arbitrarily divided into four squares), and assessed for locomotor activity (running times, number of squares crossed) as well as for grooming, rearing, and defecations. Activity level testing was carried out for three daily 5-min sessions, and 24 days later for an additional 5-min session. Before the 24-day test session, the groups received the same treatments used earlier, thus testing activity level under conditions comparable to those used for visual discrimination training and retention testing. Locomotor activity data (square crossings and rearings) were analyzed by a repeated measures MANOVA, which evaluated across-group changes over the 5-min period. ANOVAs were then computed for defecations, groomings, and running times. Postmortem examinations after the completion of retention testing veriﬁed cannula placement in the VH, and showed that the area of diffusion of the injected antiserum in the ﬁve test animals remained within the conﬁnes of the VH. Results of the various analyses of the learning and retention test data can be summarized as follows: (1) the absence of signiﬁcant differences in original learning among the subsequently deﬁned treatment groups indicated that learning performance was not an inﬂuential factor in the group differences observed in the retention test; (2) both control groups (Sal and NRS treatments) exhibited a substantial amount of spontaneous forgetting (poor retrieval) and moderate relearning; their relearning performance was comparable to that obtained in session 2 of original learning; (3) AVP microinjected into the VH reduced the spontaneous forgetting that normally occurs in this task, that is, the levels of both retrieval and relearning for this treatment group were comparable to those for session 3 of original learning; (4) neutralization of VH vasopressin impaired retrieval and prevented relearning. Thus, mice receiving anti-VP serum microinjections into the VH performed no better in retention performance than they had at the onset (session 1) of original learning, suggesting that they had forgotten the previously learned discrimination; and (5) in contrast, the NRS-treated mice, although exhibiting poor performance in the retrieval trial, did relearn the discrimination over subsequent go/no-go trials, demonstrating some savings. Results of the analysis of the locomotor activity scores in experiment 1 indicated that neither AVP nor anti-VP serum treatment affected locomotor activity (i.e., squares crossed per unit time, rearings, or autonomic

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activity), with the exception that both these treatment groups exhibited increased running times relative to their corresponding controls. The authors attributed this locomotor decrement to an interaction between treatment and the food-deprived/nonrewarded status of the animal, rather than to treatment-induced hypoactivity. This interpretation was consistent with the following: (1) although the subjects were food deprived during performance of both the discrimination task and the locomotor activity test, they were rewarded in accordance with the reinforcement contingency during the former task, but were never rewarded during the latter test; and (2) there were no signiﬁcant differences between the treatment group and the corresponding controls on the number of squares traversed in the locomotor test. In experiment 2 IHC staining (incubation with VP antiserum) was used postmortem to localize the VP-ergic neuronal cell bodies and ﬁbers in the amygdala–hippocampal pathway that mediated the behavioral performance observed in this study. The brains of naive BALB/c mice from three groups were studied postmortem: (1) group 1 [six normal mice whose brains were studied by IHC staining to locate VP-immunoreactive (VPir) ﬁbers in the VH]; (2) group 2 [2 days before examination of VP neurons in AME, eight mice received an intracerebroventricular injection of colchicine (12, 24, or 48 g) and the remaining four mice received no colchicine pretreatment]; and (3) group 3 (ﬁve mice received a unilateral AME lesion 22 days before postmortem staining for VP-ergic ﬁber endings in the VH). The results of experiment 2 were as follows: (1) in group 1, VPir ﬁbers were detected in the CA4–dentate gyrus region, but were less dense than in the CA1–ventral hippocampal ﬁeld. Thus, VP ﬁbers from the AME appeared to reach the VH via the amygdalohippocampal area and entered CA1 and CA2 pyramidal cell layers; (2) in group 2, IHC neuronal staining enhancement was best after the 24-g dose of colchicine, whereas the VP neurons in the brains of non-colchicine-pretreated mice could not be stained; and (3) in group 3, the AME lesions were similar in size to those given to mice in experiment 3. IHC staining for VP indicated an almost complete disappearance of VPir ﬁbers in the CA1 and CA2 ﬁelds of the VH ipsilateral to the lesion; however, VPir ﬁbers were intact contralaterally, and also bilaterally, in the CA4–dentate gyrus region. Taken together, these results indicated an ipsilateral VP-ergic projection from the AME that originated in the dorsal portion of the structure, passed through the amygdalo–hippocampal area, and ended in CA1 and CA2 ﬁelds and in the ventral subiculum of the VH. However, this VP-ergic input into the VH may not be exclusively from the AME, because the lesion may have destroyed some VP-ergic ﬁbers of passage originating in other brain structures. In summary, the results of experiment 2 indicate that the AME is the source of VP innervation to the CA1–CA2 VH ﬁelds and to the ventral subiculum regions of the VH, but not to the CA4–dentate gyrus region of this structure.

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Experiment 3 used the go/no-go visual discrimination task to test the effects of AVP injected into the VH on retrieval and relearning processes of mice previously lesioned in the medial amygdala. The apparatus and procedure for both the visual discrimination task and the locomotor activity test were the same as for experiment 1. AME-lesioned and sham operates were tested in the visual discrimination task. Three treatment subgroups were formed for the AME-lesioned mice, depending on intra-VH injection: noninjected, saline injected, or AVP injected (25 pg bilaterally). First, comparison between the performance of the AME-lesioned group and sham operates indicated signiﬁcant impairment of retrieval, but not rate of relearning, in the lesioned subjects. Second, comparison between VP- and saline-injected AME-lesioned mice indicated that VP injection into the VH enhanced both retrieval and relearning. Taken together, these two results suggest that VP-ergic projections from the AME to the VH affect retrieval by means of inﬂuencing postsynaptic receptors that remain intact after the AME lesion. The fact that the AME lesion did not affect relearning suggests that VP inputs from sites other than the AME, and ending in the CA4– dentate gyrus region (not affected by the AME lesion), may mediate the inﬂuence of VP on relearning. The results of the locomotor activity test indicated that the sham operates exhibited greater running times than did the lesioned group, whether treated or nontreated; thus AVP treatment after an AME lesion did not increase running times as it did for the nonlesioned subjects in experiment 1. In summary, the results of this study indicated that (1) increasing VP levels in the VH, by microinjection of the peptide, enhanced both retrieval and relearning of the successive B/W discrimination (experiment 1); (2) decreasing the level of endogenous VP in this structure, by immunoneutralization, markedly impaired both retrieval and relearning in this task (experiment 1); and (3) VP-ergic cell bodies in the medial amygdala project to the CA1–CA2 ﬁelds in the ventral hippocampus and contribute to retrieval by means of postsynaptic inﬂuence (experiments 2 and 3).

B. The Septal Area 1. Effects of AVP and AVP Receptor Antagonists on Memory Processing in an Active Avoidance Paradigm a. Selected Study: Engelmann et al. (1992a) Engelmann et al. (1992a) investigated the effects of enhancing or reducing intraseptal AVP activity on the acquisition of a pole-jump avoidance response in male Wistar rats. The subjects were trained in a pole-jump avoidance task for 3 successive days (one 30-min session per day, 10 trials/session). In this task, the animals were scored for the number of trials per session in which they successfully performed an avoidance response (completed a pole jump before the end of

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the 8-s auditory conditioned stimulus). Group differences in average number of successful avoidance responses per session were statistically analyzed by a one-way ANOVA followed by the Newman–Keuls test for individual group comparisons. Treatment solutions were infused into the mediolateral septum (MLS) of rats via a microdialysis probe that had been implanted after training session 1 [see Engelmann et al., 1994 (Chapter 13), for a discussion of the microdialysis technique]. A nontreatment group served as a control for the effect of the microdialysis probe on task performance. The rats with probe implants were infused via the MLS with one of the following treatment solutions during training sessions 2 and 3: (1) artiﬁcial cerebrospinal ﬂuid on its own (aCSF treatment group), (2) aCSF containing synthetic AVP (approximately 0.2 ng; AVP treatment group), (3) aCSF containing the V1 receptor antagonist d(CH2)5[Tyr(Me)]AVP (approximately 5.0 ng; V1ant treatment group), or (4) aCSF containing the V1/V2 receptor antagonist [1-(-mercapto-,-pentamethylenepropionic acid)-2-(O-ethyl)-d-tyrosine, 4-valine]arginine vasopressin [d(CH2)5[d-Tyr(Et)]VAVP, approximately 5.0 ng; subsequently referred to as the V1/V2ant treatment group]. The results were as follows: (1) histological study of implant localization carried out at the end of each experiment indicated that the probes had been correctly placed in the MLS; (2) implantation of the microdialysis probe per se did not inﬂuence task performance [i.e., there were no signiﬁcant differences between the aCSF-treated rats and the untreated rats in number of conditioned responses (CRs) during any of the training sessions]; (3) the infusion of AVP into the MLS did not inﬂuence the rate of acquisition in this pole-jump test (i.e., the number of CRs per session performed by the AVP-treated rats did not signiﬁcantly differ from those shown by the aCSFtreated rats); (4) intraseptal infusion of both the V1 and V1/V2 antagonists signiﬁcantly impaired acquisition of the pole-jump response (i.e., both the V1ant and the V1/V2ant treatment groups exhibited signiﬁcantly fewer successful CRs per session than did the untreated and aCSF-treated groups during sessions 2 and 3); and (5) the V1/V2 antagonist was equal in potency to the V1 antagonist in its impairment of avoidance learning (there were no signiﬁcant differences between the two antagonist-treated groups in the number of CRs per session during sessions 2 and 3). In discussing these ﬁndings the authors made the following comments: ﬁrst, increasing intraseptal AVP, by microdialysis of synthetic AVP, beyond that produced by endogenous release in this stressful situation did not signiﬁcantly inﬂuence pole-jump avoidance learning. Bohus et al. (1978b) also found that centrally (intracerebroventricularly) injected AVP failed to inﬂuence pole-jump avoidance learning but observed that it did enhance retention in this task. Their ﬁndings were interpreted as consistent with the proposal of De Wied and colleagues that AVP does not play an important role in the acquisition phase, but it does in the consolidation phase of

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memory processing (see Chapter 2). However, the ﬁndings in the present study suggest that V1-mediated VP-ergic neurotransmission in the MLS does contribute to learning, at least in the pole-jump avoidance paradigm, because blocking this transmission impaired this learning. As an alternative explanation for the failure of centrally administered AVP to enhance avoidance learning, Engelmann et al. (1992a) suggested that the presence of the footshock during acquisition induced the release of central AVP that was sufﬁcient for learning. An increase in this level by treatments such as an intraseptal infusion of AVP may raise it above the optimal value and therefore fail to improve acquisition. This would not pertain during extinction when centrally applied AVP might be expected to improve retention as observed by Bohus et al. (1978b). Second, the present ﬁndings could not rule out the possibility that, in addition to a V1 receptor, a V2 receptor is also involved in mediating the septal–AVP inﬂuence on active avoidance learning. This latter possibility is consistent with evidence obtained from in vitro studies that, in addition to the V1subtype of septal AVP receptor (Raggenbass et al., 1987; Shewey and Dorsa, 1988), a V2 receptor subtype is present, and may produce a positive feedback action of AVP on its own release (Landgraf et al., 1991a). The authors concluded that their ﬁndings support the following hypothesis: ‘‘following its release in the septum, endogenous AVP may be involved in the facilitation of the acquisition and storage of information in stressful situations represented by the acquisition period of pole-jumping behavior in rats’’ (Engelmann et al., 1992a, p. 56). 2. Effects of AVP and/or AVP Receptor Antagonists on Memory Processing in a Spatial Learning Task a. Selected Studies i. Engelmann et al. (1992b) Engelmann et al. (1992b) studied the effects of increasing and decreasing septohippocampal AVP neurotransmission on spatial learning in Long-Evans hooded male rats tested with the Morris water maze (MWM). Treatment solution was delivered into the mediolateral septum (MLS) via previously implanted microdialysis probes. Behavioral testing began 2 days after probe implantation and consisted of 3 days of acquisition training (1 session of 12 trials/day) in the MWM. In addition to an untreated group, three treatment groups received either artiﬁcial cerebrospinal ﬂuid alone (aCSF group), aCSF containing AVP (AVP group), or aCSF containing the V1 receptor antagonist d(CH2)5[Tyr(Me)]AVP (referred to as the AAVP group). The treatment perfusion began 7 min before the ﬁrst trial and continued at a rate of 3 l/ min throughout the session, resulting in the delivery of about 0.2 ng of AVP or 5.0 ng of AAVP into the MLS over the 30-min perfusion interval. In a given learning trial, the rat was placed in the pool at one of four ﬁxed starting positions and scored for escape latency (time required to reach

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the underwater target platform hidden from view by the opacity of the water). The average escape latency scores of each 12-trial session for each subject were used for statistical evaluation by a two-way ANOVA (treatment days) with repeated measures on the last factor followed by post hoc paired t tests. At the end of behavioral testing, microdialysis probe location was histologically veriﬁed. The ANOVA indicated signiﬁcant main effects of treatment and days but no signiﬁcant interaction. Follow-up t tests indicated more speciﬁc ﬁndings. These statistical comparisons indicated that (1) all groups learned the task as indicated by the signiﬁcant decrease in escape latency between the ﬁrst and second, and the second and third, sessions; (2) microdialysis probe infusion per se did not interfere with acquisition of the MWM because the aCSF-infused group learned the maze at the same rate as did the untreated subjects; (3) decreasing septal–VP transmission had no effect on maze learning (i.e., no signiﬁcant difference between AAVP- and aCSF-treated rats on escape latency within and across training sessions); (4) increasing the level of intraseptal AVP impaired maze performance as indicated by (a) a signiﬁcantly lengthened escape latency in AVP-infused rats, relative to the other groups tested, during each of the three training sessions; and (b) a signiﬁcantly slower rate of maze learning by the AVP treatment group relative to the other groups on all 3 days. The present results were discussed in relation to other relevant ﬁndings in the research literature. First, failure of the microdialysis technique itself to interfere with normal performance in this spatial learning task has also been observed with an active avoidance task (Engelmann et al., 1992a; this chapter). Second, the inability of the VP receptor antagonist to inﬂuence maze acquisition suggests that V1 receptor-mediated VP-ergic transmission in the septal area is not critically involved in spatial memory processing, at least as measured in this task. In contrast, this VP-ergic neurotransmission appears to be important for mediating learning/memory in an active avoidance paradigm (Engelmann et al., 1992a; this chapter) and in a test of social recognition memory (Dantzer et al., 1987; see Chapter 13). Third, the disturbance to spatial learning/memory in the MWM by intraseptally infused AVP has not been observed for other types of memory processing. Intraseptal administration of exogenous AVP did not impair retention of an active avoidance response (Engelmann et al., 1992a), and improved it when tested with a passive avoidance task (Kovacs et al., 1979a; see Chapter 4) and the social recognition memory test (e.g., Dantzer et al., 1988; see Chapter 13). The authors concluded that endogenous AVP (at least that inﬂuencing the V1 receptor in the MLS) is not essential for spatial learning in the MWM, whereas the excessive presence of synthetic AVP interferes with it. These ﬁndings, together with others noted in their discussion, were interpreted as consistent with other evidence reviewed by O’Keefe and Nadel (1978) in support of the thesis that different brain structures and

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hence neurotransmitter/neuromodulatory systems contribute to spatial and nonspatial learning. ii. Everts and Koolhaas (1999) Everts and Koolhaas (1999) infused a V1/V2 receptor antagonist into the lateral septum (LS) of male Wistar rats to examine the role of septal VP-ergic neurotransmission in spatial memory processing tested in the MWM. The V1/V2 receptor antagonist used in this study, d(CH2)5[d-Tyr(Et)]VAVP, was shown to be as potent as the most commonly used V1 antagonist, d(CH2)5[Tyr(Me)]AVP, in a pole-jump avoidance task by Engelmann et al. (1992a). The former blocks both V1 and V2 types of vasopressin receptor and was selected for study because of the demonstration of the V2 receptor in the hippocampus and other brain sites (Hirasawa et al., 1994; Kato et al., 1995), and the suggestion that it may also be present in the septum (Engelmann et al., 1992a; Landgraf et al., 1991a; Ramirez et al., 1990). Saline or the V2/V1 antagonist (2 ng/l, enough to ensure a total blockade of both receptor types) was bilaterally infused into the LS via a preimplanted cannula/osmotic minipump assembly throughout behavioral testing. The MWM (a circular polyester pool containing opaque water and divided into four ‘‘imaginary’’ quadrants) was placed in a large observation room provided with a number of extramaze cues. Twelve training trials (3 trials/day; 1-h intertrial interval) were given over 4 days. The animals were allowed 2 min to ﬁnd the hidden escape platform located in quadrant A. If the platform was not found within this time limit, the rat was placed on the platform for 30 s. During trial 10 the platform was switched to a new location for the rest of the test. The ﬁrst 9 trials assessed rate of learning the location of the platform relative to extramaze cues, and trials 10–12 assessed memory for its former location and relearning its new one. In each trial the animals were assessed on the following dependent measures: (1) the time taken and distance swum until 2 min had passed; (2) time required to reach the platform (escape latency), and (3) time spent in each of the four quadrants. The results were as follows: (1) both treatment groups reached asymptotic performance on the dependent measures by trial 9, and in trial 10 showed retention of the platform’s original location by their disturbed behavior (increase in travel distance and swimming speed) when it was moved to a new location, which they quickly learned; and (2) statistical testing indicated that the VP antagonist did not impair learning/retention in this task [no signiﬁcant differences between the two treatment groups on any of the measures of performance (traveled distance, average swimming speed, latency to reach the escape platform, or time spent in any of the four quadrants)]. In discussing their ﬁndings the authors noted that whereas research has supported a role for the lateral septum in spatial memory processing

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(M’Harzi and Jarrard, 1992), only more recently has attention been directed to a possible septal VP contribution. These ﬁndings are in conformity with those of Engelmann et al. (1992b), who found that microdialysis application of a V1 receptor antagonist into the mediolateral septum during spatial learning in the MWM was without effect. Taken together, this observation and the present results suggest that septal AVP is not causally involved in spatial learning, at least that mediated by either V1 or V2 receptors.

C. The Parvocellular Hypothalamic VP-ergic System 1. Introductory Comments There are two sets of parvicellular (small-sized) VP-ergic neurons in the paraventricular nucleus (PVN) of the hypothalamus, which inﬂuence neuroendocrine and autonomic output during stress (these neurons and their projection pathways are described in Chapter 1). One set sends ﬁbers to the median eminence and inﬂuences adrenocorticotropic hormone (ACTH) release from the pituitary gland. A second set projects to autonomic nervous system (ANS) centers in the brainstem and spinal cord and inﬂuences sympathetic and parasympathetic outﬂow, especially in connection with cardiovascular regulation. These systems are activated by certain stressors and undoubtedly contribute to neuroendocrine and physiological activities conducive to adaptive responding to these stressors. Feedback from these peripheral activities during stress could inﬂuence arousal level and thereby contribute to the learning and memory effects of AVP hormonal treatment, in accordance with theoretical views of Koob and associates (see Chapter 6) and Sahgal and associates (see Chapter 7). However, it is the inﬂuence of the extrahypothalamic VP-ergic system, present in the midbrain–limbic structures of the central nervous system, which may be most relevant to the thesis of De Wied et al. (see Chapters 2–5) that AVP exerts a direct inﬂuence on memory storage and retrieval, independent of its role in central arousal. Because the parvicellular VP-ergic systems in the PVN and the extrahypothalamic VP-ergic systems are separately localized in the brain, they can be independently manipulated. This strategy was used in the research described below. 2. Selected Study: Herman et al. (1991) Herman et al. (1991) designed a study to determine whether parvicellular VP-ergic neurons in the hypothalamic PVN contribute to memory processing by means of arousal-related behavioral processes. Adult female rats of the selectively bred Roman high-avoidance (RHA) strain were used as subjects to permit comparison with a previous study (Herman et al., 1986b). The parvicellular VP-ergic neurons were selectively destroyed by ibotenic acid (IBO) lesions whereas the adjacent magnicellular VP-ergic

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neurons, involved in ﬂuid and electrolyte balance, were spared. Twelve of the 18 subjects received the IBO lesion; the remaining 6 were given sham operations. After recovery from surgery the subjects were tested for open ﬁeld behavior, acquisition of a multimodal sensory discrimination, spatial short-term memory, and approach-avoidance behavior. Open ﬁeld behavior was tested over four consecutive days (1 trial/day) during which the subjects were assessed for number of premarked central and perimeter squares entered, number of rearings, amount of grooming (mild stress), and autonomic signs of stress (number of fecal boli). After open ﬁeld testing, the subjects were placed on a food-restricted diet; 1 week later they were adapted for 8 days to enter and eat in the accessible goal box of a T-maze apparatus; and then they were successively trained and tested in this apparatus using paradigms designed to test sensory discrimination learning, short-term spatial memory in a delayed nonmatching-to-sample paradigm, and approach-avoidance behavior. The discriminant stimulus for the sensory discrimination task was a plastic grid ﬂoor insert that provided tactile, visual, and perhaps olfactory cues for the subject; depending on the trial, it was placed over the pressboard ﬂoor of one or the other goal box. The number of correct choices on each of 4 training days (12 trials/day) was tabulated for each subject. Training in the delayed nonmatching-to-sample (DNMS) paradigm began on the day after completion of sensory discrimination testing. For this task, six trials (each trial consisted of an information run followed 20 s later by the choice run) were given each day for 10 days. During the information run (the sample) one door was open to permit entry into a goal box where food was available. During the choice run, both doors were open but food was available only in the goal box not containing it during the information run. For analysis the data were grouped in blocks of 12 trials. The number of correct choices per 12-trial block (session), and the number of sessions to reach the accuracy criterion (10 correct responses out of any 12 consecutive trials), were tabulated for each subject. Training in the approach-avoidance task began on the day after completion of the DNMS task. For this task, the pressboard inserts were removed to expose the metal grid ﬂoor, enabling delivery of electric shock, and the left goal box was closed off throughout testing. During the approach phase (six trials/day for 3 days), the subjects were trained to run rapidly and consistently to the right goal box and eat the food reward within 15 s, and were doing so by the third day. After the eighteenth trial, an intense shock (1.5 mA) was delivered across the grid ﬂoor and the metal food cup for each contact with the food cup during the 60-s conﬁnement in the goal box. During postshock testing, which began 24 h later and lasted for six consecutive days (three trials/day), food was available in the goal box but no shock was given. The subjects were scored for latency to eat in the goal box (up to a default time of 60 s) in the last preshock and ﬁrst postshock trials, and for

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running speed [(1/latency) 100] in the last preshock trial and during each day of the postshock (avoidance response extinction) period. The results were as follows: (1) histological examination indicated that 8 of the 12 ibotenic acid-lesioned rats met the criterion for a successful lesion (elimination of the vast majority of identiﬁable parvicellular PVN neurons while sparing signiﬁcant numbers of magnicellular neurons). These subjects were included in the behavioral analyses; (2) lesioning affected neither drinking behavior nor body weight maintenance, verifying that hypothalamic magnicellular VP-ergic secretory neurons were spared; (3) total ambulation, ambulation in the centrally located squares, and number of rearings in the open ﬁeld test were greater in the lesioned mice than in the sham operates; (4) both groups of subjects met the sensory discrimination learning criterion, but the lesioned group exhibited a nonsigniﬁcant tendency toward poorer performance (reduced percentage of correct discriminations) relative to the sham operates on each of the daily test sessions; (5) both groups achieved criterion performance in the DNMS task [performed the task at above-chance levels by session 5 (ﬁfth block of 12 trials)], but the lesioned subjects performed more poorly (fewer correct choices per session) than the sham operates in each daily session, a difference that reached statistical signiﬁcance in sessions 2 and 3; and (6) both groups were similar in the avoidance response exhibited in the ﬁrst postshock trial (i.e., signiﬁcant increase in goal approach response latency relative to preshock levels) and in their rates of extinguishing the learned avoidance response. The authors inferred that the lesion reduced fear and cautiousness in the novel open ﬁeld environment because the greater levels of total and central ambulation and incidence of rearing behavior observed in the lesioned subjects, has been found to be negatively correlated with indices of ‘‘fear’’ in rats (e.g., Archer, 1973). This ﬁnding suggests that the parvicellular VP system in the PVN is normally involved in mediating fear, hence an increased arousal level. On the other hand, this system does not appear to play any essential role in memory processing because the lesion did not prevent criterion performance in the sensory discrimination or the DNMS tasks, retention of the shock experience (postshock avoidance responding), or subsequent extinction of the avoidance response. The lesion did slightly retard acquisition of the discrimination and impair short-term memory in the DNMS task, which was interpreted as reduced behavioral efﬁciency due to a lesion-induced reduction in behavioral arousal. Earlier research by Herman et al. (1986b; see Chapter 3) tested a group of RHA rats genetically deﬁcient in endogenous AVP (i.e., homozygous for diabetes insipidus; RHA-DI) in several of the behavioral tasks used in this study. In comparison with normal RHA rats, RHA-DI rats exhibited behaviors similar to those observed for the lesioned mice in this study (i.e., increased levels of central and peripheral ambulation and incidence of rearing in an open ﬁeld; good memory for the shock experience in the

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approach-avoidance paradigm and impaired performance in the DNMS task). Because both the RHA-DI rats and the lesioned rats were deﬁcient in parvicellular VP in the PVN, their behavioral commonalities suggest a role for this VP-ergic system in both emotional behavior (open ﬁeld test) and performance efﬁciency in cognitive tests (multimodal discrimination learning, spatial short-term memory). In summary, the results of this study are consistent with the hypothesis that the VP-ergic parvicellular system in the hypothalamic PVN inﬂuences cognitive performance via an inﬂuence on behavioral arousal, an effect that occurs in conjunction with the system’s joint inﬂuence on endocrine and autonomic responsiveness to a variety of environmental stressors.

III. VP and/or OT Interaction with Central Neurotransmitter Systems and Memory Processing

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De Wied and colleagues have obtained supportive evidence that the inﬂuence of vasopressin on long-term memory is mediated by interaction of the peptide with catecholaminergic (CA-ergic) mechanisms (see Chapter 4). Most of this research has focused on the locus coeruleus–noradrenergic (LC–NA-ergic) projection system, although a few studies included midbrain dopamine (DA) projections. Investigators outside the immediate De Wied et al. sphere of inﬂuence extended the research on the DA system, and also initiated study on acetylcholine (ACh) mechanisms in the effect of vasopressin on memory processing, as indicated in the following sections.

A. The Nigrostriatal DA System The possibility that VP released in the caudate nucleus may interact with the striatal DA system in its inﬂuence on memory processing was suggested by research observations reported in Chapter 4 (e.g., Kovacs et al., 1977; Van Heuven-Nolsen and Versteeg, 1985). 1. Selected Study: Hamburger-Bar et al. (1984) Hamburger-Bar et al. (1984) pursued this line of inquiry in an investigation of the effect of vasopressin on shuttlebox avoidance learning in DAlesioned male rats. At 5 days of age, rats in the DA lesion group received an intracisternal injection of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA), after an intraperitoneal injection of desipramine that spared noradrenergic neurons. This treatment had previously been shown to result in a persistent reduction of brain DA content (70% reduction in the whole brain, 95% reduction in the striatum) (Smith et al., 1973). Rats in the nonlesioned control group were injected with vehicle.

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At 2 months of age, both groups of rats received subcutaneous injections of physiological saline, LVP (1 g/rat), or the VP derivative 1-desamino-8d-arginine vasopressin (DDAVP, 20 g/rat), 1 h before the 15-trial learning session on each of 4 consecutive days of acquisition training. The doses selected for use were based on the 20:1 ratio for behavioral activity of the peptide and its derivative reported by Walter et al. (1978). Extinction testing was carried out on day 7, 3 days after the rats reached a learning criterion of 50% avoidance responding and the last treatment. The authors had previously shown that this VP treatment did not increase open ﬁeld locomotor activity (Hamburger-Bar et al., 1983). An independent group of DA-lesioned and nonlesioned rats that had been treated with DDAVP or saline and tested as described above were killed 1 month after acquisition training (3 months of age). Brain tissue samples were extracted and tested for AVP levels in the pituitary, hypothalamus, hippocampus, and caudate nucleus. The behavioral results for the nonlesioned rats and the lesioned rats were as follows: (1) the saline-treated nonlesioned group demonstrated better learning performance than did the saline-treated lesioned group [signiﬁcantly greater number of conditioned avoidance responses (CARs) in the latter group on test days 1 and 2]. However, the lesioned group had ‘‘caught up’’ by the third day of testing and the two groups showed no further signiﬁcant differences in learning or in subsequent extinction testing; (2) comparisons between the treatments given to the nonlesioned rats indicated that, relative to saline treatment, LVP, but not DDAVP, signiﬁcantly enhanced learning on test day 4 and retarded extinction (signiﬁcantly greater number of CARs on day 7); and (3) comparisons between the treatments given to the DA-lesioned rats indicated that relative to saline treatment, LVP and DDAVP signiﬁcantly enhanced learning on each day of training and retarded extinction. The histological testing indicated that (1) the DA lesions changed VP levels only in the pituitary, where VP content was signiﬁcantly reduced. DDAVP treatment helped to restore a normal level of pituitary VP, and it was suggested that this effect was probably due to compensatory changes in VP turnover induced by the interference of DDAVP with water metabolism (Boer and Swab, 1983); and (2) the amount of VP in the caudate nucleus was signiﬁcantly correlated with learning averaged over the 4 days of training in the nonlesioned controls (r ¼ 0.69) as well as in the saline-treated (r ¼ 0.64) and DDAVP-treated (r ¼ 0.60) lesioned rats (correlations between caudate levels of VP and rate of extinction were not tested). The authors interpreted their ﬁndings as follows: (1) comparison of the learning/retention performance of the lesioned and nonlesioned rats suggested normal levels of brain DA are important for early acquisition but not for the elaboration and maintenance of memory processing in this task; (2) the effects of LVP on learning/extinction were in accord with the De Wied et al. proposal that whereas VP consistently facilitates memory consolidation and retrieval, it has no important inﬂuence on learning except in

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circumstances in which learning is impaired (see Chapters 2 and 3); (3) the failure of DDAVP to inﬂuence memory processing in the normal controls may have been due to the weak behavioral efﬁcacy of the drug relative to LVP (i.e., the dose may have been sufﬁcient for enhancing learning in VP-sensitive DA-damaged rats, but not in normal controls); and (4) the correlational data combined with the ability of both VP analogs to enhance memory processing in the DA-lesioned rats suggest that, although VP release in the caudate nucleus inﬂuences one or more phases of memory processing in this task, DA is not an essential mediator in this inﬂuence. The following commentary on this study seems warranted. First, although these ﬁndings indicated that DA had no important role in regulating the release of VP in the caudate nucleus as it did for VP in the pituitary gland, they did not rule out the converse. In fact, evidence that VP is able to modulate the release of DA in the caudate nucleus was reviewed in Chapter 4 (Van Heuven-Nolsen and Versteeg, 1985; Versteeg et al., 1979). Second, the ﬁnding that the DA-lesioned rats given LVP treatment showed enhanced retention relative to those injected with saline could signify that VP increased DA release from whatever DA ﬁbers remained intact in the nigrostriatal projection system after the lesion. However, it could also signify that the LVP treatment enhanced retention by means other than a VP–DA interaction, for example, by a VP–NA interaction (Kovacs et al., 1979b; see Chapter 4) or by a VP–ACh interaction (Baratti et al., 1989; Faiman et al., 1987, 1988, discussed below). Third, the absence of a signiﬁcant difference in retention between the saline-injected lesioned versus saline-injected nonlesioned rats suggests that DA was not importantly involved in memory consolidation as it was during early acquisition in this paradigm.

B. The Cholinergic System 1. VP–ACh Interactional Effects and Memory Processing in a Passive Avoidance Paradigm a. Introductory Comments Faiman and colleagues (1987) noted that cholinergic mechanisms have been implicated in the modulation of memory processing (Flood et al., 1981), that cholinergic input inﬂuences the release of hormonal vasopressin (Hatton and Mason, 1985), and that before 1987 there were no studies examining a potential interaction between VP and ACh mechanisms in memory processing. Accordingly, beginning in 1987, these researchers (Baratti et al., 1989; Faiman et al., 1987, 1988, 1991) carried out a number of studies designed to investigate this possibility. In these studies, the experimental protocols examined VP–cholinergic interactional effects using peripherally administered vasopressin, and cholinergic agonists and/or antagonists for the two major types of cholinergic receptors: muscarinic and nicotinic receptors. The subjects, adult male Swiss mice,

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were trained in a single-trial passive (inhibitory) avoidance task with footshock as the negative reinforcer and tested for reentry latency 48 h after the posttraining treatment. The VP–ACh aspects of these studies are described below; other aspects of these studies were reviewed in Chapter 9. b. Selected Studies i. Faiman et al. (1987) Faiman et al. (1987) formed 10 independent treatment groups that received posttraining subcutaneous injections of either physiological saline, LVP (0.03 g/kg), 1 of 4 cholinergic antagonists [methylatropine (0.5 mg/kg, peripheral muscarinic antagonist), atropine (0.5 mg/kg, central muscarinic antagonist), hexamethonium (5 mg/kg, peripheral nicotinic antagonist), mecamylamine (5 mg/kg, central nicotinic antagonist)], or each of these cholinergic antagonists combined with LVP. The subjects were tested for reentry latency 48 h after the inhibitory learning trial and the posttraining treatment. The results indicated that LVP, on its own, facilitated memory consolidation (i.e., signiﬁcantly prolonged reentry latency relative to saline controls), whereas none of the cholinergic antagonists given alone did so. When coinjected with LVP, only the central nicotinic receptor antagonist mecamylamine prevented the LVP-induced facilitation of memory consolidation. That mecamylamine (the central nicotinic receptor antagonist) but not hexamethonium (the peripheral nicotinic receptor antagonist) prevented the LVP-induced facilitation of retention indicates that the interactional effect occurred at a central and not a peripheral level. However, the speciﬁc nature of this vasopressin–cholinergic interaction was not clear from the results. The authors noted that: either (1) the nicotinic receptor antagonist interacted with some central effect produced by the pressor or aversive properties of peripherally acting LVP, which itself was unable to cross the blood–brain barrier (BBB) and directly interact with central VP-ergic systems (Ermisch et al., 1985a; and see Chapter 14); or (2) LVP did reach central VP receptors, as surmised by De Wied and colleagues (1984a, 1991; see Chapter 5), and the antagonist interacted with these centrally activated VP-ergic systems. ii. Faiman et al. (1988) Faiman et al. (1988) examined a potential vasopressin–cholinergic interaction in memory retrieval in BALB/c mice tested in the passive (inhibitory) avoidance paradigm (see Chapter 9). This study employed the same four cholinergic blockers and dose levels used by Faiman et al. (1987). Independent groups of mice were subcutaneously injected with either saline, LVP (0.03 g/kg), one of the four cholinergic blockers, or LVP combined with each cholinergic antagonist. The injection was administered 20 min before the 48-h retention test. LVP facilitated memory retrieval (prolonged reentry latency) in this task. At the dose levels used, none of the cholinergic antagonists, injected alone, was effective.

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When coinjected with LVP only the central nicotinic antagonist mecamylamine prevented the LVP-induced facilitation of memory retrieval. These ﬁndings complement those obtained in the earlier study (Faiman et al., 1987), demonstrating that cholinergic–vasopressinergic interactions operate at the central rather than the peripheral level in mediation by the peptide of retrieval as well as consolidation of memory for the single-trial experience in this inhibitory avoidance paradigm. iii. Baratti et al. (1989) Baratti et al. (1989) designed a study to investigate the ability of an osmotic stimulus to inﬂuence retention in an inhibitory avoidance task and, if so, to determine whether cholinergic blocking agents can antagonize this effect. The osmotic stimulus, intraperitoneal injection of hypertonic saline, had previously been shown to release endogenous hormonal and central vasopressin in rats [Koob et al., 1985a (see Chapter 6); and see Lebrun et al., 1987]. An initial experiment tested the effects of various doses of hypertonic saline on retention in shocked and unshocked mice tested in this paradigm. Four treatment groups received a subcutaneous injection of physiological saline immediately after the training trial and 10 min later, depending on the group, an intraperitoneal injection of physiological saline (controls) or hypertonic saline (0.25, 0.50, or 1.00 M NaCl). Reentry latency was tested in the apparatus, under nonshock conditions, 48 h later. The results indicated that for the shocked mice, hypertonic saline, at a dose of 1.00 M, enhanced retention (prolonged reentry latency) relative to the saline controls. A subsequent experiment tested the effects of each of four cholinergic antagonists on the enhanced retention induced by 1.00 M NaCl. The cholinergic receptor antagonists and dose levels used in this study were the same as those previously used by these researchers (Faiman et al., 1987, 1988). Independent groups of mice received an intraperitoneal injection of hypertonic saline (1.00 M NaCl solution) 10 min after a subcutaneous injection of physiological saline, or 10 min after a subcutaneous injection of each of the four cholinergic blocking agents. Given alone, the osmotic stimulus released sufﬁcient endogenous vasopressin to facilitate memory consolidation, as it had done in the initial experiment. This memory enhancement effect remained intact when the osmotic stimulus was combined with three of the four cholinergic antagonists; it was reversed only by mecamylamine, the centrally acting cholinergic nicotinic receptor antagonist. The authors interpreted these results as suggesting that (1) endogenous vasopressin released by the osmotic stimulus enhanced retention of the learning trial and (2) this vasopressin was localized at central receptors that interacted with cholinergic mechanisms contributing to memory formation in this task. These results, together with the observation that vasopressin may act as a modulator for catecholaminergic transmission in this task (Versteeg and Van Heuven-Nolsen, 1984; and see Chapter 4,

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Section III), are consistent with the proposal that central vasopressin might interact with both cholinergic and catecholaminergic mechanisms during memory processing and require further study. iv. Faiman et al. (1991) Faiman et al. (1991) designed several experiments to further clarify the nature of the interaction between vasopressin and cholinergic mechanisms in memory consolidation of inhibitory (passive) avoidance responding in male Swiss mice. The rationale for these experiments was based on the proposition that if peripherally administered vasopressin inﬂuences memory consolidation by activating nicotinic cholinergic mechanisms, then the following experimental outcomes should be expected: (1) a peripherally administered nicotinic receptor agonist (nicotine) should exert the same effect as LVP on inhibitory avoidance retention (experiments 1 and 2); (2) a nicotinic-induced inﬂuence on retention should be blocked by a nicotinic receptor antagonist (experiment 3); (3) posttraining treatment with LVP and a nicotinic agonist should produce similar time gradient effects on retention (experiment 4); (4) subthreshold doses of LVP and a nicotinic agonist should have additive effects on retention (experiment 5); and (5) LVP-induced retention effects should be blocked by pretreatment with either a V1 receptor antagonist or a nicotinic receptor blocker, whereas a nicotinic-induced retention effect should be blocked by a nicotinic receptor blocker but not by a V1 receptor antagonist (experiment 6). Experiments 1 and 2 tested retention effects induced by immediate posttraining subcutaneous injections of physiological saline or various doses of LVP (0.003–1.00 g/kg, experiment 1) or of nicotine (1.00–30.00 g/kg, experiment 2). These treatments were given to mice that received a footshock, as well as to nonshocked mice, to test for possible nonspeciﬁc effects of the drugs. LVP and nicotine, when given to the shocked mice, produced similar inverted U-shaped dose–response curves for retention of the passive avoidance (PA) task. Thus, a posttraining injection of a midrange dose of LVP or of a central nicotinic receptor agonist (nicotine) signiﬁcantly facilitated PA retention (i.e., prolonged reentry latencies) whereas higher and lower levels had no signiﬁcant effect on these scores. These treatments did not affect posttraining reentry latency for the nonshocked subjects, indicating an absence of nonspeciﬁc pharmacological effects on this behavior. Experiment 3 demonstrated that nicotinic cholinergic mechanisms were involved in memory processing in this avoidance task paradigm. Different groups of mice received an immediate posttraining subcutaneous injection of physiological saline, nicotine (10.0 g/kg), a cholinergic antagonist [i.e., atropine (0.5 mg/kg), methylatropine (0.5 mg/kg), hexamethonium (5.0 mg/kg), or mecamylamine (5.0 mg/kg)], or each of the cholinergic antagonists plus nicotine (10.0 g/kg) as a single injection. Nicotine enhanced memory consolidation, but none of the anticholinergic agents

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inﬂuenced retention when injected alone. When combined with nicotine, the central nicotinic receptor antagonist mecamylamine prevented the nicotinic retention effect. However, neither the nicotinic receptor antagonist hexamethonium, which crosses the blood–brain barrier poorly, nor the muscarinic receptor antagonists atropine and methylatropine, prevented the inﬂuence of nicotine on retention. Time-dependent effects were tested in experiment 4. Separate groups of mice received posttraining physiological saline, LVP (0.03, g/kg, subcutaneous), or nicotine (10.0 g/kg, subcutaneous) either 0, 30, or 180 min after the single training trial. Posttraining injections of either LVP or nicotine prolonged reentry latencies relative to saline controls if given within 30 min, but not when delayed 180 min after training. Thus, similar time gradient effects on retention were found for LVP and a nicotinic agonist. In experiment 5, separate groups of mice were injected subcutaneously with physiological saline, LVP (0.003 g/kg), physostigmine (35.0 g/kg), nicotine (1.5 g/kg), or LVP combined with physostigmine or nicotine as a single injection, immediately after training. Physostigmine, an anticholinesterase, is a cholinergic agonist because it inactivates the enzyme (acetylcholinesterase) that destroys acetycholine. When given alone none of these drugs, at these dose levels, signiﬁcantly affected reentry latency. However, when given with the ineffective dose of LVP each of the cholinergic agonists, physostigmine and nicotine, signiﬁcantly increased reentry latency. Thus, combining subthreshold doses of LVP and either cholinergic agonist did produce additive effects on retention. In experiment 6, six independent groups of mice received, immediately posttraining, a subcutaneous injection of physiological saline, LVP (0.03 g/kg), a V1 antagonist (0.01 g/kg), mecamylamine (5.0 mg/kg), or LVP combined with either the V1 antagonist or mecamylamine as a single injection. Six independent groups of mice received the same treatments cited above except that nicotine (10.0 g/kg) instead of LVP was tested. When injected alone, neither the V1 antagonist nor mecamylamine inﬂuenced reentry latency. The retention effect of posttraining LVP was prevented when it was combined with either the V1 antagonist or the cholinergic blocker mecamylamine. On the other hand, the prolonged reentry latency produced by nicotine when given alone was prevented by mecamylamine but not by the V1 antagonist. The fact that mecamylamine by itself, at the dose level used, did not affect retention but blocked the LVP-induced facilitation of retention, indicated that the antagonism between LVP and the nicotinic receptor blocker was not due to a nicotinic blockade of the release of endogenous vasopressin. The ﬁnding that mecamylamine at a higher dose level (10 mg/kg, subcutaneous) impaired retention (Faiman, 1990, as cited in Faiman et al., 1991) is consistent with the participation of nicotinic cholinergic receptors in memory modulation.

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The authors concluded that a cholinergic system is involved in inhibitory avoidance retention and suggested that the enhanced consolidation induced by vasopressin treatment was due, at least in part, to action of the peptide on central nicotinic cholinergic mechanisms important to memory formation. 2. OT–ACh Interactional Effects and Memory Processing in a Passive Avoidance Paradigm a. Introductory Comments Two separate lines of evidence stimulated the study presented below. First, numerous ﬁndings have been consistent with the proposition that, whereas VP and VP fragments enhance memory consolidation and retrieval in appetitive and avoidance learning paradigms, OT tends to attenuate this memory processing (see Chapters 2–5). Two studies by Boccia, Baratti, and colleagues, using mice, conﬁrmed the proposed OT amnestic role in memory processing. Boccia and Baratti (1999) showed that OT impaired retention of a ‘‘nose poke’’ habitation response. Boccia et al. (1998) observed that when immediately administered after the footshock learning trial, OT impaired, whereas a selective OT receptor antagonist on its own enhanced, retention in this single-trial inhibitory avoidance paradigm. These effects were dose dependent, producing signiﬁcant effects at midrange dose levels and produced reciprocal U-shaped dose– response curves. Pretreatment with a dose of the OT antagonist, which did not affect retention by itself, prevented the effects of OT on retention. This latter ﬁnding, combined with the observation that pretreatment with a V1a vasopressin receptor antagonist failed to inﬂuence the effect of OT on retention, led the authors to propose that the OT amnestic effect in this paradigm was probably due to an interaction of OT with a speciﬁc OT type of receptor (but see De Wied et al., 1991; Chapter 5). Second, there have been a number of studies indicating that OT, like VP, modulates synaptic transmission in catecholaminergic transmitter pathways implicated in memory processing in limbic subcortical and cortical structures (see Chapter 4). Given the evidence obtained by this research team suggesting that the facilitative role of VP in retention of learned inhibitory behavior may be mediated, at least in part, by its positive modulatory inﬂuence on central cholinergic mechanisms involved in memory processing (see above), it was deemed feasible to investigate the possibility that OT might also interact with these cholinergic mechanisms, and if so to characterize the nature of this interaction. b. Selected Study: Boccia and Baratti (2000) Boccia and Baratti (2000) investigated potential interactive effects on memory storage between oxytocin (OT) and an OT receptor antagonist and drugs known to affect the cholinergic system centrally and/or peripherally. Elands et al. (1988b) considers the arginine vasotocin (AVT) analog (CH2)5[Tyr(Me)2,Thr4,

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Tyr(NH2)9]OVT (OVT, ornithine vasotocin) to be a highly potent OT receptor antagonist. It was used as the putative OT receptor antagonist (AOT) in this study. Three major experiments were conducted using independent treatment groups of adult male Swiss mice. In all the experiments with OT or the vasotocin analog AOT, the drug was subcutaneously injected after the training trial and tested for its effects on PA retention 48 h later. The ﬁrst set of experimental tests were attempts to replicate earlier ﬁndings by these investigators concerning the effect of OT, AOT, and their interaction on PA retention (Boccia et al., 1998; see Chapter 9). In this experiment, independent treatment groups of mice were used in each of three experimental tests. The ﬁrst test examined the effects of OT, AOT, or their interaction on PA retention when treatments were given within 10 min of the training trial. Speciﬁcally, each of ﬁve pairs of test solutions was subcutaneously injected: the ﬁrst, immediately after the training trial, the second, 10 min later. Depending on the group, the two sets of injected test solutions were as follows: (1) saline–saline (saline controls), (2) saline–OT (0.10 g/kg); (3) AOT (0.03 g/kg)–saline; (4) AOT (0.30 g/kg)–saline, or (5) AOT (0.03 g/kg)–OT (0.10 g/kg). The second test examined the effects of posttraining subcutaneously injected saline, OT (0.10 g/kg), or AOT (0.30 g/kg) on PA retention when the training-treatment delay was extended to 3 h. The third test examined the possibility that nonspeciﬁc proactive effects of these peptides lasted more than 48 h. For this test, three treatment groups were trained under nonshock conditions and subcutaneously injected with saline, OT (0.10 g/kg), or AOT (0.30 g/kg) immediately after the nonshock training trial. The results were as follows: (1) when administered within 10 min of the footshock training trial, OT signiﬁcantly retarded, and the higher dose of AOT signiﬁcantly enhanced, PA retention (reduced and increased reentry latencies, respectively) relative to saline controls. Moreover, pretreatment with AOT at a dose (0.03 g/kg) that had no PA retention effect on its own blocked the amnestic effect of OT in this paradigm (reentry latency of OT under this condition did not differ from that of saline controls). This ﬁnding conﬁrms the ability of this AVT agonist to serve as a highly effective and perhaps selective OT receptor antagonist (Boccia et al., 1998; Manning and Sawyer, 1993); (2) when administered 3 h after the footshock training trial, neither OT nor its receptor antagonist inﬂuenced PA retention (reentry latencies did not signiﬁcantly differ among OT-, AOT-, and saline-treated groups). Taken together, the ﬁndings of the ﬁrst and second experimental tests are consistent with the idea that these peptides affect a process underlying storage of recently acquired information (McGaugh, 1989); and (3) neither peptide inﬂuenced reentry latency when the subjects were trained under nonshock conditions, indicating that nonspeciﬁc proactive effects of OT and AOT on retention were not responsible for the results obtained in the ﬁrst experimental test.

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The second experiment examined the possibility that the amnestic action of OT, observed in the ﬁrst experiment, might involve an interaction between OT and a peripheral and/or central cholinergic transmitter system. To this end, central (physostigmine) and peripheral (neostigmine) anticholinesterase drugs were tested for their effects on PA behavior and for their ability to reverse the amnestic action of OT on this behavior. Anticholinesterase drugs promote cholinergic activity at postsynaptic sites because they prevent the hydrolysis of acetycholine produced by action of the enzyme acetylcholinesterase (Taylor, 1996). Ten treatment groups were formed. Each of ﬁve groups of mice received a posttraining subcutaneous injection of saline followed 10 min later by intraperitoneally injected saline, physostigmine (35, 70, or 150 g/kg), or neostigmine (150 g/kg). Each of ﬁve additional treatment groups received a subcutaneous injection of OT (10 g/kg) immediately after the PA training, followed 10 min later by an intraperitoneal injection of saline, physostigmine, or neostigmine at the dose levels designated above. For this second experiment, statistical comparisons indicated that (1) OT (saline–OT group) produced its expected amnestic effect (signiﬁcantly decreased reentry latencies) in the 48-h PA retention test; (2) the low dose of physostigmine (saline–physostigmine low-dose group) had no effect on retention but produced a partial (not signiﬁcant) attenuation of the amnestic effect of OT (OT–physostigmine low-dose group); (3) the two higher doses of physostigmine (saline–physostigmine high-dose group) signiﬁcantly enhanced PA retention, and fully reversed the PA amnestic action of OT when injected 10 min after OT (OT–physostigmine high-dose group); (4) neostigmine had no effect on PA behavior when given alone, nor did it modify the amnestic action of OT when accompanying OT treatment. Taken together, these ﬁndings suggest an OT–cholinergic interaction at central but not peripheral cholinergic receptor sites. The third experiment tested the ability of centrally and/or peripherally acting cholinergic receptor antagonists to impair the memory-facilitative effect of AOT on PA behavior observed in the ﬁrst set of experiments. Depending on the treatment group, the subject received an intraperitoneal injection of saline (controls), atropine (0.5 mg/kg), methylatropine (0.5 mg/kg), mecamylamine (5.0 mg/kg), or hexamethonium (5.0 mg/kg) immediately after the training trial, followed 10 min later by a subcutaneous injection of saline or AOT (0.30 g/kg). These researchers cited evidence indicating that atropine and mecamylamine, but not methylatropine and hexamethonium, can readily cross the blood–brain barrier. Accordingly, the former act at central, and the latter at peripheral, cholinergic receptor sites. Comparisons with the saline control condition indicated that (1) AOT signiﬁcantly enhanced retention (increased reentry latencies) in the 48-h PA retention test; (2) neither anticholinergic drug on its own, at the dose levels used here, inﬂuenced PA retention; (3) pretreatment with the centrally, but

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not the peripherally, acting cholinergic receptor antagonists prevented enhancement by AOT of PA retention. Taken together, the ﬁndings that only (1) centrally acting anticholinesterases were able to reverse the OT-induced PA amnestic action (experiment 2) and (2) centrally acting cholinergic antagonists prevented the AOTinduced facilitated memory effect on PA behavior (experiment 3) strongly suggest that an OT–cholinergic interaction contributes to the OT inﬂuence in memory processing. It can be concluded that the pharmacological evidence obtained in the second and third experiments is clearly consistent with the authors’ speculation that ‘‘oxytocin negatively modulates the activity of central cholinergic mechanisms during the posttraining period that follows an aversively motivated learning experience, leading to an impairment of retention performance of the inhibitory avoidance response’’ (Boccia and Baratti, 2000, p. 217).

IV. Endogenous AVP and/or OT and Memory Processing

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A. Endogenous AVP and Avoidance Learning 1. Selected Study: Engelmann et al. (1992a) Engelmann et al. (1992a) performed two experiments in their investigation of the effects of increasing and decreasing endogenous levels of peripheral and central AVP on pole-jump footshock avoidance learning. Experiment 1, reported in Section II.B.1, involved pharmacological manipulation of intraseptal AVP neurotransmission during acquisition of the avoidance response. That experiment showed that increasing septal AVP concentration beyond its normal level had no inﬂuence on avoidance learning, but that blocking normal VP receptor transmission in this brain site impaired it. In experiment 2, herein reported, central and peripheral levels of endogenous VP were increased by osmotic stimulation (peripherally administered hypertonic saline), and central as well as peripheral VP receptor transmission was blocked by a peripherally administered lipophilic V1 receptor antagonist. The procedure for training and performance evaluation in experiment 2 was the same as described for experiment 1 (Section II.B.1). After completion of the ﬁrst training session, the subjects tested in this experiment were randomly assigned to one of four treatment groups. The animals in each group received three intraperitoneal injections containing two types of test solutions [the solution mentioned ﬁrst was injected twice (immediately before the second and third training sessions), the one mentioned second was injected once (immediately after the second training session)]: (1) Iso-Iso

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group [isotonic saline (0.14 M NaCl)–isotonic saline]; (2) V1ant-Hyper group [V1 antagonist (10 g)–hypertonic saline (2.0 M NaCl)]; (3) V1antIso group (V1 antagonist–isotonic saline); and (4) Iso-Hyper group (isotonic saline–hypertonic saline). It was found that all four treatment groups improved their performance in this avoidance learning across the successive training sessions. Statistical analysis indicated no signiﬁcant differences between the four treatment groups in number of CRs per session performed in any of the training sessions. The authors related these experimental ﬁndings to relevant studies conducted by other researchers as well as to those obtained in experiment 1 of this study. It was noted that the failure of peripherally administered hypertonic saline to inﬂuence pole-jump acquisition behavior appears to be at variance with ﬁndings by Koob et al. (1985a), in which similar treatment facilitated retention (delayed extinction) in the same behavioral task. Because the osmotic stimulus is known to release endogenous AVP in both central and peripheral compartments (Landgraf et al., 1988), the retention effect observed by Koob et al. (1985a) could theoretically have been mediated by activation of AVP in either or both compartments (see Chapter 6). However, they attributed the enhanced retention effect to the pressor-associated arousal action of peripheral AVP, released by the osmotic stimulus. In support of this interpretation was their further observation that pretreatment with a subcutaneously injected V1 antagonist that blocks the peripheral pressor effects of AVP also prevented the memory-enhancing effect of osmotic stimulation (Koob et al., 1985a). However, one major difference between the two studies is that the ﬁndings of the present study are relevant to acquisition of the avoidance response, whereas those of Koob et al. (1985a) concerned retention of this learned behavior. The ﬁndings of experiment 2 are consistent with a number of experimental ﬁndings by De Wied and colleagues, and with their viewpoint that suggests that, although endogenous VP has a deﬁnitive role in mediating memory storage and retrieval, it is not important for learning except under special circumstances (see Chapter 2). The ﬁndings of experiment 2 are also partly in accord with those obtained in experiment 1 of this study. Thus, increasing the concentration of AVP centrally (experiments 1 and 2) as well as peripherally (experiment 2) did not affect acquisition behavior. Engelmann et al. (1992a) proposed that the failure of intraseptal infusion of AVP to inﬂuence avoidance learning (experiment 1) might have been due to an increase in the concentration of septal VP beyond that conducive to learning. This explanation would seem equally applicable to endogenous VP released by the osmotic stimulus. As to the failure of the lipophilic V1 antagonist to inﬂuence this behavior, the view held by De Wied and colleagues, that VP is not normally implicated in learning as it is in memory storage and retrieval, could equally

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explain this result. However, the results of experiment 1 suggested the converse, that is, that endogenous VP, at least that present in certain brain sites, does have a role in avoidance learning because interfering with the normal neurotransmission of the peptide impaired this learning (experiment 1). If so, then the failure of the peripherally administered V1 antagonist to impair avoidance learning (experiment 2) might have been due to the fact that the amount of the V1 antagonist that reached central receptors was insufﬁcient to interfere with the VP contribution to this behavior.

B. Endogenous AVP and OT and Memory Processing in an Aversive Paradigm 1. Selected Study: Ermisch et al. (1986) Ermisch et al. (1986) examined endogenous levels of peripheral and central AVP and OT in male Wistar rats selected for high and low learning/ memory performance in a Y-maze aversive brightness discrimination task. These subjects were tested in this footshock avoidance discrimination task during 4 sessions (22 trials/session). The ﬁrst session was termed the ‘‘training’’ (T) session and the following three were termed ‘‘relearning’’ (R1, R2, and R3) sessions. R1, R2, and R3 occurred 1 day, 5 days, and 6 weeks after T, respectively. The number of errors in the training session (Te) and in the relearning sessions (Re1, Re2, and Re3) were used to calculate the relearning index [i.e., RI ¼ (Te – Re/Te) 100]. The results indicated (1) progressive improvement (reduced errors) over the successive relearning sessions for all tested rats, including those selected from the highest and lowest end of the learning/relearning performance gradient (the high- and low-performance groups); and (2) these two groups signiﬁcantly differed from each other in terms of RI in each relearning session. Two months after completion of behavioral testing, the high- and lowperformance groups were killed and examined postmortem for endogenous AVP and OT levels in plasma, and in the posterior pituitary and four areas of the brain (the motor cortex, hippocampus, septum/striatum [contained the septal nuclei, bed nucleus of the stria terminalis, caudate nucleus, putamen, and pallidum], and the hypothalamus). The results of this postmortem analysis were as follows: (1) the AVP levels in the septum/striatum and the posterior pituitary of the high-performance group exceeded those of the lowperformance group; (2) relative to the low-performance group, the OT level in the high-performance group was signiﬁcantly higher in the septum/striatum and lower in the hippocampus; and (3) there were no signiﬁcant differences between the two groups in OT or VP peptide level in the motor cortex, the hypothalamus, or the plasma. The authors commented on the results of this study as follows: (1) the peptide levels observed at postmortem probably reﬂected genetically

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determined differences between the two performance groups rather than a differential response to task-related stressful events, because 2 months intervened between the end of behavioral testing and the postmortem analysis; (2) the pattern of high endogenous AVP and OT levels in septum/striatum neurons and low OT levels in hippocampal neurons might be prerequisites for high performance in the brightness discrimination task; and (3) although endogenous AVP and OT are likely involved in memory processing in a brightness discrimination learning task, they are not necessarily similarly involved in other learning tasks. For example, in a preliminary study involving a pole-jump active avoidance paradigm, these authors observed no evidence of the extremely high and low performance scores exhibited in this brightness discrimination test. In addition, the authors noted that a number of other research groups have also attempted to relate high and low performance in learning/memory tasks to differences in VP and/or OT levels in various brain areas. Lecesse (1983) reported that in mice, drugs that enhanced avoidance performance also resulted in increased AVPir levels in the lateral septum and dorsal raphe nucleus. Hamburger-Bar et al. (1984; this chapter) found that in rats, improved learning ability in an active avoidance task was correlated with enhanced levels of AVP in the caudate nucleus. In their studies with rats, discussed in Chapter 3, Laczi and colleagues observed that immediately after the retention trial in a passive avoidance (PA) task, rats that showed good PA behavior also exhibited decreased levels of AVP in the lateral septal nucleus (Laczi et al., 1983a) and dorsal hippocampus (Laczi et al., 1983b). The decreased levels were thought to reﬂect task-associated increases in AVP release at peptidergic terminals (Laczi et al., 1983a,b, 1984). However, unlike the ﬁndings of Lecesse (1983), Hamburger-Bar et al. (1984), and Laczi et al. (1983a,b, 1984), which probably reﬂected AVP responses to a fearful situation, the ﬁndings of this study were thought to reﬂect differences in ‘‘genetically determined potency of neurons to produce, transport and release AVP and OT’’ (Ermisch et al., 1986, p. 27).

C. Endogenous OT and Spatial Memory 1. Selected Study: Ferguson et al. (2000) Ferguson et al. (2000) compared male mice mutant for the OT gene (OT /) with wild-type mice, which are normal for the OT gene (OT þ/þ) on social recognition memory (SRM). As discussed in Chapter 13, the OT/ group was impaired in SRM. The two groups were also tested on spatial memory to determine whether it is dependent on a normal OT genotype, as had been found for SRM. Separate subgroups were tested in the Morris water maze (MWM), and in a two-trial Y-shaped maze under red light illumination (see Fig. 1).

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FIGURE 1 Performance in spatial memory test. Morris water maze data depict the decline in the mean (1 SEM) distance [(a) effect of repeated testing: F(4, 290) ¼ 26.4, p < 0.05] and latency [(b) effect of repeated testing: F(4, 290) ¼ 20.5, p < 0.05] required for mice mutant for the oxytocin gene (Oxt/; open symbols) and wild-type mice (Oxtþ/þ; solid symbols) to locate a submerged platform averaged over four repeated trials within ﬁve successive daily sessions. We detected genotype-dependent differences for latency [F(1, 290) ¼ 4.28, p < 0.05] and swim speed [F(1, 290) ¼ 9.04, p < 0.05], but not for distance traveled. Analysis did not detect interaction effects on any measures of water maze performance. Also portrayed is the amount of time spent in the platform quadrant during a 2-min probe trial on day 6 (c). We did not detect genotype-dependent differences. (d) Performance in the two-trial Y-maze test. We did not detect signiﬁcant genotype-dependent differences in the behavior of mice during the familiarization trial. During the test trial, subjects showed a preference for exploring the new arm expressed as amount of time (mean 1 SEM) and activity allocated to the new versus two familiar arms during the recall trial [effect of arm preference—duration, F(2, 28) ¼ 6.62, p < 0.05; effect of arm preference-distance traversed, F(2, 28) ¼ 5.87, p < 0.05]. We did not detect signiﬁcant genotype-dependent differences. *Signiﬁcant difference between the new arm and arm 1; þsigniﬁcant difference between the new arm and arm 2. Source: Ferguson et al., 2000 (Fig. 3, p. 286). Copyright ß 2000 by the Nature Publishing Group. Reprinted with permission.

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Spatial navigation in the MWM was observed over ﬁve successive days [4 trials/day with a 10-min intertrial interval (ITI)]. Initial placements in the opaque water varied systematically across the four trials. Time and distance required to locate the submerged platform were recorded as the dependent measures of learning. A 2-min probe trial with no platform present was given on day 6. Although OT / mice swam faster and exhibited shorter latencies than did OT þ/þ mice, the two genotypes were comparable in learning the maze (signiﬁcant decline in time and distance required to reach the platform over successive trials), and in remembering the location of the platform (both genotypes spent equal amounts of time in the platform quadrant in trial 5). The OT / and OT þ/þ mice tested in the Y-shaped maze (made of clear Plexiglas with guillotine doors isolating each of the three arms) were given two trials separated by a 30-min ITI. During the ﬁrst trial (familiarization phase), one arm of the Y-shaped maze was closed off, and the mice were placed in one of the two remaining arms and allowed to explore the maze for 5 min. During the second trial (retrieval phase), the door was removed and the mice were allowed free access to all three arms. A signiﬁcant preference for the novel arm was interpreted as evidence of spatial recognition (Contarino et al., 1999). There was no signiﬁcant difference between the two genotypes in Y-maze performance, and OT / as well as OT þ/þ mice exhibited spatial recognition (each genotype showed a signiﬁcant preference for the novel arm relative to each of the two remaining arms). Taken together, the results of this study indicated that mice genetically deﬁcient in OT were not impaired in spatial memory. The authors concluded that their data ‘‘indicate that OT is necessary for the normal development of social memory in mice and support the hypothesis that social memory has a neural basis distinct from other forms of memory’’ (Ferguson et al., 2000, p. 284). One should not conclude from this study that endogenous OT has a physiological role in modulating only memory underlying SRM. There is evidence that OT is involved in modulating avoidance retention (relevant evidence cited in various chapters of this text, especially in Chapters 2–5). Moreover, the failure of mice lacking endogenous OT to show a speciﬁc memory deﬁcit does not preclude the possibility that endogenous OT has a physiological role in modulating that memory system under normal circumstances. A host of other classic neurotransmitters and peptides interact in complex ways to inﬂuence spatial memory and may compensate for the absence of OT. Finally, for a number of other reasons caution is warranted in interpreting ﬁndings from research using genetic knockout models (see Section II of Chapter 3, and Bohus and De Wied, 1998). Chapter 11 summarizes the research studies presented herein and relates their ﬁndings to the theoretical views described in earlier chapters of this text.

Barbara B. McEwen

Expansion of Vasopressin/ Oxytocin Memory Research III: Research Summary and Commentary on Theoretical and Methodological Issues

I. Introductory Remarks

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The researchers cited in Chapters 9 and 10, although differing in their speciﬁc objectives, collectively contributed in a number of ways to the vasopressin (VP) and oxytocin (OT) memory research literature. They both replicated earlier ﬁndings and greatly increased the scope of the previous experimental paradigms, adding tests of acquisition, short-term memory (STM), long-term memory (LTM), and ethologically oriented as well as standard laboratory experimental manipulations in their animal research studies. Although several of these investigators used aversive motivation, the majority employed appetitive tasks to make up for the relative paucity of studies using low-stress environmental conditions, thus further testing the generality of the reported vasopressin and oxytocin inﬂuences on memory processing. None of these researchers speciﬁcally challenged the major theoretical views expressed by De Wied and colleagues (see Chapters 2–5), as had Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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been done by Koob, Ettenberg et al. (see Chapter 6), and Sahgal et al. (see Chapter 7). Nevertheless, many of their ﬁndings have relevance for the VP/OT research program and theorizing of De Wied and colleagues as well as that of opposing theoretical approaches. Their studies have also provided insights into the types of variables that may inﬂuence experimental outcomes and contribute to discrepant ﬁndings in the VP/OT memory research literature, thus ultimately furthering the attempt to test theoretical positions. The animal research presented in Chapter 9 examined the effect of peripherally administered VP or OT on both the early (learning) and late components (consolidation) of memory storage, as well as on the process of memory retrieval. In the studies with human subjects, a variety of neuropsychological tests were used to assess the nature of the inﬂuence exerted by these neurohypophysial peptides on early and later stages of memory processing as well as on other cognitive processes. The research studies discussed in Chapter 10 were designed to investigate in more detail central aspects of VP-ergic, and in a few cases putative OT-ergic, involvement in memory processing. A primary focus of this summary is to examine the ﬁndings with respect to the light they shed on the contribution of these neurohypophysial peptides to learning and memory and to relate them to the ﬁndings and views of the major theoretical positions.

II. Research Summary 1: Peripherally Administered VP and/or OT and Memory Processing—Studies Reviewed in Chapter 9

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A. Animal Research The following discussion of the animal research presented in Chapter 9 centers on whether VP and/or OT has an important role in learning and/or the later phases of memory processing (consolidation and retrieval), and whether the inﬂuence of these peptides occurs in appetitive as well as aversive learning contexts. This research also has relevance for theoretical issues concerning the mechanisms by which these neurohypophysial peptides, especially VP, may inﬂuence memory processing. 1. Vasopressin and/or Oxytocin and Learning De Wied and colleagues have theorized that, with certain exceptions, neither VP nor OT has an important role in the learning phase of memory processing. A failure of these peptides to inﬂuence learning has been observed for VP in avoidance and appetitive learning tasks in intact [e.g., Bohus, 1977; De Wied and Bohus, 1966; Kovacs et al., 1978; Vawter and Van Ree, 1995 (see Chapter 2)] as well as in vasopressin-deﬁcient rats [e.g., De Wied et al., 1988; Van Wimersma Greidanus et al., 1975a (see

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Chapter 3)], and for OT in active [Bohus et al., 1978b; Schultz et al., 1974 (see Chapter 2)] and passive (Kovacs et al., 1978; see Chapter 2) avoidance paradigms. The exception to this generalization, at least for VP, is the ability of the peptide to modulate learning when cognitive ability is low or deﬁcient, as in rats in which the pituitary gland has been removed (De Wied, 1965; see Chapter 2), or when the VP dose level produces aversive (Gaffori and De Wied, 1985; see Chapter 3) or arousal-incremental (Skopkova et al., 1991; see Chapter 3) effects. The six aversive motivational studies, which tested VP and/or OT inﬂuence on learning (see Chapter 9), were generally consistent with the conclusions of De Wied and colleagues. First, with respect to the acquisition of conditioned taste aversions (CTAs), two sets of ﬁndings indicated that (1) peripherally administered desglycinamide-arginine vasopressin (DG-AVP) failed to inﬂuence the acquisition of a CTA to ingested saccharin paired with LiCl treatment (Vawter and Green, 1980), and (2) pairing saccharin intake with a subcutaneous injection of OT or its antiserum failed, respectively, to induce a CTA to the saccharin solution or to prevent the CTA induced by an intraperitoneal injection of LiCl (Verbalis et al., 1986). The ﬁndings of the latter study indicate that peripheral OT is not causally involved in acquiring a CTA even though treatment with LiCl and other agents that produce CTAs also increases plasma levels of OT. Second, with respect to active and passive avoidance paradigms, several studies demonstrated a failure of VP and/or OT to inﬂuence learning in normal, brain-intact mice and rats. Thus either acute and/or chronic pretreatment with peripherally administered VP analogs failed to inﬂuence acquisition of a shuttlebox avoidance response in four of six inbred strains of mouse (Hamburger et al., 1985), an ethologically relevant passive avoidance response in mice (Leshner and Roche, 1977), or a shuttlebox avoidance response in rats (Hamburger-Bar et al., 1985). Moreover, chronic application of peripherally administered OT before the onset of training failed to inﬂuence shuttlebox avoidance acquisition in rats (Uvnas-Moberg et al., 2000). Third, with respect to avoidance learning in cognitively and/or emotionally impaired laboratory rats, two studies showed that (1) acute treatment with the nonpressor V2 receptor analog 1-desamino-8-d-arginine vasopressin (DDAVP) improved the rate of shuttlebox avoidance learning in braindamaged rats (Hamburger-Bar et al., 1985); and (2) chronic treatment with OT for 5 days before the onset of conditioning trials signiﬁcantly improved acquisition of a shuttlebox avoidance response in rats characterized by high emotional reactivity, poor avoidance learning, and low plasma OT levels (Uvnas-Moberg et al., 2000). The study by Hamburger-Bar et al. (1985) did indicate that chronic treatment with DDAVP produced a slight but statistically signiﬁcant improvement in learning in the normal intact subjects, a ﬁnding at variance with the

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results obtained by Bohus and De Wied (1966), who observed no learning effect in intact rats chronically treated with the vasopressin preparation Pitressin tannate. Inconsistent ﬁndings characterized the results of seven appetitive studies, discussed below, that examined the role of VP in learning an autoshaped operant response and a variety of visual discriminations (see Chapter 9). DG-AVP facilitated acquisition of an autoshaped operant response in Holtzman albino rats (Messing and Sparber, 1983) and in Long-Evans hooded rats (Messing and Sparber, 1985), and was even more effective when task difﬁculty was increased by insertion of a delay between the response and the reward (Messing and Sparber, 1985). However, Mundy and Iwamoto (1987) performed two replicate experiments with the same task, treatment procedure, and VP analog as that used by Messing and Sparber (1983), but found no evidence of a DG-AVP enhancement of learning in the Sprague-Dawley rats that served as subjects in this task. Moreover, in a separate experimental test with this paradigm, Mundy and Iwamoto (1987) observed no learning impairment in Brattleboro rats homozygous for diabetes insipidus (HODI rats, totally lacking in brain vasopressin) relative to their heterozygous littermates (HEDI rats, somewhat decreased in brain vasopressin but no disruption of water balance). De Wied and colleagues (see Chapter 3) have also reported that HODI rats exhibit normal (De Wied et al., 1975) or nearly normal (Bohus et al., 1975; De Wied et al., 1988) rates of learning in avoidance paradigms. Thus, overall there was no consistent evidence of a VP inﬂuence on learning in this operant task. Inconsistent ﬁndings and rather complex interactional effects were also reported in studies investigating a vasopressin effect on visual discrimination learning (see Chapter 9). Hostetter et al. (1977) observed that presession treatment with the vasopressin analog Pitressin had no effect on the rate of acquiring a black /white T-maze discrimination, whether the black or white goal arm was rewarded. Sara et al. (1982) reported that presession lysine vasopressin (LVP) treatment did not inﬂuence the more easily learned discrimination in a semiautomated Y-maze (dark goal arm, positive), but did accelerate the rate of learning the more difﬁcult discrimination (bright goal arm, positive). Two types of interactions affected the experimental outcome in two of the appetitive studies cited in Chapter 9. In one type, a high dose of AVP, which has both aversive and memory-enhancing effects, improved or disrupted learning performance depending on whether it was given early or later in the course of task training (Alescio-Lautier and SoumireuMourat, 1990). The second type of interaction between vasopressin and subject factors is relevant to the occasional report of a vasopressin-induced bimodal effect on learning (e.g., Sahgal and Wright, 1983). Alescio-Lautier and Soumireu-Mourat (1990) observed a bimodal treatment effect on

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discrimination learning after a pretraining dose of AVP on the second day of training. 2. Vasopressin and/or Oxytocin and Memory Consolidation and Retrieval In their ‘‘VP/OT Central Memory Theory,’’ De Wied and colleagues propose that VP facilitates, and OT attenuates, memory consolidation and retrieval (propositions 1 and 2, respectively; see Chapter 2). As noted below, the data obtained from studies examining the effects of these neurohypophysial hormones on these stages of memory processing have ﬁrmly supported proposition 1. However, the evidence relevant to proposition 2 has been of a more complex and less consistent nature. More speciﬁcally, these researchers have repeatedly observed prolonged extinction in a multitrial active avoidance (AA) task, and longer reentry latencies in a single-trial passive avoidance (PA) task, after peripheral administration of either the parent peptide (AVP, LVP) or one of the nonpressor analogs [e.g., DG-AVP, AVP(4–9), AVP(4–8)]. Depending on whether the VP analog was given posttraining, or before AA extinction testing or the PA retention test, the performance effects were interpreted as indicating a VP enhancement of memory consolidation and retrieval, respectively. In contrast, the comparatively fewer studies that have examined the inﬂuence of OT on memory storage and retrieval have indicated that, depending on the dose level, peripherally administered OT has been observed to facilitate, exert no effect, or attenuate retention in both active and passive avoidance tasks. Many of the studies relevant to these propositions are presented in Chapter 2. Focusing on the role of VP in memory processing, Koob and colleagues (see Chapter 6) used task and treatment paradigms and subject selection procedures similar to those of De Wied and colleagues, as well as appetitive paradigms (e.g., water-ﬁnding task), in their research studies. Their ﬁndings were interpreted as indicating a facilitation of memory consolidation and retrieval by the pressor-inducing parent peptides (AVP, LVP) but not by the nonpressor, nonaversive analog DG-AVP. The comparatively few VP/memory research studies that Sahgal et al. (see Chapter 7) conducted provided some evidence of a vasopressin-induced bimodal effect rather than an overall (average) facilitation of passive avoidance retention. a. Aversive Paradigms The researchers cited in Chapter 9 employed a diverse set of aversive paradigms to explore the inﬂuence of VP on memory consolidation and /or retrieval. As discussed below, many of the VP studies tended to replicate and extend the generality of the retention effects of VP as reported by De Wied and colleagues (see Chapters 2 and 3) and by Koob and associates (see Chapter 6). However, some of these researchers found that

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whether or not VP facilitated retention often depended on complex task-, treatment-, and subject-related variables and interactions that are discussed more fully in a subsequent section of this chapter. Vawter and Green (1980) demonstrated that chronic injections of desglycinamide-lysine vasopressin (DG-LVP), given 1 h before the conditioning and/or extinction trials in a conditioned taste aversion paradigm, prolonged extinction of the learned aversion. Speciﬁc group comparisons indicated that the peptide facilitated both memory consolidation and retrieval in this task. Hagan (1983) found that an injection of LVP, given after completion of shuttlebox avoidance training, strengthened behavioral suppression in the presence of the avoidant conditioned stimulus (CS) 24 h after avoidance training. On the assumption that fear mediated the behavioral suppression, the results indicated that vasopressin enhanced the fear learned in association with the avoidant CS. However, the mechanism responsible for the LVPinduced enhancement of the learned fear was not discernible from the evidence, and interpretations conforming to the ‘‘VP/OT Central Memory Theory,’’ the ‘‘VP Dual Action Theory,’’ and the ‘‘VP Central Arousal Theory’’ were all deemed plausible. Leshner and Roche (1977) and Roche and Leshner (1979) demonstrated that LVP enhanced both memory consolidation and retrieval in mice trained to avoid a future encounter with an aggressive ﬁghter mouse. Moreover, comparison between treatment effects on this behavioral paradigm indicated that LVP produced long-term, whereas adrenocorticotropic hormone (ACTH) produced relatively short-term, effects on retention, consistent with observations by De Wied and colleagues of laboratory rats tested in footshock avoidance paradigms (e.g., De Wied, 1965, 1971; see Chapter 2). With mice as subjects and the single-trial passive avoidance (PA) task as the experimental paradigm, Faiman and colleagues (Baratti et al., 1989; Faiman et al., 1987, 1988, 1991) corroborated results reported by De Wied and colleagues (see Chapters 2–5). Taken together, their ﬁndings speciﬁcally demonstrated (1) a vasopressin facilitation of memory consolidation and retrieval after treatment with exogenous vasopressin (Faiman et al., 1987, 1988) or by increasing endogenous levels of the peptide (Baratti et al., 1989); (2) a V1 receptor mediation of these effects (Baratti et al., 1989; Faiman et al., 1987, 1988); and (3) dose- and time-dependent effects of the peptide on PA retention (Faiman et al., 1991). Rigter (1982), using a memory consolidation design, observed a dosedependent effect of AVP on retention of a PA response when treatment was given immediately, but not when delayed for 90 min after the footshock trial. This study also demonstrated that this retention effect depended on adequate pretraining. The degree of pretraining found to be effective by Rigter (1982) has been typically used in the successful demonstration of a vasopressin enhancement of PA memory consolidation and retrieval by

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De Wied and colleagues (e.g., Ader and De Wied, 1972; De Wied, 1971; see Chapter 2), but was absent in the PA study by Hostetter et al. (1980), who used a retrieval design and observed no LVP inﬂuence on retention. Hamburger-Bar et al. (1985) found that chronic but not acute treatment with DDAVP enhanced retention in a shuttlebox avoidance task for both learning-impaired /brain-damaged rats and normal intact rats. Using mice as subjects, Hamburger et al. (1985) found that AVP prolonged extinction in a shuttlebox avoidance task in one of the six inbred strains of mouse tested in this task. Because only one dose level of the peptide was used in this study, it is possible that other dose levels would have inﬂuenced retention in one or more of the other mouse strains. Alescio-Lautier and Soumireu-Mourat (1990) found that AVP did not inﬂuence PA retention when given immediately after the footshock (FS) trial, but prolonged reentry latency when given 20 min before the 24-h retention test. The authors were reluctant to interpret these results as a true AVP modulation of memory consolidation and retrieval, respectively, for two reasons. First, it was questionable whether the FS had been of sufﬁcient intensity to promote passive avoidance learning given the short reentry latencies for the majority of vehicle controls. Second, the prolonged reentry latencies observed in the retrieval design were considered more likely a drug-induced hypoactive state rather then a true memory retrieval effect given the behavioral depression observed during the independent test of the inﬂuence of AVP on locomotor activity. One study presented in Chapter 9 focused on the role of OT in memory consolidation. Boccia et al. (1998) found that, on their own, peripherally administered OT and a selective OT receptor antagonist (a vasotocin analog) produced dose-dependent effects on PA behavior in mice. The U-shaped dose–response curves were mirror images of each other, with the former attenuating and the latter facilitating memory consolidation in this task. Tests for OT interactions with the selective OT receptor antagonist and with a potent V1a receptor antagonist, and for VP interactions with these receptor antagonists, supported the selectivity of the OT receptor that mediated the OT attenuation of PA behavior in mice tested in this paradigm. However, these latter ﬁndings were not consistent with those reported by De Wied et al. (1991; see Chapter 5) for laboratory rats. b. Appetitive Paradigms The appetitive tasks described in Chapter 9 had in common the feature of multiple acquisition and extinction trials. In these studies a vasopressin effect on memory consolidation was tested by treatment during or after the completion of training. An effect on memory retrieval was tested by treatment given before or during the course of extinction testing. Inconsistent ﬁndings were common both within and between the various appetitive studies. Thus, depending on the speciﬁc learning task or study, vasopressin treatment enhanced, impaired, or had no effect on memory

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consolidation and/or retrieval. In addition, several studies indicated complex subject–treatment interactive effects for both the parent peptide (AVP, LVP) and the nonpressor, nonaversive metabolite AVP(4–9). Messing and Sparber (1983) reported that DG-AVP, administered before each of 2 days of acquisition and 1 day of extinction training, facilitated retention (prolonged extinction) in an autoshaped operant response task. The design features of this study made it impossible to dissociate the effect of the peptide on consolidation from that on retrieval. In a subsequent study, Messing and Sparber (1985) observed that DG-AVP given during early phases of learning enhanced learning in a later phase when the peptide treatment had been discontinued. This peptide facilitation of subsequent learning, found in both the easy and more difﬁcult versions of the task, was interpreted as indicative of facilitated memory consolidation. Mundy and Iwamoto (1987) failed to observe a retention effect with the same peptide analog, treatment regimen, task, and training procedure used by Messing and Sparber (1983). The discrepant results may have been due to differences between the two studies in treatment (dose level) and/or subject variables (strain and/or age of the subjects). Using rate of extinction as the measure of retention, Hostetter et al. (1977) studied the effect of chronic treatment with Pitressin on either memory consolidation or retrieval for a learned black Sþ or white Sþ discrimination in a T-maze (Sþ, stimulus signaling availability of reinforcement). The peptide had no effect on memory consolidation for either discrimination and facilitated retrieval only for the more easily learned black Sþ discrimination. Sara et al. (1982) used savings on a 19-day retention test to assess the effect of LVP treatment on memory for a previously learned Y-maze brightness discrimination in Sprague-Dawley rats. A single injection of LVP was given before either acquisition or retention testing in this paradigm. Appropriate subgroup comparisons indicated no peptide effect on memory consolidation in rats trained to enter either the bright or dark goal arm, or on memory retrieval in rats trained to enter the bright goal arm (retrieval not tested for the dark/positive discrimination). Mulvey et al. (1988) used both rate of extinction and savings to test an LVP effect on memory consolidation, and savings alone to test the effect of the peptide on memory retrieval in Sprague-Dawley rats trained in a Y-maze brightness discrimination task. The two measures of retention indicated a vasopressin-induced enhancement of memory consolidation but the savings measure indicated no peptide inﬂuence on memory retrieval. Alescio-Lautier and Soumireu-Mourat (1990) reported differential effects of AVP depending on the dose level used for treatment. The high dose facilitated memory consolidation and retrieval whereas the low dose impaired memory consolidation and had no effect on retrieval in

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mice trained in a go/no-go black/white discrimination task. The memory consolidation effect was inferred from the improved or impaired learning that occurred on day 2 of training after AVP treatment given at the end of day 1 of training. The retrieval effect was based on comparisons between saline and treatment groups tested for savings in the 24-day retention test, 20 min after receiving saline or AVP. The evidence was interpreted as indicating that the high dose of AVP produced both aversive/learning-disruptive and memory-enhancing effects on performance. The authors did not clarify the impaired retention effects produced by the low dose of the peptide, which gave no evidence of an aversive effect. Strupp (1989) found that posttrial AVP(4–9) interacted with baseline proﬁciency in its inﬂuence on long-term memory, enhancing memory consolidation in subjects low, but not high, in task proﬁciency under nontreatment conditions. By way of explanation, Strupp (1989) hypothesized that under control (baseline) testing conditions, the less and more proﬁcient subjects may be characterized by suboptimal and optimal levels of endogenous vasopressin, respectively. Vasopressin treatment improves retention in the less proﬁcient by increasing the level of endogenous vasopressin toward a more optimal level. However, the increment added by the peptide treatment to already optimal levels in highly proﬁcient subjects produces no further beneﬁt and may even disrupt performance in accordance with an inverted U dose–response curve. Strupp et al. (1990) observed that AVP(4–9) enhanced retrieval of socially transmitted information concerning food preferences provided the retention interval was of sufﬁcient duration to produce signiﬁcant forgetting in the vehicle controls. By using a task paradigm designed to minimize stress and an AVP metabolite lacking the effects on the pituitary–adrenal axis that characterize the parent peptide, these researchers were able to reduce at least some potential ‘‘cueing’’ effects that could contribute to the retrieval effect observed in aversive paradigms. In a subsequent study with this paradigm, Bunsey and Strupp (1990) observed that the inﬂuence of the vasopressin metabolite on retrieval depended on the degree of retention exhibited under control conditions. That is, at the dose level used in this study, the peptide improved memory retrieval at a time when memory accessibility was low [after a retention interval (RI) during which signiﬁcant forgetting occurred in vehicle controls], impaired it when memory accessibility was high (after an RI during which memory was excellent in the vehicle controls), and had no effect on retrieval when memory accessibility was intermediate in value (after an RI at which recall was intermediate in vehicle controls). The authors suggested that this pattern of effects may be related to a vasopressin interaction with underlying biochemical events at synaptic storage sites that correspond to accessibility of the memory [for further discussion and speculation concerning these effects see Bunsey and Strupp (1990)].

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3. Vasopressin and Short-Term Memory Although De Wied and colleagues have not used tasks speciﬁcally designed to assess the effect of vasopressin on short-term memory (operationally deﬁned as trial-speciﬁc information in animal learning studies), some of their observations led to the conclusion that, as in learning (which De Wied suggests involves short-term memory processing), vasopressin is not importantly involved in mediating short-term memory processes (e.g., King and De Wied, 1974; see Chapter 2). Several of the researchers cited in Chapter 9 investigated the effect of the peptide on short-term memory (STM), tested either in a radial maze or in a delayed matching to sample (DMS) discrimination task. Buresova and Skopkova (1980, 1982) tested the effect of peripherally administered AVP and of a number of d- and l-isomers of the peptide on STM in a radial maze task. None of the peptides inﬂuenced the duration of STM, which the authors suggested may be optimal under nontreatment conditions for efﬁcient working memory (Buresova and Skopkova, 1980). On the other hand, Buresova and Skopkova (1982) demonstrated enhanced capacity of STM after treatment with the desglycinamide derivative of one of the d-isomers; this occurred for both young adult and middle-aged rats. However, this effect was not observed when the procedure was replicated with the same subjects 2 weeks later. The authors suggested that the original effect may have been due to a peptide enhancement of arousal, which was not subsequently observed because of adaptation to this drug effect. Van Haaren et al. (1986) observed that presession treatment with a dose of AVP (1 g/rat, subcutaneous) that frequently facilitates retention in aversive conditioning tasks, severely depressed choice activity in an eight-arm radial maze. Barring this performance deﬁcit there was no peptide inﬂuence on STM errors per se.

B. Human Research Of the four studies with human subjects reviewed in Chapter 9, two of them (Ferrier et al., 1980; Kennett et al., 1982) reported that a prolonged infusion of a high dose of OT, used for inducing therapeutic abortion, did not inﬂuence learning (immediate recall) but did impair memory (delayed recall after an intervening memory task) in a paired associate learning task. This was so whether comparisons were made between pre- and post-OT treatment conditions for the same patients (Ferrier et al., 1980), or between these patients and a non-OT-treated external control group (Kennett et al., 1982). OT-induced performance deﬁcits were also observed in the picturematching and recognition task: Ferrier et al. (1980) observed that the OT infusion rendered the patients less decisive in their choice behavior, and Kennett et al. (1982) observed that the OT-treated patients performed less well in this test of immediate memory than did the external control group.

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In the remaining two studies, the subjects received a single intranasal injection of either physiological saline or a low dose of either OT or AVP and were evaluated for their performance on a verbal learning/memory task (Fehm-Wolfsdorf et al., 1984), and in a neuropsychological test battery that tested for mood status as well as a number of cognitive variables (vigilance, attention, learning and memory of abstract words, visual memory for faces, performance in the Sternberg paradigm) (Bruins et al., 1992). Fehm-Wolfsdorf et al. (1984) observed no signiﬁcant effect of either VP or OT on immediate or delayed recall in the verbal memory paradigm used in their study. The slight VP-induced improvement did not signiﬁcantly differ from the equally slight OT-induced impairment of immediate memory. This failure to reach statistical signiﬁcance may have been due, in part, to a prominent placebo-induced facilitative action on this variable. That neither peptide signiﬁcantly inﬂuenced delayed recall (long-term memory) may have been a joint result of an inadequate dose level of these peptides and manipulation of a behavioral variable (retrieval strategy) that appears to modulate the effect of the peptides on memory processing (Fehm-Wolfsdorf et al., 1983). Bruins et al. (1992) found that (1) ‘‘vigor,’’ the only variable affected by peptide treatment in the mood status test, was reduced by OT but not inﬂuenced by DG-AVP treatment; (2) of the cognitive variables tested, vasopressin /oxytocin comparisons indicated that DG-AVP improved and OT attenuated learning in a verbal learning/memory test of abstract words, and treatment/placebo comparisons indicated that DG-AVP improved recognition /memory of the abstract words learned a week earlier, whereas OT had no effect on this measure; and (3) DG-AVP increased the rate of perceptual and motor activity in the Sternberg paradigm, an effect previously observed by Beckwith et al. (1983) and interpreted as a VPinduced enhancement of ‘‘attention’’ (see Chapter 8). In contrast to laboratory animals (see Chapters 2–5), the studies reviewed above and those cited by Beckwith and colleagues (see Chapter 8) suggest that, in humans, these peptides play as signiﬁcant a role in learning and attendant cognitive processes as they do in long-term memory processing. On the other hand, the opponent effects of these neuropeptides on memory processing consistently observed in laboratory animal research (e.g., see Chapters 2–5) have also been indicated in the human research studies (e.g., Chapter 9) reviewed in this text. The OT-induced decrease in vigor (Bruins et al., 1992), and the increased arousal experienced after VP application (Fehm-Wolfsdorf et al., 1984), also suggest that these neuropeptides induce opposite effects at the arousal level, with VP increasing and OT decreasing arousal level. Numerous experimental ﬁndings from animal laboratory research are consistent with the proposals of Koob et al. (see Chapter 6) and Sahgal et al. (see Chapter 7) that treatment with exogenous vasopressin increases the subject’s level of arousal. Along parallel lines,

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the writings and research ﬁndings of Uvnas-Moberg and colleagues suggest an OT-induced dearousal effect on behavior (e.g., Uvnas-Moberg, 2000; see Chapter 9).

C. Peripherally Administered VP and/or OT, and Memory Processing: General Conclusions The ﬁndings from both animal laboratory and human research studies discussed in Chapter 9 offer considerable support for the proposals of De Wied and colleagues (see Chapters 2–5) that in normally functioning intact subjects, neither VP nor OT exerts an important inﬂuence in learning (the rate of learning acquisition) but both neurohypophysial hormones do inﬂuence long-term memory consolidation and retrieval, but in opponent ways. The following observations from the animal research studies are consistent with the views of De Wied and colleagues on vasopressin and the rate of learning: (1) HODI rats failed to show a learning impairment (i.e., Mundy and Iwamoto, 1987); (2) normal intact rats treated with exogenous vasopressin showed no improvement in learning in both aversive (Hamburger et al., 1985; Leshner and Roche, 1977; Vawter and Green, 1980; Verbalis et al., 1986) and appetitive (Hostetter et al., 1977; Mundy and Iwamoto, 1987) paradigms; (3) shuttlebox avoidance learning was improved in cognitively impaired rats after chronic treatment with DDAVP during the training sessions (Hamburger-Bar et al., 1984), and with OT before the onset of training (Uvnas-Moberg et al., 2000); similar courses of treatment failed to inﬂuence the rate of learning in cognitively normal rats (Hamburger-Bar et al., 1984; Uvnas-Moberg et al., 2000); and (4) VP failed to inﬂuence performance on tasks assessing STM (Buresova and Skopkova, 1980, 1982; Van Haaren et al., 1986), consistent with De Wied’s suggestion that the short-term memorial processes involved in learning are not affected by peripherally administered vasopressin (King and De Wied, 1974). It may also be noted that studies reporting a vasopressin inﬂuence on learning were compromised by subsequent failure of replication (Messing and Sparber, 1983) or by the results being conditional on certain task-related (Sara et al., 1982) or subject-related (Alescio-Lautier and Soumireu-Mourat, 1990) variables. Support from the human research studies include the observations that (1) infusion of a high dose of OT over a 4-h and/or 8-h period failed to inﬂuence learning (immediate recall) of aurally presented word pairs (Ferrier et al., 1980; Kennett et al., 1982) or STM for face pairs in a picture recognition and matching task under conditions in which the patients served as their own controls (Ferrier et al., 1980); and (2) a single intranasally administered lower dose of either DG-AVP or OT failed to inﬂuence STM assessed in a visual memory test with photographs of faces, or memory scanning tested in the Sternberg paradigm (Bruins et al., 1992).

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In general, results of the aversive motivational paradigms discussed in Chapter 9 indicated that VP facilitated, and OT impaired, memory consolidation and /or retrieval. More speciﬁcally, a vasopressin positive inﬂuence was observed for (1) a conditioned taste aversion in rats (Vawter and Green, 1980); (2) a conditioned shuttlebox avoidance response in both braindamaged and brain-intact mice (Hamburger-Bar et al., 1984); (3) a passive avoidance response to footshock punishment in rats (Rigter, 1982) and in mice (Baratti et al., 1989; Faiman et al., 1987, 1988, 1991); and (4) a passive avoidance response to aggressive attack from a conspeciﬁc male in mice (Leshner and Roche, 1977; Roche and Leshner, 1979). An OT-induced amnestic effect was indicated in a study by Boccia et al. (1998), which reported that in mice: (1) peripherally administered on their own, OT impaired and OT antiserum enhanced memory storage in a PA paradigm, in accordance with a U-shaped dose–response function; and (2) tests for peptide–receptor antagonist interactional effects strongly suggested that an OT receptor mediated this amnestic effect of the peptide. For the animal studies presented in Chapter 9, a VP-facilitated retention effect was less consistently observed in appetitive than in aversive paradigms. The VP-induced facilitated effects on retention in an autoshaped operant learning task reported by Messing and Sparber (1983, 1985) were not subsequently conﬁrmed by Mundy and Iwamoto (1987), with a similar type of operant learning task. Sara et al. (1982) found no VP inﬂuence in tests of either memory consolidation or retrieval for a visual discrimination, whereas Mulvey et al. (1988) reported a positive effect on memory consolidation but not retrieval, and Hostetter et al. (1977) found a peptide improvement of retrieval for only the easier learned (black positive) discrimination. An enhancement of memory consolidation and retrieval of a learned go/no-go discrimination in mice was observed for the high dose of AVP, whereas the low dose impaired consolidation and had no effect on retrieval (Alescio-Lautier and Soumireu-Mourat, 1990). In the human research literature discussed in Chapter 9, a vasopressin inﬂuence on long-term memory was not consistently observed in the two studies that tested its effects on verbal learning. Fehm-Wolfsdorf and colleagues (1984) found no AVP inﬂuence on free recall of previously learned lists of words tested either at the end of a 2-h test session, or several months later. On the other hand, DG-AVP enhanced recall tested 1 week after original performance in the verbal learning/memory task used by Bruins et al. (1992). The inconsistent ﬁndings might have been related to differences between these studies with respect to treatment (VP analog, dose level) and / or task factors (verbal material memorized, conditions under which recall was tested, or the learning–retention interval). Of the four studies that examined the effect of OT on long-term memory, two studies showed that prolonged infusion of a high dose of OT impaired recall of a previously learned list of word pairs (Ferrier et al., 1980; Kennett et al., 1982). The two

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remaining studies found no evidence that acute treatments with lower doses of OT inﬂuenced long-term memory for previously learned verbal materials whether tested 2 h or several months after learning numerous lists of monosyllabic words (Fehm-Wolfsdorf et al., 1984) or 75 min or 1 week after learning a list of 15 abstract words (Bruins et al., 1992).

III. Research Summary 2: Central Aspects of VP-ergic and OT-ergic Involvement in Memory Processing—Studies Reviewed in Chapter 10

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The discussion below centers on subjects investigated in the studies reviewed in Chapter 10. These include (1) localizing the brain structures that mediate the inﬂuence of VP and /or OT on memory processing in a variety of learning tasks; (2) examining putative interactions between VP and /or OT and aminergic and cholinergic neurotransmitter systems that have been implicated in memory processing; and (3) determining the generality of involvement of central VP-ergic and /or OT-ergic systems in various types of learning and memory tasks.

A. Central Structures That Mediate VP and/or OT Influences on Memory Processing 1. The Hippocampal Area Hippocampal involvement in memory processing has been a topic of major interest in both human clinical research (Milner et al., 1968; Penﬁeld and Milner, 1958; Squire and Zola-Morgan, 1988) and animal experimental research (e.g., Eichenbaum et al., 1988, 1989; Mishkin and Appenzeller, 1987; O’Keefe and Nadel, 1978; Squire et al., 1993). The presence of VP-ergic nerve terminals within the hippocampal area (Sofroniew, 1985a; see Chapter 1) together with experimental ﬁndings by De Wied and colleagues (Chapter 4) are consistent with the thesis that hippocampal vasopressin plays a contributory role in memory consolidation and retrieval. Speciﬁcally, study with laboratory rats tested in active and passive avoidance paradigms has demonstrated that (1) lesions within the hippocampus block the retention enhancement induced by exogenous vasopressin in a pole-jump avoidance task (Van Wimersma Greidanus and De Wied, 1976b; see Chapter 4); (2) AVP and OT, microinjected into the dentate gyrus of the hippocampus, respectively, facilitate and inhibit memory consolidation in a PA task (Kovacs et al., 1979a; Chapter 4); and (3) neutralizing vasopressin within the dorsal or ventral hippocampus impairs memory consolidation and retrieval in the single-trial PA task (Veldhuis et al., 1987; see Chapter 4). Ibragimov (1990) studied the effects of LVP and OT, or their respective metabolites DG-AVP and prolyl-leucyl-glycinamide (PLG), on memory

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processing in a shuttlebox avoidance task in CFY rats. These peptides were administered either intracerebroventricularly (LVP) or were microinjected into the ventral hippocampus (OT, DG-AVP, and PLG) during acquisition training. LVP weakly facilitated learning, and OT strongly retarded it at all dose levels. On the other hand, LVP signiﬁcantly enhanced retention (retarded extinction) at all dose levels and OT weakly attenuated retention (approaching signiﬁcance only at the highest dose level). DG-AVP and PLG inﬂuenced learning and memory in a manner similar to that of their parent peptides, but their opposing effects reached a level of statistical signiﬁcance only during retention (extinction) testing. Because these peptides were chronically injected 1 h before daily testing throughout acquisition and extinction training, it is not possible to dissociate their effects on learning, memory consolidation, and retrieval. Despite this interpretational difﬁculty, it is clear that the directional inﬂuence of these neurohypophysial peptides on memory processing is consistent with the ‘‘VP/OT Central Memory Theory.’’ The ﬁnding that in this study, PLG produced OT-like effects on memory processing, rather than the VP-like effects reported by other investigators (e.g., Gaffori and De Wied, 1988; see Chapter 2), is puzzling and clearly needs further study for clariﬁcation. Alescio-Lautier, Metzger, and associates used BALB/c mice as subjects and the appetitive go/no-go black /white discrimination task as the experimental paradigm in their studies of the hippocampus and vasopressin in memory processing. The ﬁndings of these studies indicated that (1) the dorsal hippocampus is important for mediating the memory retrieval effect induced by intracerebroventricularly administered AVP, but endogenous vasopressin in this brain area is not essential to this memory processing because its immunoneutralization did not impair memory retrieval (Alescio-Lautier et al., 1989); (2) AVP directly microinjected into either the dorsal or the ventral hippocampus facilitated memory retrieval relative to saline controls, although the ventral hippocampus was superior in this respect (Metzger et al., 1989); and (3) memory retrieval and relearning were enhanced by increasing levels of ventral hippocampal vasopressin via microinjected AVP, and impaired by decreasing these levels via microinjected AVP antiserum (Metzger et al., 1993). Moreover, VP-ergic cells originating in the medial amygdala and terminating in the ventral hippocampus appear to inﬂuence this retention and are essential for the retrieval, but not the VP-mediated relearning, effects (Metzger et al., 1993). Taken together, the ﬁndings of these latter three studies were interpreted as indicating that, for the mouse strain used in this appetitive experimental paradigm, both the dorsal and ventral hippocampus contribute to mediating the retention effects of exogenous AVP. Whereas dorsal hippocampal vasopressin plays no important role in memory retrieval, ventral hippocampal vasopressin makes a signiﬁcant contribution to memory processing and this is expressed in both enhanced retrieval and relearning in this discrimination

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task. The experimental results obtained with CFY rats by Ibragimov (1990) are consistent with this interpretation. 2. The Septal Area Studies by De Wied and colleagues support the proposition that the septal area is another limbic brain site in which vasopressin acts to inﬂuence memory processing assessed in avoidance paradigms (see Chapters 4 and 5). Relevant evidence has been obtained from lesion, microinjection, and electrophysiological studies. First, Van Wimersma Greidanus et al. (1975b; see Chapter 4) reported that a bilateral lesion that destroyed the medial septal nucleus completely, and the lateral septum and nucleus accumbens partially, impaired the VP-enhanced retention observed in a pole-jump avoidance task. Second, Kovacs et al. (1979a; Chapter 4) reported that AVP microinjected into the dorsal septal nuclei facilitated memory consolidation in a passive avoidance paradigm. Microinjection studies with the one-trial passive avoidance paradigm have shown that, when locally injected into the dorsal septal nuclei, AVP facilitated memory consolidation (Kovacs et al., 1979a), whereas AVP antiserum impaired memory retrieval but had no effect on memory consolidation (Veldhuis et al., 1987; see Chapter 4). This latter ﬁnding suggests that endogenous septal VP is essential for only the retrieval phase of memory processing in this paradigm. Third, electrophysiological studies by Joels and Urban (1982, 1984a, 1985; see Chapter 5) support the proposal that neuromodulatory actions in septal VP-ergic neurons enhance glutamatergic synaptic activity in the septohippocampal system; the linkage between the results of these in vivo single-cell studies of the putative role of septal VP in memory processing is that glutamate is considered the neurotransmitter that mediates long-term potentiation (LTP) (Teyler and DiScenna, 1987) and the LTP paradigm is used by numerous researchers in their attempts to clarify the synaptic underpinnings of memory storage (see discussion in Carlson, 1998). Moreover, an in vitro study by Van den Hooff et al. (1989; see Chapter 5) has obtained evidence indicating the importance of septal VP for the maintenance of LTP induced in the septohippocampal system. Studies reviewed in Chapter 10 are also relevant to the role of septal VP in memory processing. Engelmann et al. (1992a) observed that blocking septal VP neurotransmission by intraseptal infusion of either a V1 or a V1/V2 antagonist slowed the rate of learning a pole-jump avoidance response, whereas application of exogenous AVP to this brain site was without effect. The interference with normal learning produced by central application of the antagonists indicates that endogenous septal VP is an important inﬂuence on this learning. It was proposed that the inability of intraseptally infused AVP to enhance learning was due to the fact that the footshock

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present during training had already increased central AVP to a level conducive to learning, and the further increase by AVP treatment raised the level above an optimal value and was consequently ineffective. Two studies investigated the putative role of endogenous septal AVP in memory processing underlying efﬁcient spatial navigation in the Morris water maze (MWM). These studies showed that interference with septal VP-ergic neurotransmission by intraseptal infusion of a V1 antagonist (Engelmann et al., 1992a; Everts and Koolhaas, 1999) or a V1/V2 antagonist (Everts and Koolhaas, 1999) did not inﬂuence learning (Engelmann et al., 1992a; Everts and Koolhaas, 1999) or retention (Everts and Koolhaas, 1999) of this maze. Taken together, these latter results, and those of others, suggest that whereas septal VP is important for retention of a conditioned active (Van Wimersma Greidanus et al., 1975b) and passive (Kovacs et al., 1979a; Veldhuis et al., 1987 ) avoidance response, and for olfactory-based recognition of a preencountered conspeciﬁc (Dantzer et al., 1988; see Chapter 12), it is not essential for spatial memory. 3. The Hypothalamic Parvocellular VP-ergic System The research ﬁndings of Herman et al. (1991), described in Chapter 10, are relevant to the controversy over the role of the arousal system in VP/ memory research. The paraventricular nucleus of the hypothalamus (PVN) contains VP-ergic magnocellular neurons (which secrete hormonal vasopressin into the peripheral circulation) and VP-ergic parvocellular neurons [which inﬂuence ACTH–adrenocortical and sympathetic nervous system (SNS)–adrenomedullary output during environmental stress]. A chemical lesion was induced that selectively destroyed PVN parvocellular VP-ergic neurons while sparing the majority of magnocellular VP-ergic neurons. Compared with the sham operates, the lesioned animals demonstrated (1) reduced fear and timidity and increased exploration-oriented behaviors in the open ﬁeld test; (2) impaired performance in learning a sensory discrimination task and solving a delayed matching to sample task; and (3) normal memory of a footshock experience and extinction behavior in an approach-avoidance paradigm. Taken together, these results support the conclusions that an intact parvocellular VP-ergic system normally contributes to the heightened arousal elicited by stressors such as those involved in the open ﬁeld test, and contributes the incremental arousal/alertness that beneﬁts discrimination learning and short-term memory performance. On the other hand, it has no essential role in the long-term memory processing occurring under the high-stress conditions of the approach-avoidance paradigm. However, participation of extrahypothalamic VP-ergic circuitry in the long-term memory effects expressed in this paradigm was possible because the lesion did not affect this circuitry.

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B. AVP and Perhaps OT Interact with Catecholamine and Acetylcholine Neurotransmitters in Memory Processing De Wied and colleagues have proposed that VP interacts with the catecholamines, especially noradrenaline (NA), in the role(s) they serve in memory processing. Several lines of experimental support for this proposition were presented in Chapter 4 and included the demonstration that (1) lesioning the coeruleus–telencephalic NA-ergic pathway prevented the facilitation of PA retention observed with posttraining peripherally administered AVP (Kovacs et al., 1979b); (2) pretreatment with an enzyme that inhibits catecholamine (CA) synthesis prevented the retention effect induced by AVP treatment in a bench-jump PA task (Kovacs et al., 1977); (3) pretreatment with the CA synthesis-inhibiting enzyme indicated that microinjected AVP modulated NA utilization (hence neurotransmission) in several structures implicated in memory processing (Kovacs et al., 1979a); and (4) neutralization of endogenous AVP, by microinjected VP antiserum into certain limbic structures (e.g., dorsal and ventral hippocampus, dorsolateral septum), attenuated both memory consolidation and retrieval in the single-trial PA task (Veldhuis et al., 1987). Studies carried out in other laboratories have extended this research and investigated a potential VP interaction with catecholaminergic (dopamine) and also with cholinergic projections to selected forebrain structures in which VP has been shown to inﬂuence some phase of memory processing. The results of these investigations, reviewed in Chapter 10, are summarized and discussed below. 1. The Nigrostriatal DA System Several researchers have obtained evidence in support of a role for the striatal DA system in learning and memory of instrumental behavior (see Beninger, 1983; Jog et al., 1999; Packard and White, 1991; Packard et al., 1989, 1994). Two ﬁndings discussed in Chapter 4 suggested that VP and its derivatives enhanced memory retention and retrieval, respectively, by an interaction with DA systems projecting to the caudate nucleus (Kovacs et al., 1977) and to the amygdala (Bohus et al., 1982; Van Heuven-Nolsen et al., 1984b). Several biochemical studies provided fairly convincing evidence that VP does inﬂuence DA neurotransmission in the nigrostriatal system. For example, peripherally or intracerebroventricularly injected vasopressin increased alpha-methylparatyrosine-induced disappearance of striatal DA (Kovacs et al., 1977; Tanaka et al., 1977a; see Chapter 4) whereas an intracerebroventricular injection of its antiserum reduced it (Versteeg et al., 1979; see Chapter 4). Moreover, Van Heuven-Nolsen and Versteeg (1985; see Chapter 4) demonstrated that the site at which VP inﬂuenced striatal DA neurotransmission occurred in the caudate

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nucleus (target for the striatal DA projection system) rather than in the substantia nigra (origin of the DA projection to the caudate nucleus). However, the meaning of these biochemical ﬁndings is not clear-cut because VP (Meisenberg and Simmons, 1983) and DA (Beninger, 1983) have been implicated in mediating motor behaviors, as well as memory processing. Further information about a VP interaction with the nigrostriatal DA system was provided by Hamburger-Bar et al. (1984) and described in Chapter 10. Their behavioral ﬁndings were interpreted to indicate that striatal VP has a role in mediating memory processing in an instrumental learning task (shuttlebox avoidance), but that the nigrostriatal DA projection system is not essential for this effect. This latter statement was based on the ﬁnding that DA lesioning at 5 days of age did not prevent vasopressin from enhancing retention tested in 3-month-old rats. However, these results do not rule out the possibility that a VP–DA interaction is normally involved in this memory processing. It is possible that compensatory changes occurred over the long interval between the onset of the lesion and the behavioral testing. For example, other neurotransmitter systems [e.g., NA and acetylcholine (ACh)] that interact with VP may have compensated for the loss of the normal VP–DA interactional inﬂuence on this memory processing. Compensatory mechanisms have been suggested as possible explanations for the lack of expected learning/memory deﬁcits after severe depletions in neurotransmitter systems implicated in this processing on the basis of other lines of evidence (e.g., see discussion in Harley, 1987). 2. The Cholinergic System Using mice as subjects and the single-trial passive avoidance (PA) task as the experimental paradigm, Faiman, Baratti, and colleagues reinforced and extended the research ﬁndings and theoretical views of De Wied and associates by indicating that, in its mediation of memory processing, vasopressin (1) interacts with cholinergic as well as the noradrenergic neurotransmitter systems and (2) this interaction is mediated by central rather than peripheral cholinergic receptor sites (see Chapter 10). Speciﬁcally, the results of these studies have shown that (1) at the dose level used, neither of the two peripheral or two central cholinergic receptor antagonists, on their own, inﬂuenced PA retention, but when combined with vasopressin treatment the central nicotinic antagonist, mecamylamine, prevented the peptide-induced facilitation of memory consolidation (Faiman et al., 1987) and retrieval (Faiman et al., 1988); (2) endogenous vasopressin released by an osmotic challenge facilitated memory consolidation in the PA task and this effect was blocked by pretreatment with the central nicotinic antagonist mecamylamine (Baratti et al., 1989); and (3) at the dose level used, neither of the cholinergic agonists (physostigmine or nicotine) on their own inﬂuenced PA retention, but when either agonist was coinjected with an

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ineffective dose of LVP the combination facilitated memory consolidation in this task (Faiman et al., 1991). Taken together, these observations are consistent with the thesis that, in addition to its interaction with central noradrenergic systems, vasopressin also interacts with central cholinergic neurotransmission to inﬂuence memory consolidation and retrieval. Findings by Boccia and Baratti (2000) have indicated that OT, as well as VP, interacts with central cholinergic mechanisms in mediating its inﬂuence on memory processing in the PA learning paradigm.

C. Endogenous VP and OT, and Memory Processing Engelmann et al. (1992a; see Chapter 10) increased endogenous vasopressin by a peripheral injection of hypertonic saline (osmotic stimulus) and decreased it by a lipophilic V1 receptor antagonist. These treatments, administered during acquisition training, were assessed for their ability to inﬂuence learning of a pole-jump footshock avoidance response. First, the osmotic stimulus had no effect on learning. Considering its ability to increase peripheral levels of AVP (Landgraf et al., 1988), this result supports the theory that endogenous AVP in peripheral circulation does not contribute to avoidance learning [De Wied, 1965 (see Chapters 2 and 3); Engelmann et al., 1992a]. Relevant to its ability to increase central AVP levels (Landgraf et al., 1988), the ﬁnding is consistent with that obtained with intraseptal infusion of AVP, and was explained on the same basis (see Section III.A.2). Second, the peripherally injected lipophilic V1 receptor antagonist did not impair normal learning of this task, a result also obtained by Koob et al. (1981; see Chapter 6) using a similar experimental protocol. Unlike AVP, there is little controversy about the ability of the lipophilic receptor antagonist to cross the blood– brain barrier (Koob et al., 1981). The ability of the receptor antagonist to impair avoidance learning when intraseptally injected (experiment 1; see Section III.A.2), but not when peripherally injected (experiment 2; Koob et al., 1981; see Chapter 6), may reﬂect the fact that insufﬁcient amounts of the peripherally injected antagonist reached appropriate central targets. Ermisch et al. (1986) selected rats for high and low performance in learning and relearning a footshock avoidance brightness discrimination in a Y-maze. At postmortem the brains of these rats were examined for VP and OT levels in the plasma, posterior pituitary, and various brain structures (motor cortex, hippocampus, septum/striatum, and hypothalamus). The comparisons indicated the following signiﬁcant differences: relative to the low performers, the high performers exhibited a higher level of VP and OT in the septum/striatum, a higher level of VP in the posterior pituitary, and a lower level of OT in the hippocampus. It was concluded that the peptide

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levels represented genetic differences because 2 months intervened between testing and the postmortem examination. Ferguson et al. (2000) compared a mutant strain of mouse, lacking endogenous OT, with a wild-type mouse strain, normal for this peptide, in two tasks designed to assess spatial learning and memory. The normal performance observed for the OT-mutant mice suggests that this peptide has no important role in memory processing during spatial navigation. This contrasts with evidence that endogenous OT is important for memory modulation in avoidance paradigms (e.g., Bohus et al., 1978b; see Chapter 2) and in olfactory-based recognition of conspeciﬁcs (see Chapter 13). It is interesting to note that, like OT, endogenous AVP has an important role in memory processing tested in avoidance [Kovacs et al., 1979a; Van Wimersma Greidanus et al., 1975a (see Chapter 3); Van Wimersma Greidanus et al., 1975b (see Chapter 4); Veldhuis et al., 1987 (see Chapter 4)] and social recognition (e.g., Dantzer et al., 1988; see Chapter 12) test paradigms, but not in that tested in spatial learning tasks (Engelmann et al., 1992b; Everts and Koolhaas, 1999).

IV. Controversial Issues Concerning Interpretation of the Influence of Peripherally Administered VP on Learning/Memory Tasks

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A. Pressor/Aversive Properties of Peripherally Administered AVP: Essential for VP-Enhanced Memory Processing? De Wied and colleagues take the view that endocrinological effects of the peptide are not essential to the ability of the peptide to facilitate either memory consolidation or retrieval from long-term memory stores (see Chapters 2–5). Koob, Ettenberg, and associates (see Chapter 6) argue that they are essential because they increase arousal level, the mechanism by which the peripherally circulating AVP affects task performance. Although several researchers cited in Chapter 9 have reported positive effects of nonpressor, nonaversive vasopressin analogs on memory processing, several inconsistencies, discussed below, prevent full support for either side of this theoretical controversy. Consistent with the De Wied et al. position, a positive inﬂuence on memory processing was found in several studies that used acute or chronic treatment with nonpressor, nonaversive peptide analogs. Peripherally administered DG-LVP facilitated memory consolidation and retrieval in a conditioned taste aversion task (Vawter and Green, 1980). Chronic but not acute treatment with DDAVP enhanced retention in a shuttlebox avoidance task (Hamburger-Bar et al., 1984). AVP(4–9) improved memory retrieval in

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a task involving social learning related to food preferences, when signiﬁcant forgetting had occurred under control conditions (Bunsey and Strupp, 1990; Strupp et al., 1990). It also facilitated memory consolidation of information learned in a radial maze in subjects exhibiting low but not high task proﬁciency when tested under baseline (nondrug) conditions (Strupp, 1989). Acute, intranasally applied DG-AVP enhanced long-term memory for previously learned abstract words in young, healthy human females (Bruins et al., 1992). However, there were several equivocal ﬁndings concerning the effectiveness of these nonpressor analogs on memory processing. Messing and Sparber (1983, 1985) reported evidence of enhanced retention after DGAVP treatment in an autoshaped operant learning task, but subsequent study by Mundy and Iwamoto (1987) failed to replicate these ﬁndings. Buresova and Skopkova (1982) observed only a transient (nonreplicated) facilitation of short-term memory in the more complex 24-arm radial maze after treatment with the desglycinamide derivative of the d-isomer, an effect attributed to a possible inﬂuence on the subject’s arousal level. Even this ﬁnding of a transient facilitation may not be relevant to the issue because it concerns effects on short-term rather than long-term memory processing.

B. Peripherally Administered VP and the Postulated Arousal–Performance Efficiency Relationship: Relevant Findings For Sahgal and associates (see Chapter 7), vasopressin increases the subject’s basal arousal level and thereby affects a variety of cognitive processes [including short-term memory (STM) as well as long-term memory (LTM)] in accordance with the putative inverted U-shaped arousal–performance efﬁciency relationship (Hebb, 1955; Hebb and Donderi, 1987). It is not the aversive effects per se of a large dose of peripherally administered AVP (LVP), or a smaller dose to overaroused subjects, but the ﬁnal level of arousal that determines the effect on performance. Similarly, low doses of peripherally or centrally administered peptide that give no evidence of discomfort may facilitate, have no effect, or even disrupt retention depending on the resulting arousal level produced by the peptide–basal arousal interaction [for further discussion, see Sahgal (1984) and Chapter 7 in this text]. Sahgal and colleagues (e.g., Sahgal and Wright, 1983, 1984; see Chapter 7) observed a vasopressin-induced bimodal effect in their studies using the passive avoidance paradigm and have interpreted the effect as support for the ‘‘VP Central Arousal Theory.’’ Two studies described in Chapter 9 found depressed locomotor activity after pretraining treatment with AVP (Alescio-Lautier and SoumireuMourat, 1990; Van Haaren et al., 1986). The depressed activity may be inferred as due, in part, to an aversive-induced arousal aspect of the peptide

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treatment (Bluthe et al., 1985a,b; see Chapter 6); as Sahgal has pointed out, depressed behavior (immobility) can sometimes signify increased rather than decreased arousal, as when an animal freezes in extreme fear, and in his experience ‘‘the immobile crouching and staring caused by high doses of VP reﬂects an alert state distinct from the type of sedation seen after treatment with tranquillizers and anesthetics’’ (Sahgal, 1984, p. 223). In addition, male medical student volunteers, pretreated with an intranasally applied 10-IU dose of AVP 24 and 1 h before the behavioral test session, reported more feelings of arousal than did placebo- and OTtreated subjects in their item ratings on the posttest 15-item questionnaire (Fehm-Wolfsdorf et al., 1984). The peptide-induced depressed activity discussed in Chapter 9 was associated with impaired performance in a radial maze task (Van Haaren et al., 1986) and in a go/no-go visual discrimination task (Alescio-Lautier and Soumireu-Mourat, 1990). For the former study, the impairment may have been due to an aversive/arousal effect of the peptide. The performance impairment referred to the inability of the peptide-treated subjects to attain the full complement of rewards in this task because of failure to visit all the baited arms of the maze. On the other hand, there was no evidence of impairment in cognitive processing per se because neither STM nor LTM error rates were greater for the AVP-treated subjects than for the placebo controls. Aside from a lack of general vasopressin-associated cognitive impairment, there was no reported AVP-induced bimodal effect on cognitive performance as would be predicted by the ‘‘VP Central Arousal Theory.’’ Alescio-Lautier and Soumireu-Mourat (1990) did observe a learning impairment after presession AVP treatment on the go/no-go discrimination task, which could have been a result of an AVP-associated aversive/arousal effect. The problem with this interpretation is that the peptide treatment was not consistently disruptive in this study. When presession treatment was administered before any task experience, learning was profoundly disturbed, but when given after some degree of experience with the task the presession treatment produced a bimodal effect, impairing learning in some subjects but enhancing it in others. When given after the completion of learning, the presession treatment enhanced retrieval. Moreover, when posttraining treatment was given at an early phase of learning, the aversive dose of the peptide enhanced memory consolidation, as indicated by improved performance on the subsequent day of training (i.e., 24 h after the treatment). Presuming that the posttraining treatment on day 1 of training was as aversive as the pretraining treatment on day 1, it is difﬁcult to explain how the arousal effect induced by pretraining treatment interfered with cognitive activity (learning) whereas the same effect given posttraining operated retroactively to improve rather than to impair cognitive activity (i.e., enhanced memory consolidation of the learning on day 1). Whereas AVP, administered before the learning session on day 2, may have interacted with the subject’s

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baseline arousal level to produce the bimodal learning, why was this effect not observed after presession treatment on day 1 of training or on the day of retention testing? An ‘‘arousal’’ explanation of these data is less convincing than the interpretation offered by the authors of the study, that is, that vasopressin exerted two effects: an aversive/arousal-inducing effect that interfered with cognitive performance and a memory-enhancing effect, and the effect that prevailed at a given time was determined, in part, by the amount of learning undergone at the time of treatment. Strupp (1989) also observed a bimodal vasopressin effect on memory consolidation that depended on earlier established proﬁciency in task retention under nonpeptide conditions. AVP(4–9) enhanced retention in the least proﬁcient animals and had no effect or impaired it in the highly proﬁcient rats. It is possible, in the context of the ‘‘VP Central Arousal Theory,’’ that the bimodal effect on task performance represented a peptide interaction with the subject’s baseline arousal level. However, the effect is highly unlikely to be due to the pressor/aversive effect-induced heightened arousal as suggested by Koob and associates (see Chapter 6) because the vasopressin metabolite used has neither pressor nor aversive effects. The least and most proﬁcient animals may have differed in behavioral arousal, but lacking an independent measure of this subject variable, it is equally plausible that these subgroups differed in brain levels of endogenous vasopressin released under control conditions, as suggested by Strupp (1989).

V. Methodological Issues: Subject, Treatment, and/or Task Variables May Affect the Outcome or Interpretation of Experimental Studies on VP/Memory Research

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The studies reviewed in Chapter 9 make it clear that discrepant results in the vasopressin/memory-processing research literature (e.g., vasopressin does not always improve performance in learning/memory tasks) were especially prevalent as experimental paradigms went beyond the standard active and passive avoidance tasks employed by De Wied and colleagues (see Chapters 2–5). Several factors potentially responsible for the discrepancies were revealed by previous research cited in Chapters 2–7. Their importance has been conﬁrmed by one or more studies cited in Chapter 9, which have also provided evidence of other determining factors not suggested by the earlier research. These inﬂuences on experimental results in vasopressin/ memory research fall into three major categories: subject variables (e.g., pharmacogenetic factors, age differences, individual differences in task proﬁciency and memory accessibility), treatment variables (e.g., dose- and timedependent treatment effects, acute versus chronic treatment regimens), and

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task variables (e.g., pretraining/training experience in the task, level of task difﬁculty, motivational factors, measures of retention).

A. Subject Variables 1. Pharmacogenetic Factors A pharmacogenetic factor (genetically based endogenous factor inﬂuencing the degree of one’s reactivity to vasopressin) may account for performance differences observed between strains of a given species, as well as for intrastrain variation. A pharmacogenetic inﬂuence may have contributed to the differential effects of AVP on learning and extinction of a shuttlebox avoidance response observed by Hamburger et al. (1985). Thus, depending on the inbred strain(s) of mouse tested, AVP, at the dose level used in this study, either exerted no effect on acquisition or extinction, facilitated acquisition and retarded extinction, or impaired acquisition and had no effect on extinction. A pharmacogenetic inﬂuence may also have been responsible for the higher dose of DG-AVP needed to improve learning in an autoshaping operant task in Long-Evans hooded rats (Messing and Sparber, 1985) compared with Holtzman albino rats (Messing and Sparber, 1983). It may also have contributed, in part, to the discrepant results obtained by Messing and Sparber (1983) and Mundy and Iwamoto (1987), given that the latter research group tested Sprague-Dawley albino rats in their study. A pharmacogenetic inﬂuence accounting for within-strain variation appears to have occurred in the study by Strupp (1989), who observed that Long-Evans male rats differed in which of several dose levels of the AVP(4–8) metabolite was optimal for retention performance in a radial maze task. This inﬂuence may also have contributed to the peptide-induced bimodal effects observed by Alescio-Lautier and Soumireu-Mourat (1990) because the bimodal effect could not be attributable to differences in baseline learning proﬁciency in this study. 2. Age Differences Age differences in susceptibility to vasopressin analogs (Turkington and Everitt, 1976) as well as in the ability to synthesize and secrete vasopressin (Tang and Phillips, 1978) suggest the potential relevance of this subject variable to results obtained in vasopressin/memory studies. Age differences were suggested as a possible contributing factor to the discrepant results obtained by Messing and Sparber (1983) and Mundy and Iwamoto (1987) in their investigations of DG-AVP and learning/retention in the autoshaped touch task. On the other hand, Buresova and Skopkova (1982) found no age-related differences in STM performance in a 24-arm radial maze after treatments with various l- and d-isomer analogs of AVP in a study that used young adult (6 months of age) and middle-aged (19 months of age) male hooded rats of the Drucker strain as subjects.

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3. Individual Differences in Baseline Task Proficiency and in Memory Accessibility The research on vasopressin and selective attention by Beckwith and colleagues (see Chapter 8), and on vasopressin and memory consolidation in a radial maze task by Strupp (1989; see Chapter 9), suggests an interaction between baseline task proﬁciency and vasopressin treatment. Both research groups observed that vasopressin treatment improved cognitive performance in the less proﬁcient subjects to a greater degree than in the more proﬁcient subjects whether these subjects were human college volunteers (Beckwith et al., 1984; Till and Beckwith, 1985) or laboratory rats (Strupp, 1989). Strupp et al. (1990; see Chapter 9) reported an interaction between memory accessibility and vasopressin treatment in a social learning task. The interaction indicated that using a retention interval where signiﬁcant memory occurred in controls, vasopressin enhanced retrieval in subjects exhibiting poor memory, and impaired it in those with excellent memory under nontreatment conditions. It was suggested that the two groups may have differed in levels of endogenous vasopressin released by the task environment during learning.

B. Treatment Variables 1. Dose-Dependent and Time-Dependent Effects Dose- and/or time-dependent effects have been found in numerous studies evaluating the inﬂuence of vasopressin on memory processing. An inverted U-shaped dose–response curve has been observed in studies using rats (e.g., Ader and De Wied, 1972; Bohus et al., 1972; De Wied, 1971; Hagan et al., 1982) as well as mice (e.g., Faiman et al., 1991). In this context, as dose level either increases or decreases from a level that is optimal for learning or memory in a given task, the vasopressin-induced facilitation of performance is correspondingly reduced, and performance may even be impaired. The importance of both this dose–response curve and pharmacogenetic subject factors suggests the value of using more than one dose level in a given study. Time-dependent effects have been observed for both memory consolidation and retrieval in neurohypophysial peptide–memory research studies with rats by De Wied and colleagues (e.g., Bohus et al., 1972, 1978a; Hagan et al., 1982). These studies showed that the greater the delay between vasopressin treatment and the completion of training (consolidation design), or the initiation of retention testing (retrieval design), the less the retention effect. Thus De Wied (1971) found that the inhibitory effect of the peptide on extinction of an active avoidance response was less complete when posttraining treatment was delayed 1 h than when given at zero delay, and

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when delayed for 6 h there was no effect on extinction. Faiman et al. (1991) replicated these ﬁndings with mice as subjects, and observed signiﬁcant retention in a passive avoidance task when posttraining treatment was given immediately or 30 min, but not 3 h, after the learning trial. The time-dependent effects observed for vasopressin treatment in these studies are consistent with the view that the peptide inﬂuences both the consolidation and retrieval stages of memory processing. The absence of tests for time-dependent effects for vasopressin treatment in the appetitive studies described in Chapter 9 indicates the need for this type of study. 2. Acute versus Chronic Treatment Regimens and Long-Term Memory De Wied and associates (see Chapters 2–5) found that vasopressin facilitated retention in multitrial avoidance tasks whether using chronic (two or more treatments, at a single dose level, during acquisition and/or extinction) or acute (single treatment given before or after acquisition or before extinction) treatment regimens. However, Hamburger-Bar et al. (1985) found that, of the two types of regimens, chronic treatment with DDAVP was clearly superior for enhancing retention in both brain-impaired and normal intact rats in a multitrial shuttlebox avoidance task. The ﬁndings by Hamburger-Bar et al. (1985) are interesting for their potential relevance to human clinical research and in accounting for discrepant results in the animal literature. Aside from Hamburger-Bar et al. (1985), none of the studies cited in Chapter 9 directly tested the relative merits of the chronic versus the acute treatment regimen in their vasopressin/memory research studies. Of the researchers who used chronic treatment, two groups reported successful memory-enhancing effects (Messing and Sparber, 1983, 1985; Vawter and Green, 1980), two other groups found successes and failures, depending on the version of the task (Hostetter et al., 1977) or the strain of the animal tested (Hamburger et al., 1985), whereas Mundy and Iwamoto (1987) failed to replicate the positive ﬁndings reported by Messing and Sparber (1983). Of the eight research groups that used a single treatment regimen, most reported that vasopressin facilitated memory consolidation and/or retrieval (e.g., Alescio-Lautier and Soumireu-Mourat, 1990; Boccia et al., 1998; Hagan, 1983; Leshner and Roche, 1977; Mulvey et al., 1988; Roche and Leshner, 1979; Strupp, 1989), whereas one group observed no vasopressin effect on either memory process (Sara et al., 1982). The vasopressin research ﬁndings cited in Chapter 9 do not permit direct comparisons between the beneﬁts of chronic versus acute treatment regimens. Nevertheless, a simple count of success and failures obtained by studies employing one or the other regimen does not unequivocally support the value of chronic to acute treatment as a means of investigating the ability of the peptide to enhance memory consolidation and retrieval. Although

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this point may be applicable to the animal research literature, it does not argue against the value of chronic treatment in clinical trials designed to test the efﬁcacy of the peptide in treating a human learning or memory disorder. Along similar lines, the human research reviewed in Chapter 9 suggests that the treatment regimen and/or dose levels are important treatment factors regarding the OT-induced amnestic effect on memory processing. Thus delayed recall for earlier presented verbal stimuli was impaired in females who were intravenously infused with a high dose of the peptide over a 4- to 8-h period (Ferrier et al., 1980; Kennett et al., 1982), but not in males after one or two intranasal applications of a much lower dose level of the peptide (Bruins et al., 1992; Fehm-Wolfsdorf et al., 1984).

C. Task Variables 1. Amount of Pretraining and Training Experience in the Task One factor inﬂuencing the experimental outcome in vasopressin/memory research is the degree of task-related learning experience obtained before peptide treatment. This includes both pretraining experience as well as the amount of actual learning experience attained during the task. Hostetter et al. (1980) and Rigter (1982) both obtained evidence suggesting that adequate pretraining experience in a single-trial passive avoidance task is important for demonstrating a vasopressin enhancement of retention. The amount of task learning experience appears to interact with the peptide treatment to inﬂuence experimental outcome. De Wied and colleagues (King and de Wied, 1974; see Chapter 2) were ﬁrst to make the point that some minimal degree of associative strength for a given task must be present to demonstrate a vasopressin facilitation of memory consolidation. Data obtained by Alescio-Lautier and Soumireu-Mourat (1990) suggested that some degree of associative strength in the task is especially necessary both to beneﬁt from the memory-enhancing effects and to avoid the learning-disruptive effects associated with aversive dose levels of vasopressin. 2. Level of Task Difficulty Task difﬁculty is another important variable in vasopressin/memory research. Unless the task is sufﬁciently difﬁcult to prevent ceiling effects there may be no room for performance improvement in subjects given vasopressin treatment. Thus, Sara et al. (1982) observed that pretraining vasopressin facilitated learning in a visual discrimination for the more difﬁcult brightnesspositive discrimination, but not for the easier darkness-positive discrimination. Mulvey et al. (1988) used only the brightness-positive discrimination and observed a vasopressin facilitation of memory consolidation.

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3. Motivational Factors a. Need for Sufficient Motivational Incentives to Promote Efficient Learning Detecting the inﬂuence of a drug on memory consolidation and/or retrieval requires that appropriate motivational incentives be in use to promote efﬁcient appetitive or avoidance learning in the placebo control subjects. Although this is typically assumed to be so, the failure of Alescio-Lautier and Soumireu-Mourat (1990) to obtain a VP-induced enhancement of memory consolidation in their mouse subjects was attributed to an insufﬁcient level of footshock intensity necessary to produce efﬁcient passive avoidance learning in this task. b. Arousal Effects and the Interpretation of VP/Memory Research Aversive/ arousal effects associated with the peripheral administration of the pressorinducing peptides AVP and LVP (Bluthe et al., 1985a; Ettenberg et al., 1983a) make it difﬁcult to interpret the ﬁndings of studies that use this route of treatment application. The study by Alescio-Lautier and Soumireu-Mourat (1990) provided evidence suggesting that peripherally applied AVP produces both direct memory-inducing effects as well as arousal-associated inﬂuences on task performance and the sets of effects are difﬁcult to disentangle. Because arousal level has been postulated as the major means by which vasopressin induces its effects on memory processing in both of the theoretical positions that challenge the De Wied et al. theoretical position, it may be preferable to avoid, insofar as is possible, procedures that act to increase baseline arousal level. Thus, it may be preferable to use either a central route of application with the parent peptides, AVP (LVP), or to employ nonpressor, nonaversive vasopressin analogs when using peripheral administration. This latter approach appears to have been adopted by a number of researchers whose studies were presented in Chapter 9 (Hamburger-Bar et al., 1984; Messing and Sparber, 1983, 1985; Mundy and Iwamoto, 1987; Vawter and Green, 1980) and efforts to reduce arousalassociated variables in experimental design were especially adhered to by Strupp, Bunsey, and colleagues in their vasopressin/memory research studies (Bunsey et al., 1990; Strupp, 1989; Strupp et al., 1990). Parenthetically, it is notable that Skopkova et al. (1991; see Chapter 3) showed that even when a subcutaneously administered nonpressor, nonaversive VP analog (DG-AVP) increased the baseline arousal level of preselected ‘‘excitable’’ subjects to an extent that proved detrimental to learning performance, the peptide nevertheless facilitated long-term memory storage.

D. Measures of Retention Reentry latency is typically used as a measure of retention in a passive avoidance (PA) paradigm, but Sahgal and colleagues have questioned its use as the sole measure of this variable. These researchers included response

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choice along with reentry latency in several of their studies on PA behavior and argued that response choice may be a preferable measure in vasopressin/ memory research study (Sahgal and Wright, 1984; and see Chapter 7). In support of this view, Alescio-Lautier and Soumireu-Mourat (1990) provided data suggesting that reentry latency may not be a valid measure in studies of memory retrieval because a dose of the peptide that produces aversive as well as memory-enhancing effects may induce a hypoactive state (increased latency) erroneously interpreted as a positive retention effect. These lines of evidence, together with that of Koob and associates (e.g., Bluthe et al., 1985a,b; Ettenberg et al., 1983a; see Chapter 6), indicating the aversive nature of peripherally administered AVP, suggest the desirability of including an alternative test of PA retention (e.g., choice behavior) and/or an independent measure of the effect of vasopressin on general activity in future tests of the effect of the parent peptide on memory retrieval. Resistance to extinction is often used as a measure of retention in behavioral pharmacology. However, it can be argued that this variable may reﬂect not only the strength of previous task learning but also the rate of new learning that occurs during successive extinction trials. In an aversive motivational task the subject experiences the new contingency only when it fails to make the learned avoidance response, suggesting that increased resistance to extinction may be a fair measure of memory for the previous learning. However, in appetitive tasks each extinction trial informs the subject of the changed response–reinforcement contingency and thus it is debatable whether increased resistance to extinction should be interpreted as reﬂecting strong memory of the previously learned contingency, or impaired learning of the new contingency. It has been argued that vasopressin treatment given before extinction in an appetitive task should facilitate extinction of the appetitive response if the peptide improves memory consolidation of learning occurring during the extinction procedure (Packard and Ettenberg, 1985). A number of appetitive studies summarized in Chapter 9 used extinction as a measure of retention. Mulvey et al. (1988) found that LVP increased retention in a visual discrimination task when assessed by resistance to extinction and also when assessed by the savings method. This argues in favor of resistance to extinction as a valid measure of retention in an appetitive task. However, Mundy and Iwamato (1987) reached the opposite conclusion when they observed that the rate of extinction failed to increase as predicted, after a second cycle of acquisition/extinction in an autoshaped lever touch task.

E. Methodological Issues: Conclusions The numerous subject-, treatment-, and task-related variables that have been observed to inﬂuence the outcome of vasopressin/memory studies permit an appreciation of the difﬁculties involved in comparing

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studies differing in one or more of these variables and hence of resolving the discrepancies that occur in this literature. An increasing knowledge of the speciﬁc ways in which these variables operate on their own or through their interactions with each other may provide insights that help to increase our understanding of the role of vasopressin in memory processing and, additionally, help guide our choice of paradigms and design of vasopressin/memory research for future study. Although the issues raised and the relevant evidence cited in this section have been limited to studies with vasopressin, it is undoubtedly the case that the conclusions drawn from this discussion are equally applicable to memory-processing research with oxytocin.

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Part VI

Research Contributions of Dantzer, Bluthe, and Colleagues to the Study of the Role of Vasopressin in Olfactory-Based Social Recognition Memory

I. Overview

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The studies reviewed in this chapter represent a later phase in the vasopressin/memory-processing research of Koob and associates, with Dantzer, Bluthe, and colleagues at Neurobiologie Integrative in Bordeaux, France playing a key role in directing the course of this research. The De Wied–Koob controversy was difﬁcult to resolve because, as Dantzer subsequently noted, ‘‘the acquisition and extinction of active and passive avoidance responding, which were commonly used for assaying the memory-enhancing effects of vasopressin, were inevitably contaminated by performance factors such as arousal and emotionality’’ (Dantzer, 1998, p. 409). To limit these problems, these researchers chose a species-typical type of rodent social memory for study. Speciﬁcally, they examined the putative roles of vasopressin, and on occasion oxytocin, in olfactory-based rodent social recognition, employing a paradigm originally used by Thor Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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and Holloway (1982). It has long been appreciated that numerous rodent species, including the Norway rat, recognize members of their colony by the individually speciﬁc olfactory cues that identify each member of the colony (Carr et al., 1976), probably through numerous investigative encounters with them. In their early studies these researchers investigated effects of peripherally administered vasopressin (VP) and oxytocin (OT), and of centrally administered VP, on this form of social memory. Comparisons were made between these results and those obtained from study of similarly treated rats in conventionally used avoidance/appetitive paradigms. In subsequent studies they focused more speciﬁcally on the role played by the sexually dimorphic extrahypothalamic VP circuitry in the social memory paradigm. The ‘‘VP Dual Action Theory’’ derived from research study of the role of VP in long-term memory tested in various types of appetitive and avoidance learning situations (see Chapter 6). Its relevance for the species-typical form of memory investigated in the studies presented in this chapter is discussed in later sections of this chapter.

II. The Olfactory-Based Social Recognition Memory Task

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The olfactory-based social recognition memory (SRM) task developed for assessing conspeciﬁc social recognition permits the subject to encounter a conspeciﬁc stranger juvenile for a 5-min exposure period during which active investigation of the juvenile, characterized by close following and close snifﬁng of the anogenital area, provides the odor cues for forming the transient memory that allows subsequent recognition. The use of a juvenile avoids the intrusion of sexual and aggressive provocation by the test animal. Normally, the adult can retain memory for the conspeciﬁc for less than 1 h (30 to 60 min) after the single-exposure trial. The procedure employed by Dantzer, Bluthe, Koob, and associates is similar to that initially used by Thor and Holloway (1982). The rats are tested in their home cages in an experimental room equipped with the same light cycle as the home room and with a background white noise; testing occurs in the dark (active) portion of the diurnal cycle. A video camera, equipped with an infrared spotlight, records the behavior of the animal throughout the communicative interchange. Social recognition is exhibited when the duration of investigating a preencountered juvenile is signiﬁcantly less than that observed during the original encounter. Comparing the investigatory behavior directed toward a familiar versus a novel juvenile during the second encounter can help rule out nonmemorial factors (e.g., adaptation and satiation effects) as explanations for the shorter duration of time spent investigating the familiar

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juvenile. Experimental treatments are given within 1 min of the initial 5-min encounter with the juvenile. Thus, the ability of AVP to prolong the retention interval (e.g., from 1 to 2 h) is interpreted as indicative of the ability of the peptide to facilitate memory storage in this task.

III. Research Findings

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A. Effects of Peripherally and Centrally Circulating VP and OT on Conspecific SRM 1. Selected Studies The two studies described below tested the validity and reliability of this behavior paradigm for the study of memory in behavior pharmacology and demonstrated that the ﬁndings relating peripherally injected vasopressin to this form of memory are similar to those observed in tasks of long-term memory. a. Dantzer et al. (1987) Dantzer et al. (1987) used the olfactory-based social memory task to accomplish two main objectives: (1) to determine whether this memory could be modulated by retroactive facilitation and inhibition induced by an interpolated experience with the same or a different test juvenile as the one investigated in the initial encounter; and (2) to determine the effect on social recognition of AVP, OT, and the antagonist 1-deaminopenicillamine-2-(O-methyl)tyrosine-arginine vasopressin (i.e., [dPTyr(Me)]AVP), given on its own or in conjunction with AVP. These treatments were administered just after the initial investigative encounter. The ﬁrst objective was accomplished by presenting a second juvenile test stimulus for a second 5-min exposure period, 5 min after completion of the initial 5-min encounter with the original juvenile. For the retroactive facilitation condition, the same juvenile was presented for both exposure periods and the retention test was given 120 min after the original encounter (a time when the original encounter is normally forgotten). For the retroactive inhibition condition, a different juvenile was presented during the second exposure period and the retention test was given 30 min after the original encounter (a time when the original encounter is normally remembered). Control groups (retroactive nonfacilitation and noninterference conditions) were tested for retention at comparable times but received no interpolating experience with a juvenile test animal. The results demonstrated that an interpolated experience with the appropriate stimulus animal produced the expected retroactive facilitatory and inhibitory effect on the duration of social memory for the originally encountered juvenile. Memory was facilitated (exhibited in the 120-min retention test) when the same juvenile was presented during the second exposure period, and impaired (memory absent in the 30-min retention test) when a different juvenile was presented during the second exposure period.

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The second objective was accomplished by peripheral injection of each of the following treatments to independent groups of subjects: AVP (0 or 6 g/kg, subcutaneous); OT (0 or 6 g/kg, subcutaneous), and the VP antagonist [dPTyr(Me)]AVP (0 or 30 g/kg, subcutaneous). These treatments were given alone, or in the case of testing for an antagonist–agonist interaction effect, an injection of the AVP antagonist was injected 5 min before AVP (experimental condition) or saline (control condition) (i.e., [dPTyr(Me)]AVP–AVP or [dPTyr(Me)]AVP–saline). Treatments were given 1 min after the initial encounter. Retroactive facilitation of social memory after AVP treatment, as well as the ability of the antagonist to block this effect, were tested after a retention interval of 120 min; the OT- and [dPTyr(Me)]AVP-treated rats were tested for their ability to retroactively inhibit memory storage after a retention interval of 30 min. The possibility that a reduced time of investigation during the retention test could signify toxicity rather than memory effects was evaluated by control tests in which a novel juvenile stimulus was presented in the retention test. The results indicated that (1) AVP retroactively facilitated social memory, and this effect was blocked by pretreatment with the V1 pressor antagonist [dPTyr(Me)]AVP. Thus, unlike the saline controls, the subjects treated with AVP after the initial social exposure period spent signiﬁcantly less investigative time with the original juvenile in the 120-min retention test then during the initial encounter; (2) OT and the V1 antagonist, each on its own, retroactively inhibited social memory. Thus, the subjects given postencounter injections of OT or [dPTyr(Me)]AVP failed to exhibit a signiﬁcant reduction of investigation time characteristic of saline controls in the 30-min retention test; and (3) the toxicity test indicated no reduction in the duration of time spent investigating a novel juvenile stimulus in the 120-min retention test. Thus, the retroactive facilitation effect of AVP treatment could not be attributed to a nonspeciﬁc decrement in performance. The authors made the following points in discussing these results. First, the demonstration that the persistence of investigation observed during the retention test is subject to the effects of retroactive facilitation and inhibition further supports the hypothesis that the behavior tested in this paradigm is a function of memory for the initial social encounter. Second, in the retroactive enhancement procedure, AVP facilitated memory at a dose level found to have mnemonic effects on various aversive and appetitive tasks of longterm memory (see Chapter 6). Moreover, the ability of the V1 antagonist [dPTyr(Me)]AVP to reverse the memory-facilitatory action of peripherally administered AVP on transient memory tested in the SRM task is consistent with that reported for long-term memory tested in aversive conditioning tasks (see Chapter 6) and represents an extension to an appetitive memory paradigm. Third, the retroactive interfering effects of OT treatment are consistent with previous research reports that posttraining OT impairs

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memory in aversive conditioning tasks (see Chapters 2–5). Fourth, the observation that the effects of the antagonist, injected on its own, produces effects opposite to those of AVP suggests involvement of endogenous vasopressin receptors in this form of memory. However, because this antagonist can presumably cross the blood–brain barrier (Koob et al., 1985b) the locations of these receptors were not illuminated by this study. b. Le Moal et al. (1987) Le Moal et al. (1987) tested the effects of centrally (intracerebroventricularly) administered AVP on conspeciﬁc social recognition in adult male Wistar rats with prior breeding experience. Preliminary testing subjected an independent group of rats to ﬁve sessions, one per day, using retention intervals of 5, 10, 30, 120, and 120 min. This testing was done to make sure that normal recognition occurred after a retention interval of 30 min or less and that no recognition occurred after the 120-min retention interval. Treatment sessions (i.e., ﬁve sessions) began 2 days later with an intersession delay of 2 days. Each subject received physiological saline or AVP at each of four dose levels (0.25, 0.50, 1.0, and 2.0 ng/rat, intracerebroventricular) immediately after the initial encounter. The order of treatments over the ﬁve treatment sessions was as follows: half the rats received saline on day 1 and the 0.5-ng dose of AVP on day 3; the remaining half received these treatments in the reverse order. The same dose schedule was used for the 1.0and 2.0-ng doses of AVP on days 5 and 7. All the subjects were given the 0.25-ng dose of AVP on day 9. The results were computed for each daily session so that the investigative time for the retention test (second encounter) was compared with that observed for the initial encounter. Comparisons were also made between each of the treatment conditions for the investigative time spent in the retention test. To test for possible motor activation effects after these treatments, separate groups of rats were tested in photocell activity cages during the light or dark phase of the diurnal activity cycle. AVP (0, 0.5, 1.0, or 2.0 ng/rat, intracerebroventricular) was administered immediately after a 90-min habituation period in the activity cage, after which the subject was replaced in the cage where locomotor activity was monitored every 5 min for the subsequent 2 h. The results obtained during preliminary testing indicated that nontreated subjects exhibited social recognition for all intervals of 30 min or less (i.e., investigative activity was of signiﬁcantly shorter duration than that for the initial encounter). On the other hand, when tested after a retention interval of 120 min, there was no evidence of memory for the juvenile, because the time spent in social investigation was even longer than that for the original encounter. Centrally injected AVP facilitated memory in this task for each dose level except for the lowest dose of 0.25 ng/rat. Thus, in contrast to saline controls, those subjects that received effective dose levels

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of the peptide (i.e., 0.5, 1.0, or 2.0 ng/rat, intracerebroventricular) exhibited a signiﬁcantly shorter duration of investigative activity after the 120-min retention interval than during the initial encounter. Moreover, the AVP-induced facilitation was dose dependent, in accordance with an inverted U-shaped dose–response curve, with the 1-ng dose being most effective. None of these dose levels signiﬁcantly inﬂuenced locomotor activity level for either the dark (active) or light (inactive) phase of the diurnal activity cycle. In discussing these ﬁndings, these investigators noted that the efﬁcacy of the 1-ng dose level for this ethologically relevant transient memory has also been observed for long-term memory assessed in the pole-jump avoidance task (Koob et al., 1981; see Chapter 6) and for the VP-induced changes in electroencephalograpic (EEG) activity (Ehlers et al., 1985; see Chapter 6). Moreover, the highly effective 1-ng dose level of the peptide demonstrates neither aversive nor pressor effects (Koob et al., 1986; see Chapter 6). These researchers concluded that this behavioral paradigm is a useful and reliable tool for the study of learning and memory in behavioral pharmacological paradigms, and that increasing vasopressin levels in the brain can improve this form of transient memory.

B. Brain Sites Involved in VP-Mediated Olfactory-Based SRM 1. Selected Study: Dantzer et al. (1988) Having established a role for central VP-ergic circuitry in this form of rodent social memory, these researchers turned their attention to seeking more information about the brain sites involved. Dantzer et al. (1988) microinjected AVP and a hydrophilic V1 receptor antagonist into the septal nucleus of adult male Wistar rats and determined the potential involvement of this brain site in AVP-mediated conspeciﬁc recognition. The V1 receptor antagonist desGly-NH2,d(CH2)5[Tyr(Me)]AVP was selected for study because its hydrophilic nature makes it extremely unlikely that it can cross the blood–brain barrier to inﬂuence peripheral V1 receptors. The septal nucleus was chosen for study because it receives VP-ergic terminals (De Vries and Buijs, 1983) and contains an abundance of V1-binding sites (Baskin et al., 1983). In addition, experimental study has demonstrated that AVP, microinjected into this structure, modulates the response of septal neurons to other transmitter inputs (Joels and Urban, 1984a; see Chapter 5) and also inﬂuences catecholaminergic neuronal activity and retention in an active avoidance task (Kovacs et al., 1979a; see Chapter 4). The subjects were given 2 weeks of initial training to familiarize them with the task requirements and to adapt them to the handling involved in the microinjection procedure. Treatment sessions then began and were carried out at 3- to 4-day intervals. Treatments were given immediately after the 5-min initial social encounter and each subject received all treatments in the

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following sequence: AVP (0, 0.01, and 0.1 ng in random order and with a 120-min retention interval), the V1 antagonist (0, 0.5, and 5 ng in random order and with a 30-min retention interval), and a 0.1-ng dose of AVP (with a 120-min retention interval and a different juvenile for retention testing than the one presented during the initial exposure interval). The results were as follows: (1) AVP at the 0.1-ng, but not the 0.01-ng, dose level facilitated social recognition (i.e., reduced investigative time compared with the original encounter) in the 120-min retention test; (2) this behavioral effect could not be attributed to nonspeciﬁc effects on activity level because there was no interference with the normally occurring investigation of a different juvenile presented at the 120-min retention test interval; and (3) the V1 antagonist prevented the social recognition that was observed in the saline control condition in the 30-min retention test; neither the low nor high dose of this peptide produced a signiﬁcant reduction in investigative activity during the second encounter, and, in fact, the higher dose level resulted in a signiﬁcant increase in this activity. The primary ﬁndings of this study indicated that elevating septal vasopressin levels immediately after the original social encounter enhanced the formation of social memory, whereas blocking septal V1 receptors at this time prevented normal expression of this memory. Taken together, these ﬁndings were interpreted as suggesting that ‘‘endogenous septal AVP is physiologically involved in the regulation of social memory in male rats’’ (Dantzer et al., 1988, p. 146). The authors further suggested that septal vasopressin circuitry may mediate the effects of androgens in conspeciﬁc recognition because VPergic projections from the bed nucleus of the stria terminalis to the septal nucleus in the brain of male rats is androgen dependent. This is indicated by the observation that vasopressin present in this circuitry is reduced by castration and restored by testosterone replacement therapy (De Vries et al., 1984). Thus, the intraseptal injection of the V1 receptor antagonist may produce the same effect as that induced by castration (i.e., a reduction of AVP neurotransmission in this circuitry).

C. Central Circuitry Mediating the Effect of VP on Olfactory-Based SRM Is Androgen Dependent 1. Selected Studies The three studies described below established the androgen dependency of the central vasopressinergic circuitry involved in mediating the memory assessed in this olfactory-based conspeciﬁc recognition task. a. Bluthe et al. (1990) Bluthe et al. (1990) designed several experiments to test possible interactions between androgens and central vasopressin in social recognition. The rationale for this study was 2-fold and based on the

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ﬁndings discussed above. First, participation of central VP-ergic circuitry in olfactory-based social recognition in the male rat is suggested by the observation that the peripherally injected antagonist [dPTyr(Me)]AVP, on its own, impaired recognition (Dantzer et al., 1987) whereas an intraseptal microinjection of AVP improved it (Dantzer et al., 1988). Second, the possibility that the participant VP-ergic circuitry is androgen dependent is suggested by the observations that (1) some vasopressin-containing central neurons are androgen dependent, such as those projecting from the bed nucleus of the stria terminalis to the septal nucleus (De Vries et al., 1984), and (2) castrated male rats, depleted of androgen, are unable to use olfactory cues to recognize a juvenile conspeciﬁc (Sawyer et al., 1984). Experiment 1 tested the effects of castration on social recognition. In an initial test, comparisons between castrated and sham-operated rats were made in the social recognition test on three occasions: 1 week before castration, and then 1 and 2 weeks after castration. Social recognition was normal before surgery, was temporarily disrupted when tested 1 week after surgery, and was again normal 2 weeks after surgery. A second test provided experience in the task every other day after surgery (i.e., tested 1, 3, and 7 days after surgery). Under these test conditions, social recognition was normal 1 week after surgery. The authors interpreted the results as indicating that the decline in testosterone that follows castration reduced the androgendependent vasopressin that normally contributes to social memory in the intact male rat. The temporary disruption in social memory was viewed as suggestive of ‘‘a gradual shift from a vasopressinergic mediated neurotransmission to a non-vasopressinergic one in the neural circuit(s) involved in social recognition, following the drop in plasma testosterone levels due to castration’’ (Bluthe et al., 1990, p. 156). Presumably this shift is accelerated with intervening experience, as indicated by the progressive improvement observed when the animals were tested on postsurgery days 1, 3, and 7. Experiment 2 assessed the sensitivity of castrated rats to the memoryblocking effects of the peripherally administered V1 antagonist [dPTyr (Me)]AVP. Intact male rats and castrated rats were injected with saline or the antagonist at a dose (30 g/kg, subcutaneous) that has been shown to block recognition in intact male rats (Dantzer et al., 1987). The results indicated that the V1 antagonist, given alone, blocked social recognition in the 30-min retention test in sexually intact, but not in castrated, male rats. This result is consistent with the interpretation that androgen-dependent vasopressin circuitry plays a signiﬁcant role in mediating social recognition in sexually intact adult male rats, but not in castrated male rats. Experiment 3 was designed to learn whether the failure of the antagonist to block social recognition in castrated male rats was due to castrationinduced interference with the pharmacological properties of the antagonist. Accordingly, castrated rats were tested for their sensitivity to the memoryfacilitating effect of peripherally injected AVP (6 g/kg, subcutaneous) on

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this social memory task and to its reversal by the V1 antagonist [dPTyr (Me)]AVP (30 g/kg, subcutaneous). Each rat served as its own control and received all treatments (i.e., saline–saline, saline–AVP, and V1 antagonist– AVP) in randomized order. Treatment was given immediately after the ﬁrst exposure to the juvenile and the retention test was given 3 h later (this retention interval was selected because the social memory of castrated male rats is longer than that of intact male rats). AVP facilitated social recognition, and this effect was blocked by pretreatment with the V1 pressor antagonist. It was concluded that castration does not interfere with the ability of peripherally administered AVP to facilitate social recognition or with the pharmacological properties of the antagonist that block the AVPinduced social recognition effect in male castrates just as it does in intact male rats (Dantzer et al., 1987, cited earlier). Experiment 4 tested the effects of testosterone replacement therapy on the lack of sensitivity exhibited by the male castrates to the memoryblocking effect of the V1 antagonist. Nine castrated male rats were implanted with a testosterone-ﬁlled capsule 3 weeks after castration. Controls included 10 castrated males and 10 sexually intact male rats, which received an empty capsule. Responses to the V1 antagonist (30 g/kg, subcutaneous) were assessed 16 days after implantation of the capsule. The V1 antagonist, on its own, prevented social recognition in the 30-min retention test in the intact, but not in the castrated, male rats. Testosterone replacement therapy restored male castrate sensitivity to the V1 antagonist. These results replicated the insensitivity of the male castrate to the memoryblocking effects of this V1 antagonist, and demonstrated its reversal by testosterone replacement therapy. Finally, the results of an immunohistochemical analysis, carried out after completion of behavioral testing, indicated that vasopressin-positive ﬁbers were markedly reduced in the lateral septum in all castrated animals compared with intact controls. Other brain areas, such as the horizontal limb of the diagonal band of Broca and the centromedial nucleus of the thalamus, also exhibited decreased VP-ergic ﬁber density (i.e., AVP content was absent in many of the VP-containing neuronal ﬁbers normally present in the area). Changes in ﬁber density were not observed in those hypothalamic nuclei known to contain VP-ergic cells that are engaged in secreting hormonal AVP, controlling adrenocorticotropic hormone (ACTH) release, or inﬂuencing autonomic nervous system (ANS) centers in the brainstem and spinal cord. Nor were changes in VP-ergic density observed in the amygdala. Taken together, the results of these experiments were interpreted as supporting the proposition that androgen-dependent VP-ergic neurotransmission is operative in the central circuits that play a role in conspeciﬁc social recognition in the mature intact male rat. The observation that peripherally administered AVP facilitates conspeciﬁc memory and that this effect is reversed after pretreatment with the V1

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antagonist in both intact and castrated male rats suggests that the peripheral mechanism contributing to social recognition is the same as that responsible for other forms of memory (i.e., the pressor/aversive induced arousal effect). However, the validity of this generalization requires further study involving, for example, the agonist–antagonist interactional tests carried out for the learning/memory tasks presented in Chapter 6. b. Bluthe and Dantzer (1990) Bluthe and Dantzer (1990) performed two experiments to determine whether there are sex differences in performance of the olfactory-based social recognition task and, if so, whether sexually dimorphic VP-ergic circuitry contributes to this difference. Adult male and female Sprague-Dawley rats served as subjects in this study. The ﬁrst experiment compared males and females in task performance. The second experiment tested the effects on female task performance of peripherally administered AVP and the V1 receptor antagonist [dPTyr(Me)]AVP. In experiment 1, preliminary testing permitted selection of retention intervals sufﬁciently short to ensure memory, and sufﬁciently long to ensure forgetting, of the preencountered juvenile. On the basis of this testing, the following retention intervals were chosen for this task: 5, 30, and 120 min for the males, and 30, 120, and 180 min for the females. Exposure to a juvenile other than the one presented during the original encounter provided the control condition necessary to reduce the likelihood of misinterpreting a nonspeciﬁc drug effect for an inﬂuence on retention behavior. The results indicated that (1) social recognition occurred after retention intervals of 5 and 30 min for the male rats and after retention intervals of 30 and 120 min for the female rats. Thus, at these time intervals the duration of social investigation of the reencountered juvenile was signiﬁcantly decreased in comparison with that of the different juvenile; and (2) male/female differences occurred with respect to both the persistence of the original social investigation and the duration of the olfactory memory. That is, in comparison with the mature male, the female was less persistent (i.e., spent less time) in investigating the juvenile during the original encounter but exhibited a more durable memory for the juvenile (i.e., lasted for 120 min in the female rat, and for 30 min in the male rat). Experiment 2 tested the effects on social recognition of peripherally administered AVP (0 or 6.0 g/kg, subcutaneous) and of the peripherally administered V1 antagonist [dPTyr(Me)]AVP) (0 or 30 g/kg, subcutaneous) in a group of naive female subjects. This antagonist, which can presumably cross the blood–brain barrier and act centrally (Le Moal et al., 1982; see Chapter 6), was tested for its ability to block the potential contribution of endogenous central vasopressin to this form of memory processing. These peptides were injected immediately after the initial 5-min exposure period. The retention test, given 30 min after the initial encounter, tested the potential memory-blocking effect of the V1 antagonist, and 180 min after

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this encounter test, the memory-facilitating effect of AVP. During the retention test, the subject was presented with either the same or a different juvenile; the latter condition evaluated nonspeciﬁc pharmacological effects of the peptide. The results were as follows: (1) relative to the saline treatment condition, AVP signiﬁcantly reduced the time spent investigating the reencountered juvenile in the 180-min retention test. Thus, peripherally injected AVP facilitated social recognition in the female as it does in the mature male rat (Dantzer et al., 1987); (2) the AVP treatment effect was not due to nonspeciﬁc pharmacological effects because the peptide-treated rats did not differ from saline controls in their investigation of the stranger juvenile in the 180-min retention test; and (3) on its own, the 30-g/kg dose of the peripherally injected V1 receptor antagonist did not block the memory for the reencountered juvenile in the 30-min retention test. The authors drew three major conclusions from these ﬁndings. First, male and female rats differ in their patterns of performance in this conspeciﬁc memory task; in contrast to the mature male rat, the mature female rat shows less persistence (attention, motivation) during social investigation of a juvenile stranger but exhibits a more durable memory of the preencountered test animal. The authors cited evidence indicating that the reduced persistence in the initial investigation shown by the female, relative to the male, is hormonally related because androgenized females treated with testosterone as adults investigated a novel juvenile conspeciﬁc as long as did the sexually intact mature male (Thor et al., 1982). Second, peripherally administered AVP enhances social recognition in the female as it does for both the intact and castrated male rat (Bluthe et al., 1990), and this is purportedly induced by the same peripheral mechanisms that inﬂuence memory formation in traditional avoidance and appetitive motivational tasks (i.e., an increase in arousal level induced by pressor/ aversive properties of the peptide). Third, in contrast to the mature male, central vasopressin circuitry is not involved in social recognition in the mature female rat. This conclusion was based on the ﬁnding that the V1 antagonist, on its own, peripherally administered at a dose of 30 g/kg, failed to interfere with social recognition in the 30-min retention test. Supporting this conclusion are the observations that (1) the antagonist can cross the blood–brain barrier because the peripherally administered AVP antagonist at this dose level blocks the effects of intracerebroventricularly administered AVP (Koob et al., 1986; see Chapter 6); and (2) peripheral injection of this V1 antagonist, at the same 30-g/kg dose level, blocks social memory in the sexually intact male but not in the male castrate (Bluthe et al., 1990). Proceeding further, it was hypothesized that the failure of endogenous AVP to contribute to social recognition in the female rat is related to the sexual dimorphism that characterizes central VPergic neural circuitry (Van Leeuwen et al., 1985). Thus, the female brain may

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use the same neuronal circuitry but with different transmitters, or different neuronal circuitry when processing and forming an olfactory-based social memory. c. Bluthe and Dantzer (1992) Bluthe and Dantzer (1992) further studied the role of androgen-dependent brain VP-ergic transmission in the modulation of social recognition in the rat. Whereas earlier study of the role of central vasopressin in this behavioral paradigm used acute injections of AVP and its antagonist, this study altered brain levels of AVP over a long period by implanting a device that delivered the relevant peptide directly into the cerebrospinal ﬂuid (CSF) at a constant rate without disturbing the normal ﬂow of CSF. AVP was chronically administered to male castrates, and the V1 antagonist [dPTyr(Me)]AVP was administered to sexually intact male rats. These treatments were evaluated for their effects on the duration of social investigation of a juvenile stranger as well as for their ability to inﬂuence the duration of olfactory-based social memory. The peptide was delivered into the CSF by means of an implanted Accurel minidevice, at a constant rate over a 3-week period (see Boer et al., 1984 for further information about the minidevice). For both the AVP and antagonist treatment groups, half the rats in each group received the experimental treatment (i.e., the device was loaded with AVP or the antagonist), and half received the placebo (i.e., the device was loaded with apyrogenic water). Each subject was tested for social recognition before implantation of the minidevice and again 3 weeks after recovery from implantation surgery. Before implantation, the subjects were tested on day 1 with a 30-min retention interval, and on day 2 with a 120-min retention interval. After implantation, the tests using 30- and 120-min retention intervals were separated by 1 week. The results were as follows: (1) before implantation, the duration of juvenile investigation during the initial encounter was signiﬁcantly longer in the intact males than in the castrates; chronic treatment with the centrally administered antagonist shortened this value in intact rats whereas treatment with centrally administered AVP increased it in the male castrates; (2) both the intact and castrated male rats implanted with the water-ﬁlled device exhibited memory for the familiar juvenile on the 30-min retention test, but only the castrates recognized the juvenile test animal 120 min after the original encounter; and (3) chronic exposure to their respective treatments reversed the recognition patterns displayed by the castrated and intact male rats. That is, the AVP-treated castrates no longer recognized the juvenile 120 min after the initial encounter, whereas the antagonist-treated intact males demonstrated memory for the juvenile at this retention interval. In summary, these ﬁndings indicated that chronic central administration of AVP to androgen-depleted male castrates restored behavior patterns observed in the sexually intact male in this behavioral paradigm (i.e., increased persistence with which a juvenile stranger is investigated and formation of a

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transient memory for the juvenile, exhibited in the 30-min, but not the 120-min, retention test). Conversely, chronic central administration of the antagonist to the sexually intact male rat resulted in a behavior pattern typical of the male castrate (i.e., decreased persistence in the initial investigation of the juvenile but a more durable memory for the juvenile). Along with earlier ﬁndings (Bluthe et al., 1990; Dantzer et al., 1988), the results of this study are further supportive of a role for androgen-dependent VP-ergic circuitry in olfactory-based social memory in the intact but not the castrated male rat.

D. The Vomeronasal System and VP-ergically Modulated Olfactory-Based SRM 1. Introductory Remarks The study described below demonstrated the essential participation of the vomeronasal organ (VNO) in juvenile recognition by the sexually intact male rat. The VNO is accessory to the main olfactory system and processes nonvolatile chemoceptive stimuli such as sex pheromones. The VNO has its own segregated projection system to the brain; it projects to the accessory olfactory bulb (AOB) and from there to the medial and posteromedial nucleus of the amygdala with a minor input to the bed nucleus of the stria terminalis (BNST). The medial nucleus of the amygdala, in turn, sends massive projections to the BNST and the medial preoptic area (MPOA). Moreover, the vomeronasal system (VNO, AOB, and its limbic system projections) is sexually dimorphic (larger in male than in female rats), and this dimorphism is androgen dependent for its development and maintenance (Dantzer and Bluthe, 1993; Madeira and Lieberman, 1995). One known function of this system is to mediate the inﬂuence of chemoceptive stimuli over reproductive behavior (Simerly, 1990). This fact is consistent with the pathway’s projection to the MPOA, a major hypothalamic area involved in mediating reproductive behavior. 2. Selected Study: Bluthe and Dantzer (1993) Bluthe and Dantzer (1993) designed two experiments to assess the role of the VNO in the dependence of olfactory-based social recognition in VPergic transmission in the adult male rat. Participation of the VNO in processing nonvolatile chemosensory cues that identify a juvenile is suggested by characteristics of the subject’s investigative behavior during the initial social encounter (e.g., close physical contact, licking the anogenital area, lack of snifﬁng at a distance, etc.) (Sawyer et al., 1984). Linkage of the VNO to the androgen-dependent VP-ergic circuitry is suggested by the observation that the vomeronasal pathway is sexually dimorphic (larger in males than in females), is modulated by circulating gonadal hormones, and includes as

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two of its components the bed nucleus of the stria terminalis (BNST) and the medial amygdala (MA), which in turn contain androgen-dependent VP-ergic neurons (De Vries et al., 1984; Simerly, 1990). The ﬁrst experiment tested the inﬂuence of androgen-dependent VPergic and VNO processing on social recognition. Social recognition was tested before and after castration or surgery involving the VNO (i.e., sham operation or surgical removal of the VNO). The second experiment tested the memory-blocking effect of the V1 antagonist [dPTyr(Me)]AVP in castrated and VNO-ablated animals. For each of the two experiments, adult male Wistar rats were randomly assigned to one of the following treatment groups: castrated, VNO-X (surgical removal of the VNO), and VNO-sham (subjected to the initial surgical procedure but without removal of the VNO). In experiment 1, the subjects were tested in the social recognition task on each of four occasions: 8 days before surgery (VNO-X, VNOsham, or castration) as well as 8, 14, and 21 days after this surgery. For each of the four occasions, social recognition was evaluated by comparing the duration of juvenile investigation after the 30-min retention interval with that occurring during the initial encounter. The results were as follows: (1) the VNO-sham operates exhibited normal social recognition on each of the four test days; (2) social recognition in the VNO-X and castrate groups was normal 8 days before surgery, impaired 8 days after surgery, and once again normal 14 and 21 days after surgery; moreover, the temporary impairment in social recognition observed on postsurgery day 8 could not be attributed to postsurgery debilitation because both the VNO-X and male castrates exhibited normal investigative behavior during the original encounter on that day; (3) compared with VNO-sham animals, the time spent in original investigation of the juvenile was reduced on postsurgery days 14 and 21 for castrated rats and on postsurgery day 21 for VNO-X rats; and (4) testosterone levels in the plasma did not differ between the VNO-sham and VNO-X rats, but were not detectable in the castrates. In experiment 2, the VNO-X, VNO-sham, and castrated rats were tested in this behavioral paradigm before surgery and again 3 weeks after surgery. Both before and after surgery the subjects were tested on two occasions separated by a 3-day interval: once after treatment with physiological saline and again after treatment with the antagonist, [dPTyr (Me)]AVP (30 g/kg, subcutaneous). The treatments were given in a counterbalanced order, with half the subjects in each experimental group receiving the saline ﬁrst, and the remainder receiving the antagonist ﬁrst. The subjects were injected immediately after the initial encounter and tested 30 min later for social memory. The results of experiment 2 indicated that the VNO-sham rats exhibited normal sensitivity to the memory-blocking effects of the antagonist in the 30-min retention test both before and subsequent to surgery. On the other hand, the castrates and the VNO-X

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subjects, although exhibiting normal sensitivity to the blocking effect of the antagonist before surgery, did not do so when tested 3 weeks after surgery. Histological analysis indicated that the VNO ablation did not produce gross damage to the olfactory bulbs in any of the VNO-X animals. In conclusion, removal of the VNO in the sexually intact rats rendered their behavioral performance highly similar to that observed in the male castrates (i.e., decreased persistence in their investigative behaviors toward newly encountered conspeciﬁcs, temporary impairment of their ability to form a social memory, and loss of sensitivity to the memory-blocking effects of the V1 receptor antagonist). The temporary impairment of the VNO-X subjects indicates that, over time, the contribution to social recognition of this accessory olfactory system can be replaced by the main olfactory system. Because the VNO ablation did not result in lowered levels of circulating androgens, it is unlikely that the androgen-dependent VP-ergic neurons in the bed nucleus of the stria terminalis were altered by this surgery. However, the insensitivity of the VNO-ablated rats to the memory-blocking effects of the V1 antagonist led to the conclusion that, for these subjects, the VP-ergic neurons were no longer involved in mediating the effects of VNO processing on social recognition. The loss of interest (persistence) displayed by the VNO-ablated subjects during their investigation of the juvenile stranger was attributed to the possible interruption of an interaction between circulating androgens and VNO processing, normally important for full expression of the response persistence effects of testosterone (Andrew, 1978).

E. AVP Neurotransmission Is Involved in Olfactory-Based SRM in Mice as well as in Rats 1. Selected Study: Bluthe et al. (1993) Bluthe et al. (1993) tested the involvement of androgen-dependent AVP neuronal transmission in social recognition in mice. The social recognition task, regularly used for rats, was employed to observe social memory processing in sexually intact as well as castrated male mice. The sexually mature intact male mice exhibited social recognition patterns similar to those of male rats. These similarities included (1) recognition of a juvenile male on the second encounter after a postexposure interval of 20 to 60 min, but not after 120 to 180 min; (2) enhanced social recognition after peripherally administered AVP (0.4 g/mouse, subcutaneous); and (3) impairment of normal social recognition (i.e., at a 20-min retention interval) after a peripheral injection of the V1 pressor antagonist (injected at a dose ﬁve times the AVP dose). Comparisons between the sexually intact and castrated male mice revealed differences similar to those observed for rats. Like rats, castrated male mice differed from sexually intact mice in the investigative behavior displayed during an original encounter with a conspeciﬁc juvenile (less

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persistent and less intense exploration of the juvenile), duration of the social memory formed on the basis of this encounter (memory more durable in the castrate), and in their reaction to the V1 antagonist [dPTyr(Me)]AVP (castrates insensitive to the memory-blocking effect of the antagonist). The authors concluded that the involvement of vasopressin neurotransmission in social recognition is modulated by sex steroids in mice as well as in rats. Moreover, this cross-species similarity increases conﬁdence in the generality of this phenomenon and its physiological signiﬁcance.

IV. The ‘‘VP Dual Action Theory’’ and Olfactory-Based SRM

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A. Interpretation of SRM Effects Produced by Pharmacological Doses of Peripherally and Centrally Administered Vasopressin As discussed in Chapter 6, the ‘‘VP Dual Action Theory’’ was formulated primarily to explain the retention effects observed in various avoidance and appetitive tasks after vasopressin treatment. It explained the retention effect as secondary to an increase in the subject’s arousal level induced by behaviorally active doses of either peripherally or centrally administered vasopressin but achieved via different mechanisms. Peripherally injected vasopressin produced pressor/aversive effects that in turn increased the arousal level, and centrally injected vasopressin activated the brain substrate for arousal (Ehlers et al., 1985; see Chapter 6). Early studies using the olfactory-based social memory paradigm (Dantzer et al., 1987; Le Moal et al., 1987) had been useful in assessing the pharmacological effects of VP. In this paradigm, peripherally or centrally administered AVP produced retention effects similar to those observed in active and passive avoidance tasks at equivalent dose levels. Because earlier observation had indicated that these dose levels also had arousal effects, these researchers concluded that the pharmacological effects of peripherally and centrally administered VP on the social memory paradigm were also explainable by the ‘‘VP Dual Action Theory.’’ The relevant evidence is as follows: (1) Dantzer et al. (1987) observed that peripherally administered AVP (6 g/kg) facilitated social memory (prolonged its duration) at a dose level known to increase blood pressure (Le Moal et al., 1981; see Chapter 6) and to produce an activational EEG proﬁle, interpreted as indicating a VP-induced arousal effect (Ehlers et al., 1985; see Chapter 6); (2) moreover, the AVP-induced social memory effect observed by Dantzer et al. (1987) was blocked by pretreatment with the V1 antagonist (30 g/kg, subcutaneous) at an antagonist–agonist dose ratio that blocked the pressor activity of AVP (Le Moal et al., 1981; see Chapter 6); (3) these agonist–antagonist

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interactional effects have been observed not only in sexually intact male rats (Dantzer et al., 1987), but in castrated male rats (Bluthe et al., 1990), female rats (Bluthe and Dantzer, 1990), and sexually intact and castrated male mice (Bluthe et al., 1993); (4) a 1.0-ng dose level of centrally administered AVP proved most efﬁcacious in facilitating retention in this social memory paradigm (Le Moal et al., 1987), similar to the results obtained in the pole-jump avoidance task (e.g., Koob et al., 1981, 1986; see Chapter 6); and (5) centrally administered AVP at a 1.0-ng dose level was also most effective in inducing the EEG activational proﬁle observed by Ehlers et al. (1985). Although the ﬁndings cited above indicated the importance of an AVP/ arousal effect in mediating social recognition, and hence the applicability of the ‘‘VP Dual Action Theory’’ for these ﬁndings, subsequent research suggested an additional vasopressin inﬂuence, independent of its interaction with the arousal system.

B. A Role for Endogenous VP in Olfactory-Based SRM Independent of VP-Induced Arousal Effects A role for endogenous vasopressin in SRM has been demonstrated in both rats (Dantzer et al., 1987, 1988) and mice (Bluthe et al., 1993). A peripheral injection of a V1 receptor antagonist ([dPTyr(Me)]AVP) at a dose that blocks facilitation by VP of this form of transient memory, impairs it when given on its own in sexually mature male rats (Dantzer et al., 1987) and mice (Bluthe et al., 1993). Because this antagonist can presumably cross the blood–brain barrier (Koob et al., 1985b), it can block central VP receptors involved in the normal mediation of SRM. Dantzer et al. (1988) observed that normal social recognition was prevented by an intraseptal injection of a hydrophilic V1 antagonist that is unable to cross the blood–brain barrier and enter the peripheral circulation. Moreover, SRM was facilitated by elevating the intraseptal level of AVP, an effect inhibited by pretreatment with an intraseptally administered V1 antagonist (Dantzer et al., 1988). Subsequent studies with this paradigm identiﬁed the androgen-dependent nature of the central VP-ergic system involved in the mediation of social memory, and showed its independence from a VP-induced arousal effect. Relevant observations were as follows: (1) castration of male rats (Bluthe et al., 1990) and mice (Bluthe et al., 1993), which depletes normal levels of circulating testosterone, temporarily impaired social memory that was restored by testosterone replacement therapy (Bluthe et al., 1990); (2) castrated male rats (Bluthe et al., 1990) and mice (Bluthe et al., 1993), as well as female rats (Bluthe and Dantzer, 1990), are insensitive to the memory-blocking effects of a peripherally administered V1 receptor antagonist that can reach central VP receptors. This effect was reversed by testosterone replacement therapy (Bluthe et al., 1990); and (3) social memory in both castrated and sexually

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intact rats was facilitated by peripherally injected AVP, and this effect was blocked by pretreatment with the V1 antagonist (Bluthe et al., 1990). Thus, despite the castration-induced depletion of vasopressin in this androgen-dependent circuitry, transient memory in this task, like longterm memory in others, was sensitive to the pharmacological properties of the peptide, suggesting that a different mechanism was responsible for this effect (Bluthe et al., 1990). Further characterization led these researchers to conclude that this androgen-dependent VP-ergic circuitry is a component of the vomeronasal olfactory pathway, which is involved in processing the chemoceptive input that identiﬁes the conspeciﬁc individual. Major support for this conclusion comes from ﬁndings of a study that examined the role of the vomeronasal system in VP-ergic modulation of social recognition in the male rat (Bluthe and Dantzer, 1993). First, although VNO ablation did not disturb the level of circulating androgens, the operates were no longer sensitive to the memory-blocking effect of the V1 antagonist, indicating that an intact VNO normally contributes to the social recognition mediated by VP-ergic circuitry. However, neither the VNO nor its VP-ergic projections are the sole contributors to this form of rodent memory because VNO ablation, as well as castration, produces only a temporary impairment.

C. Two Functionally Distinct VP-ergic Systems in the Rodent Brain Taken together, the ﬁndings from the avoidance and appetitive research studies reviewed in Chapter 6, and those from the social memory studies described in this chapter, led these researchers to propose that two functionally distinct VP-ergic systems are present in the brains of male rats and mice: ‘‘an androgen-dependent system involved in the processing of olfactorybased social memory, and an androgen-independent system involved in the modulation of arousal’’ (Dantzer, 1998, p. 413). The ‘‘VP Dual Action Theory’’ pertains to the latter system only. The androgen-dependent VPergic circuitry is postulated to mediate normal social recognition in males only. The arousal system in both sexes mediates the retention effects of pharmacological doses of the peptide in the olfactory-based social memory task as well as in all other learning paradigms discussed in this text.

V. Commentary I: Roles of VP in Mediating Olfactory-Based SRM

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A. The ‘‘VP Dual Action Theory’’ and Olfactory-Based SRM The proposition that peripheral vasopressin facilitates transient conspeciﬁc memory by means of increased arousal induced by the pressor/aversive

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properties of the hormone appears open to the same challenges discussed in connection with long-term memory (see Chapter 6). For example, peripheral injection of DG-AVP and [pGlu4, Cyt6]AVP(4–8), which lack the pressor and aversive properties of the parent peptide, nevertheless facilitates transient memory tested in the olfactory-based SRM paradigm (Popik et al., 1991; see Chapter 13) these metabolites similarly enhance long-term memory tested in avoidance (e.g., De Weid et al., 1987; see Chapter 5) and appetitive (e.g., Strupp, 1989; see Chapter 9) paradigms. These observations indicate that the pressor/aversive effects of AVP are not essential for the inﬂuence of vasopressin on either type of retention.

B. Sexual Dimorphy and Olfactory-Based SRM Engelmann et al. (1998; see Chapter 13) found support for, and extended, the Bluthe and Dantzer (1990) proposal that central VP-ergic circuitry does not mediate conspeciﬁc recognition in female rats. They reported that, on its own, an intracerebroventricular injection of an OT receptor, but not a VP receptor, antagonist impaired the social recognition normally shown at 60 min. This suggests that in female rats, OT-ergic, rather than VP-ergic, circuitry normally contributes to the social memory tested in this paradigm. In addition, when tested over various stages of the estrus cycle, under nontreatment conditions, both estrous and nonestrous females were comparable in their ability to recognize conspeciﬁc juveniles 30 and 120 min after a previous brief investigative encounter. Thus, this OT-ergic circuitry appears to be independent of gonadotropic hormonal control. Engelmann et al. (1998) have developed a ‘‘working model’’ that is in accord with several ﬁndings presented in this chapter. Using an ontogenetic framework, they suggest that OT-ergic circuitry mediates olfactory-based social recognition during early development in both sexes. OT-ergic circuitry continues this role in the female, and as it comes under the inﬂuence of female gonadal steroid hormones it becomes involved in social recognition associated with mating and maternal behavior. In males, however, the mediation of social recognition becomes subject to the developing androgendependent VP-ergic circuitry that also regulates male social and territorial behavior. This proposal accounts for the female-like social recognition patterns of the male castrate, which presumably express the ontogenetically earlier OT-ergically mediated pattern of social investigation and retention behavior. It is also in accord with the observation that long-term treatment with the intracerebroventricularly injected vasopressin V1 antagonist caused sexually intact male rats to behave like castrates in their ability to recognize a conspeciﬁc juvenile for a longer interval than control males. Both observations suggest that the later-maturing androgen-dependent VP circuitry suppressed an earlier-appearing OT-ergic system involved in prepubertal social recognition.

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Studies of prairie vole social behavior also support the thesis that OT-ergic and VP-ergic circuitry have different roles in olfactory-based social recognition during mating and parental behavior. OT-ergic circuitry is important for mate recognition and preference in female prairie voles (Insel and Hulihan, 1995; Witt et al., 1990). In contrast, male prairie voles depend on VP-ergic circuitry for social recognition involved in postmating pair bonding and aggressive mate guarding (Winslow et al., 1993a) and in paternal behavior (Bamshad et al., 1994; Wang et al., 1994).

C. The Vomeronasal Organ and the Contribution of AVP to Olfactory-Based SRM This research group has provided evidence that the VNO is necessary for the contribution of endogenous vasopressin to juvenile recognition in the sexually intact male rat. This was indicated by the following two observations: (1) VNO ablation temporarily impaired conspeciﬁc recognition, which presumably was then mediated by processing in the main olfactory system and non-VP-ergic neural circuitry (Bluthe and Dantzer, 1993); and (2) removal of the VNO did not inﬂuence circulating androgens, and therefore left intact the dimorphic VP-ergic pathway from the BNST and amygdala to the septal area. It did, however, prevent this pathway from making its normal contribution to conspeciﬁc recognition, because recovered social recognition in VNO-ablated rats was no longer sensitive to the blocking effects of the VP antagonist (Bluthe and Dantzer, 1993). These data suggest that the vomeronasal pathway is the dominant system mediating juvenile recognition in the sexually intact male rat. Of speciﬁc interest is the nature of the vasopressin contribution and where this may occur within the vomeronasal system: the VNO (the receptor region for chemosensory stimulus input), the AOB (the accessory olfactory bulb, which processes this input received from the VNO), and central sites to which the AOB projects, including the amygdala and BNST and their VPergic outputs to septal and hippocampal brain sites. Dantzer and Bluthe (1993) suggested the possibility that the VP-ergic circuitry contributed to social recognition by processing socially relevant odors, rather than mediating social recognition per se. Another possibility is that vasopressin contributes to memory engram formation in the AOB itself and/or at one or more sites in the androgen-dependent VP-ergic neuronal systems receiving chemosensory inputs processed by the AOB. Although no direct evidence is available, a VP release in the AOB may contribute to alterations in this circuitry similar to those produced by OT released in the main olfactory bulb (MOB) during mother–offspring bonding in sheep, as found by Levy, Keverne, and colleagues. Brieﬂy, these researchers provided evidence that oxytocin, released during parturition, interacts with NA-ergic and cholinergic inputs to the olfactory bulb to

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bring about synaptic alterations in MOB circuitry associated with the ewe’s recognition of her offspring (Da Costa et al., 1996; Kendrick, 2000; Kendrick et al., 1992; Levy et al., 1995). Given the importance of the VNO and the AOB in conspeciﬁc recognition in male rats, it would be of interest to learn whether AOB circuitry stores chemosensory information necessary for individual conspeciﬁc identiﬁcation, and if so, whether androgen-dependent VP inﬂuences this storage. In addition to a VP inﬂuence in the beginning of the vomeronasal pathway, an important VP contribution may also occur at more central sites receiving vomeronasally processed inputs. Thus, numerous studies have indicated a role in male rat juvenile recognition for androgen-dependent VP-ergic systems within the septum [Dantzer et al., 1988; Engelmann and Landgraf, 1994 (see Chapter 13); Van Wimersma Greidanus and Maigret, 1996], as well as in the dorsal and ventral hippocampus (Van Wimersma Greidanus and Maigret, 1996; see Chapter 13).

VI. Commentary II: Contribution of the California/Bordeaux Research Teams in VP Memory Research

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As noted in the preface and Chapter 1, VP and OT have many functions, some of which are concerned primarily with self-preservation and homeostatic maintenance of bodily activity whereas others are related to species preservation and social behavior. These investigators have covered both functional categories in their vasopressin/memory research studies. The putative role of vasopressin in learning/memory pertinent to self-preservation is investigated in the appetitive and avoidance tasks used in the earlier conducted studies described in Chapter 6. Their adoption of the olfactorybased social memory paradigm for this line of study marked a signiﬁcant transition to the putative role of the peptide in memory processing relevant to reproductive and social behaviors. The social memory paradigm has also provided a constructive link between laboratory-generated research and ethologically relevant ﬁeld investigation. This being so, the research presented in this chapter is relevant to vasopressin and oxytocin social-oriented research that has been carried out with hamsters (Albers and Bamshad, 1998; Albers et al., 1992), prairie voles (Cho et al., 1999; Wang et al., 1998; Winslow et al., 1993b), and sheep (Kendrick, 2000). The research ﬁndings indicating a role for VP-ergic circuitry in the conspeciﬁc recognition of male but not female rodents is an intriguing ﬁnding and bears further comparative study to determine (1) whether this sexually dimorphic functioning occurs in other mammalian groups and, if so, (2) does oxytocin play a parallel role for female mammals? Their theoretical position linking an androgen-independent

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circuitry to an arousal-mediated inﬂuence on retention behavior and an androgen-dependent circuitry to VNO chemosensory processing has already received commentary. Further discussion relating the theoretical construct of central arousal to memory processing is given in the ﬁnal chapter because the arousal construct has an important role in the theoretical orientation held by numerous investigators outside of the De Wied et al. theoretical camp.

Barbara B. McEwen

Expansion of Olfactory-Based Social Recognition Memory Research: The Roles of Vasopressin and Oxytocin in Social Recognition Memory

I. Introductory Remarks

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This chapter updates research on the roles of vasopressin (VP) and oxytocin (OT) in rodent olfactory-based social recognition memory (SRM). As noted in Chapter 12, Dantzer, Bluthe, and colleagues launched this burgeoning ﬁeld of inquiry in the late 1980s. After a brief description of the test paradigms used to assess SRM, evidence is discussed that conﬁrms and expands the research ﬁndings presented in Chapter 12.

II. Test Paradigms for Assessing SRM

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A. Social Recognition Test The conventional paradigm used to test SRM in rats and mice is the social recognition test (SRT) described in Chapter 12. A signiﬁcant reduction in the duration of social investigatory behavior of a stranger juvenile when it Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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is reintroduced in a second 5-min exposure period after a designated interexposure interval (IEI) is operationally deﬁned as SRM. Control test sessions, in which a novel juvenile is presented after the IEI, are generally included to ensure that any reduced investigative behavior of the same juvenile by the subject is not merely the result of satiation, fatigue, or nonspeciﬁc drug effects. Preliminary testing in this paradigm is typically included for the purpose of subject selection and can be illustrated by reference to a study by Sekiguchi et al. (1991a): the adult rat was presented with the juvenile twice daily for 5 days. The IEI on days 1 and 2 was 5 min, on days 3 and 4 it was 30 min, and on day 5 it was 120 min. The same juvenile was presented in the second presentation trial, except for day 4, when a different juvenile was used. Only the animals that reliably investigated the juveniles and did not display aggressive or sexual behavior toward them were used. As a further precaution against the danger of aggressive behavior in the resident subjects, the juveniles may have been periodically replaced (e.g., every 10 days or so) (Arletti et al., 1995). Under nontreatment conditions, intact adult male mice and rats recognized the reencountered juvenile after an IEI of 30 but not 120 min; whereas female rats appeared to recognize the juvenile after IEIs of 120 but not 180 min (Bluthe and Dantzer, 1990).

B. The Social Discrimination Test Engelmann et al. (1995) developed the social discrimination test (SDT) as an alternative paradigm for testing this type of memory. In the SDT the familiar juvenile (presented in the ﬁrst investigative encounter) and a novel (different) juvenile are simultaneously presented during the second investigative encounter. SRM is indicated when the time spent investigating the familiar juvenile is signiﬁcantly less than for the novel juvenile. It is argued that the advantage of this procedure is that the use of the novel juvenile as a comparative test stimulus provides a built-in control that reduces the number of sessions needed for a given experimental series. Like the SRT, this procedure has veriﬁed that a single investigative exposure of the juvenile results in a short-term memory that is present after an IEI of 30 but not 120 min the male rat.

C. The Multitrial Social Recognition Test A more recently developed paradigm, the multitrial recognition test, has been used to test SRM in both male and female mice (Ferguson et al., 2000). The mouse in its home cage is presented with the same conspeciﬁc social test stimulus in each of four 1-min social investigative trials (10-min intertrial interval, ITI). SRM is operationally deﬁned as a signiﬁcant decline in the duration of social investigative activity over the successive 1-min encounters

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with the same social stimulus. A different conspeciﬁc social stimulus is presented in a ﬁfth trial as a control for nonspeciﬁc effects. Prolonged social investigation of the stranger is interpreted as renewal of interest that had waned with increasing familiarity of the previous conspeciﬁc. Another control for nonspeciﬁc effects used by Ferguson et al. (2000) with this paradigm was the presentation of a different conspeciﬁc test stimulus in each of the four 1-min presentation trials. The conspeciﬁc test stimuli used by Ferguson and colleagues included adult male mice, as well as ovariectomized and intact female mice.

III. Effects of Peripherally and/or Centrally Administered VP, OT, or Their Metabolic Fragments on SRM in Laboratory Rats and Mice

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A. Vasopressin and Related Peptides 1. General Comments The pioneer studies by Dantzer, Bluthe, and colleagues (see Chapter 12), which showed that peripherally administered AVP enhances SRM in male rodents, have been conﬁrmed and extended by the research described below. Popik et al. (1991) have drawn parallels between the effects of arginine vasopressin (AVP) in the social recognition paradigm and in other learning paradigms in their studies using AVP derivatives. Sekiguchi et al. (1991a) further clariﬁed the contribution of exogenous AVP to this form of memory by their analysis of time–effect, structure–activity, and dose–response relationships. In structure–activity testing, the investigator attempts to learn which part of the peptide molecule is speciﬁcally responsible for the physiological or behavioral effect under study. Popik and Van Ree (1992) also carried out a structure–activity analysis to further characterize the memory-enhancing effect of exogenous AVP in the social recognition test. Their ﬁndings led them to suggest that social recognition depends on two types of memory processes, short-term and long-term, and they appear to be differentially sensitive to the facilitating effects of various AVP-related peptides. 2. Peripheral Administration a. Selected Studies i. Popik et al. (1991) Popik et al. (1991) investigated the effects of peripherally administered desglycinamide-arginine vasopressin (DG-AVP) and AVP(4–8) on SRM in Wistar male rats. It was of interest to determine whether these AVP derivatives, which lack the endocrine effects of the parent peptide, would facilitate this SRM as they were shown to with avoidance retention [De Wied et al., 1972, 1987; Gaffori and De Wied, 1986; Kovacs et al., 1986 (see Chapter 5)].

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The SRT, with varying IEIs, was used to assess SRM in three experiments. Two measures of social investigative behavior were used: (1) the total duration of social investigation, which included proximate orientation to the juvenile and direct contact activity (e.g., snifﬁng, social grooming, inspection of body surface); and (2) duration of each of the following social behaviors: close following, explorative snifﬁng other than that of the anogenital area, anogenital snifﬁng, and social grooming. Drugs or placebo were subcutaneously injected 1 min after the ﬁrst investigative encounter. Differences in investigative time between the ﬁrst and second 5-min encounters were computed separately for the rats presented with the same and different juveniles during the second exposure period. The ﬁrst experiment was designed to examine the effects of IEI duration (15, 30, 60, or 120 min) on SRM. The results were as follows: (1) social investigation of the same juvenile during the second encounter was signiﬁcantly reduced compared with that of the ﬁrst encounter for an IEI of 15 or 30 min, but not of 60 or 120 min; and (2) no signiﬁcant difference in investigative time between the two encounters was found when a different juvenile was presented during the second encounter. In the second experiment the investigators used the SRT with IEIs of 60 and 120 min to examine the effect of two AVP derivatives on SRM. A peripheral injection of placebo, DG-AVP (6.0 g/kg, subcutaneous), or AVP(4–8) (1.0 g/kg subcutaneous) was given 1 min after the ﬁrst presentation trial. A pilot experiment had found that AVP (3 g/kg, subcutaneous) enhanced SRM [signiﬁcantly reduced social investigative time (SIT) in the second investigative trial, but not for the placebo-treated rats]. The results of experiment 2 were as follows: (1) placebo-treated rats exhibited no difference in SIT between the ﬁrst and second encounters when either the same or a different juvenile was tested; (2) DG-AVP produced a small but signiﬁcant decrease in SIT during the second encounter, when the same juveniles were presented after an IEI of 120 min, but not 60 min; (3) the AVP(4–8)-treated rats exhibited signiﬁcantly decreased SITs in the second encounter with the same juvenile after both IEIs; and (4) no effect of either peptide was found when a different juvenile was presented in the second investigative trial. In the third experiment the investigators examined the degree to which each of the four types of social investigative behaviors monitored in this study (anogenital exploration, close following, snifﬁng, and grooming) was associated with the inﬂuence of DG-AVP on SRM. The subjects were presented with the same or a different juvenile during the second presentation trial after an IEI of 30, 60, or 120 min. The results indicated that (1) half of the ﬁrst presentation trial was spent in social investigation of the juvenile, and most of this involved anogenital snifﬁng (75%), with considerably less time involved with the remaining social behaviors [close following (2%), snifﬁng (13%), and grooming (10%)]; (2) placebo-treated rats signiﬁcantly reduced their social investigative behavior of the familiar juvenile after a

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30-min, but not a 60- or 120-min, IEI, and this was entirely attributable to a reduction in anogenital snifﬁng. The SIT was not reduced when a different juvenile was presented during the second investigative trial; and (3) DGAVP-treated rats spent signiﬁcantly less time investigating the same juvenile after a 30- or 120-min IEI, but not after a 60-min IEI. This reduction in investigative behavior was entirely due to a decrease in anogenital exploration, because time spent on other social behaviors was not decreased. DGAVP did not produce changes in the duration of investigative behavior when a different juvenile was tested during the second presentation trial. Taken together, these results have conﬁrmed the short-term duration of SRM observed in laboratory rats (e.g., Thor and Holloway, 1982) (experiment 1), and the importance of olfactory investigation of the anogenital area for this recognition (Carr et al., 1976; Sawyer et al., 1984), because this was the major social behavior observed during the ﬁrst presentation trial and was the one reduced during the second presentation of the familiar juvenile (experiment 3). Moreover, they have shown that other measures of social investigative activity, such as grooming, snifﬁng per se, and following the juvenile, appeared not to be important for SRM because their duration did not change from the ﬁrst to the second presentation trial (experiment 3). The study further showed that, in addition to peripherally administered AVP (pilot test), its derivatives DG-AVP (experiments 2 and 3) and AVP(4–8) (experiment 2) enhanced SRM and AVP(4–8) was a far more potent enhancer than DG-AVP (induced a stronger effect at a six-times lower dose level) (experiment 2). The study also showed that the AVP-induced enhancement of SRM was not dependent on its peripheral endocrine effects because DGAVP and AVP(4–8), which lack these effects, also enhanced SRM. Moreover, this ﬁnding is in accord with similar ﬁndings for memory tested in other retention paradigms [e.g., Bohus, 1977; Vawter and Van Ree, 1995; Vawter et al., 1997 (see Chapter 2); and De Wied et al., 1987; Gaffori and De Wied, 1986; Kovacs et al., 1986 (see Chapter 5)]. The failure of DG-AVP-treated rats, in contrast to AVP- and AVP(4–8)treated rats, to enhance SRM after a 60-min IEI could not be explained. This ﬁnding was even more puzzling given the ability of DG-AVP to enhance SRM after a longer (120 min) IEI. The authors noted that Gaffori and De Wied (1986) reported differences between various VP analogs with respect to their time-dependent effects on avoidance retention. However, the time-dependent effects pertained to training-treatment intervals over which the peptides demonstrated memory facilitation, and may have resulted from differences among the peptide analogs in ‘‘metabolism, rates of distribution to the various body compartments or brain structures involved in the behavioral effects of the peptides’’ (Popik et al., 1991, p. 1033). This explanation does not seem applicable to the present study, because the training-treatment interval was the same for all the tested peptides, and it does not explain the ability of DG-AVP to enhance SRM tested with a 120-min, but not a 60-min, IEI.

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ii. Sekiguchi et al. (1991a) Sekiguchi et al. (1991a), noting the reports of the ability of exogenous AVP to facilitate SRM in the male rat (Dantzer et al., 1987, 1988; Le Moal et al., 1987; see Chapter 12), designed several experiments to further analyze this inﬂuence by studying time–effect, structure–activity, and dose–response relationships. Adult male Wistar rats were tested in the SRT with male juvenile rats (4–5 weeks of age) as social test stimuli. The duration of investigative behavior in each presentation trial was determined by adding the times engaged in each of the following speciﬁc activities directed toward the juvenile: close following, social snifﬁng of the body surface other than the anogenital area, anogenital investigation, and social grooming of the juvenile’s body other than the anogenital area. Experiment 1 tested time–effect relationships. Placebo (physiological saline) or DG-AVP (6 g/kg, subcutaneous) was injected immediately after the ﬁrst investigative trial and the same juvenile was re-presented in the second trial after an IEI that differed for independent groups of rats (1, 2, 4, 6, 8, 24, or 48 h). The results indicated that DG-AVP treatment resulted in social recognition for all IEIs except for those of 1 or 48 h (investigative time during the second trial was signiﬁcantly reduced after IEIs of 2, 4, 6, 8, or 24 h). In contrast, the placebo controls showed no signiﬁcant reduction in the duration of second trial investigative behavior for any of these IEIs. Experiment 2 tested structure–activity relationships. After the ﬁrst investigative trial the subjects were subcutaneously injected with placebo or a 6-g/kg dose of one of the following AVP analogs: AVP(4–8), AVP(4–9), AVP (5-8), or AVP(5–9). The same juvenile was re-presented in the second trial after a 120-min IEI. Three of the tested analogs, AVP(4–9), AVP(5–8), and AVP(5–9), enhanced SRM (investigative behavior of the same juvenile in the second trial was signiﬁcantly reduced from that in the ﬁrst trial). The effect of AVP(4–8) on SRM was in the expected direction but did not reach statistical signiﬁcance. Placebo treatment did not facilitate SRM. Experiment 3 tested dose–response relationships. DG-AVP (0.0, 0.2, 0.6, 2.0, 6.0, or 20.0 g/kg) or AVP(4–8) (0.0, 0.2, 0.6, 2.0, or 6.0 g/kg, subcutaneous) was injected 1 min after the ﬁrst presentation trial with a 120-min IEI. DG-AVP at the two highest dose levels (6.0 and 20.0 g/kg), and AVP(4–8) at a dose level of 2.0 g/kg enhanced social recognition (SIT was signiﬁcantly reduced from the ﬁrst to the second trial). Placebo-treated controls did not recognize the reencountered juvenile. The following points were made during discussion of these results: 1. DG-AVP (6 mg/kg, subcutaneous) induced a long-term enhancement of SRM, extending it from its normal duration of 30 min (Popik et al., 1991; Thor and Holloway, 1982) to 24 h (experiment 1). Other attempts to prolong recognition time, such as increasing the duration of the ﬁrst presentation, or the number of encounters on one day or on several subsequent days, have not been successful (Sekiguchi et al., 1991b). These observations

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together with the present ﬁndings suggest that long-term action of DG-AVP is more related to the memory processes involved than to the amount of information received. 2. It is noteworthy that this long-term effect on SRM required that the juveniles and adults remain in the same experimental room during the 24-h IEI. The effect was absent when the animals were returned to the animal house, and this may have been due to interference from other environmental stimuli encountered during the IEI, that is, an example of the retroactive inhibitory effect demonstrated by Dantzer et al. (1987). Although not stated by the authors, the continued presence of background stimuli (visual, auditory, airborne odors) in attendance at the initial social encounter might also have interacted with the peptide to keep SRM processing active during the IEI. 3. The social recognition enhancement induced by DG-AVP was also observed for the AVP analogs AVP(4–9), AVP(5–8), and AVP(5–9) (experiment 2). These observations suggest that portion 5–8 of the vasopressin molecule is the active site for this effect, and is in accord with ﬁndings on structure–activity relationships reported in studies with active and passive avoidance paradigms [De Wied et al., 1987; Kovacs et al., 1986 (see Chapter 5)]. 4. Peripherally administered AVP(4–8) was somewhat more potent than DG-VP in facilitating SRM, enhancing it at a lower dose level (2 g/kg) than did DG-AVP (6.0 and 20.0 g/kg) (experiment 3). This, too, is consistent with ﬁndings on active and passive avoidance tasks, although the difference in potency between these two peptides is more pronounced in these two latter paradigms. iii. Popik and Van Ree (1992) Popik and Van Ree (1992) used the SRT with a 24-h IEI to determine whether peripherally administered AVPrelated peptides can extend the duration of SRM over a 24-h period, and if so, to characterize the active part of the molecule responsible for this ability. The design of this study was stimulated, in part, by observations that SRM in wild rats (Thor, 1979), after a single encounter with a conspeciﬁc juvenile, may endure signiﬁcantly longer then the 30-min interval suggested by the study of nontreated laboratory-bred rats (Thor and Holloway, 1982). It is possible that in the laboratory SRM lasts longer than 30 min, but is too weak to be demonstrated by the paradigm used. The design of the study was also inﬂuenced by the memory-modulating effects of AVP and OT in the SRM paradigm (e.g., Dantzer et al., 1987) and in particular by the demonstration by Sekiguchi et al. (1991a) that peripherally administered DG-AVP extended SRM in male rats from 30 min to 24 h, thus producing the VPinduced memory persistence observed in other task paradigms [Bohus, 1977 (see Chapter 2); Kovacs et al., 1986 (see Chapter 5)].

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The subjects were sexually experienced adult male Wistar rats, and the social test stimuli were male juvenile (3- to 4-week old) conspeciﬁcs. The juvenile was removed after the ﬁrst presentation trial, and SRM was evaluated after a 24-h IEI in the second presentation trial with the same or a different juvenile as the social test stimulus. Peptide or placebo was given immediately after the ﬁrst presentation. The peptides tested were as follows: AVP(1–9), numerous AVP fragments [AVP(1–8), AVP(1–7), AVP(1–6), AVP(1–5), AVP(4–9), AVP(4–8), and AVP(7–9)], OT(1–9), and the OT fragment OT(1–6). With the exception of OT(1–9) and AVP(7–9) each of these peptides was injected at two dose levels (0.3 and 3.0 g/rat, subcutaneous). OT(1–9) was tested at the 3.0-g/rat dose level and also at a dose level of 0.75 ng/rat (subcutaneous), on the basis of the observation that low doses of OT facilitated SRM after an IEI of 2 h (Popik et al., 1992). The subjects in each peptide dose group served as their own placebo controls (i.e., ‘‘each peptide treatment was placebo controlled’’; p. 568). More speciﬁcally, this was accomplished by a cross-over design whereby half the subjects in a peptide dose group received the peptide, and the remainder received the placebo (physiological saline) on the ﬁrst test day; the same subjects received the reversed treatment on the second test day. A placebo-controlled recognition index was computed for each peptide treatment. The social investigative time (SIT) during the ﬁrst and second encounters was determined for each resident rat. These data were entered into a formula that comprised the recognition index (RI) for each rat: RI ¼ ([SITsecond encounter (peptide)/SITﬁrst encounter (peptide)] – [SITsecond encounter (placebo)/ SITsecond encounter (placebo)]) 100. A signiﬁcant negative value indicated SRM after peptide treatment as compared with placebo treatment. These values were averaged for each experiment (peptide treatment/placebo control) and statistically analyzed in a two-way analysis of variance (ANOVA). The results of the analysis of the placebo-controlled RIs of the subjects treated with different peptide fragments indicated that (1) SRM (a signiﬁcant decrease in SIT during the second encounter) was present after treatment with the 3.0-g dose of the following peptides: AVP(1–8), AVP(1–6), and AVP(1–7), all of which contain the covalent ring structure of the peptide molecule; (2) of these peptides, only AVP(1–6), the covalent ring structure of the AVP molecule, induced social recognition at the low dose level (0.3 g/ rat); (3) those AVP peptide molecules that lacked an intact covalent ring [AVP(1–5), [pGlu4,Cyt6]AVP(4–8), [pGlu4,Cyt6] AVP(4–9), and AVP(7–9)] did not inﬂuence social recognition (i.e., did not signiﬁcantly reduce SIT during the second encounter); and (4) treatment with OT(1–6), which does contain an intact ring structure, did not result in social recognition, thereby indicating the speciﬁcity of the inﬂuence of VP on social recognition; moreover, neither the 0.75-ng nor 3.0-g dose of OT(1–9) affected the placebocontrolled RI values after an IEI of 24 h.

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In discussing these ﬁndings the authors noted the following points: 1. Several of the VP peptides exerted long-term effects on SRM, as has been demonstrated for retention in avoidance paradigms (De Wied, 1971; and see Chapters 2–5), ‘‘supporting the assumption that memory processes are involved in social recognition’’ (Popik and Van Ree, 1992, p. 570). 2. The observed importance of the covalent ring structure for the longterm effects of VP on SRM supports the proposal that it contains the primary site for the memory-enhancing activity of the molecule, although a second active site for this process may be present in the linear part of the molecule (Van Ree et al., 1978). More speciﬁcally, Van Ree et al. (1978) have reported that AVP(1–6) enhances memory consolidation in avoidance paradigms, but in contrast to the C-terminal linear component, does not facilitate memory retrieval (i.e., prevent experimentally induced amnesia). 3. Earlier ﬁndings have indicated the ability of AVP(4–8) and AVP(4–9) to enhance memory consolidation in avoidance learning tasks at considerably lower doses than that of the parent peptide (De Wied et al., 1987; see Chapter 5), and to promote social recognition after an IEI of 2 h [Popik et al., 1991; Sekiguchi et al., 1991a (see above)]. These observations along with the present evidence of their inability to enable SRM to extend over a 24h IEI led the authors to postulate that SRM involves two different memory processes, one short-term and another long-term in nature, which are differentially sensitive to the facilitating effects of various VP-related peptides. This interpretation receives support from the authors’ unpublished observation that the covalent ring structure of AVP was not active when an IEI of 2 h was used.

B. Oxytocin and Related Peptides 1. Section Overview The amnestic property of peripherally and/or centrally administered OT in active and passive avoidance conditioning tasks has been well documented (see Chapter 2), and has also been reported for the nonstressful social recognition test (Dantzer et al., 1987). Moreover, a memory-impairing action for endogenous OT has been demonstrated in avoidance paradigms after peripheral and central administration of OT antiserum [Bohus et al., 1978b (see Chapter 2); Kovacs et al., 1979a (see Chapter 4)]. A study by Popik and Vetulani (1991) found a similar effect on SRM after peripheral administration of high doses of two OT receptor antagonists. Whereas the foregoing evidence supports postulated amnestic properties of OT, there have been reports that OT does not always impair memory because the neuropeptide prevented puromycin-induced amnesia (Walter

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et al., 1975; see Chapter 2) and exerted a bimodal effect on avoidance behavior (Gaffori and De Wied, 1988; see Chapter 2). Results of most of the studies described below suggest a dose-dependent modulation of SRM, whereby low doses of exogenous OT facilitate SRM, and the higher doses, more frequently used in behavior study, impair it (Popik et al., 1992). Structure–activity analyses, used to clarify this dose–response effect of exogenous OT on SRM, have led to proposals of physiological mechanisms that may be responsible for it (Popik et al., 1996). The study by Arletti et al. (1995) conﬁrmed this dose-dependent SRM enhancement effect of OT, and demonstrated its effectiveness in aged as well as young rats. Using centrally injected OT, Benelli et al. (1995) found the same low-dose enhancement effect of OT that had been observed after peripheral administration of the peptide. 2. Peripheral Administration a. Selected Studies i. Popik and Vetulani (1991) Popik and Vetulani (1991) used the SRT to learn whether peripheral administration of two OT antagonists, [10 -(10 -thio-40 -methylcyclohexane)-acetic acid1]-oxytocin (MeCAOT) and [10 -(10 -methyl-40 -thiopiperidine)-acetic acid1]-oxytocin (MePAOT) act by themselves as memory-enhancing factors, and interfere with the amnestic action of OT when combined with this peptide. MeCAOT appears to be a peripheral OT antagonist, because it and not MePAOT blocked the action of OT in an isolated rat uterus preparation (Rekowski et al., 1987). This study deﬁned a peptide-induced memory-impairing effect as loss of social recognition after a 20-min IEI, and a memory enhancement effect as the presence of juvenile recognition after a 60-min IEI. The putative OT amnestic effect was tested with a 20-min IEI; the putative memory-enhancing effect of the antagonistic treatment on its own was tested with a 60-min IEI. Both antagonists were peripherally administered at two dose levels (12 and 24 g/ kg, subcutaneous). A given subject received one or the other dose of the peptide. When tested on its own the OT antagonist was injected immediately after the ﬁrst encounter. For antagonist–OT interactional effects on SRM, the lowest memory-disrupting dose of OT (determined by pretest results), was injected 2 min after injection of the OT antagonist (administered 1 min after the ﬁrst presentation trial). The four main results were as follows: (1) injected on their own, the high dose level of both OT antagonists enhanced SRM (i.e., SIT was signiﬁcantly reduced during the second investigative trial when the same juvenile was presented after a 60-min IEI); (2) there was no change in SIT between the ﬁrst and second investigative trials if a different juvenile was presented after the 60-min IEI, even if the subjects received the high dose of the OT antagonists; (3) injected on its own, OT, over a dose range of 6 to 750 mU/kg, impaired normal SRM (no signiﬁcant reduction in SIT between

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the ﬁrst and second investigative trials, a reduction that occurred for placebo controls when the same juvenile was presented after the 20-min IEI); and (4) MePAOT at both dose levels, and MeCAOT at the higher dose level, antagonized the amnesia induced by the least effective amnestic dose of OT (6 mU/kg). The following points were made during the discussion of these results: (1) the results veriﬁed the OT-antagonistic property of MePAOT and MeCAOT. This was indicated by their memory-enhancing effects when injected alone, and their ability to interfere with the amnestic effects induced by appropriate doses of peripherally administered OT; (2) the results support a physiological role for OT in the modulation of SRM as appears to occur for memory tested in other learning paradigms. That is, the enhancement of SRM induced by peripheral injections of the OT antagonists was presumably due to interference with OT-ergic neurotransmission, just as the enhancement of memory in avoidance paradigms induced by intracerebroventricularly injected OT antiserum was presumably due to the resulting reduction of endogenous OT (Bohus et al., 1978b; see Chapter 2); and (3) the greater effectiveness of MePAOT in antagonizing the centrally mediated amnestic action of OT, together with its inability to inﬂuence a peripheral action of the hormone (Rekowski et al., 1987), indicates a distinction between OT central and peripheral actions, and suggests that separate and possibly independent mechanisms are involved. ii. Popik et al. (1992) Popik et al. (1992) used the SRT to examine the effects of a wide dose range of peripherally administered OT on SRM in male rats. This study was stimulated by the observation that the doses of peripherally administered OT that have been observed to attenuate SRM (i.e., greater or equal to 24 ng/kg; Popik and Vetulani, 1991) probably result in rather high plasma levels of OT, compared with physiological levels of the hormone (Mens et al., 1983). Accordingly, this study used doses of OT lower than those effective in attenuating SRM. Also, OT and AVP have been shown to have opponent effects on memory tested in avoidance learning paradigms (e.g., Bohus et al., 1978b), as well as in the SRT (Dantzer et al., 1987). Therefore, AVP and arginine vasotocin (AVT), the ancestral neurohypophysial peptide, were included in this study for comparative purposes. AVP and AVT were tested at two dose levels (1.5 and 6.0 ng/kg, subcutaneous), and OT was tested over a dose range of 0.09 to 24.0 ng/kg (i.e., 0.09, 0.36, 1.5, 6.0, or 24.0 ng/kg) in experiments in which the same juveniles were presented in the second encounter, and at two dose levels (1.5 and 6.0 ng/kg) in those experiments using novel juveniles. The same 16 subjects were tested under all treatment conditions. Injections were given immediately after the ﬁrst encounter, and 48 h intervened between successive treatments. Attenuated SRM was deﬁned as the inability to recognize the preencountered juvenile after an IEI of 30 min, and facilitated SRM as the ability to

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recognize the juvenile after an IEI of 120 min. The criterion for SRM in a group of residents was a statistically signiﬁcant (paired t test) shortening of the SIT on the second encounter with the familiar juvenile. Paired t tests indicated no change between SIT for ﬁrst and second encounters with the same juvenile under placebo conditions, or at an OT dose level of 24 ng/kg, whereas OT treatment at doses ranging from 1.5 to 6.0 ng/kg facilitated SRM (induced a signiﬁcant reduction in SIT during the second encounter). A one-way ANOVA of the SIT scores (SIT ¼ difference in SIT between the ﬁrst and second encounter) indicated a signiﬁcant treatment effect, and individual comparisons revealed a signiﬁcant reduction in SIT during the second encounter relative to the placebo treatment condition, after OT doses of 1.5 and 6.0 ng/kg. The results of the control experiments with the novel juvenile indicated that an OT dose of neither 1.5 nor 6.0 ng/kg decreased SIT during the second encounter. In experiments with an IEI of 30 min, placebo treatment resulted in normal SRM (i.e., SIT was signiﬁcantly reduced in the second, relative to the ﬁrst, encounter with the juvenile). The rats also showed normal SRM after treatment with the two lower doses (1.5 and 6.0 ng/kg, subcutaneous) of OT but not with the higher dose (24 ng/kg, subcutaneous). In addition, the ANOVA indicated a signiﬁcant difference in SIT scores between placebo treatment and OT treatment with the 24-ng/kg dose, but not with the 1.5- and 6.0-ng/kg doses for this experiment. In the experiments with the same juvenile and the 120-min IEI, comparison between placebo treatment and each of the dose levels used for AVP and AVT treatment conditions indicated no signiﬁcant differences for the SIT scores obtained in the second encounter. The most important result of this study was the dose–response curve indicating that peripherally administered OT facilitates SRM when given in low doses, and impairs it in high doses. Noting that the passage of these peptides across the blood–brain barrier and their uptake by brain tissue is still under debate (see Chapter 14), these authors nevertheless interpreted their data as evidence that the peptide interacted with central brain sites implicated in the processing of SRM. Evidence that a central action of OT mediates this form of memory has been demonstrated in studies using intracerebroventricularly and microinjected peptides into speciﬁc brain structures (discussed below). Two other points were made in discussing these results. The ﬁrst was their relevance for a physiological action of the peptide in SRM. Although the plasma level of OT was not directly measured in this study, it was suggested that the low dose levels used in this study produced plasma levels within the range of physiological values observed in animals and/or humans during or after a variety of self- and species-preservative encounters. These include increases in OT plasma level after restraint (Gibbs, 1984), during sexual activity (McNeilly and Ducker, 1972; Murphy et al., 1987), parturition

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(Fitzpatrick, 1961; Higuchi et al., 1985), and nursing of young (Higuchi et al., 1985). The second point was the generalizability of this dose–response effect to retention in a variety of situations involving adaptive behavior (including learning). The authors proposed that a mild increase in plasma OT, such as might result during an encounter with a conspeciﬁc juvenile, facilitates retention of the experience. On the other hand, high levels of circulating OT, such as those observed during the stressful experience of parturition in animals and humans (Fitzpatrick, 1961; Higuchi et al., 1985), attenuate retention of the experience. High levels of OT during labor (Fitzpatrick, 1961) and memory impairment for the pain experienced at this time (Kennett et al., 1982) have both been reported in humans. iii. Popik et al. (1996) Popik et al. (1996) used the SRT to investigate dose-dependent memory-facilitating and -attenuating effects of subcutaneously injected OT(1–9) and several OT-derived peptides in male rats. The OT-derived peptides were as follows: desglycinamide-OT [OT(1–8)]; tocinamide [OT(1–6)]; [pGlu4,Cyt6]OT(4–9) [OT(4–9)]; [pGlu4,Cyt6]OT(4–8) [OT(4–8)]; [Pro-Leu-Gly-NH2]OT(7–9) [PLG]; [Leu-Gly-NH2]OT(8–9) [LG], and glycine. The doses used for assessing SRM-facilitating effects were 0.6 and 6.0 ng/kg (except glycine, which was used at doses of 1.0 and 10.0 ng/kg). The SRM-attenuating effects were assessed with doses of 0.6 and 6.0 g/kg. The selection of these doses was based on previous studies [Popik and Vetulani (1991) and Popik et al. (1992); see above]. The subjects were tested under all treatment conditions in a cross-over design that ensured placebo-controlled treatment conditions; 2 days intervened between successive test treatments. Placebo or peptide was subcutaneously injected at the end of the ﬁrst presentation trial. The same juvenile was reintroduced after a 30- or 120-min IEI. The change in social interest, expressed as the recognition index (RI), was calculated as the social investigative time (SIT) in the second presentation trial divided by that of the ﬁrst trial and multiplied by 100: (second SIT/ﬁrst SIT) 100. The placebocontrolled RI was calculated for each subject and equaled the RI of placebo treatment minus the RI of peptide treatment. Thus, positive values for this RI indicated SRM facilitation, and negative values indicated attenuation. The results were as follows: (1) social investigation of the juveniles by the residents was vigorous and lasted between 80 and 120 s (i.e., about 30% of the presentation interval); (2) an OT amnestic action on normal SRM was found after treatment with both high doses (0.6 and 6.0 g/kg) of OT(1–9), OT(1–8), OT(7–9), and OT(4–9), and with only the 0.6-g/kg dose of OT(1–6) and OT(4–8), whereas neither of the high doses of OT(8–9) inﬂuenced this form of memory; and (3) SRM of the preencountered juvenile occurred after an IEI of 120 min in rats treated with low doses (0.6 and

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6.0 ng/kg) of the following OT-related peptides: OT(1–9), OT(7–9), and OT(8–9), whereas the other peptides and glycine were ineffective in this respect. The discussion points were as follows: 1. These ﬁndings together with those obtained by Popik et al. (1992; see above) support the concept that SRM is facilitated by low but not by high doses of peripherally administered OT. The observation that, unlike a low dose of OT(7–9), a high dose failed to enhance SRM (120-min IEI) offered further support for this concept. 2. The structure–activity component of this study indicated that different parts of the OT molecule are responsible for its dose-related attenuating and facilitating of memory effects. The memory attenuation induced by high doses of OT was mimicked by OT-related peptides with and without the C-terminal glycinamide. The SRM attenuation induced by OT(1–9), OT(1–6), OT(7–9), OT(4–9), and OT(4–8), but not by OT(8–9), indicates that the amino acid residues in region 5–7 of the OT molecule are particularly important for this amnestic action. The SRM facilitation induced by low doses of OT was mimicked only by peptides with the C-terminal glycinamide, such as OT(1–9), OT(7–9), and OT(8–9), suggesting that region 8–9 of the OT molecule was important for this function. However, the failure of low doses of OT(4–9) to facilitate SRM is not consistent with this interpretation and requires clariﬁcation. 3. The SRM-attenuating and -facilitating effects of OT and its Cterminal metabolites have been observed in other learning paradigms. Memory-attenuating effects for OT(1–9) and its C-terminal metabolites OT(4–8) and OT(4–9) have been observed in tests of retention involving active and passive avoidance behavior [Bohus et al., 1978b (see Chapter 2); De Wied et al., 1987 (see Chapter 5)]. Small C-terminal peptides of OT, such as PLG and LG, had memory-facilitating effects: reduced puromycin-induced amnesia in mice, and PLG facilitated reversal learning in rats in a brightness discrimination task (Rigter and Popping, 1976). In other studies, PLG and related peptides exhibited attenuating effects on retention, such as the facilitation of extinction in a conditioned taste aversion (Rigter and Popping, 1976). All these studies, however, used much higher dose levels than those used in the present study. 4. The effectiveness of low doses of PLG [OT(7–9)] and LG [OT(8–9)] in facilitating SRM in this study led these researchers to postulate that these, or structurally related metabolites of OT, are physiologically involved in memory processes. They cited evidence that such peptides may be normally generated in the brain by enzymatic processes (Burbach et al., 1983b; see Chapter 5) as consistent with this postulate. However, two facts detract from the strength of this postulate: (a) neither this study nor that of Popik et al. (1992) obtained an independent measure of the plasma levels of OT and OT metabolites produced by their treatments; and (b) given the

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blood–brain barrier (see Chapter 14), it is not clear that these peripherally administered peptides entered the brain and exerted direct effects on brain structures involved in SRM processing. iv. Arletti et al. (1995) Arletti et al. (1995) designed a study to test both SRM-enhancing effects and antidepressant effects of OT in aged male Wistar rats. Only the testing pertinent to SRM is discussed here. They noted that memory function in humans is usually compromised during normal aging (Rapp and Amaral, 1992) and that, in rats, OT improves SRM (Benelli et al., 1995; Popik et al., 1992). The subjects (26-month-old Wistar male rats) were tested for memory in the SRT (IEI of 120 min) with 4- to 5-week-old male juvenile conspeciﬁcs as social test stimuli. Placebo or OT (1.5, 3.0, 6.0, or 15.0 ng/kg; intraperitoneal) was peripherally administered at the end of the ﬁrst presentation trial. The difference in the time spent investigating the juvenile (proximally oriented toward, or in direct contact with the juvenile) between the ﬁrst and second presentations was calculated as SIT (second SIT – ﬁrst SIT); a negative SIT value means a reduction in SIT during the second trial. The criterion for enhanced SRM was a statistically signiﬁcant shortening in mean SIT in the second relative to the ﬁrst presentation for a given treatment group. Placebo treatment did not enhance SRM (no signiﬁcant SIT after a 120min IEI). OT treatment at the dose levels of 3 and 6 ng/kg, but not at the dose levels of 1.5 or 15 ng/kg, enhanced SRM memory (signiﬁcant SIT after the 120-min IEI). Thus, depending on the dose, OT enhanced SRM in these aged rats as it has been observed to do in young rodents when administered peripherally (Popik et al., 1992; see above) or centrally (Benelli et al., 1995; see below). 3. Central Administration a. Selected Study: Benelli et al. (1995) Benelli et al. (1995) studied the effect of a wide dose range of intracerebroventricularly injected OT (1 ng to 1000 ng/rat) on SRM, and the ability of a selective OT antagonist, d(CH2)5[Tyr(Me),Orn8]vasotocin (VT), to block this effect. A decrease in SIT during the second encounter with the same juvenile after a 120-min IEI indicated improved memory. Experimental rats received a speciﬁc dose level of OT immediately after removal of the juvenile at the end of the ﬁrst encounter; control rats received physiological saline by the same route, at the same infusion rate, and at the same time as the experimental rats. Tests were separated by a minimum of 48 h. In tests with the OT antagonist, the two peptides were injected either alone [i.e., OT (1 ng or 500 ng/rat, intracerebroventricular) or the OT antagonist (1 ng or 500 ng/rat, intracerebroventricular)] or together (OT, 1 ng plus OT antagonist, 1 ng; or OT, 500 ng plus OT antagonist, 500 ng) after removal of the juvenile in the ﬁrst encounter.

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When injected together, the injection of the antagonist preceded that of OT by 5 min. The results were as follows: (1) OT at the low end of the dose range used in this study (10 ng to 1 ng) signiﬁcantly improved SRM but had no effect or slightly impaired it at the high end (above 10 ng). The failure of these low doses to reduce SIT time when a novel juvenile was presented after a 120min IEI supports a mnemonic effect rather than a spurious nonspeciﬁc treatment effect; and (2) injected on its own, the OT antagonist at either dose level had no effect on SRM, but pretreatment with the antagonist at the same dose as the agonist blocked the memory-improving effect of the low dose (1 ng) of OT, and reversed the slight memory impairment induced by the high dose (500 ng) of OT. In the discussion, the authors commented on (1) the implication of these results for a physiological role of OT in the modulation of SRM in the male rat, and (2) possible reasons for opponent effects of high and low doses of OT on this memory. The effectiveness of minimally active doses of OT (within the range of physiological values) in preserving STM suggested that centrally released OT plays a physiological role in this processing, although the failure of the OT antagonist to block SRM when given alone did not corroborate this. Nevertheless, the ability of the antagonist to block the facilitated SRM induced by OT treatment makes clear that central OT receptors mediated this mnemonic effect. The opposing effects of the low and high doses of OT on SRM could be attributed to a number of causal factors. Thus, the two dose ranges may each have recruited or activated different circuitries with opposite effects on memory processes, or activated different OT receptor subpopulations mediating opposing actions on social memory, or these effects may have resulted from dose-related opposing effects of OT on certain neurotransmitter systems involved in memory processes (see discussion by Kovacs, 1986 and Chapter 4).

IV. Sex Differences and the VP/OT Influence on SRM

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A. General Comments ‘‘Anatomical sexual dimorphy’’ characterizes the extrahypothalamic VP-ergic circuitry in the brains of rats. Anatomical studies with VP-staining techniques have shown that the amount of AVP mRNA in cell bodies of the medial amygdala and bed nucleus of the stria terminalis (BNST), and density of VP-ergic ﬁber projections from these nuclei, are greater in male than in female rats (De Vries and Al-Shamma, 1990; De Vries et al., 1985; Miller et al., 1989b; Van Leeuwen et al., 1985; see Chapter 1). Studies of De Vries, Wang, and colleagues (cited in Chapter 1) provided evidence that

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the steroidal hormonal environment during prenatal development exerts an organizational effect on this sexually dimorphic circuitry. Further support has been added by observations that the birth of AVP cells within various subdivisions of the BNST occurred in close proximity to the gestational days when testosterone level increased in the male fetus (Al-Shamma and De Vries, 1996; Baum et al., 1991), and that prenatal exposure to ﬂutamide (androgen antagonist) reduced AVP immunoreactivity (AVPir) in the BNST of male rats (Axelson et al., 1993). Bluthe and Dantzer (1990; see Chapter 12) demonstrated a ‘‘functional sexual dimorphy’’ for SRM by showing that only male rats were dependent on extrahypothalamic VP-ergic circuitry for normal SRM (i.e., a peripherally applied AVP receptor antagonist disrupted this memory in male but not in female rats). In addition, they demonstrated that AVP-mediated SRM in the male was androgen dependent because social recognition in castrated male rats was as insensitive to the effects of AVP antagonists as that in intact females. The research discussed below conﬁrms and extends these ﬁndings of Bluthe and Dantzer (1990): (1) Axelson et al. (1999) furthered our understanding of the inﬂuence of the prenatal hormonal environment on the ‘‘functional sexual dimorphy’’ operating in SRM; (2) Van Wimersma Greidanus and Maigret (1996) and Landgraf et al. (1995) indicated the importance of VP-ergic sexually dimorphic circuitry for SRM in the male; and (3) Engelmann et al. (1998) provided evidence that OT may be signiﬁcant for SRM in the female. 1. Selected Studies a. Axelson et al. (1999) Axelson et al. (1999) carried out two experiments: in experiment 1 they determined the degree to which VP-mediated SRM in the male rat is inﬂuenced by previous sexual experience, and in experiment 2 they investigated the organizational actions of circulating androgens in the prenatal environment on SRM. In experiment 1, sexually experienced intact males (‘‘breeders,’’ which successfully copulated with sexually primed and receptive ovariectomized females during each of three test trials) and sexually naive intact males (‘‘virgins’’; denied copulation by nonreceptive ovariectomized females during such test trials) were tested in the SRT with a 30-min IEI. Each subject was tested for SRM under three testing/treatment conditions: familiarcontrol, unfamiliar-control, and familiar-antagonist. These three conditions differed as to whether a familiar (preencountered) or an unfamiliar (novel) juvenile was presented after the 30-min IEI, and whether the subject received a subcutaneous injection of physiological saline or a 30-g/kg injection of the AVP receptor antagonist [deamino-Pen1,O-Me-Tyr2,Arg8]vasopressin (AVP-Ant treatment condition) immediately after the initial presentation. These test conditions were given to all subjects in a counterbalanced order, with each subject tested under a given condition every other day over 6 days.

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The SIT of the second presentation trial was scored as a percentage change from that of the ﬁrst presentation trial. The results of experiment 1 showed that sexually experienced and virgin males behaved similarly in this SRT: (1) after vehicle treatment, both breeders and virgins recognized the familiar juvenile. Both groups signiﬁcantly reduced SIT in the second relative to the ﬁrst investigative trial with the familiar but not the unfamiliar juvenile (percent reduction for breeders and virgins was 69 and 64%, respectively); and (2) in the AVP-Ant treatment condition, both breeders and virgins failed to recognize the previously encountered juvenile. SIT during the second trial with the familiar juvenile did not differ signiﬁcantly from that with the unfamiliar juvenile (SITs during the second encounter with the same juvenile were 94 and 87% of the level observed during the ﬁrst encounter for the breeders and virgins, respectively). In summary, the results of experiment 1 indicated that prior sexual experience was not a requirement for either olfactory-based conspeciﬁc recognition, or for the role of AVP in this behavior. Both virgins and breeders recognized the familiar juvenile after a 30-min IEI and this depended on normal VP-ergic transmission, because AVP-Ant treatment blocked this SRM. In experiment 2, SRM was tested in four groups of subjects: (1) ﬂutamide-TP males, male offspring of females that received an injection of the androgen antagonist (ﬂutamide) on each day of the last 10 days of gestation; these offspring were treated with testosterone proprionate (TP; 50 g/rat, subcutaneous) within 8 h of birth; (2) ﬂutamide-control males, male offspring of ﬂutamide-treated females; these offspring received saline instead of TP after birth; (3) control males, nontreated offspring of females that were injected with saline instead of ﬂutamide during gestation; and (4) normal females. At 25 days of age the subjects were weaned and at a later date serum samples were assayed for total testosterone and immediately thereafter each male was castrated and implanted with crystalline testosterone to provide physiological levels of the hormone (Smith et al., 1977). At 90 days of age the subjects were tested for SRM with the same treatment and test procedures used in experiment 1. The major ﬁndings of experiment 2 were as follows: (1) after vehicle treatment, both the control males and females recognized the familiar juvenile after the 30-min IEI (relative to the ﬁrst encounter, the SIT with the familiar, but not the unfamiliar juvenile, signiﬁcantly decreased by 45 and 57% in the control males and females, respectively); moreover, SRM did not differ between estrous and nonestrous females; (2) AVP-Ant treatment blocked normal SRM in the control males, but not in the females (AVPAnt-treated male controls increased SIT spent with the familiar juvenile during the second encounter relative to the ﬁrst encounter, as also occurred with the novel juvenile after vehicle treatment. However, AVP-Ant-treated females recognized the familiar juvenile, as reﬂected by a 72% reduction in SIT); (3) whether or not they received day 1 treatment with vehicle or TP, the

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offspring of ﬂutamide-treated females recognized the familiar juvenile during the second encounter, as did their male controls and female counterparts; and (4) however, like the females, both the ﬂutamide-control males and ﬂutamide-TP males were insensitive to the effects of AVP-Ant treatment, and recognized the familiar juvenile after the 30-min IEI (they decreased their SITs by 47 and 33%, respectively, during the second encounter). Taken together, the results of experiment 2 replicated the earlier ﬁndings of Bluthe and Dantzer (1990; see Chapter 12) that normal males show more interest than females in their initial investigation of juvenile conspeciﬁcs, and that unlike males, SRM in females is not dependent on sexually dimorphic extrahypothalamic VP-ergic circuitry. These results further showed that prenatal androgens were important for sex differences in VP-dependent SRM, but not for sex differences in interest shown during initial investigation of the juvenile. Flutamide-induced antagonism of normal androgen release in the prenatal environment prevented the VP-dependent SRM normally observed in intact males, and rendered their performance on the SRT equivalent to that of their female counterparts. However, this antagonism did not inﬂuence the sex differences in juvenile investigative behavior during the initial encounter. These ﬁndings and previous studies demonstrating that prenatal ﬂutamide treatment reduced AVP immunoreactivity within the BNST (Axelson et al., 1993), and that septally released AVP is important for SRM in the male rodent [Bluthe et al., 1990; Dantzer et al., 1988 (see Chapter 12)] provide evidence that sex differences in AVP dependency are linked to sex differences in AVP content in cells of the BNST and their projections to the lateral septum. b. Engelmann et al. (1998) Engelmann et al. (1998) designed a study to determine whether endogenous OT is involved in the SRM of female rats. These animals were tested in their home cage during the activity phase of the light–dark cycle with the social discrimination paradigm (SDP) described earlier. Brieﬂy, the resident was given an initial 4-min investigative trial with a juvenile conspeciﬁc (23–35 days old, both sexes) that was promptly removed from the resident’s cage at the end of the trial. After an IEI of 30, 60, or 180 min it was reintroduced to the resident female, along with a novel juvenile, for a second 4-min investigative trial. SRM was judged to be present if the duration of investigation of the familiar juvenile was signiﬁcantly shorter than that of the novel one during the second trial. All subjects were tested under three types of testing conditions: (1) nontreatment sessions with IEIs of 30, 120, and 180 min; (2) treatment with intracerebroventricularly injected OT (1 ng/rat) or vehicle (Ringer’s solution), and IEIs of 120 or 180 min; and (3) treatment with intracerebroventricularly injected vehicle, the OT receptor antagonist desGly-NH2,d(CH2)5[Tyr(Me)2Thr4]OVT (100 ng), or the V1 receptor antagonist d(CH2)5[Tyr(Me)]AVP (100 ng), and IEIs of 60 min. Treatments were administered via previously

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implanted cannulas immediately after removal of the juvenile during the ﬁrst investigative trial. The 60-min IEI was chosen for testing the effects of the antagonists on SRM because this IEI was within the time interval during which the nontreated residents consistently recognized the preencountered juvenile, and it allowed a more sensitive detection of the effects of a blockade of OT-ergic and VP-ergic neurotransmission than the 30-min IEI. Investigative behavior was compared for the estrous versus the anestrous condition during nontreatment and treatment test sessions. The results were as follows: (1) the SIT during the ﬁrst presentation was signiﬁcantly shorter in the estrous than in the anestrous state; however, there was no signiﬁcant difference in SRM between the two hormonal states; (2) the effects of the various treatments were also independent of the stage of the estrous cycle; (3) in the vehicle control condition, the SITs were signiﬁcantly shorter in duration with the preencountered compared with the novel juvenile after IEIs of 30 and 120 min, but there were no SIT differences after an IEI of 180 min; (4) there were no signiﬁcant differences between OT-treated females and vehicle controls in SRM tested with either the 120- or 180-min IEI; (5) in contrast to vehicle treatment, the OT antagonist blocked SRM after the 60-min IEI (i.e., OT treatment abolished the signiﬁcant SITs for the familiar versus the unfamiliar juvenile observed for the vehicle control condition after the 60-min IEI); and (6) unlike OT, the VP antagonist failed to block SRM after this 60-min IEI. The investigators related these results to other ﬁndings and offered several explanations for their causes. First, the reduced duration of investigation during estrus was likely due to increased time spent in proceptive (ear wiggle, darting, and hopping) and receptive (lordosis) sexual behavior. Although the sex of the juvenile might have inﬂuenced this behavior, this was not thoroughly investigated in the study. Despite their reduced investigative curiosity, these estrous females showed normal recognition ability. In addition, the duration of the IEIs during which female rats demonstrated SRM for preencountered conspeciﬁcs (30–120 min), or not (180 min), replicate observations reported by Bluthe and Dantzer (1990; see Chapter 12) and further indicate that the duration of SRM is twice as long in female than in male rats tested under similar conditions (Engelmann et al., 1995; Landgraf et al., 1995). Second, the inability of OT treatment to inﬂuence SRM may have been due to a saturation of relevant binding sites by endogenous OT during the ﬁrst encounter, so that reinforcement of this OT-ergic neural communication by supplemental exogenous OT was of no further consequence. If so, it is not clear why higher doses of OT with different treatment schedules have been shown to alter social behavior (Witt et al., 1992). In addition, it is not clear why, in contrast to OT, peripherally and centrally administered AVP should improve SRM in females (Bluthe and Dantzer, 1990; Engelmann and Wotjak, unpublished observations), as well as in males (Bluthe and Dantzer, 1990). According to Dantzer and colleagues (see Chapter 12) the

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improvement in SRM induced by exogenous AVP is due to activation of a non-androgen-dependent AVP that produces its effects on SRM via an interaction with the central arousal system. Third, the inability of the centrally administered V1 receptor antagonist to interfere with normal social recognition conﬁrms the results obtained by Bluthe and Dantzer (1990) with this VP analog when peripherally administered. Further, the differential effects of the VP and OT receptor antagonists on formation of SRM implicate causal involvement of OT, but not VP, receptors in this processing. These ﬁndings, together with evidence that both VP and OT receptor antagonists show partial antagonism on each other’s receptors when present in the same brain structures (De Wied et al., 1991; see Chapter 5), raise a question concerning the brain site(s) at which endogenous OT acts to inﬂuence this processing in the female rat. It was proposed that whereas endogenous VP in the septum is important for mediating social recognition in male rats [Dantzer et al., 1988 (see Chapter 12); Landgraf et al., 1995], endogenous OT in the medial preoptic area might perform this role in the female rat. Evidence that this brain site is important in mediating social behavior in the female rodent (Popik and Van Ree, 1991; see below) is consistent with this suggestion. It was further proposed that local administration of the OT receptor antagonist via inverse microdialysis (retrodialysis) might clarify this issue. In concluding remarks, the authors explained the sex-differentiated dependency of SRM on VP and OT by proposing the following ‘‘working hypothesis’’: OT becomes progressively involved in SRM during ontogeny, and in females this situation persists and may be further developed and ﬁne-tuned by female sex steroids, which contribute to the characteristic aspects of maternal behavior (Argiolas and Gessa, 1991; Pedersen and Prange, 1979; Pedersen et al., 1994). However, the increasing production by males of male sex steroids results in the greater degree of VP synthesis observed within their limbic brain, and this sexually dimorphic AVP is likely to be among the regulatory mechanisms involved in male social and territorial behavior (Bluthe et al., 1990; Compaan et al., 1993; Koolhaas et al., 1991). Consequently, ‘‘these mechanisms (including AVP) may dominate the original ones (including OT) in regulating social recognition abilities in male rats as well’’ (Engelmann et al., 1998, p. 93). The investigators cite a number of observations supportive of this working thesis (for further discussion see Engelmann et al., 1998).

V. Influence of Septal–Hippocampal VP and OT on SRM

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A. General Comments Evidence cited in earlier chapters has indicated that VP and OT, present in the septal–hippocampal system, inﬂuence memory processing in selfpreservative learning paradigms, and that the effects of these peptides on

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retention may involve an interaction with classic catecholamine (see Chapter 4) and cholinergic (see Chapter 10) mechanisms implicated in information processing, as well as via enhancement of glutamatergic neurotransmission (see Chapter 5). The studies presented below provide support that these peptide systems also play a role in memory processing underlying conspeciﬁc recognition, which is clearly important for reproductively related social interaction. Dantzer et al. (1988) were the ﬁrst to demonstrate that exogenous AVP (0.1 ng) injected into the septal area enhanced (prolonged the duration of) normal SRM, and this ﬁnding was subsequently replicated by others (Popik et al., 1992) and led to the demonstration of similar but more potent effects of VP metabolites (Popik et al., 1992). Dantzer et al. (1988) also provided the ﬁrst supportive evidence that endogenous septal AVP has an important role in SRM in the male rat (i.e., an intraseptal injection of a V1 receptor antagonist on its own prevented the normal expression of SRM). The research ﬁndings discussed below conﬁrm and extend these research ﬁndings of Dantzer, Bluthe, and colleagues. 1. Selected Studies a. Van Wimersma Greidanus and Maigret (1996) Van Wimersma Greidanus and Maigret (1996) injected anti-AVP serum or anti-OT serum, intracerebroventricularly or locally, into several limbic brain sites to examine the putative involvement of endogenous AVP and OT in SRM processing in the male rat. This technique was previously used to investigate the role of endogenous VP and OT in avoidance learning paradigms [Bohus et al., 1978a; Van Wimersma Greidanus and De Wied, 1976a (see Chapter 2); Van Wimersma Greidanus et al., 1975b (see Chapter 4)]. Resident male rats were tested in the SRT. Separate groups received an intracerebroventricular or local injection of anti-AVP serum (AVP antiserum), anti-OTserum (OTantiserum), or normal rabbit serum (NRS; controls). Each intracerebroventricularly injected substance was delivered via an implanted cannula into the left lateral ventricle in a volume of 3 l (1:10 or 1:20 dilution) or 2 l (1:10 dilution) for AVP antiserum, and of 2 l (1:10 or 1:30 dilution) for OT antiserum. Local injections of AVP antiserum (2-l volume, 1:20 dilution) or OT antiserum (2 l, 1:10 dilution) or NRS (1:20 or 1:10 dilution as controls for peptide antiserum treatments) were delivered via bilaterally implanted cannulas into the dorsal hippocampus (DH), ventral hippocampus (VH), dorsal septal region (DSR) or olfactory nucleus (ON). Treatment was administered immediately after removal of the juvenile in the ﬁrst investigative trial. An IEI of 30 min was used with AVP antiserum treatment, and of 120 min with OT antiserum treatment, to test for impairment and preservation of normal SRM, respectively. A signiﬁcant reduction in mean SIT in the second relative to the ﬁrst encounter for a given treatment group (assessed by t tests for paired data)

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indicated SRM. The mean SIT score for a given treatment group was calculated by subtracting the average SIT in the ﬁrst encounter from that in the second trial (a negative value indicated an average reduction of SIT in the second trial). Comparisons between selected antiserum treatment groups and their NRS controls on mean SITs were evaluated by the Newman–Keuls test (i.e., a signiﬁcantly more negative mean SIT value in a treatment versus a NRS control group indicates a treatment-induced improvement in SRM, whereas a signiﬁcantly less negative value indicates the converse). Cannula placements were histologically veriﬁed at the end of the experiments. Results of the experiments with intracerebroventricularly injected NRS, AVP, and OT antisera were as follows: (1) all NRS-treated rats recognized the familiar juvenile after the 30-min IEI (signiﬁcant reduction in SIT in the second presentation trial); (2) AVP antiserum at a dose of 3 l (1:10 or 1:20 dilution) but not at a dose of 2 l (1:10 dilution) impaired SRM after the 30-min IEI (i.e., compared with NRS treatment, the higher dose of AVP antiserum signiﬁcantly increased SIT during the second trial and signiﬁcantly decreased the negative SIT scores); (3) treatment with 2 l of OT antiserum at a dilution of 1:10, but not 1:30, preserved SRM after a 120min IEI (i.e., decreased SIT during the second encounter, and induced a mean SIT that was signiﬁcantly more negative than that in NRS controls); and (4) the increased SIT observed with anti-OT serum at the 1:10 dilution during the second trial with the same juvenile did not occur in a control experiment that presented a different juvenile during that trial. Results with the locally injected substances indicated that (1) AVP antiserum (2 l in a 1:20 dilution), injected into the VH, impaired normal SRM (30-min IEI) (i.e., no signiﬁcant difference in SIT between the ﬁrst and second encounters with the same juvenile, and the SIT value was signiﬁcantly less negative than that for the NRS control group); (2) OT antiserum (2 l in a 1:10 dilution) preserved SRM (120-min IEI) when injected into the VH (i.e., compared with the NRS control condition, anti-OT serum signiﬁcantly reduced SIT during the second relative to the ﬁrst encounter, and produced a signiﬁcantly greater negative mean SIT value); (3) AVP antiserum (2 l, 1:20 dilution), injected into the DH, impaired normal SRM (30min IEI) (i.e., compared with NRS treatment, AVP antiserum signiﬁcantly lengthened SIT during the second relative to the ﬁrst encounter and produced a signiﬁcantly less negative SIT); (4) OT antiserum (2 l, 1:10 dilution), injected into the DH, did not preserve SRM (120-min IEI) (no signiﬁcant difference from controls with respect to either SIT during the second relative to the ﬁrst encounter, or SIT); (5) AVP antiserum (2 l, 1:20 dilution), injected into the DSR, impaired normal SRM (30-min IEI) (compared with NRS treatment, AVP antiserum produced a signiﬁcantly smaller reduction in SIT during the second relative to the ﬁrst encounter, and a signiﬁcantly smaller SIT value); (6) OT antiserum (2 l, 1:10 dilution), injected into the DSR, did not signiﬁcantly inﬂuence SIT or SIT relative to

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NRS controls; and (7) when injected into the ON, neither antiserum inﬂuenced SRM [no signiﬁcant differences in SIT during the second relative to the ﬁrst encounter, or in SIT scores in AVP antiserum (2 l, 1:20 dilution)treated subjects relative to NRS controls (tested with a 30-min IEI) or in OT antiserum (2 l, 1:10 dilution)-treated subjects relative to NRS controls (tested with a 120-min IEI)]. Taken together, these results showed that (1) anti-AVP serum, injected intracerebroventricularly or microinjected into the DH, VH, and DSR, impaired SRM after a 30-min IEI, a time when NRS controls recognized the previously encountered juvenile; (2) anti-OT serum, injected intracerebroventricularly or microinjected into the VH, but not into the DH or DSR, preserved SRM for the 120-min test interval, a time when it was absent in NRS controls; and (3) microinjections of either antiserum into the ON did not inﬂuence SRM in the test paradigm. In their discussion of these results the authors made the following comments: (1) OT, released in the local limbic structures studied in this investigation, seems less involved than AVP in SRM; thus OT located within or released from the VH (this study) and the medial preoptic area (Popik and Van Ree, 1991), but not from the DH, DSR, or ON, was shown to have a role in SRM; (2) in those limbic areas where OT does exert an effect on SRM, its role is rather complex in nature. Thus, depending on the dose level, central administration of this peptide enhances, impairs, or has no effect on this form of memory (Benelli et al., 1995; Popik and Van Ree, 1991); (3) given the importance of olfactory cues in this paradigm (Carr et al., 1976; Popik et al., 1991; Sawyer et al., 1984), and the presumed importance of an intact vomeronasal system in VP-ergic modulation of SRM in rats (Bluthe and Dantzer, 1993), it was surprising to observe that apparently neither VP nor OT in the ON appears to be physiologically important in conspeciﬁc recognition memory; and (4) comparison between the present study and those investigating the physiological roles of VP and OT in avoidance paradigms has shown that, at least for VP, release of this peptide within the DH, VH, and septum is of physiological importance in mediating memory processing tested in the olfactory-based social recognition paradigm as well memory processing tested in aversive learning paradigms. b. Engelmann et al. (1994) Engelmann et al. (1994) noted that it had previously been shown that osmotic stimulation of the hypothalamic supraoptic nuclei (SON) caused it to release endogenous AVP (Landgraf and Ludwig, 1991). They designed two series of experiments to determine (1) whether this stimulation would also release endogenous AVP from a brain site (i.e., mediolateral septum, MLS) other than the SON, and (2) if so, to determine whether this increase in endogenous AVP was associated with enhanced SRM in the male rat. Osmotic stimulation was effected by administration of hypertonic artiﬁcial cerebrospinal ﬂuid (aCSF containing

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1 M NaCl) via microdialysis into each SON. Endogenous AVP released in the dialysates collected from the SON during microdialysis, and in the perfusates collected from the MLS during push–pull perfusion, was measured by radioimmunoassay (RIA). Two surgical procedures were performed: (1) bilateral implantation of U-shaped microdialysis probes into each hypothalamic supraoptic nucleus (SON) for osmotic stimulation and measurement of AVP released from the SON and (2) implantation of the push–pull cannula into the MLS for measurement of AVP released from this brain site in response to the osmotic stimulation. Probe placements were conﬁrmed by postmortem histology. An initial series of experiments tested the effects of osmotic stimulation of the SON on AVP release from the SON, and from the MLS in anesthetized rats. Endogenously released AVP (picograms per sample) was measured by RIA in dialysate and perfusate samples collected simultaneously over successive periods of 30 min. During osmotic stimulation, isotonic aCSF (0.15 M NaCl) was replaced by hypertonic aCSF (1.0 M NaCl). Figure 1 depicts the AVP content in the SON dialysates (Fig. 1A) and MLS perfusates (Fig. 1B) simultaneously collected during microdialysis of the SON with isotonic aCSF (samples 1 and 2), with hypertonic aCSF (sample 3), and during the poststimulation period (samples 4 and 5). Osmotic stimulation of

FIGURE 1 (A) AVP contents in 30-min dialysates sampled continuously (means þ/ SEM; data are pooled from left and right SON) in urethane-anesthetized male rats (n ¼ 12). Isotonic (0.15 M) was replaced with hypertonic aCSF (containing 1 M NaCl) during collection period of sample number 3. Note the typical ‘rebound’ increase in AVP release during the poststimulation period (**p < 0.01 vs all other dialysates, ANOVA; see also Fig. 2). (B) Simultaneously collected push-pull perfusates (means þ/ SEM) from the mediolateral septum (perfusion medium: isotonic aCSF). *p < 0.05 vs perfusions 1, 2 and 5, ANOVA. & microdialysis; & push-pull perfusion. Source: Engelmann et al., 1994 (Fig. 1, p. 392). Copyright ß 1994 by Blackwell Science. Reprinted with permission.

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the SON with microdialysis-applied hypertonic aCSF produced a nonsignificant increase in the content of AVP released from the SON, and this was followed by a signiﬁcant increase in this content, associated with the robust ‘‘rebound’’ effect that occurred in the poststimulation period (dialysis medium was changed from hypertonic to isotonic aCSF) (Fig. 1A). Concomitantly released AVP within the MLS was signiﬁcantly increased during and after osmotic stimulation of the SON (Fig. 1B, samples 3 and 4). The authors noted that previous observations (Engelmann, Ludwig, and Landgraf, unpublished results) ruled out the probability that diffusion of AVP from the SON was responsible for the increase in AVP content in the MLS. In the second series of experiments the effect of osmotic stimulation of the SON on SRM was investigated. For these experiments, separate groups of rats were implanted with a U-shaped microdialysis probe either (1) into the right SON alone, (2) in combination with a concentric microdialysis probe into the MLS, or (3) in combination with a guide cannula for microinjection into the medial nucleus of the ipsilateral amygdala. After a 2-day period of recovery from surgery, the microdialysis probes were connected with a microperfusion pump via polyethylene tubing suspended over the center of the home cage and dialysates were collected in vials during concomitant behavioral testing. The subjects were tested in the conventional SRT with juvenile (20- to 25-day-old) rats of both sexes as social test stimuli, and with a 120- or 30min IEI. SRM was assessed by the reduction of SIT with the familiar juvenile, expressed as the ratio of SITs during the second and ﬁrst presentation trials [RID (ratio of investigation duration) scores]. Pilot studies had shown that untreated male rats recognize a familiar juvenile after a 30-min IEI (RID range, from 0.5 to 0.6), but not after a 120-min IEI (RID, approximately 1.0). In the ﬁrst experimental test (described below), those rats that showed enhanced SRM were retested the next day with a different juvenile during the second presentation to ensure that the signiﬁcant reduction in investigative time with the familiar juvenile was due to factors speciﬁc to the individual juvenile (i.e., expected RID, approximately 1.0). Two types of experimental tests were performed with the SRT. In the ﬁrst test SRM was assessed in rats that were also monitored for release of AVP within the SON in response to osmotic stimulation of this brain site. SON microdialysis probes were perfused for two consecutive periods, each lasting 30 min. The ﬁrst perfusion was started 35 min before the ﬁrst presentation with the juvenile, and the second perfusion followed immediately thereafter during the ﬁrst presentation. The rats were assigned to one of four groups (n ¼ 9 rats/group) depending on whether the ﬁrst presentation trial occurred in the absence of SON microdialysis (untreated group), during microdialysis of the SON with isotonic aCSF (isotonic group) or with aCSF containing 1 M NaCl (hypertonic group), or after microdialysis of the SON with hypertonic aCSF (after-hypertonic group).

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The results of this experimental test are presented in Fig. 2. The individual bars in Fig. 2A depict the AVP content per 30-min sample of dialysate recovered from the SON during isotonic, during hypertonic, and after hypertonic stimulation of this nucleus. As noted, osmotic stimulation (during hypertonic) tended to increase AVP release from the SON, and the amount of AVP released during the typical rebound AVP release effect that occurs during the poststimulation interval (after-hypertonic group) was signiﬁcantly greater than that observed during SON microdialysis with isotonic and hypertonic aCSF. The bar graphs (columns) in Fig. 2B represent the RID scores obtained under the different osmotic treatment conditions used in this test. Analysis of the data presented in Fig. 2B indicated that SRM was improved only during the rebound release of AVP within the SON in the 30 min after osmotic stimulation. Moreover, reexposure to a different juvenile (toned column in Fig. 2B) showed that the memory-enhancing effect was speciﬁc to the familiar juvenile. Figure 2C depicts the relationship between RID scores and AVP levels in SON dialysates collected simultaneously from the same rats. The weak but signiﬁcant correlation between the two variables indicated that the better the SRM (the lower the RID scores) the greater the osmotically induced release of AVP from SON. For the second test additional groups of rats were used to investigate the effects of a V1 receptor antagonist, d(CH2)5[Tyr(Me)]AVP, on the SRM effects induced by osmotic stimulation of the SON. The SRT was given over a 2-day period, the ﬁrst day without the V1 antagonist (aCSF alone), the second day with the V1 antagonist (aCSF containing the V1 antagonist, 40 ng) delivered into the right SON, mediolateral septum (MLS), or the ipsilateral central amygdala during the two 30-min dialysis periods before and during osmotic stimulation of the SON. The osmotic stimulus (hypertonic aCSF) was delivered via the SON microdialysis probe 35 min before the ﬁrst presentation of the juvenile. A control group implanted with the SON dialysis probe was tested under the same conditions but was not osmotically stimulated (i.e., SON dialyzed with isotonic aCSF). Effects of the V1 antagonist delivered to the SON and the MLS are presented in Fig. 3. On day 1, SRM was enhanced during the hypertonically induced intranuclear rebound release of AVP that followed osmotic stimulation of the SON in rats that had received aCSF alone in the SON and MLS [RID scores were signiﬁcantly reduced in both osmotically stimulated groups (after-hypertonic SON and after-hypertonic MLS) relative to nonstimulated controls (isotonic)]. On day 2, the osmotically induced facilitation of SRM observed on day 1 was partially abolished by delivery of the V1 antagonist to the SON and MLS [no signiﬁcant difference in RID scores between nonstimulated untreated controls (isotonic plus aCSF) and the two osmotically stimulated V1 treatment groups (the after-hypertonic plus V1 antagonist-treated SON and the after-hypertonic plus V1 antagonist-treated

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FIGURE 2 Effects of microdialysis with isotonic or hypertonic aCSF on the release of AVP within the right SON and on performance in the social recognition paradigm. The rats (n ¼ 9 per group) were behaviorally tested in the social recognition paradigm with ﬁrst exposure to the juvenile without SON microdialysis (untreated), during SON microdialysis with isotonic or hypertonic aCSF or after SON microdialysis with hypertonic aCSF (i.e., during ‘rebound’ release of AVP). (A) Each bar represents the mean pg AVP þ/SEM recovered during 30-min microdialysis with the treatment indicated. Osmotic stimulation (hatched bar) tended to increase AVP release within the SON (during hypertonic). Note the typical ‘rebound’ release of the neuropeptide during the poststimulation interval (after hypertonic; þþp < 0.01 vs isotonic and during hypertonic, ANOVA). (B) The mean ratio of investigation duration (RID) þ/SEM is shown for treatment via microdialysis probe during the ﬁrst exposure. RID was not altered during SON microdialysis with isotonic or hypertonic aCSF. However, ﬁrst exposure to the juvenile after microdialysis with hypertonic aCSF followed by isotonic aCSF (i.e., during the ‘rebound’ release of AVP) caused signiﬁcantly improved social recognition (**p < 0.01 vs all other columns, ANOVA), whereas exposure to a different juvenile during the same period showed this to be juvenile-related memory (toned column; p < 0.01 vs same juvenile after hypertonic. (C) The correlation between RID and AVP levels in SON microdialysis samples collected simultaneously from the same animals was signiﬁcant (r ¼ 0.425. p < 0.05). Source: Engelmann et al., 1994 (Fig. 2, p. 393). Copyright ß 1994 by Blackwell Science. Reprinted with permission.

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FIGURE 3 Ratio of investigation duration (RID; means þ SEM) of adult male Wistar rats implanted with a microdialysis probe in the right SON alone (n ¼ 11, toned columns), or additionally in the mediolateral septum (n ¼ 8, hatched columns); the animals received the osmotic stimulus (hypertonic aCSF) via the SON microdialysis probe 35 min before ﬁrst exposure to a juvenile. For comparison, a control group implanted with a microdialysis probe in the SON was tested under the same conditions except that no osmotic stimulus was applied (n ¼ 9, open columns). Again, ﬁrst exposure during the intranuclear ‘rebound’ release of AVP (after hypertonic) on the ﬁrst day decreased RID (indicating improved social recognition) in both experimental groups (*p < 0.05 vs isotonic control, ANOVA). However, microdialysis administration of the V1 antagonist d(CH2)5[Tyr(Me)]AVP either into the SON (toned column, after hypertonic þ V1 antagonist) or into the septum (hatched column, after hypertonic þ V1 antagonist; 40 ng of the antagonist was delivered during 2 consecutive 30-min dialysis periods in either brain area) on the second day partially abolished this memory-facilitating effect. Source: Engelmann et al., 1994 (Fig. 3, p. 394). Copyright ß 1994 by Blackwell Science. Reprinted with permission.

MLS groups)] (see Fig. 3). Although not presented in Fig. 3, the results also showed that direct injection of the V1 receptor antagonist into the ipsilateral medial nucleus of the amygdala signiﬁcantly interfered with the improved recognition memory induced by osmotic stimulation of the SON (i.e., IEI, 120 min; RID after control injection of aCSF, 0.5; RID after injection of the V1 antagonist, 1.8; p < 0.01). The ﬁndings that osmotic stimulation of the SON increased AVP release within both the SON and MLS, and also enhanced SRM, suggested that the endogenous AVP released at this time was responsible for the enhanced SRM. The observation that blockade of VP-ergic transmission in both

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brain sites interfered with this osmotically induced memory enhancement supported this interpretation. It was suggested that direct osmotic stimulation via microdialysis activated a ‘‘functional SON–septum axis’’ (pathway from the SON to the septum) causally responsible for the observed SRM improvement. The present ﬁndings together with the observation that electrical stimulation of the SON increased septal release of AVP (DemotesMainard et al., 1986) support this suggestion. Moreover, the ﬁnding that microinjection of the V1 antagonist into the medial amygdala interfered with the SRM improvement induced by osmotic stimulation of the SON led to the proposal that this brain site is part of the ‘‘functional SON–septum axis’’ involved in VP-ergic mediation of olfactorybased SRM. The direct anatomical connections between the olfactory system and the medial amygdala (Brennan et al., 1990) are in accord with this proposal. c. Landgraf et al. (1995) Landgraf et al. (1995) designed a study to test the ability of antisense oligodeoxynucleotide (AS oligo) treatment, an AVP receptor knockdown strategy to inﬂuence SRM and anxiety in male Wistar rats when chronically infused into the septal area. Only the testing pertinent to SRM is discussed here. Application of AS oligo to a brain area interferes with synthesis of the AVP V1 receptor and is an alternative to antagonists or immunotoxins as a strategy for disrupting AVP neurotransmission in that area. The authors noted several potential advantages of this technique compared with AVP antagonists in studying the behavioral effects of endogenous AVP (see Landgraf et al., 1995, for further discussion). In this study, the AS oligo was chronically infused via an osmotic minipump into the septum of adult male rats, and Ringer’s solution (vehicle), scrambled sequence oligodeoxynucleotide (SS oligo), and sense oligodeoxynucleotide (S oligo) served as controls. The social discrimination test (SDT) was used to assess SRM, with a 4-min exposure period during the ﬁrst and second presentations and a 30- or 120-min IEI. Behavioral testing was carried out on the evenings of days 3 and 4 after implantation of the minipump–tubing– cannula device used for delivery of the treatments to the septal area. After behavioral testing the animals were killed and the brains were removed and either examined for histological veriﬁcation of the infusion site (all rats were treated with intracerebroventricular AVP) or the septal brain areas were dissected and prepared for receptor autoradiography. The ﬁrst experiment was designed as an attempt to verify that the experimental conditions per se did not interfere with social discrimination abilities. Subjects received 3 days of vehicle infused into the mediolateral septum (MLS) via the osmotic minipumps. Immediately after the ﬁrst presentation on day 3, they additionally received intracerebroventricularly administered vehicle solution (5 l infused over a 1-min period) and similarly, on day 4, either intracerebroventricularly administered V1 AVP

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receptor antagonist d(CH2)5[Tyr(Me)]AVP (100 ng/5 l) or synthetic AVP (1 ng/5 l) (IEI, 30 and 120 min, respectively). The results of the ﬁrst experiment indicated that (1) on day 3 after intraseptal implantation, intracerebroventricularly administered vehicle immediately after the ﬁrst presentation trial did not affect normal SRM [i.e., the simultaneously present novel juvenile was investigated for a signiﬁcantly longer time than the preencountered (same) juvenile during the second presentation trial after a 30-min IEI]; and (2) on day 4, the control rats intracerebroventricularly injected with the V1 receptor antagonist immediately after the ﬁrst presentation were impaired in SRM (no signiﬁcant difference in investigative time directed to the same and different juveniles during the second presentation trial 30 min after the ﬁrst one), whereas those similarly injected with AVP were facilitated in this type of memory (signiﬁcantly shorter time investigating the same compared with the different juvenile in the second presentation trial 120 min after the ﬁrst one). In a second experiment they examined the effects of oligo treatment on SRM. Independent groups of septally implanted rats received vehicle, SS oligo, S oligo, or AS oligo, and were tested in the evening of day 3 (30-min IEI). After the ﬁrst presentation period on day 4, some of the rats from the vehicle, S oligo, and AS oligo treatment groups were infused intracerebroventricularly with synthetic AVP (1 ng/5 l) over a 1-min period, and tested with a 120-min IEI. The results of the second experiment indicated that (1) intraseptally infused AS oligo over a 3-day period signiﬁcantly interfered with normal SRM (both the same and different juveniles were equally investigated after a 30-min IEI); (2) chronic intraseptal infusion with either SS oligo or the vehicle solution did not affect normal SRM (the shorter time investigating the same relative to the different juvenile after the 30-min IEI was highly signiﬁcant for both groups); (3) although the animals intraseptally infused with S oligo were still able to recognize the same juvenile after the 30-min IEI, the difference in investigative behavior directed toward the same and different juveniles was less signiﬁcant than that for the vehicle- and SS oligotreated animals; and (4) on day 4, intracerebroventricularly injected AVP given to the AS oligo and S oligo treatment groups immediately after the ﬁrst presentation trial did not preserve SRM after the 120-min IEI, although it did for the vehicle control group (i.e., both types of intraseptally infused oligos similarly interfered with the SRM-enhancing effects of the exogenous peptide). Results of the postmortem analyses performed on whole brains or septal areas were as follows: (1) histological study indicated that the infusion cannulas of the rats treated with intracerebroventricular AVP were precisely located in the MLS; (2) receptor autoradiography, which assessed [3H]AVP binding to V1 receptors in the septum, indicated that in contrast to vehicle or SS oligo, receptor density was markedly reduced by AS oligo treatment, and

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slightly reduced by S oligo treatment. It also showed that these treatment effects did not spread to the central amygdala or to the bed nucleus of the stria terminalis; and (3) RNA analysis demonstrated that after infusion of AS oligo into the septal region,V1 receptor mRNA levels were markedly increased compared with those of vehicle- and SS oligo-infused rats. The S oligo-treated rats showed a reduction in V1 receptor mRNA. Taken together, the results of this study suggested that AS oligo treatment selectively reduced synthesis of the V1 receptor in the infused limbic site (septal area) and that this receptor subtype is critically involved in the mediation of SRM. The ﬁndings also suggested that some degree of interference with the AVP–receptor interaction occurred in the septal area after local administration of S oligo. The authors noted that the antisense-targeting technique has been used to manipulate synthesis/release of AVP (Skutella et al., 1994) and OT (Neumann et al., 1994). In addition to the absence of the drawbacks associated with VP antagonist treatment (e.g., crossreacting with OT receptors, thereby also preventing behavioral effects of OT; Di Scala-Guenot et al., 1990) and the Brattleboro rat model (see Chapter 4), the present ﬁndings demonstrate that this antisense-targeting technique is a highly useful tool for revealing ‘‘relationships between local gene expression, neuropeptide–receptor interaction in distinct brain areas, and behavioral performance’’ (Landgraf et al., 1995, p. 4250). d. Everts and Koolhaas (1997) Everts and Koolhaas (1997) investigated whether the involvement of the septal VP-ergic system in SRM (Dantzer et al., 1988; Engelmann and Landgraf, 1994; Landgraf et al., 1995) also extends to the inanimate environment. To this end, they tested adult male Wistar rat subjects in their home cages for the effect of a VP receptor antagonist in the lateral septum (LS) in two comparable paradigms, one designed to test social, the other object, recognition. The object recognition task was comparable to the social memory test in both time course and test settings. The object equivalent of the same juvenile was a gray plastic food cup, that of the different juvenile, a transparent Erlenmeyer ﬂask equal in size to the food cup. Social investigative behavior consisted of anogenital snifﬁng, close following, and pawing directed toward the juvenile; object investigative behavior included object dragging, pushing, gnawing, and snifﬁng. Recognition memory was deﬁned by a signiﬁcant reduction in investigative time in the second relative to the ﬁrst encounter with the same juvenile (or object). Rats were initially tested in the two tasks under nontreatment conditions, with a 30- or 120-min IEI in tests with the same juvenile (or object), and a 30-min IEI for sessions with a different juvenile (or object). The results were as follows: (1) SRM occurred when the animals were presented with the same juvenile after the 30-min IEI. Time spent in investigative behavior (mainly anogenital snifﬁng) directed toward the same, but not the different,

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juvenile was signiﬁcantly decreased during the second versus the ﬁrst encounter; (2) as expected, SRM did not occur for the same juvenile after a 120-min IEI (no signiﬁcant reduction in social investigative time during the second encounter with the same juvenile); (3) the time spent investigating the object (mainly snifﬁng and manipulating the object) during the ﬁrst presentation was the same as that spent with the juvenile (about 180 s); (4) investigative time during the second trial was signiﬁcantly reduced from the ﬁrst trial whether the subject was shown the same or the different object (i.e., investigative time reduced by 40 s for both objects); and (5) an IEI of 120 min resulted in a slight (nonsigniﬁcant) reduction in investigative behavior toward the preencountered object during the second trial. After the completion of initial baseline testing, osmotic minipumps and brain cannula guides were implanted for bilateral infusions of the vasopressin V1 receptor antagonist [dPTyr(Et)]AVP (1 ng/0.5 l per hour) or physiological saline into the LS. Behavioral testing began 7 days after recovery from surgery. All rats were ﬁrst tested for object recognition [a ﬁrst investigative trial (5 min) followed 30 min later by a second trial with the same object]. After 1 day of rest all rats were tested for social recognition using the same paradigm (two exposures with a 30-min IEI). The data for the object recognition task indicated that (1) treatment with the VP antagonist resulted in a slight but not signiﬁcant reduction in initial object investigative behavior (saline controls tended to spend more time investigating the object than did the VP antagonist-treated rats during the ﬁrst encounter); and (2) the VP antagonist did not inﬂuence object recognition after the 30-min ITI (during the second encounter, both controls and antagonist treatment groups signiﬁcantly decreased the duration of their investigation of the reencountered object by approximately 40 s). The results of the SRT indicated that (1) the VP antagonist did not interfere with social investigative behavior during the ﬁrst encounter [both the saline- and AVP antagonist-treated rats spent equal amounts of time investigating the juvenile (about 180 s)]; and (2) the VP antagonist impaired normal SRM (when tested 30 min later with the same juvenile, the saline controls signiﬁcantly reduced their investigative time by 40 s, whereas those given the VP antagonist increased their investigative time to above 200 s). The authors made the following points in the course of discussing these results: (1) this study upheld previous ﬁndings indicating the important involvement of LS V1 receptor-mediated neurotransmission in SRM (Dantzer et al., 1987; Popik et al., 1992; Van Wimersma Greidanus and Maigret, 1996), and also showed that object recognition appears to be independent of this system; (2) however, the present ﬁndings should not be interpreted to suggest that the LS itself is not important for object recognition, because there is evidence to the contrary. Lesioning the LS reduced the animal’s preference to investigate a novel object, probably because of insufﬁcient processing of sensory information (Myhrer, 1989), and disruption of

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large parts of the septum after septal or medial frontal cortical damage impairs exploratory activity and habituation during object displacement (Poucet, 1989); and (3) relevant to the present ﬁndings is the importance of processing in the olfactory vomeronasal system (includes projections to the septal area via the medial amygdala and BNST) for social recognition memory (Bluthe and Dantzer, 1993; Popik et al., 1991; Simerly, 1990). This pathway processes species-speciﬁc olfactory cues used for identifying conspeciﬁcs, and appears not to be involved in object recognition, which instead may be mediated through the main olfactory system. e. Everts and Koolhaas (1999) Everts and Koolhaas (1999) tested the effect of infusing a V1//V2 receptor antagonist into the lateral septum (LS) on SRM (tested with the SRT), spatial memory (tested with the Morris water maze, MWM), and anxiety-related behavior (tested with elevated plus maze) in male Wister rats. Only the testing for SRM is reported here. The V1/V2 receptor antagonist [1-(-mercapto-,-pentamethylenepropionic acid)-2-(O-ethyl)-d-tyrosine, 4-valine]arginine vasopressin [d(CH2)5[dTyr(Et)]VAVP] used in this study was shown to be as potent as the most commonly used V1 antagonist d(CH2)5[Tyr(Me)]AVP (Engelmann et al., 1992a,b). The former blocks both V1 and V2 types of vasopressin receptor and was selected for study because of the demonstration of the V2 receptor in the hippocampus and other brain sites (Hirasawa et al., 1994; Kato et al., 1995) and the suggestion that it may be present in the septum as well (Engelmann et al., 1992a; Landgraf et al., 1991a; Ramirez et al., 1990). Saline or the V2/V1 antagonist (2 ng/l, sufﬁcient to ensure a total blockade of both receptor types) was bilaterally infused into the LS, via a preimplanted cannula/osmotic minipump assembly, throughout behavioral testing. The rats were tested in the SRT with juvenile male conspeciﬁcs as social test stimuli and with a 30-min IEI. The time spent investigating the same juvenile (anogenital snifﬁng, close following, and pawing) in the second relative to the ﬁrst trial was the measure of SRM. The results of this testing were as follows: (1) both treatment groups were comparable in the time spent investigating the juvenile in the ﬁrst presentation trial (about 180 s); and (2) unlike the saline-treated controls, which decreased investigative activity by 40 s on reencountering the juvenile 30 min later, the rats treated with the VP antagonist increased it by 20 s (signiﬁcant treatment exposure interaction on ANOVA, and signiﬁcant difference between the two groups conﬁrmed by post hoc t testing). These ﬁndings offer additional support for a role for septal AVP in SRM in the male rat, and also indicate that a VP antagonist relevant to V2 as well as V1 receptors blocks this form of memory. Moreover, additional behavioral testing with the MWM (see Chapter 10) suggested that the LS VP system is fairly speciﬁc for SRM, because it appears to be involved neither in spatial learning and memory, nor in object recognition memory (Everts and Koolhaas, 1997).

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A. General Comments Dluzen and coworkers (1998a,b, 2000) carried out several studies with the social discrimination test (SDT) to examine the role of olfactory bulb (OB) VP and OT in mediating SRM in the male rat (described below). Several lines of evidence stimulated their interest in this research question. First, these peptides act in various brain sites to modulate SRM (Popik and Van Ree, 1991; Popik et al., 1992; Van Wimersma Greidanus and Maigret, 1996). Second, this memory appears to be heavily reliant on the olfactory system (Sawyer et al., 1984). Third, the peptides (Dogterom and Buijs, 1980; Halasz and Shepherd, 1983), as well as VP-binding sites (Levy et al., 1992), are present in the OB. Fourth, these peptides are released within the OB of ewes during social interactions involving recognition processing (Levy et al., 1995). 1. Selected Studies a. Dluzen et al. (1998a) Dluzen et al. (1998a) examined the effects of infusions of VP, OT, and their antagonists into the olfactory bulb (OB) on SRM in male rats. The subjects, adult male Wistar rats, were tested in the SDT with male or female juvenile (21–30 days of age) social test stimuli. Depending on the agent tested, the IEI was either 30 or 120 min, because SRM is typically present with the former and absent with the latter (e.g., Dantzer et al., 1987; Thor and Holloway, 1982). On the day of testing, the vehicle or peptide was infused into the OB over a 10-s interval via two infusion cannulas inserted through previously implanted guide cannulas. Behavioral testing began within 1 min of infusion. At the conclusion of behavioral testing, the animals were killed and their brains were visually inspected to verify cannula placement. The rats in a given treatment group received a 1-l solution of one of the following agents: sterile Ringer’s solution (vehicle), AVP (0.5 ng/l), OT (0.5 ng/l), the V1 antagonist d(CH2)5[Tyr(Me)]AVP (AVP-Ant, 5.0 and 50.0 ng/l), or the OT receptor antagonist desGly-NH2d(CH2)5[Tyr(Me)2, Thr4]OVT (OT-Ant, 5.0 and 50 ng/l). The duration of the IEI for each treatment was as follows: vehicle, 30 and 120 min; AVP and OT, 120 min; AVP-Ant and OT-Ant, 30 min. The 120-min IEI in the AVP and OT treatment conditions assessed the recognition-preserving effects of the peptides, and the 30-min IEI used in the AVP-Ant and OT-Ant treatment conditions tested the putative recognition-interference effects of these antagonists. A t test determined whether the tested males exhibited SRM under each treatment condition (i.e., directed signiﬁcantly more investigation time to the novel juvenile, compared with the preencountered juvenile, during the second trial). A separate sample of urethane-anesthetized subjects received

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an infusion of 1 l of radiolabeled AVP (125I-labeled AVP) into the OB to determine the approximate area of peptide diffusion through this structure and into the frontal cortex, septal area, and cerebrospinal ﬂuid (CSF). The results of the statistical analyses indicated that (1) the vehicle control subjects recognized the preencountered juvenile after the 30-min, but not the 120-min, IEI (i.e., a signiﬁcantly greater amount of time was directed to the novel versus the same juvenile after an IEI of 30, but not 120, min); (2) infusion of a 0.5-ng dose of either AVP or OT into the OB facilitated SRM after an IEI of 120 min (i.e., these subjects spent signiﬁcantly more time investigating the novel juvenile, compared with same juvenile, after an interval when control animals spent similar times investigating both types of juveniles); (3) infusion of AVP-Ant at both the 5- and 50-ng dose levels did not interfere with normal SRM tested at the 30-min IEI (i.e., at both dose levels the subjects spent signiﬁcantly more time investigating the novel juvenile, compared with the same juvenile, in the second trial after a 30-min IEI, as did the controls); and (4) as with AVP-Ant, OB-infused OT-Ant at both the 5- and 50-ng dose levels failed to interfere with normal social recognition tested with a 30-min IEI. The results of the spread of diffusion of the 125I-labeled AVP bilaterally infused into the OB, expressed as the mean percentage of total activity relative to that in the OB, were as follows: frontal cortex, 23.1%; septal area, 0%; and CSF, 0%. This indicates that the peptide infused into the OB remained primarily localized within that structure. Most important for interpreting the present results is that although the septal area is particularly responsive to the modulatory effects of these peptides [e.g., Dantzer et al., 1987, 1988 (see Chapter 12); Engelmann and Landgraf, 1994; Engelmann et al., 1994; Everts and Koolhaas, 1997], they were unlikely to have activated this area and thus its complicating effects can be ruled out. The following points were made during discussion of these results: 1. These ﬁndings revealed that the OB, like the septal area, is a target structure that mediates memory-modulating effects of these peptides on SRM. Although these data may represent only pharmacological effects of the peptides, it was noted that in ewes the release of OB AVP and OT occurs under physiological conditions, and evidence suggests that OT release in this structure may be involved with promoting maternal recognition of her lamb offspring (Levy et al., 1995). 2. The lack of an effect on normal SRM after AVP-Ant and OT-Ant infusion into the OB was compared with the reported results of infusions of these antagonists into the septal area. This comparison indicated similar ﬁndings for intraseptal infusions of OT-Ant (Van Wimersma Greidanus and Maigret, 1996), but opposite ﬁndings for AVP-Ant, which interferes with normal SRM [Dantzer et al., 1988 (see Chapter 12); Engelmann and Landgraf, 1994; Everts and Koolhaas, 1997].

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3. The failure of these OB-infused antagonists to interfere with normal SRM (a 30-min IEI) was contrary to what would be expected if their presence in this brain site played a physiological role in this type of memory processing. Two possibilities may have accounted for this failure: (a) the dose levels of the antagonists were insufﬁcient to produce a behavioral effect. However, two facts argue against this explanation: (i) the two dose levels used were separated by a 10-fold difference in range, and (ii) the 5-ng/ l dose of AVP-Ant, which did not block normal SRM (tested with a 30-min IEI) when infused into the OB, did so when injected into the lateral septum (Dantzer et al., 1988); and (b) at the time of treatment, there was insufﬁcient ongoing activity in the VP and OT receptor neurons in the OB to be inﬂuenced by the blocking action of the antagonists. This condition was considered analogous to results obtained when AVP-Ant was infused into the SON. Under basal conditions (analogous to the present study), the antagonist was ineffective in inﬂuencing SRM, but blocked this memory under circumstances (i.e., osmotic stimulation) that enhanced AVP activity in the SON (Engelmann and Landgraf, 1994). Whatever the reasons for the asymmetric agonist–antagonist effects observed in this study, these researchers noted that, in general, SRM is ‘‘preserved’’ (extended in its normal duration from 30 to 120 min) after infusion of VP and/or OT agonists into relevant brain sites (e.g., Dantzer et al., 1988; Engelmann and Landgraf, 1994; Van Wimersma Greidanus and Maigret, 1996), whereas their antagonists do not in all cases block the ‘‘display’’ of normal SRM tested with the 30-min IEI (Engelmann et al., 1998; Popik et al., 1992) despite what might be expected. Taken together, these ﬁndings led to the suggestion that ‘‘the underlying mechanisms by which peptides function within the olfactory bulb differ as a function of whether they are involved with the display versus the preservation of recognition responses’’ (Dluzen et al., 1998a, p. 999). b. Dluzen et al. (1998b) Dluzen et al. (1998b) selectively depleted noradrenaline (NA) from the OB and assessed its effect on AVP- and OTinduced preservation of SRM in the male rat. The rationale of this study was based on several ﬁndings, which together suggested that when AVP and OT are infused into the OB, activation of the NA system in this site may be one mechanism by which they act to preserve SRM (i.e., extend its duration from 30 to 120 min). These ﬁndings include (1) the demonstration that release of NA into the OB was critical for memory/recognition responses associated with reproduction (Brennan et al., 1990; Kaba and Nakanishi, 1995); (2) the presence of VP and OT in the OB (Dogterom and Buijs, 1980; Halasz and Shepherd, 1983; Levy et al., 1995); and (3) the observation that these peptides activated NA release in the OB of sheep (Levy et al., 1995).

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The procedure and coordinates for cannula implantation in the OB were identical to those used by Dluzen et al. (1998a). OB depletion of NA was achieved by a bilateral infusion of 1.0 l of 6-hydroxydopamine (6-OHDA; dissolved in Ringer’s solution to a concentration of 20 g/l) via guide cannulas into the OB. The OB in control rats was bilaterally infused with Ringer’s solution (vehicle). During behavioral testing, the subjects received a 1.0-l infusion of vehicle, AVP (0.5 ng/l), or OT (0.5 ng/l) over a 10-s interval into the OB via infusion cannulas inserted through guide cannulas. The animals were tested in the SDT with a 120-min IEI to assess the recognition preservation effects of AVP and OT, and 1–2 days later under nontreatment conditions, with a 30-min IEI to determine the ability of each animal to demonstrate a normal recognition response. Paired t tests were used to determine whether recognition responses were present or absent under the different test conditions (i.e., differences in SIT spent with the same versus the novel juvenile during the second exposure period). At the completion of testing, the animals were killed and the brains were visually inspected to conﬁrm cannula location within the OB. The OB was then removed and prepared to determine NA concentrations by highpressure liquid chromatography (HPLC) with electrochemical detection (see Feldman et al., 1997, for a description of HPLC). The mean OB NA concentrations were calculated for each of three groups deﬁned on the basis of the status of the 6-OHDA-induced chemical lesion (i.e., NA depletion) and the peptide treatment received during behavioral testing: (1) lesioned rats given AVP treatment, (2) lesioned rats given OT treatment, and (3) nonlesioned rats given either AVP or OT treatment. The data for these three groups were statistically evaluated in a one-way ANOVA. The behavioral results were as follows: (1) when tested with the 120min IEI, AVP infusion into the OB preserved SRM in the nonlesioned rats (a longer time was spent investigating the novel versus the same juvenile), but not in the 6-OHDA-lesioned rats (no signiﬁcant difference in SIT spent with the novel versus the same juvenile); (2) when retested 1–2 days later under nontreatment conditions with a 30-min IEI, the 6-OHDA-lesioned rats displayed normal SRM (a signiﬁcantly greater amount of SIT was spent with the novel compared with the same juvenile); (3) when tested with the 120-min IEI, OT infusion into the OB preserved SRM in the nonlesioned rats (signiﬁcantly longer time spent investigating the novel versus the same juvenile), but not in the 6-OHDA-lesioned rats (no signiﬁcant difference between the times spent investigating the same and the different juvenile); and (4) retesting 1–2 days later under nontreatment conditions with a 30-min IEI indicated that the lesion itself did not impair normal SRM (a statistically signiﬁcant greater amount of time was spent investigating the novel juvenile). The results of the postmortem analysis were as follows: (1) the OB NA concentrations [picograms of NA per milligram wet tissue weight

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(mean standard error of the mean) for the lesioned rats treated with AVP, 39.2 8.7; for lesioned rats treated with OT, 47.4 8.7; and for nonlesioned rats receiving AVP or OT treatment, 205 18.3]; (2) the ANOVA and post hoc comparisons indicated that the 6-OHDA lesion signiﬁcantly reduced NA levels in the OB; and (3) speciﬁcally, there was an overall signiﬁcant difference among these three groups, and the post hoc pairwise comparisons indicated that the OB NA concentration in the nonlesioned AVP/OT group was signiﬁcantly greater than those in the two lesioned groups, which did not differ from each other. The following points were made in the discussion of these results: 1. Previous research supports the argument that the 6-OHDA lesion effects observed in this study were primarily due to depletion of NA in the OB rather than to secondary effects of lesion-induced depletion of DA or serotonin (5HT) in the OB, or NA depletion in other brain sites (Doty et al., 1988; Guan et al., 1993; Royet et al., 1983). 2. The ﬁndings that the 6-OHDA lesion per se did not impair the display of normal social recognition with a 30-min IEI under nonpeptide treatment, and that AVP and OT infusion into the OB in nonlesioned rats preserved SRM with a 120-min IEI in non-6-OHDA-treated rats, strongly suggest a speciﬁc interaction between the peptides and OB NA in social recognition preservation. 3. Moreover, the failure of the 6-OHDA lesion to interfere with the display of normal SRM (tested with a 30-min IEI) under nontreatment conditions, whereas it obliterated the ability of AVP and OT to preserve SRM (tested with the 120-min IEI), suggests that markedly different mechanisms apply to these two testing conditions. Speciﬁcally, these ﬁndings suggest that the display of normal SRM involves an OB NA-independent process, whereas the peptide-induced preservation of SRM involves an OB NA-dependent process. The ﬁndings that AVP and OT can modulate the release of NA within the OB of sheep (Levy et al., 1995) is consistent with the proposal that infusion of these peptides into the OB preserves social recognition by activating the NA system in that brain structure. 4. The possibility that the VP/OT interaction with the NA system in the OB may preserve SRM indirectly by a selective attention effect was raised in light of the popular view that the locus coeruleus NA system, from which the OB receives substantial input (Shipley et al., 1985), promotes selective attention by releasing NA within numerous sensory target systems in the brain (Robbins, 1997; Sara et al., 1994; Vankov et al.,1995). c. Dluzen et al. (2000) Dluzen et al. (2000) carried out four experiments designed to evaluate the relationship between the OB NA system and intra-OB infusion of OT in the preservation of SRM memory in the male Wistar rat. This study was instigated by previous work, done by these investigators, suggesting that an OB NA-dependent mechanism is involved in

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the ability of OT to preserve recognition responses (Dluzen et al., 1998b; discussed above). The animals in experiments 1–3 were tested in the SDT after recovery from surgical implantation of a guide cannula as done in previous investigations of the effects on SRM of bilateral infusion of OT into the OB (Dluzen et al., 1998a,b). General testing conditions, and assessment of social recognition, were also the same as those of the earlier studies. The doses used in all these experiments were selected because of their effectiveness in other related paradigms (Kaba and Keverne, 1988; Liebsch et al., 1996; Roozendaal et al., 1992), and represent attempts to approximate physiological concentrations when taking into consideration such variables as localized administration, diffusion, and permeability through membranes (experiment 4). The purpose of experiment 1 was to determine whether blocking presumptive OT receptors within the OB would abolish the ability of OT to preserve social recognition responses. Three perfusion groups received a 1-l intra-OB bilateral infusion of OT diluted in Ringer’s solution (0.5 ng/l) on its own (group 1) or coinfused with a highly selective OT receptor antagonist, desGly-NH2, d(CH2)5[Tyr(Me)2, Thr4]OVT (group 2); or with a highly selective V1 receptor antagonist, d(CH2)5[Tyr(Me)]AVP (0.5 ng/l) (group 3). These rats were then tested for OT-induced preservation of SRM in the SDT with a 120-min IEI. The rats that received the OT antagonist were retested 2 days later with no infusions, and with a 30-min IEI, to determine whether they were capable of displaying normal SRM. The results indicated that the OT-induced preservation of SRM was mediated by an OT receptor in the OB, because this facilitated memory effect was blocked by a coinfusion of OT with an OT receptor antagonist (desGlyNH2, d(CH2)5[Tyr(Me)2, Thr4]OVT), but not with a VP V1 receptor antagonist (d(CH2)5[Tyr(Me)]AVP). Experiment 2 examined whether infusion of an 2-noradrenergic agonist, clonidine, could preserve SRM, and thus ‘‘provide some potential indication for the actions of OT’’ (Dluzen et al., 2000, p. 761). To examine the speciﬁcity of its effect, a separate group of rats was similarly tested with the -noradrenergic agonist isoproterenol. The results indicated that adrenoceptors, but not -adrenoceptors, in the OB inﬂuence SRM in male rats, because infusion of an -adrenoceptor, but not a -adrenoceptor, agonist preserved SRM (rats in the former but not the latter treatment group spent signiﬁcantly less time investigating the familiar juvenile, compared with the novel juvenile, after the 120-min IEI). The ﬁndings suggested that the increased output of OB NA that results from OT appears to activate -adrenoceptors to produce this preservation in recognition because infusions of clonidine into the OB preserve recognition responses in a manner similar to that observed with OT.

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The purpose of experiment 3 was to determine whether the OT-dependent preservation of recognition was mediated by postsynaptic activation of - or -adrenoceptors. The animals received either a coinfusion of OT (0.5 ng/l) and an -adrenoceptor antagonist (phentolamine; 40 nM), or of OT (0.5 ng/l) and a -adrenoceptor antagonist (timolol; 40 nM). The animals were all tested with the 120-min IEI. A separate group of rats receiving an infusion of the -adrenoceptor antagonist (40 nM) and tested after a 30-min IEI was included. Because this antagonist was here found to block OT-dependent recognition it was important to learn whether this -adrenoceptor antagonist itself would block normal SRM. When the -adrenoceptor antagonist (phentolamine) was combined with OT and infused into the OB, it blocked the OT-induced preservation of SRM (the rats in this treatment group showed no signiﬁcant difference in investigative time spent with the same versus the different juvenile after the 120-min IEI). On the other hand, when the -adrenoceptor antagonist (timolol) was coinfused with OT into the OB, it had no effect on the OTinduced preservation of SRM (rats in this treatment group spent signiﬁcantly less time investigating the same juvenile, compared with the different juvenile, after the 120-min IEI). The control test with the adrenoceptor antagonist indicated that, when infused into the OB on its own, there was no impairment of normal SRM (these rats spent signiﬁcantly more time investigating the novel juvenile, compared with the same juvenile, after the 30-min IEI). Experiment 4 was designed to determine whether OT exerts any direct effect on the output of NA within the OB. Each animal was infused with either OT, OT-Ant, or normal Ringer’s solution through preimplanted microdialysis probes (retrodialyzed) to determine whether these agents directly alter OB NA output. The results indicated that OB OT is directly responsible for the release of NA in the OB by activating -adrenoceptors in this structure. NA output (measured by a microdialysis probe) was signiﬁcantly greater during the 15-min collection interval in which OT was infused in the OB (2–3 ng) than during control intervals; moreover, this increase was not found during treatments with either the OT antagonist or vehicle. In their discussion the authors related these ﬁndings to studies of maternal behavior in sheep by Keverne, Levy, and associates (Kendrick et al., 1988a,b; Levy et al., 1995), as well as to their own research on rats (Dluzen et al., 1998a–c). The sheep research indicated that OT released within the OB at parturition, and in response to vaginocervical stimulation (Kendrick et al., 1988a,b), appears to be a primary agent for the onset of maternal behavior and the associated recognition of offspring required for selective bonding. An OT interaction with NA released from brainstem NA-ergic ﬁbers projecting to the OB is an important mechanism underlying

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the OT inﬂuence. That is, in sheep, the OT-induced release of NA activates -adrenoceptors that results in offspring recognition (Levy et al., 1995). These researchers proposed that, in the male rat, a similar cascade of events might be necessary for the social recognition response to occur. Earlier studies in their laboratory support this proposal: (1) OT infusion into the OB preserves SRM in the male rat (Dluzen et al., 1998a); (2) this process is abolished by depletion of OB NA (Dluzen et al., 1998b) but not after depletion of OB serotonin (Dluzen et al., 1998c), indicating that the OT-dependent preservation of SRM involves an OB NA-mediated process; (3) additional support is provided by the present ﬁndings, and includes the demonstration that (a) OT must activate OT receptors within the OB (experiment 1), and (b) this OT-induced increase in OB NA activates the -adrenoceptor system (experiments 2 and 3), resulting in preservation of the social recognition responses; and (4) ﬁnally, the authors noted that although the present ﬁndings are based on pharmacological manipulations, they might nevertheless indicate the operation of physiologically signiﬁcant processes. For example, during copulation endogenous OT is released within the male rat brain (Hughes et al., 1987) and such an increase in the release of endogenous OT within the OB may contribute to an enhanced ability of male rats to discriminate between conspeciﬁcs.

VII. VP and OT in the Medial Preoptic Area and SRM

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A. General Comments The medial preoptic area (MPA) is an anterior extension of the hypothalamus that has been implicated in the regulation of gonadal hormone secretion (Hart and Leedy, 1985; Macrides, 1976), and in male (Edwards and Einhorn, 1986; Kondo et al., 1990) and female (Caldwell et al., 1986, 1989) sexual behavior. Both OT-ergic and VP-ergic mechanisms are present in this brain site (Caldwell et al., 1989). Moreover, the MPA is regarded as one of the brain centers involved in processing olfactory information (Macrides, 1976; Pfaff and Pfaffmann, 1969). Given the crucial importance of olfactory processing in rodent SRM, Popik and Van Ree (1991; see below) designed a study to investigate whether VP and/or OT injected into this brain site may inﬂuence this processing and therefore SRM. 1. Selected Study: Popik and Van Ree (1991) Popik and Van Ree (1991) used the SRT, with a 120-min IEI, to examine the effect of local injections of VP and OT into the MPA on SRM in the male Wistar rat. A signiﬁcant decrease in the duration of investigative behavior (anogenital snifﬁng) in the second relative to the ﬁrst encounter operationally

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deﬁned SRM. The anogenital investigative time (AIT) during the second encounter was divided by that during the ﬁrst encounter, and the resulting value was multiplied by 100 to obtain the ratio of investigation duration (RID). RIDs were used in a one-way ANOVA, followed by least signiﬁcant difference (LSD) tests to compare effects of different doses of the peptides on SRM. In a given treatment session, physiological saline (placebo) or one of the following peptides was bilaterally injected into the MPA: OT (0.03, 0.3, 3, 30, 300, or 1000 pg), AVP (0.03, 0.3, or 3 pg), AVP(4–8) (200 pg), AVP(4–9) (200 pg), or OT (300 pg) plus the OT antagonist desGly-(NH2)9, d(CH2)5[Tyr(Me)2Thr4]OVT (3000 pg). Each subject was tested under all treatment conditions, and successive treatments were separated by intervals of at least 48 h. Two additional experiments included: (1) a control test (presentation of a different juvenile during the second encounter) to rule out nonspeciﬁc inﬂuences that might be mistaken for peptide-induced memory effects, and (2) determining whether a peptide treatment that facilitated SRM when locally injected into the MPA did so when injected into the septal area. All treatments were given immediately after removal of the juvenile during the ﬁrst presentation. The results were as follows: (1) placebo-treated subjects were unable to recognize the same juvenile after a 120-min IEI (investigative time during the second encounter was similar to that of the ﬁrst encounter); (2) all but the lowest dose of MPA-injected OT dose dependently enhanced SRM of the same juvenile after a 120-min IEI (all dose levels of OT, except the 0.03-pg dose, signiﬁcantly reduced RID ratios relative to those obtained by the placebo-treated rats); (3) control experiments, which presented a different juvenile in the second trial, ensured that this OT dose-dependent memory effect was not attributable to nonspeciﬁc drug factors [RID scores for OT (3.0 and 1000 pg)-treated subjects were not signiﬁcantly different from those of placebo controls]; (4) OT dose levels (100 and 1000 pg) that enhanced SRM of the same juvenile after the 120-min IEI, when injected into the MPA, did not do so when injected into the septal area (RID scores for these OT-treated subjects did not signiﬁcantly differ from those of placebo controls); (5) pretreatment with an OT antagonist did not block the SRM effect induced by a local injection of OT in the MPA (RID scores were signiﬁcantly reduced from control values in both the placebo plus OTtreated group, and the OT-Ant plus OT-treated group, tested with the same juvenile after a 120-min IEI), (6) in contrast to OT, local injection of AVP into the MPA did not enhance SRM of the same juvenile after the 120-min IEI (no signiﬁcant difference in RID scores between placebo- and AVPtreated subjects); and (7) when injected into the MPA, neither AVP(4–8), nor AVP(4–9), at the dose level used here (200 pg), enhanced SRM of the same juvenile after the 120-min IEI [RID scores for AVP(4–8) and AVP(4–9) groups did not signiﬁcantly differ from control values].

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The following points were made during discussion of these results: 1. The ﬁnding that OT, microinjected into the MPA at a low dose range, dose dependently facilitated SRM resembles that reported for peripherally administered OT (Popik et al., 1991). 2. The facilitation of SRM induced by the injection of OT into the MPA appears to be speciﬁc, for three reasons: (a) the control experiments with a different juvenile ruled out nonspeciﬁc drug effects; (b) although VP is present in the MPA, and is involved in mediating certain behavioral effects [e.g., expression of sex behavior in female rats (Caldwell et al., 1989) and social dominance in hamsters (Ferris et al., 1984)], neither it nor its behaviorally active metabolites [AVP(4–8) and AVP(4–9)] inﬂuenced SRM when injected into the MPA; and (c) identical doses of OT that facilitated SRM, when injected into the MPA, were ineffective when injected into the septal area. 3. The failure of the OT receptor antagonist used in this study to block OT enhancement of SRM suggests that the MPA receptors mediating this effect are site and/or task speciﬁc because (a) this antagonist attenuated the effects of OT on passive avoidance retention after intracerebroventricular administration (De Wied et al., 1991), and (b) peripheral injection of other OT antagonists blocked OT-induced attenuation of SRM, and facilitated this memory when injected alone (Popik and Vetulani, 1991). 4. It was suggested that the SRM effects of MPA-injected OT might have some relation to the purported role of the peptide in sexual behavior. The following two sets of observations provide supportive evidence of a role for central OT in male and female sexual behavior: (a) OT microinjected into the MPA increases sexual receptivity in female rats, and OT immunoreactive levels in the MPA are higher in receptive females that were mounted by males than in control animals (Caldwell et al., 1989); and (b) OT facilitates male sexual behavior (Arletti et al., 1985), and central OT-ergic systems in males respond to mating (Hughes et al., 1987). 5. It was concluded that the mechanism underlying the MPA/OT-induced memory effect observed in this study may involve ‘‘enhancement of the olfactory signal and/or modiﬁcation of the processing of olfactory information’’ (Popik and Van Ree, 1991, p. 559). This suggestion is consistent with evidence implicating MPA involvement in processing of olfactory information (Pfaff and Pfaffmann, 1969).

VIII. VP and OT Genetic Knockout Models and SRM

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A. General Comments Two types of genetic neuropeptide ‘‘knockout’’ models (genetic mutations that deplete the organism of brain VP or OT) have been used in studies with SRM. The ﬁrst, the Brattleboro homozygous diabetes insipidus (HODI)

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rat, is a natural knockout model, characterized by a VP deﬁciency that results from a natural genetic mutation. Its use in VP/memory research began in the 1970s with studies by De Wied and colleagues (see Bohus and De Wied, 1998, for discussion of this work). The second, the OT knockout mouse, is unable to synthesize OT and has been ‘‘created’’ by genetic techniques used in laboratory-based stem cell research. It is important to recognize the difﬁculties inherent in interpreting the results of studies with these models (see Bohus and De Wied, 1998; and Chapter 3). In general, an observed deﬁcit could be due to any number of secondarily arising conditions that act to obscure the speciﬁc contribution of the neuropeptide under study. Further, the absence of a clear-cut behavioral outcome may reﬂect the fact that CNS functions such as learning and memory are under the concurrent inﬂuence of many peptidergic, aminergic, and cholinergic chemical messengers and not just the one studied. Nevertheless, the results obtained from experiments with knockout models, together with those on intact rats and mice, have provided useful information in neuropeptide/ behavioral study. The research discussed below illustrates the use of VP and OT knockout animals in the study of SRM. 1. Selected Studies a. Engelmann and Landgraf (1994) Engelmann and Landgraf (1994) investigated the role of septal AVP in SRM, using homozygous Brattleboro (HODI) and normal Long-Evans (LE) rats. These authors compared the performance of HODI and LE rats in the SRT before and after microdialysis administration of AVP or a V1 receptor antagonist, d(CH2)5[Tyr (Me)]AVP, into the mediolateral septum (MLS). Juveniles (20–25 days old) of both sexes were used as social stimuli. The two 5-min presentations were separated by either a 30- or 120-min IEI, and the same or a different juvenile was presented during the second presentation trial. Untreated behavioral performance was tested in the ﬁrst week [two sessions: 30-min IEI, same juvenile; and 30-min IEI, different juvenile; for the LE rats another session (120-min IEI, same juvenile) was added]. Subsequent to this test, the microdialysis probe was implanted with its tip end directed to the MLS. Thirty hours later treatment sessions began, during which the perfusion ﬂuid [artiﬁcial CSF (aCSF), or aCSF containing either AVP (0.2 or 2.0 ng/rat) or the V1 receptor antagonist (5.0 ng)] was infused into the septum at a ﬂow rate of 3 l/min. Infusion began 5 min before the ﬁrst presentation and lasted 30 min. The ﬁrst 5-min presentation trial occurred during this perfusion interval. Investigative behavior (anogenital snifﬁng, licking, pawing, and close pursuing of the juvenile) was timed during the ﬁrst and second presentation trials. The microdialysis site was histologically conﬁrmed on completion of behavioral testing. The results for the untreated sessions were as follows: (1) investigative duration during the ﬁrst presentation with the same or a different juvenile

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was signiﬁcantly longer for the HODI rats than for the LE rats; (2) the untreated HODI rats did not exhibit normal SRM after the 30-min IEI (SIT with the same juvenile was of comparable duration as that with the different juvenile during the second exposure); (3) in contrast to the HODI rats, the untreated normal LE rats recognized the same juvenile after the 30min IEI (SIT with the same juvenile, but not the different juvenile, was signiﬁcantly reduced during the second relative to the ﬁrst presentation trial); and (4) the LE rats did not recognize the same juvenile after the 120-min IEI (no signiﬁcant difference between the investigation durations of both presentation trials). The results for the treated sessions indicated: (1) implantation of the microdialysis probe into the MLS and its perfusion with aCSF did not by itself inﬂuence the behavioral performance of either rat strain, compared with that in untreated sessions; (2) infusion of either dose of AVP into the HODI rats resulted in normal SRM when tested after a 30-min IEI (SIT was signiﬁcantly less during the second relative to the ﬁrst presentation trial with the same juvenile, indicating a juvenile-speciﬁc recognition, whereas presentation of a different juvenile resulted in investigative behavior comparable to that of untreated LE rats or aCSF-perfused rats); (3) infusion of AVP into the MLS of LE rats resulted in SRM after a 120-min IEI, and this effect was juvenile speciﬁc; (4) infusion of the V1 antagonist into the MLS of LE rats interfered with normal SRM tested after a 30-min IEI (in contrast to aCSFperfused LE rats, there was no signiﬁcant difference in SIT between the ﬁrst and second presentation with the same juvenile); and (5) a comparable failure to recognize juveniles after a 30-min IEI was also observed in untreated or aCSF-perfused HODI rats. In the discussion, the authors noted that the importance of intraseptally released AVP for normal SRM was supported by the ﬁndings of this study: (1) unlike the normal LE rats, untreated HODI rats were unable to recognize a preencountered juvenile after a 30-min IEI; (2) an intraseptal infusion of AVP via the virtually stress-free microdialysis probe mimicked AVP release patterns in the septum better than intraperitoneal or intracerebroventricular injections of the neuropeptide, improved SRM in the HODI rats to the level of untreated or aCSF-treated normal LE rats, whereas the converse occurred after MLS infusion of the V1 receptor antagonist into the brain of normal LE rats; and (3) an increase in the MLS level of AVP in normal rats, induced by microdialysis of synthetic AVP, signiﬁcantly improved their performance and indicated that SRM may be manipulated over a relatively wide range. It was concluded that strain differences per se were not accountable for the results obtained in this study because the untreated Long-Evans rats did not behave differently from males of the Wistar strain (Bluthe et al., 1990; Dantzer et al., 1987) or Sprague-Dawley strain (Bluthe and Dantzer, 1990). Moreover, although the Brattleboro and LE rats were obtained from two different breeding farms, which could result in secondary causal effects for

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the observed behavioral differences (Ambrogi Lorenzini et al., 1991; and see Chapter 3), pilot studies using HODI rats from other breeders conﬁrmed the impaired recognition regardless of the inbred rat strain used (Engelmann, unpublished results, cited in Engelmann and Landgraf, 1994). Therefore, it seems clear that the lack of central AVP in the HODI rats was responsible for the impaired acquisition, storage, and/or recall of olfactory cues. In addition to the present ﬁndings, observations from other behavioral pharmacological experiments, autoradiographic observations, and electrophysiological studies provide direct or indirect supportive evidence for the following: (1) excess intraseptal levels of AVP, induced by locally administered synthetic AVP, improves SRM memory (Dantzer et al., 1988; Popik et al., 1992); (2) V1 receptor blockade of septal AVP receptors interferes with normal SRM in adult male Wistar rats (Dantzer et al., 1988); (3) highly speciﬁc AVP-binding sites are present in the septal brain area of HODI rats (Shewey and Dorsa, 1986) and stimulation of these binding sites by intraseptally administered AVP (Shewey et al., 1989) probably mediated the improved SRM demonstrated in this study; and (4) observations with electrophysiological recording techniques indicate that AVP treatment increases the ﬁring rate of neurons in hippocampal slices from HODI rats (Dreifuss and Muhlethaler, 1982). Altogether, the ﬁndings of this study and the above-cited observations support the importance of intraseptal AVP for SRM. b. Ferguson et al. (2000) Ferguson et al. (2000) compared male mice mutant for the OT-encoding gene (OT /) with those normal for this genotype (OT þ/þ or wild-type mice) in a number of tests of SRM, and in follow-up tests to analyze nonmnemonic factors that may contribute to the genotype-dependent differences observed in SRM. SRM was tested in two paradigms [a multitrial social recognition task (MSRT) and the SRT] with the subjects remaining in their home cages. The social stimuli used in this study were wild-type ovariectomized (OVX) female mice (used in each paradigm) or wild-type intact male or female mice (used only in one test with the MSRT). SRM was indicated by a reduction in olfactory investigation on prolonged exposure or repeated encounters with the same conspeciﬁc (Kendrick et al., 1997; Keverne and Brennan, 1996; Thor and Holloway, 1981). Several experimental tests were run with the MSRT. In the ﬁrst test, the investigators measured the duration of social investigation time (SIT) directed toward the same OVX female reencountered during four successive 1-min trials, and that directed toward a different OVX female presented in the ﬁfth 1-min trial. At the end of each 1-min trial the stimulus mouse was removed from the resident’s home cage and returned to an individual holding cage for the 10-min intertrial interval (ITI). SIT signiﬁcantly declined over prolonged exposure to the same OVX female for the OT þ/þ subjects,

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but not for the OT / subjects. There were no genotype-dependent differences in SIT in trial 5, when a new female stimulus was presented. The investigative behavior shown by the OT þ/þ males can be interpreted as a renewal of interest in the novel social stimulus. The second test was designed to examine the possibility that changes in the female’s behavior associated with repeated encounters with males per se may have contributed to the decline in SIT scores observed in the OT þ/þ males. To this end, the females were rotated so that new males in each of the four trials investigated each female, and a different female was presented to a given subject in each 1-min trial. There was no decline in SIT for either OT þ/þ or OT / mice, supporting a social recognition explanation for the decline in SIT scores observed in the ﬁrst test. In a third test, the resident mouse was presented with the same social stimulus (a reproductively intact male or female mouse) in each of four 1-min trials. The results showed that the SRM deﬁcit of the OT / mice was not limited to OVX female stimuli (i.e., there was a signiﬁcant decline in SIT over the repeated presentations for both types of social stimuli in the OT þ/þ mice, whereas the OT / mice showed persistent interest over the course of this test). The SRT with a 30-min IEI was used as a second measure of SRM. The OT þ/þ mice, but not the OT / mice, recognized the reencountered OVX female (SIT signiﬁcantly declined during the second relative to the ﬁrst presentation trial for the former but not the latter genotype). Subgroups of OT / and OT þ/þ mice tested for SRM were subsequently used in experimental tests designed to determine whether the observed deﬁcits in SRM might have been due to impairments in olfactory function (olfactory foraging task) or to behavioral inhibition (habituation in olfactory and acoustic startle tests). For the olfactory foraging task, the mice were initially familiarized with the taste of chocolate chip rewards that were eaten in the home cage. The test proper consisted of four trials with a 10-min ITI. For each trial the resident mouse was removed to a holding cage while a chocolate chip was placed in its home cage, either on the surface of the bedding (trial 1) or hidden in different positions beneath leveled bedding (trials 2, 3, and 4). The latency to locate the food reward on its return to the home cage was recorded. Both the OT þ/þ and OT / mice learned to locate the buried food as rapidly as they located food placed on the surface of the cage bedding. The olfactory habituation/dishabituation task consisted of ﬁve 1-min trials (10-min ITI) in which a perforated tube containing lemon-scented cotton (trials 1– 4) or lemon plus vanilla-scented cotton (trial 5) was placed in the home cage of each mouse. The amount of time spent investigating the scent (nasal contact with the tube) during each trial was recorded. The results indicated that whereas OT / mice spent more time investigating the lemon-scented object than did the OT þ/þ mice, both genotypes rapidly

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habituated to the scent (investigative activity signiﬁcantly declined over the four successive trials) and dishabituated when the scent was changed (investigative behavior increased in trial 5). The test for habituation to an acoustic startle stimulus consisted of 200 presentations of the acoustic stimulus (40 ms, 118 dB) regularly presented at intervals of 10 s. The investigators computed mean startle response amplitude data for successive blocks of 20 stimulus presentations for each subject. The results indicated that whereas the OT þ/þ mice exhibited signiﬁcantly higher startle responses during habituation to the acoustic stimulus than did OT / mice, both genotypes habituated to the stimulus. Moreover, when the data were analyzed and expressed as a percentage of the average response measured in the ﬁrst block of 20 trials, the rate of habituation was identical for both genotypes. Pharmacological experiments, carried out with a separate group of OT þ/þ and OT / mice implanted with intraventricular cannulas, were designed to learn the effect of OT, AVP, and an OT antagonist on SRM. The MSRT described earlier (the same female social stimulus presented for four 1-min trials, a new female presented in trial 5; 10-min ITI) was used in these experimental tests. Testing began 3 or 4 days after recovery from surgery. On successive treatment days (intertreatment interval, 48–72 h) the subject received an intracerebroventricular injection of artiﬁcial cerebrospinal ﬂuid (aCSF, the vehicle), or a 1-ng dose of OT, AVP, or an OT antagonist (OT-Ant) and was tested 2 min later. The vehicle control trial preceded the ﬁrst and third, and followed the last, peptide administration. Each subject received OT and AVP as its ﬁrst and second peptide dosing. OT and OT-Ant comprised the third and fourth peptide dosing and were administered according to a counterbalanced design within subjects. Only data from the mice showing correct intracerebroventricular cannula placement on histological veriﬁcation were included for analyses. Acute treatment with intracerebroventricularly injected OT completely restored social memory in the OT / mice, as indicated by the signiﬁcant decline in SIT with the same female on presentation trials 2, 3, and 4 relative to trial 1, and by the recovery of interest when a new female was presented. OT-Ant treatment did not inﬂuence SRM in the OT / mice, but impaired it in the OT þ/þ mice (relative to the vehicle control condition, intracerebroventricularly injected OT-Ant had no measurable effect on olfactory investigation in the former, but signiﬁcantly delayed its decline in the latter). At the dose tested, AVP had no signiﬁcant effect on SRM of either genotype group (no signiﬁcant difference in behavior between AVP and CSF vehicle treatment conditions for OT þ/þ or OT / mice). In their discussion, these investigators reported that similar testing for SRM ability in OT þ/þ and OT / females resulted in a pattern of deﬁcits resembling that shown by the males. However, it had been much more difﬁcult to ﬁnd a robust deﬁcit for the females because their initial level of

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investigation was less vigorous than that of the males in this paradigm. A comparable sex difference in social investigative behavior has been observed in the conventional social recognition paradigm (Bluthe and Dantzer, 1990; see Chapter 12). Taken together, the results of this study have shown that male mice genetically deﬁcient in OT (OT /) do not have the SRM abilities shown by OT-intact wild-mice (OT þ/þ). The importance of OT to this olfactory-based memory processing was further substantiated by the ﬁndings that OT, but not AVP, stimuli tested in habituation/dishabituation and food-foraging paradigms, or in tests of spatial memory treatment, repaired the SRM deﬁcit observed in the OT / mice, and the OT receptor antagonist produced a social amnesia-like effect in OT þ/þ mice. Moreover, the neural processing underlying SRM appears to be independent of that required for olfactory foraging and olfactory habituation involving nonsocial stimuli, and habituation to an acoustic startle stimulus, because these abilities were intact in OT / mice.

IX. Chapter Summary and Commentary

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A. General Comments The contributions of the investigators whose works are discussed in this chapter have been of both methodological and substantive value. Two techniques applied to this ﬁeld of study, the antisense oligonucleotide treatment used by Landgraf et al. (1995) and the OT genetic knockout model used by Ferguson et al. (2000), permitted precise strategies for removing (1) targeted VP receptors in selected brain sites in the rat, and (2) OT in the OT / mutant mouse. Yet another methodological advance has been the microdialysis technique, which provides a means of applying exogenous VP/OT and of collecting endogenous VP/OT released from activated brain structures with negligible interference with normal activity in the conscious behaving animal [Engelmann and Landgraf, 1994; Engelmann et al., 1994; and see Landgraf et al. (1998) for further discussion of this technique as applied to VP/OT memory research]. The remainder of this section summarizes and discusses the contents of this chapter in terms of relevance to the views and ﬁndings of Dantzer, Bluthe, and colleagues, who pioneered the study of VP/OT and SRM, and of De Wied and colleagues, who pioneered the general ﬁeld of VP/OT and memory processing.

B. Peripherally Administered AVP and SRM Dantzer, Bluthe, and colleagues carried out several studies (see Chapter 13) showing that peripherally administered VP facilitated SRM in male rats (Dantzer et al., 1987) and female rats (Bluthe and Dantzer, 1990) and in

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male mice (Bluthe et al., 1993). In contrast, peripheral administration of an AVP V1 receptor antagonist on its own impaired SRM in sexually intact male rats (Bluthe and Dantzer, 1990) and mice (Bluthe et al., 1993), but not in female rats (Bluthe and Dantzer, 1990), castrated male rats (Bluthe et al., 1990), or castrated male mice (Bluthe et al., 1993). Noting that a peripherally administered dose of behaviorally effective AVP is unlikely to cross the blood–brain barrier, whereas that of the AVP V1 receptor antagonist can do so, they concluded that two different VP systems account for these ﬁndings. The facilitating effect of peripherally injected AVP on SRM was thought to be due to an interaction of an androgen-independent VP system with a central arousal system, as proposed for memory tested in other learning paradigms (see Chapters 6 and 7). On the other hand, the SRM impairment induced by the V1 antagonist was attributed to an androgen-dependent VPergic system in sexually intact males, exclusively involved in the olfactory processing of conspeciﬁc social signals necessary for SRM (Dantzer, 1998; and see Chapter 12). The ﬁndings of Popik and colleagues are relevant to the view outlined above. First, while replicating the Dantzer et al. ﬁnding that peripherally injected AVP extended the duration of SRM, these researchers also showed that the endocrine (pressor) activity of AVP was not necessary for its ability to facilitate SRM. Thus, AVP derivatives lacking the endocrine effects of the parent peptide nevertheless facilitated SRM (Popik et al., 1991; Sekiguchi et al., 1991a), as they do memory tested in appetitive [Vawter and Van Ree, 1995; Vawter et al., 1997 (see Chapter 2)] and avoidance learning paradigms (De Wied et al., 1987; see Chapter 5). Second, Popik and colleagues obtained evidence that the AVP facilitation of SRM consists of both long-term and short-term memory components, which are differentially sensitive to various AVP-related peptides. That is, AVP peptides containing the covalent ring structure [e.g., AVP(1–8), AVP(1–7), and AVP(1–6)] exerted a long-term memory effect on SRM, extending its duration to at least 24 h, whereas AVP derivatives lacking this structure [e.g., AVP(4–9) and AVP(4–8)] exerted only a short-term memory effect that extended SRM for 2 h, but not for 24 h (Popik and Van Ree, 1992). Although the study of Popik and Van Ree (1992) is consistent with the possibility that two different VP mechanisms are responsible for the effects of exogenous AVP on SRM, they are not comparable to the two VP-ergic mechanisms postulated by Dantzer and colleagues. In accordance with the ‘‘VP Dual Action Theory,’’ Dantzer and colleagues postulate that the arousal-dependent VP system is peripherally associated with a pressor effect and inﬂuences SRM as it does long-term memory tested in a number of appetitive and avoidance learning paradigms. However, the ﬁndings of Popik and Van Ree (1992) suggested that the VP mechanism responsible for the long-term component of SRM is not an arousaldependent mechanism, because it was activated by peripherally injected DG-AVP, which lacks the pressor arousal effects of AVP.

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C. OT and SRM The role of OT in SRM, demonstrated in the studies reviewed in this chapter, extends the research carried out by De Wied and colleagues on the role of OT in long-term memory tested in avoidance conditioning paradigms (see Chapters 2–5). Dantzer and colleagues (see Chapter 12) studied the role of OT in SRM mainly to compare it with that of VP. In their one study with OT and SRM, Dantzer et al. (1987) showed that peripheral administration of OT and VP resulted in the same reciprocal (opponent) actions of these neuropeptides previously shown for long-term memory tested with avoidance paradigms (Bohus et al., 1978b; see Chapter 2). That is, at the dose levels used, OT impaired normal SRM, and VP facilitated it, that is, extended its duration (Dantzer et al., 1987). Subsequent research has suggested that the OT inﬂuence on olfactorybased SRM is more complicated than originally suspected. The studies reviewed in this chapter have found that exogenous OT may impair or enhance SRM depending on the dose range used in the study. That is, administered in a dose range typically used in learning/memory paradigms, OT has been found to impair memory in avoidance paradigms (Bohus et al., 1978b; see Chapter 2) and in the social recognition test (Dantzer et al., 1987; Popik and Vetulani, 1991). However, when administered at a low dose range, exogenous OT has been shown to facilitate SRM whether the peptide is administered peripherally (Arletti et al., 1995; Popik et al., 1992, 1996), intraventricularly (Benelli et al., 1995), or locally into speciﬁc brain structures (Popik and Van Ree, 1991). There is evidence indicating that endogenous OT modulates SRM as it does memory involved in avoidance behavior. Van Wimersma Greidanus and Maigret (1996) used intracerebroventricularly and locally injected anti-OT serum to reduce endogenous OT in the brain. Their ﬁndings indicated that the intracerebroventricularly injected antiserum delivered in a 2-l volume at a sufﬁciently high concentration (1:10, but not 1:30 dilution) extended the duration of SRM from the normal 30 min to at least a 120-min interval. When the antiserum was locally applied at the same volume and concentration (2 l, 1:10 dilution) used with intracerebroventricular treatment to facilitate SRM, it was similarly effective when injected into the ventral hippocampus, but was ineffective when injected into the dorsal hippocampus, septal region, or olfactory nucleus. Their ﬁndings suggested that endogenous OT in the ventral hippocampus and the brain structures reached by intracerebroventricularly injected antiserum is involved in the modulation of SRM, and opposes the facilitative SRM effect inferred for endogenous VP, as it does in avoidance learning paradigms (see Chapters 2–5). Other studies reviewed in this chapter, in accord with the abovedescribed ﬁndings, suggest a physiological role for brain OT in modulating SRM, but the ﬁndings point to a facilitative, as opposed to a retarding, effect

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[Engelmann et al., 1998; Ferguson et al., 2000 (see below)]. Engelmann et al. (1998) found that an intracerebroventricularly injected OT antagonist blocked normal SRM in female rats whereas a similarly injected AVP antagonist was without effect. Taken together, these data point to the need for more detailed study to more clearly understand the role(s) that OT plays in memory modulation. It is possible that the memory-facilitative effect of both exogenously administered and endogenous OT is speciﬁc to SRM, and is mediated by olfactory pathways and structures activated by conspeciﬁc social stimuli directly or indirectly associated with prosocial reproduction-related activities such as mate bonding and offspring nurturance. On the other hand, the memoryimpairment function of OT may be involved in reduction of the conditioned anxiety and fear that occurs in painful or stressful situations that must be reencountered for reproductive success (e.g., parturition in females, or agonistic encounters associated with defense of mates, territories, and offspring).

D. Brain Structures and Pathways Mediating the VP and OT Influence on SRM The results of the studies reviewed in this chapter have reinforced the importance of olfactory pathways containing VP-ergic and OT-ergic circuitry in mediating SRM. Bluthe and Dantzer (1993; see Chapter 12) demonstrated the importance of the vomeronasal system [accessory olfactory system, engaged in the analysis of surface-deposited rather than airborne odorous molecules (pheromones)] in mediating the effect of androgendependent AVP on SRM. The studies in this chapter detailed their beginning insight into the SRM–olfactory connection. The research ﬁndings of Dluzen et al. (1998a,b, 2000) demonstrated that (1) the olfactory bulb (OB) is a target structure that mediates the SRM enhancement effects of exogenous AVP and OT (Dluzen et al., 1998a); (2) noradrenaline released in the OB is critical for the SRM preservation effects of these peptides (Dluzen et al., 1998b); and (3) in the case of OT, 2-adrenoceptors mediated the OT–NA interaction necessary to the inﬂuence of the neuropeptide on SRM. Anatomical study has indicated that both the main and accessory components of the OB project to the amygdala and the bed nucleus of the stria terminalis (BNST), which in turn connect with the septal area. Dantzer et al. (1988; see Chapter 12) demonstrated the importance to SRM of the septally released AVP, originating from the sexually dimorphic circuitry localized in the amygdala and BNST. The importance of the septal AVP system to this type of memory processing has been repeatedly reafﬁrmed in the studies reviewed in this chapter. The VP/OT antiserum study of Van Wimersma Greidanus and Maigret (1996) provided evidence of a physiological role of

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septal AVP, but not OT, in modulating SRM. Landgraf et al. (1995) further validated the AS oligo treatment technique by showing its ability to conﬁrm the well-demonstrated septal AVP recognition memory effect. The OB is also indirectly connected with the medial preoptic area (MPA), and the MPA has been shown to be involved in processing olfactory information (Macrides, 1976; Pfaff and Pfaffmann, 1969) and in mediating the SRM-facilitative action of exogenously applied OT (Popik and Van Ree, 1991). Engelmann et al. (1994) using microdialysis and push–pull perfusion, provided evidence of a connection between the SON and the septal area in mediating a VP inﬂuence on SRM. It is noteworthy that SON AVP, which is not sexually dimorphic (see Chapter 1), is nevertheless a contributory factor to the enhancement of SRM when activated by osmotic stimulation. This may be interpreted as indirect support for the proposal by Koob, Dantzer, and colleagues that arousal-modulating, androgen-independent VP modulates SRM as it does other types of learning and memory. The VP (and presumably OT) connections with the arousal system were considered responsible by them for the opponent effects on SRT performance observed after peripherally injected AVP and OT (Dantzer et al., 1987; see Chapter 12), and for the SRM enhancement resulting from osmotic stimulation (Bluthe et al., 1991), which increases peripheral and central levels of both neuropeptides (see Koob et al., 1985a). The ability of peripherally administered V1 receptor antagonist, which decreases the pressor effect associated with high levels of peripherally circulating AVP, to block the osmotically induced SRM enhancement was interpreted as support for their proposal of a VP arousal effect on SRM (Bluthe et al., 1991). Finally, the research of Everts and Koolhaas (1997, 1999) was relevant to septal AVP involvement in SRM. The results of both studies suggested the speciﬁcity of this septal AVP memory effect, because VP antagonist treatment applied to the lateral septum produced the expected impairment in SRM but had no effect on a parallel object recognition test (Everts and Koolhaas, 1997).

E. VP/OT Knockout Models and SRM The role of endogenous brain AVP, and particularly septal VP, in SRM processing received further conﬁrmation in a study by Engelmann and Landgraf (1994). They showed that SRM was impaired in untreated Brattleboro HODI rats, but was normal in Long-Evans (LE) rats with genetically intact AVP. Moreover, microdialysis-infused AVP into the lateral septum restored SRM to its normal level in the HODI rats, whereas similar infusion of the V1 antagonist into this structure impaired SRM in AVP-intact LE rats. Ferguson et al. (2000) conﬁrmed a role for endogenous OT in rodent SRM in their study with OT-mutant male and female mice. In contrast to

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mice with OT-intact genotypes, these mice showed no evidence of SRM in a multitrial retention test or in the social recognition test, with genetically normal adult females (ovariectomized or sexually intact) and sexually intact males as social test stimuli. This suggests that the VP/OT neuropeptide effect on SRM is not limited to juveniles but extends to conspeciﬁcs of all ages. Their results also suggested that the inﬂuence of endogenous OT on SRM appears to be speciﬁcally linked to the role of the peptide in olfactory-based social memory processing because it was not essential for normal performance in tests reliant on olfactory-based processing of nonsocial stimuli tested in habituation/dishabituation and food-foraging paradigms, or on tests of spatial memory (see Chapter 10).

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Part VII

Brain–Fluid Barriers: Relevance for Theoretical Controversies Regarding Vasopressin and Oxytocin Memory Research

I. Chapter Overview

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‘‘Brain–ﬂuid barriers’’ refer to the structural and functional mechanisms that segregate the plasma from the brain (the blood–brain barrier, BBB), and from the cerebrospinal ﬂuid (blood–cerebrospinal ﬂuid barrier, blood–CSF barrier). The inﬂuence of peripherally injected vasopressin (VP) or oxytocin (OT) on central processes depends on the properties and limitations of these barriers, including the selectivity of their transport systems. This chapter ﬁrst discusses general structural and functional aspects of the brain–ﬂuid barriers and then presents evidence about the nature of the interaction of VP (and OT) with them. These data bear on the various memory theories of how peripherally circulating VP and OT (or their behaviorally active metabolites) might produce centrally mediated behavioral effects. Given the evidence, cited below, of selective barrier-mediated transport of hydrophilic (water-soluble) substances between the blood and the brain, several explanations are possible for how peripherally Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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injected hydrophilic peptides such as VP and OT may bring about their memory effects. These possibilities are discussed in Section IV.

II. Brain–Fluid Barriers: Blood–Brain Barrier and Blood–Cerebrospinal Fluid Barrier

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A. Historical Development of the Concept of Brain–Fluid Barriers Our present notion of the BBB had its origin in pharmacological research and histological studies carried out in Germany in the late nineteenth and early twentieth centuries. In these studies basic and acidic dyes (e.g., Ehrlich, 1885 as cited in Bradbury, 1979; Ehrlich, 1902 as cited in Bradbury, 1979; Goldman, 1909 as cited in Bradbury, 1979; Goldman, 1913 as cited in Bradbury, 1979), or pharmacologically active substances (Biedl and Kraus, 1898 as cited in Bradbury, 1979; Lewandowsky, 1900 as cited in Bradbury, 1979; Stern and Gautier, 1921 as cited in Davson, 1989; Stern and Gautier, 1922 as cited in Davson, 1989) were injected into laboratory animals. The tissue-staining effects of the injected dyes were examined postmortem by microscope, and the behavioral (CNS) effects of the pharmacological treatments were determined after treatment. These studies indicated that, when injected into the bloodstream, some of the tested substances entered the brain whereas others did not. However, when injected into the CSF all the test substances did so. More speciﬁcally, an intravenous injection of the acidic dye, trypan blue, stained tissues in all parts of the body, including the connective tissue of the choroid plexus and the membrane coverings of the CNS, but left neural tissue and CSF unstained (Ehrlich, 1902 as cited in Bradbury, 1979; Goldman, 1909 as cited in Bradbury, 1979). The obverse results were obtained when the dye was injected directly into the CSF; it stained CSF and the brain while leaving tissues in the rest of the body unaffected (Goldman, 1913 as cited in Bradbury, 1979). Similarly, pharmacologically active substances that demonstrated no neurological effects when injected into the bloodstream produced characteristic CNS effects when injected in the CSF (Biedl and Kraus, 1898 as cited in Bradbury, 1979; Stern and Gautier, 1922 as cited in Davson, 1989). Taken together, these studies strongly supported the concept of a barrier between the blood and the brain (BBB), between the blood and the CSF (blood–CSF barrier), and the absence of any barrier between the CSF and the brain’s extracellular ﬂuid. Further, in contrast to the acidic dyes, most of the intravenously injected basic dyes stained CNS tissue (Ehrlich, 1885 as cited in Bradbury, 1979; Ehrlich, 1902 as cited in Bradbury, 1979) and several of the pharmacologically active substances, injected directly into the blood, entered the CSF (Stern and Gautier, 1921 as cited in Davson, 1989) and also produced behavioral (CNS) effects (Stern and Gautier, 1922 as cited in Davson, 1989). These ﬁndings demonstrated that the barriers behaved

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selectively with respect to which substances were allowed entry to the brain from the blood. Ehrlich’s 1885 and 1902 ﬁndings that many basic, but few acidic, dyes could color neural tissues when injected directly into the bloodstream led some researchers to conclude that the pH value of the substance was the basis for this selectivity. However, this was later found to be an erroneous generalization, and that the lipid solubility of the test substance determined its ability to enter the brain from the blood (Bradbury, 1979; Goldstein and Betz, 1986). More speciﬁcally, lipid-soluble (lipophilic) but not water-soluble (hydrophilic) substances can freely diffuse across these barriers because, like all cell membranes, those of the endothelial cells of the brain capillaries (BBB), and of the epithelial cells of the choroid plexus (blood–CSF barrier), are composed of lipid molecules (Goldstein and Betz, 1986). In the 1960s electron microscopy and tracer enzymes such as horseradish peroxidase (HRP) became available for use in experimental study of these brain–ﬂuid barriers. As a result, experiments by Karnovsky (1967), Reese and Karnovsky (1967), and Brightman and Reese (1969) veriﬁed that the BBB could be attributed to the structural characteristics of the brain capillaries themselves and not, as some had theorized, to the astrocytes (astrocytic feet) and/or the pia-arachnoid membrane coverings associated with these capillaries. See Bradbury (1979), Davson (1989), and Goldstein and Betz (1986) for more detailed discussion of this topic.

B. Blood–Brain Barrier The BBB refers to the various structural/functional features of the brain capillary endothelium (the single layer of ﬂat epithelial cells forming the 1-m-thick capillary wall) that selectively regulate exchanges between the blood and the extracellular ﬂuid (ECF) of the brain. Like all capillaries, those in the brain are organized into networks that intervene between the blood vessels (arterioles and metarterioles) that supply the blood entering these networks, and the venules that collect the blood that leaves them. The localization of the BBB in the capillary endothelium is consistent with this being the site at which interchanges between the blood and the brain take place. In most body tissues the endothelial cells comprising the capillary wall are joined together at junctions characterized by slitlike openings (gaps) through which many substances and ﬂuids may freely pass between the blood and the ECF of these tissues. However, in barrier-protected neural tissue, these cells are tightly joined at these junctions and the gaps are absent. Viewed under the electron microscope, the external leaﬂets of the membranes of these cells are fused together, forming a seal on both the lumenal (blood-facing) and ablumenal (brain-facing) surfaces of the capillary wall. A thin-layered basement membrane (a complex matrix of collagens and other proteins) completely encloses the ablumenal surface of the capillary wall, and more than 85% of its surface is covered by the feet of astrocytic processes (glial cells whose processes contact nearby neurons as well as brain

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capillaries). Together, the basement membrane and glial foot processes provide structural support for the brain capillary (see Goldstein and Betz, 1983, 1986, for more detailed discussion). Aside from the tight junctions described above, other features of the capillary endothelium relevant to barrier function include (1) an absence of intracellular fenestrations (openings); (2) a paucity of endothelial cell pinocytes (membrane vesicles that fuse with the membrane at the surface, invaginate and engulf extracellular molecules, and then reform and transport entrapped molecules across the cell); (3) various carriers (proteins present on the lumenal and/or antilumenal surface of endothelial cell membranes) that passively or actively transport hydrophilic molecules into and/or out of the brain; (4) enzymes within an endothelial cell that may metabolically degrade certain substances that have entered the cell from the blood and thereby prevent their passage into the brain (i.e., forming an ‘‘enzymatic barrier’’); and (5) astrocytic end feet, surrounding the capillary wall, that have no barrier function themselves but may contribute to its early development (i.e., evidence from ontogenetic studies suggest that closure of the BBB is related to the maturation of glial foot processes during early development) (Johansson, 1990; Krisch and Leonhardt, 1989). The structures and features of the brain capillaries described in this section are depicted in Figs. 1 and 2 and discussed in greater detail in Oldendorf (1977), Goldstein and Betz (1983, 1986), and Johansson (1990).

FIGURE 1 The major structural differences between general and brain capillaries. The intercellular cleft, pinocytosis, and fenestrae are virtually nonexistent in brain capillaries and exchange must pass transcellularly. Solutes must enter and penetrate the cell membranes. In general capillaries any such transcellular exchange is overshadowed by the other, nonspeciﬁc routes of exchange. Source: Oldendorf, 1977 (Fig. 2, p. 179). Copyright ß 1977 by Academic Press.

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FIGURE 2 Model of brain capillary. The tight junctions (1) that join endothelial cells together in brain capillaries are continuous and complex, and they limit the diffusion of large and small solutes. Very few pinocytotic vesicles (2) are found in the cytoplasm; this potential route for transendothelial transport is inoperative in normal brain capillaries. The basement membrane (3) provides structural support for the capillary and may inﬂuence endothelial cell function. Foot processes of astrocytes (4) encircle the capillary but do not create a permeability barrier. Transport carriers (5) for glucose and essential amino acids facilitate the movement of these solutes into the brain. Active transport systems (6) appear to cause efﬂux of certain small amino acids from brain to blood. Naþ pores and NaCl carriers on the luminal surface of the endothelial cell and Naþ, Kþ-ATPase on the antiluminal surface (7) account for movement of ions across the brain capillary. Mitochondria (8) produce the ATP needed for energy-dependent transport processes. Not shown are receptor sites for agents that may regulate the permeability of this barrier. Source: Goldstein and Betz, 1983 (Fig. 1, p. 391). Copyright ß 1983 by Little, Brown and Co. Reprinted by courtesy of John Wiley & Sons, present Publishers of Annals of Neurology.

C. CSF Formation and Circulation, Blood–CSF Barriers, and the Circumventricular Organs 1. Choroid Plexus and the Formation of CSF The choroid plexus is at once the primary source of CSF formation, the major blood–CSF barrier, and a circumventricular organ (CVO), housing fenestrated ‘‘leaky’’ blood vessels. As a CVO, there are four distinct choroid plexuses, each associated with a speciﬁc brain ventricle: the lateral ventricles centrally located within each cerebral hemisphere, the third ventricle located midsagittally in the diencephalon, and the fourth ventricle on

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FIGURE 3 The choroid plexus sits within the ﬂuid-ﬁlled chambers in the brain called ventricles. It continuously secretes cerebrospinal ﬂuid (CSF), which cushions the nervous systems, carries some nutrients to brain tissues, and cleanses the brain of wastes. As new CSF is produced, old CSF is forced to ﬂow (arrows) through the ventricles, around the spinal cord, and into the subarachnoid space around the brain. As the CSF ﬂows, it exchanges substances with the interstitial ﬂuid between brain cells. Eventually it drains into blood in the superior sagittal sinus through structures called arachnoid granulations. Because the choroid plexus and the arachnoid membrane stand between the blood and the CSF, they constitute the blood–CSF barrier. Source: Spector and Johanson, 1989 (p. 69). Published by Scientiﬁc American, Inc. Copyright ß 1989 by Carol Donner and reprinted with her kind permission. (See Color Insert.)

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the dorsal surface of the lower brainstem (see Fig. 3). Viewed under the light microscope, each ‘‘choroid plexus appears as a network of densely branched fronds. Each frond consists of capillaries and other small blood vessels surrounded by a single layer of epithelial cells’’ (Spector and Johanson, 1989, p. 68). As depicted in Fig. 4, one surface (basolateral surface) of the choroidal epithelial layer is in direct contact with the blood plasma supplied by the fenestrated vessels of the plexus. Its other surface (apical surface) is in contact with ventricular CSF. Figure 4 also shows that the choroidal epithelium is continuous with the ependymal lining of the ventricle. Although both of these tissues are ontogenetic descendents of the single-cell layer that forms the wall of the neural tube, only the former contains the tight junctions that characterize it as a blood–CSF barrier (Bradbury, 1993). CSF is secreted primarily by the choroidal epithelial cells, and consists mainly of water together with selected ions derived from the blood plasma

FIGURE 4 Essential nutrients reach the neurons and glial cells in the brain by crossing either the blood–CSF barrier, which is regulated by the choroid plexus, or the blood–brain barrier of the cerebral capillaries. Water-soluble molecules cannot diffuse freely between the blood and the CSF because of impermeable tight-junction seals between the choroid epithelial cells; instead, the epithelial cells transfer certain molecules from one side of the barrier to the other. Once molecules enter the CSF, they can diffuse through the ‘‘leaky’’ ependymal layer and reach the interstitial ﬂuid around the neurons and glial cells. Similarly, wastes in the interstitial ﬂuid can pass into the CSF for disposal. The endothelial cells of the cerebral capillaries, which are also sealed by tight junctions, control the direct exchange of materials between the blood and interstitial ﬂuid. Source: Spector and Johanson, 1989 (p. 70). Published by Scientiﬁc American, Inc. Copyright ß 1989 by Carol Donner and reprinted with her kind permission. (See Color Insert.)

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within the ‘‘leaky’’ blood vessels that penetrate each choroid plexus. As illustrated in Fig. 5, mechanisms and transport systems at both surfaces of the choroidal epithelium are responsible for the ionic exchanges determining the composition of CSF. Naþ, Kþ, Ca2þ, Mg2þ, and Cl ion levels in newly formed CSF differ from those in plasma, and remain roughly constant

FIGURE 5 Flow of molecules across the blood–CSF barrier is regulated by several mechanisms in the choroid plexus. Some micronutrients, such as vitamin C, are pulled into the epithelial cells at the basolateral surface by an energy-consuming process known as active transport; the micronutrients are released into the CSF at the apical surface by another regulated process, facilitated diffusion, which requires no energy. Essential ions are also controllably exchanged between the CSF and blood plasma. Transport of an ion in one direction is linked to the transport of a different ion in the opposite direction, as in the exchange of sodium (Naþ) ions for potassium (Kþ) ions. Source: Spector and Johanson, 1989 (p. 72). Published by Scientiﬁc American, Inc. Copyright ß 1989 by Carol Donner and reprinted with her kind permission. (See Color Insert.)

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in spite of major ﬂuctuations that may occur in the peripheral circulation. Mechanisms within the choroidal epithelium can adjust the rate of ion transport from blood to CSF in response to these ﬂuctuations and thus, choroidal epithelium both secretes CSF and preserves its ionic composition. Two extrachoroidal structures also contribute to the formation of CSF: the BBB secretes extracellular ﬂuid (ECF) via coupled transport of salts and water, which drains into the surrounding CSF by a pressure-dependent mechanism (Cserr and Patlak, 1991), and the arachnoid membrane secretes CSF into the subarachnoid space (SAS) (Johanson, 1998). 2. CSF Circulation Within and Around the CNS CSF, continually replaced by newly formed ﬂuid, has a directional ﬂow path away from the pressure head of its source (choroid plexuses) and toward venous drainage sites (dural sinus). It circulates within the brain ventricles and central canal of the spinal cord, enters the surrounding SAS and ﬂows around the CNS as illustrated in Fig. 3. On reaching the superior dural sinus it is forcefully expelled from the SAS into the dural sinus via the valvelike action of the arachnoid villi (arachnoid membranous structures that penetrate the dural sinus from the SAS). The superior dural sinus is a large endothelium-lined channel formed within the bilayered dura mater (the third and outermost protective membrane that surrounds the CNS). Located within the midsagittal cleft separating the cerebral hemispheres, the dural sinus contains the venous blood and CSF that is drained from the brain and returned to the systemic circulation. During its circulation within and around the brain, CSF provides neural tissue with nutrients and removes wastes from it. CSF may also transport information-bearing molecules to various parts of the CNS along its ﬂow path. This putative transport function of CSF is discussed in Section III.C [see Spector and Johanson (1989) and Bradbury (1993) for a more detailed discussion of topics discussed in Sections II.C.1 and II.C.2]. 3. Other Brain–Fluid Barrier Structures: Arachnoid Membrane, Phagocytic Cells, and CVOs The arachnoid membrane is a multilayered structure that forms the roof of the SAS and interconnects with the pia mater (ﬂoor of the SAS) via ﬁne ﬁlamentous extensions (trabeculae). Its barrier function is based on the tight junctions that characterize the cells of its upper layers, which are in contact with the ﬂoor of the overlying superior dural sinus. This arachnoid barrier prevents exchanges between the CSF in the SAS and the blood in the dural sinuses (Johanson, 1998). Broadwell and colleagues (Broadwell, 1992; Broadwell and Banks, 1993; Broadwell and Sofroniew, 1993) have suggested that various types of phagocytic cells (macrophages, microglia, and perivascular phagocytes) may assist barrier functions by defending against infectious agents and other

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harmful substances that might breach the brain–ﬂuid barriers. One or more of these cell types is found in close proximity to all barrier structures (i.e., on the surfaces of nonfenestrated capillary endothelium and adjacent venules, choroidal plexus epithelium, and in association with other CVOs and the arachnoid membrane). The CVOs, brieﬂy described in Chapter 1, are specialized structures located in the midsagittal plane of the brain in association with each of the four ventricles, as depicted in Fig. 9 in Chapter 1. Krisch and Leonhardt (1989) have noted that CVOs occupy thin parts of the ventricle wall, thereby positioning them between the inner (ventricular) CSF and the outer (subarachnoid space) CSF. With the exception of the subcommissural organ, the CVOs discussed herein contain a hemal region (fenestrated capillaries, arterioles, and venules) and a neuroglial region (neuronal and glial cells and/or their processes). An ependymal lining (composed of ependymal cells interspersed with tanycytes) lies between the hemal/neuroglial regions and the inner CSF. Functionally, these CVOs are well known for their ability to serve as an interface between endocrine and nervous systems in their regulation of many physiological/behavioral functions. In this capacity they relay ‘‘neural and/or chemical information between or among brain, CSF and blood’’ (Johnson and Gross, 1993, p. 679). Speciﬁcally, neuronal receptor elements contacting capillaries within the hemal region of certain CVOs monitor blood-borne signals [e.g., osmolality, hormonal messages (e.g., angiotensin II, Ang II)] and transduce them into neural signals (Johnson and Gross, 1993). On the effector side, axons of neuroendocrine (neurosecretory) cells terminating on capillaries in the hemal regions of certain CVOs secrete hormones into the pituitary portal circulation [median eminence (stimulating or inhibitory anterior pituitary-releasing hormones)] or into the systemic circulation [e.g., neurohypophysis (VP or OT) and pineal gland (melatonin)]. Some CVOs [pineal gland, subfornical organ (SFO), vascular organ of the lamina terminalis (OVLT), and area postrema (AP)] may participate in both sensory transduction and neurosecretion into the bloodstream (McKinley et al., 1990). Centrally, these ‘‘hormonal–neural’’ transducer cells and neuroendocrine cells synapse with neurons that participate in neuroendocrine feedback loops and other widespread neuronal circuitry involved in the regulation of body ﬂuid homeostasis, cardiovascular functioning, and energy balance (Gross and Weindl, 1987; Johnson and Gross, 1993; McKinley et al., 1990; Phillips, 1980; Prescott and Brightman, 1998). As noted above, capillaries lacking a BBB characterize the neurohemal regions of these CVOs, and are the sites where the sensory transducer receptors respond to blood-borne signals, and where the neurosecretory effector cells release hormones into the blood. However, not all the capillaries penetrating these CVOs lack this barrier. There appear to be distinct regions, at least in some CVOs (e.g., SFO, OVLT, and AP), containing subregions of capillaries both with and without BBB features, which has

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suggested topographically specialized functions to some researchers (Gross and Weindl, 1987; Johnson and Gross, 1993). According to Krisch and Leonhardt (1989), in those regions of the CVOs where fenestrated capillaries are present, specialized ependymal cells that border the inner CSF, and arrangements of the leptomeninges (pia and arachnoid) that border the outer CSF, prevent an uncontrolled transfer of substances within the hemal milieu from entering the CSF compartment. Thus, it would seem CVOs do not provide an easy route by which peripherally circulating substances can diffuse into the brain and inﬂuence its activity. The following ﬁndings support this idea. Maness et al. (1998) used emulsion autoradiography to assess involvement of choroid plexus (CP) and SFO as potential routes by which intravenously injected radioiodinated cytokine interleukin 1 (IL-1) may enter the brain. The distribution pattern of silver grains within and around the SFO was greatly restricted to that CVO (Fig. 6), indicating that injected cytokine did not freely penetrate into the surrounding regions. In addition, they showed that this cytokine, which also localized to the CP, did not readily enter the adjacent CSF or the tissues surrounding the ventricular system. They concluded that the CVOs could not account for ‘‘total delivery of the cytokine to the brain’’ (Maness et al., 1998, p. E207). Noting previous evidence obtained from their laboratory, they suggested that carrier-mediated saturable transport is the major mechanism by which blood-borne IL-1, as well as other cytokines, are delivered to the brain.

FIGURE 6 Example of sampling ladder for image analysis. This approach was used to determine concentrations of silver grains at 10-m intervals within and away from the subfornical organ (SFO). Scale bar, 50 m. Source: Maness et al., 1998 (Fig. 1, p. E208). Copyright ß 1998 by the American Physiological Society. Figure reprinted with permission.

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D. Functional Operations of Brain–Fluid Barriers and Their Regulation Together, these barriers protect (prevent circulating toxins from entering the CNS), provide the brain with essential nutrients (substances needed by, but not synthesized in, the CNS), and maintain a stable microenvironment required for normal neuronal functioning (includes strict regulation of the substances permitted entry into the CNS, as well as removal of metabolic wastes and other substances whose build-up in this tissue would prove harmful). Given the selectivity of the brain–ﬂuid barriers against penetration of water-soluble substances, how is the extraction of essential nutrients and required substances from the blood and the ridding itself of metabolic wastes and other potentially harmful substances accomplished? The answer, in large part, is that speciﬁc transporters located on the plasma-facing surface of these barriers mediate blood-to-brain transport of substances required for brain operations. These include a number of high-capacity transporters for the following metabolites needed frequently and in great abundance by the brain: (1) hexose sugars (especially glucose, the major metabolic supplier of energy for the cellular work of the brain); (2) various groups of structurally related amino acids (AAs), such as the large neutral amino acids (LNAAs) used in protein synthesis and as precursors of certain neurotransmitters [e.g., tyrosine (for catecholamines) and tryptophan (for serotonin)]; (3) choline [acetylcholine (ACh) precursor]; and (4) nucleosides (used in the synthesis of DNA and RNA). In addition, low-capacity transporters in these barriers, especially in the choroidal epithelium, carry certain vitamins (e.g., ascorbic acid and vitamins of the B complex group) and electrolytes (e.g., sodium chloride, sodium, and potassium) from blood to brain or to CSF (Spector and Johanson, 1989). Betz (1992) has pointed out that these transport systems are characterized by (1) stereoselectivity—a speciﬁc structural arrangement of the molecular components is required for receptor recognition and binding of the test compound; for example, the transporter for hexose sugar carries d- but not l-glucose across the barrier; (2) saturable transport due to availability of only a limited number of carrier receptor sites for binding. The transporter is fully saturated when all receptor sites are occupied; (3) competitive inhibition—receptor sites for a given transporter bind any of a number of structurally related compounds that are in competition for the binding sites; and (4) symmetrical or asymmetrical transport—if the transporter is distributed on both sides of the barrier, transport is symmetrical (occurs in either direction), if limited to one side of the barrier, transport is asymmetrical (only in one direction). Thus the hexose sugar transport system is symmetrical and can transport glucose from blood-to-brain and vice versa, but the transporter for Kþ is found only on the ablumenal surface of the

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capillary endothelium and transports Kþ only from brain-to-blood. See Betz (1992), Spector and Johanson (1989), Johanson (1998), and Segal (1998) for additional discussion of these topics.

III. Origin and Fate of VP and OT within the CSF

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Studies using radioimmunoassays have demonstrated the presence of several neuropeptides (Allen et al., 1974; Joseph et al., 1975; Knigge and Joseph, 1974), including neurophysin (Robinson and Zimmerman, 1973), VP (Dogterom et al., 1978; Heller et al., 1968; Vorherr et al., 1968), and OT (Unger et al., 1974), in the CSF of several species. For example, Dogterom et al. (1978) measured immunoreactive arginine vasopressin (irAVP) in plasma and CSF samples simultaneously collected from laboratory rats [normal, homozygous diabetes insipidus (HODI), and heterozygous diabetes insipidus (HEDI)], dogs, and human hernia patients assessed for diagnostic purposes. With the exception of the HODI rats, which exhibited neither plasma nor CSF AVP, this peptide was present in both ﬂuid compartments in all subjects of each species tested. Moreover, in dogs and humans AVP levels in CSF samples were higher than in simultaneously obtained plasma samples. This consistent ﬁnding of neurohypophysial peptides circulating in the CSF raises questions about their source (plasma or brain side of the brain–ﬂuid barriers), and the destiny of their transport by the CSF. Numerous lines of evidence on their origin, cited below, support the view that these neuropeptides are directly released from brain neurons and not transported across the brain– ﬂuid barriers. As for their destiny, it has been theorized that (1) they may be transported by CSF from their sites of origin to their sites of activity in the brain (CSF ‘‘conduit function’’), and (2) the CSF may rid the brain of ‘‘used up’’ VP and OT along with other metabolic wastes and expel them into the venous circulation via the arachnoid villi (CSF ‘‘sink function’’).

A. CSF VP and OT Are of Central Origin Morphological, correlational, and experimental observations have been interpreted as supporting a central (brain), as opposed to a peripheral (plasma), origin for CSF VP and OT. Morphological support derives from immunocytochemical studies that indicate the presence of (1) extrahypothalamic VP-ergic and/or OT-ergic terminals in the ependymal lining of all four ventricles, including the cerebral aqueduct (suggesting direct contact with the ventricular space) (De Vries et al., 1985), and (2) axonal terminals with granules of neurophysin and vasopressin close to portal capillary loops (suggesting transport into the anterior pituitary gland) or protruding into the third ventricle (suggesting secretion into the ventricular system) (De Wied and Gispen, 1977).

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Correlational research also supports a central origin for CSF levels of these peptides. If CSF VP and OT were of peripheral origin, it would be reasonable to expect a signiﬁcant positive relationship between peripheral and central levels of these peptides, and that ﬂuctuations in CSF levels would reﬂect those in peripheral circulation. However, the correlational ﬁndings cited below are not consistent with this expectation. First, plasma levels of VP and OT differ from those in the CSF when simultaneously measured in either the anesthetized (Dogterom et al., 1978; Vorherr et al., 1968) or conscious (Landgraf and Gunther, 1983; Mens et al., 1980; Perlow et al., 1982; Reppert et al., 1981; Simon-Opperman et al., 1983) state. Second, AVP exhibits a circadian rhythm in the CSF, but not in the plasma, in many species of mammals including rats (Mens et al., 1982b; Schwartz et al., 1983), cats (Reppert et al., 1981), guinea pigs (Robinson and Jones, 1982), rabbits (Gunther et al., 1984), sheep (Stark and Daniel, 1989), and monkeys (Perlow et al., 1982). This rhythm is similar in both sexes (Reppert and Schwartz, unpublished observations, cited in Reppert et al., 1987). An OT circadian rhythm in the CSF, but not in the plasma, has been observed in monkeys (Perlow et al., 1982) and humans (Amico et al., 1983), but not in rats, cats, or guinea pigs (Reppert et al., 1987; Robinson and Jones, 1982). Experimental observation supports the thesis that the hypothalamic suprachiasmatic nucleus (SCN) is the source for the CSF VP rhythm, whereas a second circadian pacemaker present only in the primate brain (exact location presently unknown) is responsible for the CSF OT rhythm. Consistent with this are the observations that OT is absent from the mammalian SCN (Sofroniew and Weindl, 1980) and that removal of the SCN in the monkey has little effect on the CSF OT rhythm (Reppert et al., 1984). Additional evidence verifying an SCN origin of CSF VP in nonprimates comes from an SCN lesion study using VP-normal rats (Jolkkonen et al., 1988) and a transplant study using VP-deﬁcient Brattleboro rats (Earnest et al., 1989). Jolkkonen et al. (1988) reported that lesioning the SCN, but not the hypothalamic paraventricular nucleus (PVN), abolished the CSF VP rhythm. Earnest et al. (1989) found that a diurnal pattern of CSF circulating VP could be established in Brattleboro rat recipients of hypothalamic SCN, but not PVN, fetal tissue transplants from Long-Evans rats. Third, CSF and plasma VP typically do not ﬂuctuate together in response to numerous stimulus conditions. Mens et al. (1980), using rats as subjects, reported that water deprivation, hypertonic saline ingestion, and systemic injection with histamine or nicotine markedly increased plasma levels of VP but was without effect on CSF VP levels. Orlowska-Majdak et al. (1994), using urethane-anesthetized rats as subjects, found that sciatic and trigeminal nerve stimulation increased VP release into the bloodstream but not into the CSF. Landgraf and Gunther (1983), using conscious, unrestrained rabbits, and Mens et al. (1980), using freely moving rats, observed

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elevated plasma levels of VP and OT without concomitant increases in CSF levels after dehydration or hemorrhage. Working with chronically cannulated conscious guinea pigs, Robinson and Jones (1982) observed that intravenous infusions of OT or porcine neurophysin (Np), which achieved steady state plasma levels of each peptide, produced no detectable increase in CSF levels of either peptide, and suckling, which produced large rises in plasma levels of OT and Np, had no effect on their levels in the CSF. Working with hydrocephalic human patients, Sorensen and Hammer (1985) found that dehydration (24 h of ﬂuid deprivation), apomorphine-induced nausea, and postural changes (head-up tilt to 50 degrees for 45 min) produced a modest to marked increase in plasma VP but no changes in CSF VP. Given the blood–CSF barrier, on those occasions when increases in CSF and plasma concentrations of VP co-occurred, such as during severe hemorrhage (Szczepanska-Sadowska et al., 1983; Vorherr et al., 1968; Wang et al., 1981), endotoxin-induced fever (Kasting et al., 1983), elevated osmolality (Szczepanska-Sadowska et al., 1984), and in response to electrical stimulation of the hypothalamic PVN (Robinson and Jones, 1982) or the vagus nerve (Heller et al., 1968), the correlation can be attributed to coactivation of peripheral and central sources of AVP. A number of experimental manipulations also lend support to a central origin for CSF VP and OT. First, Dogterom et al. (1977) showed that ablation of the posterior pituitary, the means by which hypothalamusgenerated VP and OT enter the blood periphery, impaired plasma levels but not CSF levels of these neuropeptides. Comparisons between VP and OT plasma levels 4 weeks after surgery indicated a much more rapid recovery for OT than for VP, presumably due to a more effective regeneration of OT than VP ﬁbers innervating the neural lobe. Second, systemic administration of AVP, which elevates plasma concentration, is not accompanied by a simultaneous increase in CSF AVP level (Vorherr et al., 1968) and similar data have been obtained for OT (Unger et al., 1974). Third, stimulation of hypothalamic PVN and SON suggests that central release of OT and VP into the CSF originates from parvocellular but not magnocellular circuitry (Jones et al., 1983). Thus, in the anesthetized rat, retrograde stimulation of OT projections from the SON, which contains only magnocellular VP and OT cells, increases plasma but not CSF levels of OT. On the other hand, stimulation of the PVN, which contains parvocellular as well as magnocellular VP and OT neurons, increases CSF levels of OT.

B. Transport of VP and OT from Sites of Central Origin to Ventricular CSF Basically, two routes have been proposed. First, axonal endings of VPand OT-containing neurons in hypothalamic and extrahypothalamic nuclei might release their contents into the CSF via axonal terminals contacting the

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ventricular ependyma (Van Wimersma Greidanus and De Wied, 1976b). Although a number of investigators have cited earlier observations consistent with this viewpoint (e.g., Buijs et al., 1978; Kozlowski, 1986; Rodriguez, 1976; Zimmerman et al., 1975), no direct support has since been forthcoming (Sewards and Sewards, 2003; Sorensen, 1986). Second, VP and OT might reach the ventricular CSF after release within activated brain sites and subsequent transport by the brain extracellular ﬂuid (ECF) (Sorensen, 1986). This more recently proposed route of delivery is now favored by a number of researchers (De Wied et al., 1988; Robinson, 1983; Sewards and Sewards, 2003). Sewards and Sewards (2003) brieﬂy discuss evidence relevant to this proposal (discussed below). Studies using microdialysis techniques have demonstrated VP and/or OT release into the ECF of the hypothalamic PVN and/or SON (Ludwig, 1995, 1998; Ludwig et al., 1994, 1995; Wotjak et al., 1996, 1998), fulﬁlling one of the prerequisites for the Sorenson (1986) proposal. Sewards and Sewards (2003) cited research ﬁndings, consistent with the possibility that SCN VP is transported to ventricular CSF via diffusion in the ECF. Fetal transplant studies strongly suggest that the circadian locomotor rhythm in hamsters depends on a diffusible signal that is transmitted from the SCN to the brain structures that mediate this behavior: (1) Hakim et al. (1991) reported that surgical isolation of the SCN within a hypothalamic island followed by transection of SCN efferents did not prevent free-running locomotor rhythms in hamsters; and (2) Silver et al. (1996) showed that SCN tissue that had been isolated within a semipermeable polymeric capsule (prevented neural outgrowth, but allowed diffusion of humoral signals) before transplantation, restored circadian activity rhythms in hamsters whose own SCN had been ablated. This latter result was interpreted as conﬁrmation that ‘‘a diffusible signal is partially involved in the restoration of behavioral rhythms, but not necessarily of circadian rhythms in CSF concentrations of vasopressin’’ (Sewards and Sewards, 2003, p. 258). A study by Broadwell and Balin (1985) yielded more direct support for the Sorensen (1986) proposal as it pertains to SCN-generated diurnal ﬂuctuations of VP levels in ventricular CSF. They injected a retrograde tracer into the lateral ventricles and observed that it labeled neurons in a number of hypothalamic nuclei (paraventricular, supraoptic, periventricular, arcuate, and retrochiasmatic nucleus), but not in the SCN. Sewards and Sewards (2003) argued that if axonal transport to the ventricular CSF mediated the CSF VP rhythm, one would have expected that the retrograde tracer would have labeled SCN, but this did not occur (see Sewards and Sewards, 2003, for additional discussion). Thus, ECF appears to be the major medium transporting VP of SCN origin to ventricular CSF.

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C. Does CSF Serve a Conduit as well as a Sink Function in VP and OT Transport? Since their initial behavioral research with vasopressin in the mid1960s, De Wied and colleagues have maintained that the contribution of VP to memory processing is mediated by central and not peripheral receptors. Before the knowledge of VP-ergic and OT-ergic transmitter pathways in the brain and extrahypothalamic sources of VP, these researchers proposed that AVP, which was known to be synthesized in the anterior hypothalamus, was transported to central brain sites by the CSF (De Wied et al., 1976; Van Wimersma Greidanus and De Wied, 1976b). Several reported observations were interpreted as compatible with this proposed ‘‘conduit’’ role for CSF (e.g., Brownﬁeld and Kozlowski, 1977; Rodriguez, 1976). However, the subsequent research of Buijs and colleagues (Buijs and Swaab, 1979; Buijs et al., 1978) veriﬁed the presence of extrahypothalamic VP-ergic and OT-ergic neural pathways in the rat brain, thereby rendering sole reliance on CSF-mediated transport of these peptides unnecessary. De Wied and colleagues did not abandon the original proposal, but viewed a CSF-mediated volume transmission as an auxiliary means of effecting intercellular communication involving either of these two peptides. As used here, ‘‘volume transmission’’ refers to intercellular communication in which a signal molecule reaches its target site by transport through a ﬂuid medium (e.g., CSF). In this way the proposed CSF ‘‘conduit’’ function might help explain how VP and/or OT accesses receptor sites in brain regions where VP-ergic and OT-ergic axonal terminals have not been found (see Chapter 1). Until recently, however, this viewpoint has been regarded with considerable skepticism, and many researchers have favored the alternative ‘‘sink’’ function of CSF transport (Robinson and Coombes, 1993; Sorensen, 1986). In 1986, Sorensen summarized this position by noting that ‘‘although there is some evidence to suggest that injection of AVP into the lateral ventricle of rats in doses approaching the physiological range might be behaviourally active (De Wied, 1976), the role of CSF as an AVP neurosecretory pathway has not been convincingly established. Alternatively, AVP in CSF may represent peptide released on various sites within the brain and transported via the extracellular ﬂuid to the CSF, which would be in accordance with a sink action of CSF to remove substances from the target sites . . . ’’ (Sorensen, 1986, p. 95). Moreover, the following two sets of observations failed to support a conduit role for CSF: (1) intracerebroventricularly injected material, including proteins and small peptides such as AVP, is rapidly distributed throughout the CSF compartment, but does not diffuse deeply into the brain parenchyma/brain interstitial ﬂuid. Thus at a 1-mm distance from the ventricle, the concentration of injectate within the brain is often only a few

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percentage of that within the CSF (Maness et al., 1996, 1998; Rees et al., 1980); and (2) diffusion within brain tissue and from brain tissue to CSF is slow (Cserr, 1984; Cserr and Berman, 1978), and a substance injected directly into brain tissue stays mostly where it is injected. Hence, Dluzen et al. (1998a) observed that 125I-labeled AVP infused into the olfactory bulb remained primarily localized within this brain structure, with no radioactivity present in the CSF or septal area 15 min after the infusion. More recently, a number of researchers have expressed renewed interest in the possibility that CSF might provide a humoral pathway, deep within the ventricular system of the brain, by which information-bearing molecules may be transported from sites of central origin to sites of neuronal processing: Johanson (1998) noted that the arachnoid and choroidal epithelia (the blood–CSF barrier structures that form CSF) secrete trophic factors and information-signaling proteins into the CSF, which could distribute them via bulk ﬂow (pressure-driven ﬂuid transport, hence faster than diffusion) to neuronal targets. By way of example, Johanson cited references indicating that retinoic acid (growth factor), which is secreted by the fourth ventricle choroidal plexus, promotes neurite growth and development of the cerebellum. Although not speciﬁcally stated, it is presumed here that Johanson (1998) postulates that this growth factor reaches the cerebellum via CSF-mediated volume transmission. Yu et al. (1996) found that high-frequency stimulation of the hypothalamic PVN inhibited the spontaneous ﬁring rate of mitral cells and increased that of granule cells in the medial olfactory bulb (MOB) of ovariectomized female rats. These responses were mimicked by intracerebroventricular infusion or microiontophoretic application of OT, blocked by infusion of an OT antagonist into the MOB, and not affected by intracerebroventricularly infused VP. Moreover, neither infusions of a local anesthetic into the medial olfactory tract or the medial forebrain bundle, nor unilateral transections at various levels of the olfactory pathway between the bulb and PVN, inﬂuenced these responses. These results suggested that OT originating in the PVN is transported via the CSF to the MOB, where it acts to decrease olfactory processing. Sewards and Sewards (2003) developed a theory in which CSF VP plays a dominant role in the mediation of power dominance drive motivation. They postulate that, in the presence of an external drive-generating stimulus, two pathways are simultaneously activated and operate in parallel: (1) a neuronal signaling pathway that proceeds from sensory ! mnemonic ! central drive representations located in the hypothalamus, periaqueductal gray, midline thalamus, and mesial cortex; and (2) a neurohumoral signaling pathway in which CSF VP functions as the information-bearing humoral signal molecule. Speciﬁcally, stimulus activation of a hypothalamic nucleus that lies adjacent to the third ventricle leads to the release of VP into the

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ECF. The ECF transports the peptide to the ventricular CSF, which delivers it to specialized neuronal receptors located in an adjacent CVO (OVLT or SFO). These receptors transduce the volume-mediated peptide signals into neural signals, which are conveyed along neuronal pathways to the brain structures containing the central drive representations. The activated drive representation mediates the subjective experience (e.g., anger) and activates the motor circuitry that leads to the motivated behavior (e.g., attack behavior). Both the neuronal signaling pathway and the neurohumoral drivegenerating system activate the drive representation. The former is activated only in the presence of the external stimulus; the latter yields a humoral signal that outlasts the presence of the initiating stimulus, which may be necessary for completion of the consummatory response and collection of the reward.

IV. Means by Which Peripherally Injected VP and OT Might Induce Behavioral Effects

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The inﬂuence of peripherally injected VP and/or OT on retention behavior in a variety of learning and memory tasks has been well documented in this text. At issue is the means by which the peripherally circulating peptide brings this about. The various explanations can be placed in one of two major categories depending on whether or not it is proposed that the centrally induced effect involves entry of the peptide into the brain.

A. Means by Which Peripherally Injected Hormones Could Enter the Brain The view that peripherally administered VP and OT, or their active metabolites, can enter the brain in sufﬁcient quantity to inﬂuence memory processing is the cornerstone of the De Wied et al. ‘‘VP/OT Central Memory Theory’’ (see Chapters 2–5). However, how could VP or OT penetrate the brain–ﬂuid barriers given the various barrier properties discussed in previous sections of this chapter? The following avenues of entry are possible. First, the hypertensive effect induced by a supraphysiological dose of VP might result in a transient opening of the BBB, thereby permitting blood-to-brain entry of the injected peptide. In his chapter on the topic of hypertension and the blood–brain barrier, Johansson (1989) has cited a number of references that demonstrated multifocal opening of the BBB to albumin in laboratory animals whose arterial blood pressure was increased above 60 mmHg after treatment with norepinephrine, epinephrine, and angiotensin. Studies providing evidence relevant to this point (e.g., Deyo et al., 1986; Mens et al., 1983) are presented in a later section of this Chapter (Section V.A.1).

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Second, VP could enter the brain by saturated carrier-mediated transport across the brain–ﬂuid barriers. Studies by Zlokovic and colleagues, relevant to this mode of entry to the brain, are presented in Section V.A.2. Third, the peptides could enter the brain as a result of leakage to the CSF from nonfenestrated blood vessels in the brain, such as those present in the CVOs, the subarachnoid space (SAS), and in Virchow–Robin spaces (spaces between the pia-arachnoid membrane coverings and the non-barrierprotected arterioles that plunge into the brain tissue and merge with the barrier-protected brain capillaries). Taken together, these vessels comprise the major components of the ‘‘extracellular pathways that circumvent the brain–ﬂuid barriers’’ proposed by Broadwell and colleagues (see Broadwell, 1992, 1993; Broadwell and Sofroniew, 1993). Broadwell and Sofroniew (1993) found that the intravenously injected protein tracer horseradish peroxidase (HRP) labeled SFO and OVLT parenchyma, CP capillaries, and adjacent ependymal lining of the lateral ventricles, and a small amount of white matter comprising the overlying corpus callosum. In addition, the tracer probe labeled pia mater lying on the surface of the brain (ﬂoor of the SAS) and enclosing the Virchow–Robin spaces, as well as the adjacent phagocytic cells that presumably endocytosed these molecules and effected their enzymatic degradation. Taken together, the labeling was interpreted as indicative of entry into the CSF via leakages from both sets of extracellular pathways proposed to circumvent the brain–ﬂuid barriers. Although these data do demonstrate that blood-borne macromolecules as large as the protein HRP can leak from these blood vessels, the limited extent of diffusion by the probe from the sites of this leakage, together with the abundant evidence that the leaked HRP was endocytosed by adjacent phagocytic cells (the auxiliary line of barrier defense; see Section II.C.3), do not challenge the observations of Krisch and Leonhardt (1989) and Maness et al. (1998) (Section II.C.3). In this author’s opinion, it is therefore doubtful that the limited degree of such leakage is sufﬁcient to account for the central behavioral effects observed after peripheral injection of VP or OT. A ﬁnal mechanism by which blood-borne VP and/or OT could enter the brain is via transcellular passage across the capillary endothelial and choroidal epithelial cells comprising the BBB and blood–CSF barriers, respectively. Known as ‘‘transcytosis’’ (transendothelial vesicular transport), the mechanism derives from the ability of all cells to recycle old and damaged cell membranes by means of endocytosis (cellular internalization), intracellular vesicle-mediated transport, processing effected within endomembrane organelles (i.e., enzymatic degradation in secondary lysosomes and membrane repair and vesicle formation carried out in the Golgi body), and insertion of this new membrane via exocytosis. During the process of membrane endocytosis, macromolecules may be included with the endocytosed membrane either because they (1) are simply in the immediate vicinity (i.e., nonspeciﬁc bulk or ﬂuid-phase endocytosis); (2) bind to the negative charge,

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or carbohydrate-coated surface (glycocalyx) that characterizes the cell membrane (adsorptive endocytosis); or (3) bind to speciﬁc membrane receptors (receptor-mediated endocytosis). Once internalized, the macromolecules may be routed along any of a number of intracellular pathways depicted in Fig. 7.

FIGURE 7 Potential transcellular transfer of adsorptive/receptor-mediated ﬂuid phase macromolecules through endothelia (A) of the BBB and choroid plexus epithelia (B) of the blood–CSF barrier may involve direct vesicular transport (1) or indirect vesicular transport through the endosome compartment (E; 2) and the Golgi complex (G; 3). These macromolecules and those associated with ﬂuid phase endocytosis (4) also are channeled to secondary lysosomes (L). Macromolecules entering BBB endothelia do so predominantly from the blood and less so, if at all, from the brain side; hence, transcytosis through the BBB is vectorial from blood to brain. Choroid epithelia engage in endocytosis circumferentially, suggesting potential transcytosis of macromolecules through this cell type may be bidirectional. ER, endoplasmic reticulum; N, nucleus; T, tubules; TJ, tight junctional complex. Source: Broadwell, 1993 (Fig. 1, p. 139). Copyright ß 1993 by Plenum Press. Reprinted with permission.

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Broadwell (personal communication, 2003) notes that ﬁndings from HRP-labeling studies strongly suggest that the membrane behavior of the BBB endothelium and that of the choroidal epithelium are distinctly different, a difference that could argue against transcytosis across the BBB. More speciﬁcally, membrane recycling is documented at both the blood- and CSFfacing sides of the choroidal epithelium, indicating the potential for engaging in bidirectional transcytosis between blood and CSF. Conversely, membrane recycling of the BBB endothelium is demonstrable at the luminal, but not at the abluminal, surface of the BBB membrane, indicating a failure to fulﬁll the requirement necessary to complete the process of transcytosis from blood to brain (for further discussion see Broadwell, 1992, 1993; Broadwell and Banks, 1993). The absence of demonstrable recycling of the abluminal plasmalemma is an enigma for the transendothelial transfer of blood-borne macromolecules through the BBB endothelium. This issue is complicated all the more in view of the fact that Broadwell et al. (1988, 1996) have reported successful adsorptive and receptor-mediated transcytoses of speciﬁc blood-borne macromolecules through the BBB endothelium. Similar transfer from brain to blood was not documented. The presence of V1 vasopressin receptors on the luminal side of the capillary membrane (Ermisch et al., 1993) is consistent with VP cell entry via receptor-mediated endocytosis. Should this possibility be supported only future research will determine whether this represents a ﬁrst step by which VP interacts with the cell membrane to inﬂuence nutrient transport (see discussion and studies reviewed in Section VI) and/or a transcytosis-mediated entry of the peptide to the brain.

B. Means by Which VP and OT Could Induce Behavioral Effects Other than by Entering the Brain A number of possible mechanisms could explain a behavioral effect of peripherally administered VP and/or OT without their entrance to the brain (Bradbury, 1989). First, the hormone may produce peripheral effects that in turn bring about the behavioral effect. An example of this is seen in the Koob et al. ‘‘VP Dual Action Theory,’’ which proposes that the pressor/aversive actions of vasopressin bring about an increase in activity in the arousal system that accounts for its effect on memory performance. This view and relevant research are described in Chapter 6. Second, the peptide may interact with capillary endothelial transport systems to increase blood-to-brain entry of nutrients (e.g., certain amino acids, orotic acid) that are effective precursors in the synthesis of neurotransmitters or nucleic acids that assist memory consolidation and/or retrieval. This suggestion is central to the theory held by Ermisch, Landgraf,

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and colleagues explaining the means by which hormonal AVP may inﬂuence central sites involved in memory processing. This theoretical view and the associated research are described in this chapter (Section VI). Third, the hormone may activate receptors of peripheral afferent neurons that enter the CNS. This may be an important avenue by which cholecystokinin induces appetite satiety and thereby regulates eating behavior (Murphy et al., 1988), and by which stress hormone epinephrine modulates memory (McGaugh, 2000), but it does not appear to have been offered yet as a mechanism by which VP or OT may inﬂuence memory processing. Fourth, the hormone may act on receptors in one of the circumventricular organs and thereby bring about the behavioral effect. One example of a CVO role in body–brain integration is the homeostatic regulation of body ﬂuid balance. Water deﬁciency in the body brings about an increase in the level of circulating Ang II, which in turn signals receptors in the SFO, OVLT, and AP. Neural transduction at these receptor sites transmits neural impulses to brain circuitries that mediate corrective actions that restore body ﬂuid levels (including VP secretion resulting in body ﬂuid conservation, thirst-induced drinking, and increased sodium appetite) resulting in ﬂuid gain (Ermisch et al., 1993). Receptors in the AP also detect changes in levels of circulating Ang II and AVP. The resulting receptorinduced neural transduction sends neural impulses to vasomotor medullary reﬂex centers and results in baroreceptor actions that normalize blood pressure. Thus, circulating AVP acts to sensitize the baroreceptor response, thereby increasing blood pressure in response to decreased arterial pressure, while circulating Ang II acts to dampen the baroreﬂex response to increased arterial pressure (Ermisch et al., 1993). This avenue of neuroendocrine integration has been systematically studied in relation to reﬂexive corrective homeostasis of the internal environment (e.g., ﬂuid dynamics, blood pressure regulation), but has not yet been suggested as an avenue inﬂuencing memory processing. Fifth, the hormone may interact with brain blood vessels to inﬂuence blood ﬂow to brain regions mediating the observed behavioral effect. Thus, plasma VP might inﬂuence memory processing as a result of its inﬂuence on cerebrovascular circulation. There is considerable evidence that vasopressin can affect vascular dynamics in the brain as it does in the periphery (see Chapter 1). The remainder of this chapter considers research relevant to (1) the likelihood that AVP penetrates the BBB and enters the brain parenchyma to produce a central effect, and (2) the possibility that AVP interacts with the BBB to inﬂuence its permeability to the passage of substances (e.g., amino acids) into the brain, some of which may contribute to central memory processing.

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V. Research Relevant to the View That Peripherally Circulating AVP Can Penetrate Brain–Fluid Barriers

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A. Penetration of Blood–Fluid Barriers: Blood-to-Brain Transport Two types of research approaches are discussed in this section—the ﬁrst approach (type I) has been concerned mainly with evidence of whether or not peptides such as VP can penetrate the BBB and enter the brain, without making assumptions as to how this may occur. The second approach (type II) has focused on the possibility that behaviorally active peptides may interact with the brain–ﬂuid barriers and enter the brain by some type of transport carrier—that is, by interacting with either an amino acid carrier or even a carrier mechanism selective for the neuropeptide itself. The type II approach uses computational models to calculate the rate of transfer of the peptide into the brain, comparing it with that of substances to which the brain–ﬂuid barriers are either highly permeable or impermeable. 1. Type I Research Approach: Methods and Findings Both correlational and experimental studies have investigated the ability of peripherally circulating VP and OT to enter the brain tissue. The correlational work reviewed previously (Section III.A) could be interpreted as support for the view that neither plasma VP nor OT enters the brain; however, correlational data alone cannot provide a decisive answer. The strict regulation of the brain microenvironment induced by the BBB can account for the absence of a high correlation between the two ﬂuid compartments even when it is known that the substance crosses the BBB. For example, central and peripheral levels of potassium are not positively correlated even though this nutrient, needed by the brain as well as other body tissues, can be obtained only by way of peripheral ingestion (see Banks and Kastin, 1988, for further discussion of this point). Conversely, a high correlation between the two ﬂuid compartments in VP and/or OT could result from the simultaneous central and peripheral release of the circulating peptides. a. Relevant Studies The studies reviewed below were primarily concerned with determining whether or not the peptide penetrated the BBB and entered the brain. Veriﬁcation of this penetration was initially assessed by bioassay and subsequently by radioactive labeling and radioimmunoassay. i. Heller et al. (1968) In the bioassay method, CSF is collected from a species (e.g., rabbit) that has been peripherally injected with VP (or OT), and this CSF is then injected intracerebroventricularly into a second species (e.g., rat) to determine its ability to affect a function normally inﬂuenced by

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a peripheral or central injection of the peptide (e.g., antidiuretic activity in the case of VP). Heller et al. (1968) bioassayed antidiuretic activity in rats receiving an intracerebroventricular injection of CSF withdrawn from the cisterna magna of rabbits under the following conditions (depending on the experiment): (1) pentobarbital-induced general anesthesia, (2) procaine-induced local anesthesia, (3) pentobarbital-anesthetized rabbits subjected to vagal nerve stimulation, and (4) pentobarbital-anesthetized rabbits sampled for CSF and for blood (collected from jugular vein) before or 1, 2, 3, 5, or 6 min after an intravenous injection of AVP (50 mU of AVP/kg, intravenous). An antidiuretic (AD) effect in the rats was assayed by measuring inhibition of experimentally induced water diuresis. In one experiment, an effect on blood pressure was assessed for CSF drawn from generally anesthetized rabbits. The results indicated (1) an AD effect in rats that received CSF from rabbits subjected to general but not local anesthesia, vagal stimulation, and peripherally injected AVP, and (2) the AD effect was more effective in rats that received blood rather than CSF from rabbits peripherally injected with AVP; that is, AD activity in the CSF increased quickly with the rise in blood levels of AVP (within 2 min of injection), but the VP levels attained in CSF were always signiﬁcantly lower than those in blood when sampled at times the plasma VP concentration was still above normal. The authors concluded that under normal resting conditions (local anesthesia) VP levels in CSF are too low to induce an AD effect and that certain stimuli (barbiturates, vagal stimulation) increase VP levels in the CSF. The source of this AVP could be central or peripheral. The authors cited anatomical observations of terminals of neurohypophysial neurosecretory ﬁbers in the walls and ﬂoor of the third ventricle (e.g., Rodriguez, 1974), suggesting the possibility that these processes directly secrete VP into the CSF. However, the results of their experiments with peripherally injected AVP suggested that the peptide penetrated the CSF within 2 min of injection, that is, the blood–CSF barrier is permeable to AVP. It was acknowledged that a rise in systemic blood pressure caused by the large doses of VP used in this experiment may have altered the permeability of the barrier and that AVP at blood concentrations that do not produce marked vascular effects cannot penetrate into the CSF. Moreover, because the VP level in CSF never reached that in blood, it indicated that only a fraction of AVP (portion not bound to plasma protein) reached the CSF and/or that the peptide was quickly removed from the CSF. Despite these qualiﬁcations a peripheral source for CSF AVP was suggested in addition to the central source. Vorherr et al. (1968) failed to ﬁnd evidence of VP penetration of the brain–ﬂuid barriers with this method of assessment. However, this study has been criticized on the basis that the bioassay was insufﬁciently sensitive (Ang and Jenkins, 1982). Several years later radioactive labeling of neuropeptides was used to study their ability to penetrate membrane barriers separating plasma from

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brain. Zaidi and Heller (1974) sampled radioactivity in plasma and CSF after intravenous injection of anesthetized rabbits with both tritium-labeled OT and lysine vasopressin (LVP). Because these authors were interested in whether circulating AVP and OT normally contribute to CSF levels of these peptides they employed VP doses that would not increase blood pressure (BP) enough to alter permeability of the blood–CSF barrier. Little to no radioactivity was observed in the CSF, and it was concluded that neither peptide penetrated the blood–CSF barrier under the conditions of this experiment. ii. Ang and Jenkins (1982) Ang and Jenkins (1982) found evidence suggesting that much of the VP that purportedly penetrates the BBB does so in metabolically altered form. These authors peripherally administered, either intranasally or intravenously, radiolabeled AVP, 1-desamino-8-d-arginine vasopressin (DDAVP), or desglycinamide-arginine vasopressin (DGAVP) to dogs. Intravenous administration was either by a single injection or by a 60-min infusion. CSF from the cisterna magna (subarachnoid space near the fourth ventricle) and blood from the jugular vein were simultaneously collected at speciﬁed time intervals after administration of the peptide. The samples were ﬁrst analyzed for total radioactivity and then assessed, by thin-layer chromatography, for the amount of radioactivity that was associated with metabolized fragments of the peptide versus the intact peptide. For the single intravenous injection, the percentage of radioactivity in CSF relative to that in plasma was 1% for AVP, 0.5% for DDAVP, and 1.4% for DG-AVP. Thin-layer chromatography revealed that all the radioactivity of AVP and DG-VP was associated with metabolically altered peptide, whereas 86% of the total radioactivity for DDAVP was from metabolically altered peptide. Similar results were obtained for the intranasal injection. Examination of the rate of metabolism of these peptides within the plasma revealed a biphasic curve for each. For the later phase, the half-life for AVP was 13 min, for DG-AVP it was 8 min, and for DDAVP it was 50 min. These differences in metabolic breakdown may explain why some of the DDAVP (i.e., 14%) occurred in unaltered form when CSF was sampled 20–50 min after injection. These results indicate the importance of using chromatographic analysis to determine how much of the intact peptide, if any, was taken up by the brain under the conditions of the study. Two studies, cited below, utilized radioimmunoassay assessment to determine BBB or blood–CSF penetration of AVP and/or OT. Each of these studies was speciﬁcally designed to address the question of whether a single subcutaneous injection of AVP and/or OT might penetrate these barriers in amounts sufﬁcient to produce the behavioral effects so frequently reported by De Wied et al. (see Chapters 2–5) and Koob et al. (see Chapter 6).

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iii. Mens et al. (1983) Mens et al. (1983) measured immunoreactive (ir) AVP and OT concentration and time course in both plasma and CSF after administration of a given dose of subcutaneously injected AVP or OT (0.5, 2.0, or 5.0 g/rat), intravenously injected OT (5.0 g/rat), or intracerebroventricularly injected AVP (2.5 ng/rat) and OT (5.0 ng/rat). Three experiments were conducted and independent treatment groups were deﬁned depending on the route of administration, the dose level, and the time interval between injection and data collection [CSF collected from the cisterna magna (CM) and plasma, from trunk blood]. In one test series of experiment 1, CSF was collected 10 or 30 min after a subcutaneous injection of a 0.5- or 2.0-g dose of AVP or OT, or 2, 5, 10, 30, and 60 min after a subcutaneous injection of a 5.0-g dose of AVP or OT. In a second test series, CSF was collected 2, 5, 10, 30, and 60 min after an intravenous injection of 5.0 g of OT. In experiment 2, CSF was collected 2, 5, 10, 30, 60, and 90 min after an intracerebroventricular injection of 2.5 ng of AVP, and 2, 10, 30, 60, 90, and 120 min after an intracerebroventricular injection of 5.0 ng of OT. In experiment 3, testing determined immunoreactivity levels of these peptides in the plasma in order to calculate the ratio of AVP and OT concentrations in plasma and CSF. For these tests, the animals were killed and trunk blood was collected 2, 5, 10, 30, and 60 min after a subcutaneous injection of a 5.0-g dose of AVP or OT. A cannulation technique permitted the collection of CSF from freely moving rats. The results were as follows: (1) a subcutaneous injection of 5.0 g of AVP and OT increased plasma levels of irVP and irOT, respectively, within 2 min, and maximal levels were reached by 5 min after injection; (2) a signiﬁcant increase in the CSF level of AVP occurred 2 min after subcutaneous injection of the 5.0-g dose level, peaked within 5 min, and declined slowly over 60 min; no effect occurred after the 0.5-g dose level, but a signiﬁcant increase in CSF level of the peptide occurred after 10 min for the 2.0-g dose level; (3) the CSF level of OT signiﬁcantly increased within 10 min for the 5.0-g dose and only at 10 min for the 2.0-g dose whereas no effect occurred after the 0.5-g dose level. CSF OT levels also declined to nonsigniﬁcant values 60 min after the injection; (4) because of the slower decrease in peptide concentrations in CSF as compared with those in plasma, the CSF-to-plasma ratio increased with time, but at all time periods the ratio remained considerably below unity, indicating a lower peptide level in the CSF than in the plasma; (5) only a small amount [approximately 0.001% (AVP) to 0.002 to 0.003% (OT)] of the peripherally administered dose of these peptides reached the CSF within 2 min (AVP) or 10 min (OT) of injection; (6) intravenous administration of OT (5.0-g dose) signiﬁcantly increased the OT level in CSF, with a similar time course (peak level and decline) as that obtained for subcutaneous administration; and (7) intracerebroventricularly injected AVP (2.5 ng) and OT (5.0 ng) reached the cisternal cavity within 2 min of injection; both peptides were cleared from

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the CSF with terminal half-times of 26 min (AVP) and 19 (OT) min. The authors concluded that at a sufﬁciently high dose level (i.e., 2.0 and 5.0 g/animal) both VP and OT cross the BBB, albeit in small amounts (e.g., 0.001 or 0.002–0.003% of the original dose level). Nevertheless, these amounts, which are in the nanogram or picogram range, modulate memory when administered intracerebroventricularly (e.g., Bohus et al., 1978b; De Wied, 1976; see Chapters 2–5). Moreover, the resulting CSF levels remained elevated for a time period sufﬁcient to mediate central behavioral effects. iv. Deyo et al. (1986) The study by Deyo et al. (1986) is presented in Chapter 6. Brieﬂy, it was designed to determine whether peripherally injected AVP (0.05, 0.5, or 5.0 g/kg, subcutaneous) penetrated brain tissue in selected areas protected by the brain–ﬂuid barriers (thalamus–hypothalamus, amygdala–temporal cortex, hippocampus, and remaining cerebral cortex) and lying outside the blood–brain barrier (i.e., two CVO sites: median eminence and area postrema). After treatment, the rats were decapitated; trunk blood was collected and selected brain regions were removed and assessed for irVP concentration. Similar assessments were made in noninjected rats after 24 or 48 h of water deprivation. In those rats receiving the 5.0-g/kg dose of AVP, brain perfusion experiments were carried out as a control procedure to check for possible irAVP accumulations in the brain microvasculature itself. The peripherally injected peptide dose dependently increased plasma AVP, which subsequently exhibited a biphasic decline with a half-time of 25 min for the initial phase and 60 min for the subsequent phase. It also dose dependently increased irAVP levels in all brain areas tested, reaching peak levels 15 min after those in the peripheral circulation. The brain regional concentrations of irAVP were signiﬁcantly increased (three to nine times greater) over control tissue levels. Water deprivation for 48 h signiﬁcantly increased plasma AVP relative to control values, although this increase was much lower than that after peripherally injected AVP. Water deprivation for both 24 and 48 h signiﬁcantly increased AVP concentration in the hypothalamus and in the amygdala–temporal areas. Of major importance was the result of the perfusion experiments, which indicated that once the AVP trapped in the brain microvasculature was removed by this procedure, irAVP was reduced to control levels in all the brain regions protected by the BBB. It was concluded that (1) none of the irAVP that reached the blood vessels in the brain-protected areas appeared to have spread to any tissue ﬂuid in the brain, and (2) the elevated AVP in brain regions after water deprivation undoubtedly reﬂected changes of activity in VP-ergic neurons projecting to the affected brain regions. Finally, the authors noted that their ﬁndings were directly opposed to those reported by Mens et al. (1983) and

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that the larger doses of AVP used by these researchers could have produced hypertensive effects that affected the permeability of the brain–ﬂuid barriers. b. Evaluation and Commentary: Type I Approach The studies described above were equivocal concerning the question of whether peripherally administered VP can penetrate the brain–ﬂuid barriers. Three of the six studies cited reported ﬁndings supporting a VP penetration of these barriers. However, this must be interpreted with caution given certain difﬁculties that face investigation of neuropeptide penetration of the BBB (Sorenson, 1986). One problem is that injection of an extremely high, unphysiological dose of AVP may raise blood pressure enough to interfere with the normal functioning of the BBB [e.g., the studies by Heller et al. (1968) and Mens et al. (1983)]. Mens et al. (1983) administered AVP at up to 5 g/rat, which resulted in a 10,000-fold elevation of plasma AVP concentration and a 3-fold increase in CSF AVP (Sorenson, 1986). A second problem is in determining the amount of intact neuropeptide, versus a metabolic fragment, that penetrated the BBB and entered the brain. Hoffman et al. (1977) report that neuropeptide hormones, including AVP and OT, are subject to relatively rapid metabolic breakdown in the body periphery, with apparent half-lives ranging in minutes. This makes it desirable for studies investigating potential uptake by the brain to determine the percentage of the injected intact peptide that actually penetrated the brain, and often this determination is not made. Thus, Zaidi and Heller (1974) found small amounts of radioactivity in CSF after intravenous injection of radiolabeled LVP but no measure was taken (available at the time) to determine whether the radioactivity represented the intact peptide. On the other hand, such determinations were made in the study by Ang and Jenkins (1982), who noted that, with the exception of DDAVP, there was no evidence of metabolically unaltered vasopressin in the CSF after peripheral administration of either AVP or DG-AVP. Another problem confronting investigation of neuropeptide penetration of the BBB is the necessity to ensure that the neuropeptide has entered the brain tissue and has not become lodged within the blood of the capillary or bound to some portion of the BBB, such as the surface of the vascular endothelium or the inside of the endothelial cell. By including the vascular perfusion step, Deyo et al. (1986) demonstrated that the neuropeptide taken up by the brain tissue was lodged within the microvasculature of the brain and had not actually entered the brain tissue proper. In addition to the problems pointed out by Sorensen (1986), it would seem advisable that studies on VP and OT penetration of the brain–ﬂuid barriers as a basis of their inﬂuence in memory functions inject VP and OT in a dose range actually employed in memory-processing research. The study by Deyo et al. (1986) more closely approximated this range than that by Mens et al. (1983).

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2. Type II Research Approach The two research methods described below are concerned with identifying and characterizing the transport kinetics of mechanisms (e.g., transport carriers) responsible for uptake by and transport across the blood–brain or blood–CSF barriers in the direction of blood to brain. These methods have been used in vivo experiments with small animals (e.g., mice, rats, guinea pigs). The Oldendorf single-carotid injection method [also known as the ‘‘brain uptake index (BUI) technique’’] provides only a brief exposure of the radiolabeled tested substances to the BBB. As a result it is most suitable for study of substances (a variety of nutrients essential in brain metabolism) that are rapidly transported across the BBB. This method is described and discussed in Smith (1989), Segal and Zlokovic (1990), and Pardridge (1991). The vascular brain perfusion (VBP) method (also known as the continuous infusion technique) presents the radiolabeled test substance to the BBB over a period of minutes (10 to 20 min), and accordingly is suitable for study of the transport kinetics of substances (e.g., peptides and proteins) that slowly penetrate BBB. This method has been described by Segal and Zlokovic (1990). a. Oldendorf Single-Pass Technique: Methodology and Results In Oldendorf’s method, a test and a reference substance (known to readily penetrate the BBB or to not do so) are labeled with different radioactive substances and added to the solution (a 1.2-ml volume of buffered Ringer’s solution, serum, or some other vehicle) that is rapidly injected as a single bolus into the carotid artery of the tested animal. Within 5–15 s of injection the test animal is decapitated and the brain is removed, and the homogenized brain tissue is analyzed for uptake of these tracers. Because of its limited time in the cerebral circulation (15 s) there is little likelihood of metabolic degradation of the injected material and the amount of recovered radioactivity is assumed to reﬂect metabolically unaltered material. Study with this technique has indicated saturable (system is saturated when all receptors are occupied by tracer) transport systems for a variety of substances, including essential nutrients such as glucose, amino acids, and monocarboxylic acids. The basic features of this technique are diagrammed in Fig. 8. i. Landgraf et al. (1979) Landgraf et al. (1979) used the single-pass technique to assess uptake of plasma VP and OT relative to highly diffusible water and the nondiffusible substance, inulin. A solution of 125I-labeled LVP, [3H]OT, tritiated water, or [3H]inulin was injected into the right common carotid artery of an ether-anesthetized rat. After decapitation 15 s later the brain hemisphere ipsilateral to the injection was removed and dissected into 19 regions, 4 of which were outside the blood–brain barrier (i.e., the choroid plexus IV, the pineal body, and the anterior and posterior lobes of the pituitary gland). Scintillation counting permitted estimation of the

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FIGURE 8 Diagram of the brain uptake index technique. A buffered solution containing a [14C]-labeled test solute and 3H2O is injected as a bolus into the carotid artery of a rat, and then the rat is decapitated 15 sec later. Source: Smith, 1989 (Fig. 13, p. 110). Copyright ß 1989 by Plenum Publishing Corporation. Reprinted with permission.

percentage of radioactivity for each of the radiolabeled tracers recovered in each of the 19 brain regions (see Fig. 9). The results and their interpretations were as follows: (1) the concentrations of the injected peptides were supraphysiological because the resulting plasma levels were much higher than baseline values; (2) the amount of radioactivity was signiﬁcantly higher after injection of either labeled LVP or OT in comparison with [3H]inulin, indicating that the blood–brain barrier was more permeable to these tracers than to inulin; and (3) the exact localization of the labeled tracers in the brain was not ascertained. As a result, the brain uptake values do not necessarily mean that a tracer entered the brain tissue because it could have remained partially or wholly within barrier structures (i.e., in the luminal or abluminal membrane or within the cytoplasm of the endothelial cell). It was concluded that although there was evidence of some uptake of VP and OT at the BBB, it was not clear that these substances entered the brain tissue. In addition, it has been noted that the BUI is not an extremely sensitive method, but requires that a material be extracted at a high rate. For example, it can easily detect heroin uptake into brain but is too insensitive to show uptake of morphine (Oldendorf, 1974).

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FIGURE 9 Regional distribution of radioactivity after intracarotid injection of 3HOH, 125 I-labeled LVP, [3H]oxytocin, or [3H]inulin. Source: Landgraf et al., 1979 (Fig. 1, p. 79). Copyright ß 1979 by J. A. Barth, Leipzig. Reproduced with permission.

ii. Ermisch et al. (1982) Ermisch et al. (1982) cited a number of published and unpublished experiments from their laboratory that assessed radioactivity in brain tissue after an intracarotid injection of iodinated LVP or tritiated OT. These experiments indicated that the peptide either failed to penetrate the BBB (Hoffman, unpublished data) or, in those cases in which uptake did occur owing to injections of high concentrations of the peptide (Landgraf, unpublished results; Ruhle and Ermisch, 1978), it was not clear whether the radioactivity had remained in the barrier structures without entering the adjacent brain tissue. b. Vascular Brain Perfusion Technique: Methodology and Results The in situ VBP technique has been reﬁned by Takasato et al. (1984) for use with the rat (Fig. 10), and by Zlokovic and colleagues (1990, 1991, 1992) for use with guinea pigs in the study of the kinetics of BBB uptake of circulating peptides and proteins. In each case an artiﬁcial medium is used to perfuse the cerebral vasculature in the ipsilateral brain hemisphere. Extreme care is taken during the perfusion to prevent signiﬁcant mixing of perfusion medium with the

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FIGURE 10 Diagram of the technique used to perfuse the right cerebral hemisphere of a rat brain. The perfusion ﬂuid (whole blood, artiﬁcial blood, or HCO3) contains the 14C- and 3Hlabeled tracers, and is pumped into the ligated right external carotid artery at a constant rate. Key: ACA, anterior cerebral artery; MCA, middle cerebral artery; PCA, posterior cerebral artery. Source: Takasato et al., 1984 (Fig. 1, p. H485). Copyrighted ß 1984 by the American Physiological Society. Figure reprinted with permission. Legend adapted with permission.

animal’s own blood. The radiolabeled test drug may be injected directly into the perfusate. The composition of the perfusate can be precisely manipulated, thereby permitting adding and testing substances thought to inﬂuence the kinetics of BBB transport of the tested substance (e.g., competitive and noncompetitive inhibitors to transport of the tested substance). In the VBP technique used by Zlokovic and colleagues, the perfusion medium, containing the differentially labeled test substance and an impermeant reference agent (e.g., mannitol), is infused via the cannulated right carotid artery into the right cerebral hemisphere of the anesthetized guinea pig. The other arterial branches to this hemisphere are tied off. The special anatomy of the guinea pig’s cerebral vascular system precludes mixing of blood from arterial branches serving the right and left hemispheres. Immediately after the start of perfusion, the left carotid artery is tied off and both jugular veins are cut to allow free drainage of the perfusate. During perfusion, the animal maintains spontaneous breathing, normal heart rate, arterial blood pressure, acid–base status, electroencephalographic (EEG) activity, and cerebral ﬂow rate in the perfused hemisphere. Moreover, biochemical status in the perfused hemisphere exhibits normal water content, Naþ:Kþ ratio, ATP and lactate levels, and energy charge potential. Ultrastructurally, the brain tissue and capillary wall remain unchanged (Zlokovic et al., 1993).

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The perfusion is terminated by decapitation, after which samples from various brain regions are analyzed for radiotracer content. i. Zlokovic et al. (1990) Zlokovic et al. (1990) studied the kinetics of AVP uptake at the luminal side of the BBB with the VBP technique. The perfusion medium was delivered to the brain by means of a peristaltic pump at a perfusion pressure slightly above the guinea pig’s own BP. In addition to the test tracer, [3H]AVP, the perfusion medium contained one of the following test agents: (1) unlabeled VP (ranging from 0.003 to 10 M) to test for saturable carrier transport because competition for carrier receptors on the luminal side of the capillary would limit the transport of the labeled AVP (i.e., produce self-inhibition); (2) one of various peptide fragments of VP [VP(1–8), pressinoic acid [VP(1–6)], and [pGlu4,Cyt6]VP(4–9)] or a potent aminopeptidase inhibitor (bestatin) to determine whether the intact tracer or a metabolic fragment is taken up by the BBB; or (3) a substrate for the large neutral amino acid transporter (LNAA carrier), for example, l-phenylalanine or l-tyrosine at concentrations of 4.5 M and 5.0 M, to test whether AVP uses the LNAA carrier for transport across the barrier from blood to brain. In a separate series of experiments, the V1 receptor antagonist (CH2)5[Tyr(Me)2]VP, at perfusate concentrations of 1.7 and 4.5 M, was tested for its effect on the kinetic entry of [3H]VP into the perfused contralateral forebrain. At given times ranging from 0.5 to 20 min the perfusion was terminated, the guinea pig was decapitated, and the brain was removed and dissected into several regions (parietal cortex, caudate nucleus, hippocampus) for scintillation counting. Correction for the residual vascular radioactivity of the tracer ([3H]VP) in single time uptake experiments was made by washing the intravascular tracer from the brain blood vessels (i.e., perfusing the brain for 1 min with tracer-free ﬂuid). Uptake of the intravascular brain marker d-[14C]mannitol served as a standard for comparison with BBB uptake of labeled VP. The authors reported the following major ﬁndings of this study together with their interpretations of them: (1) the BBB was more permeable to AVP than to mannitol, and competitive testing demonstrated that unlabeled AVP as well as the V1 antagonist strongly inhibited BBB uptake of labeled VP in each of the three brain areas analyzed. Thus a saturable uptake carrier for intact AVP is present at the luminal side of BBB and a V1 receptor is involved in this uptake; (2) kinetic analysis indicated that this VP uptake system at the luminal surface of the BBB is of low capacity. When one considers that normal plasma levels of VP are also quite low (less than 50 fmol in the guinea pig), it is quite possible that such a system is of physiological signiﬁcance for VP homeostasis in the brain; and (3) VP inﬂux was not signiﬁcantly inﬂuenced by the presence of any of the peptide fragments [AVP(1–8),

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AVP(1–6), or AVP(4–9)], the aminopeptidase inhibitor, or the LNAA substrates (l-phenylalanine or l-tyrosine at either concentration). These ﬁndings suggest that the VP transport carrier interacts with patent peptide, but not with its metabolic fragments, and that it is not the LNAA carrier. The authors concluded, however, that although the study supports the hypothesis that intact AVP is taken up at the luminal side of the BBB, it does not rule out the subsequent metabolism of the peptide in the endothelial cell cytoplasm, at the abluminal membrane surface of the cell, or in the immediate surround of the brain parenchyma (i.e., the VP-ergic synaptic region juxtaposed to the brain microvessels). ii. Zlokovic et al. (1992) Zlokovic et al. (1992) used this technique to further characterize VP transport and receptor and enzymatic events at the luminal side of the BBB. This study also employed the capillary depletion procedure to determine the degree to which circulating AVP remains sequestered by cerebral microvessels. [3H]AVP and [14C]sucrose were simultaneously introduced into the perfusion circuitry at a slow constant rate over a 10-min perfusion period. When the aminopeptidase inhibitor bestatin was used it was preinfused into the brain 3 min before perfusion of labeled AVP and then simultaneously perfused with AVP until the end of the experiment. Correction for residual vascular radioactivity was accomplished by washing the intravascular tracer from cerebral blood vessels (perfused the brain for 1 min with cold tracer-free buffer). This method corrects completely for residual blood [3H]AVP in brain perfusion experiments (Zlokovic et al., 1990). At the end of the perfusion, the animal was decapitated and the forebrain was removed and subjected to the capillary/depletion procedure. The degree to which intact peptide, as opposed to metabolic fragments, was transported from the blood into the brain tissue was then determined by high-performance liquid chromatographic (HPLC) analysis. The major results and their interpretations were as follows: (1) there was a time-dependent and progressive uptake of labeled AVP relative to labeled sucrose in the ipsilateral forebrain homogenate and postcapillary supernatant within the 10-min period of brain perfusion. Negligible amounts of both substances were found in the vascular pellet. This ﬁnding indicated signiﬁcant BBB permeability to AVP relative to the cerebrovascular space marker sucrose; (2) the capillary depletion technique revealed that only a barely detectable portion of AVP remained sequestered and/or bound to cerebral microvessels; (3) the aminopeptidase inhibitor did not alter the initial uptake of AVP during the 10-min period. In contrast to the brain, all the radioactivity in the arterial infusate and venous drainage was found in the form of intact AVP. The absence of changes in AVP BBB transport in the presence of the aminopeptidase inhibitor, together with the recovery of intact AVP from venous drainage, indicated that intact AVP was presented

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to the BBB. On the basis of these data, the authors concluded that enzymatic cleavage does not take place at the luminal surface of the BBB, a conclusion supported by previous ﬁndings (Zlokovic et al., 1990) that demonstrated the ineffectiveness of this inhibitor to affect brain uptake of plasma AVP; and (4) the HPLC analysis indicated a progressive metabolic degradation of intact AVP (i.e., about half the AVP taken up by the brain is degraded within the ﬁrst minute of brain perfusion) with [3H]phenylalanine as the major detectable labeled metabolite. The authors surmised that the number of small peaks of radioactivity, which differed from either the intact molecule or phenylalanine alone, represented AVP metabolites that contained the phenylalanine residue. Comparisons with previously published AVP brain chromatograms (Burbach and Lebouille 1983; Burbach et al., 1983a; Stark et al., 1989) suggested the presence of [Cyt6]AVP(3–9), AVP(3–9), and AVP(2–9) fragments in this study. Thus, the transport of intact AVP into the brain is followed by its rapid metabolism, possibly in the brain parenchyma. The authors concluded that the present data in combination with those obtained by Zlokovic et al. (1990) demonstrate saturable BBB uptake of AVP and suggest that the AVP may be transported across the endothelium by a carrier similar to one previously reported for other small peptides (e.g., Banks and Kastin, 1990) and several large peptides and proteins including insulin (Pardridge et al., 1985), transferrin (Pardridge et al., 1987), and insulin-like growth factors (Frank et al., 1986). c. Evaluation and Commentary: Type II Approach The suitability of the single-pass method to study peptide transport across the brain–ﬂuid barriers and uptake by the brain has been challenged. The short exposure period in this procedure, although quite adequate for characterizing transport parameters for rapidly penetrating substances (e.g., glucose, amino acids, and monocarboxylic acids), is not so for the more slowly penetrating peptides or plasma proteins (Pardridge, 1991; Segal and Zlokovic, 1990). On the other hand, the longer tracer exposure time periods provided by the VBP method are fully sufﬁcient for the study of neuropeptide transport (Zlokovic, 1990). The published and unpublished experiments by Landgraf, Ermisch, and colleagues (Ermisch et al., 1982; Landgraf et al., 1979), which used the single-pass technique to study VP and OT penetration of the BBB, indicated a small but signiﬁcant degree of peptide uptake by the BBB relative to impermeant tracers. However, because of the absence of a capillary depletion test procedure in these experiments, it was not clear whether the peptide taken up by the barrier actually entered the interstitial ﬂuid or remained within the barrier structures. Zlokovic et al. (1990, 1992), using the VBP method, provided evidence that intact AVP is presented to the brain–ﬂuid barriers, interacts with a selective transporter other than the one for the large neutral amino acids, demonstrates no signiﬁcant capillary sequestration or degradation by barrier

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mechanisms, and is subject to rapid metabolism after barrier transport, perhaps in adjacent brain tissue. Moreover, Zlokovic et al. (1992) suggest that the metabolic fragments so produced could include one of the behaviorally active VP fragments described by Burbach et al. (1983b). The in vitro evidence that AVP is rapidly degraded by peptidase activity in brain synaptosomes (Burbach and Lebouille, 1983; Burbach et al., 1983a; discussed in Chapter 5), as well as the more recently obtained in vivo evidence demonstrating hydrolysis of AVP after local microinjection into the hippocampus (Stark et al., 1989), are in accord with this suggestion. The VBP procedure differs dramatically from methods using intravenous injection of the radioactively labeled tested solutes to study their potential for penetrating the blood–brain or blood–CSF barriers. By replacing the normal supply of blood to the hemisphere under study, the artiﬁcial perfusate solution (1) permits strict experimental control over the variables to be added to the perfusate for characterizing transport kinetics of the peptide’s interaction with the brain–ﬂuid barriers; and (2) removes endogenous factors in the systemic circulation (saturated levels of vasopressin, peptide-degradative enzymes, and perhaps binding proteins) that normally mask the potential for passage of VP across the BBB. In this manner Zlokovic et al., clearly demonstrated the presence of the carrier-mediated VP inﬂux system that promotes VP penetration of the BBB.

B. Penetration of Brain–Fluid Barriers: Brain-to-Blood Transport of AVP and OT 1. Introductory Remarks Before the studies of Zlokovic et al. (1990, 1992), Banks et al. (1987a) investigated the possibility of carrier-mediated brain-to-blood transport of AVP. Several years later, Durham et al. (1991) carried out a similar investigation for OT. These studies provided evidence of, and characterized the kinetics of, brain-to-blood transport of AVP and OT. Two independent saturable carrier-mediated transport systems mediated the efﬂux of these peptides from brain to peripheral circulation. The peptide transport system (PTS) that served OT was subsequently termed PTS-1 because, at an earlier time, it had been shown to mediate brain-to-blood transport for several OT-related peptides (Banks and Kastin, 1995). The brain-to-blood transport system for AVP and related peptides then became known as PTS-2. The general method and experimental test procedures common to both studies were as follows. 1. Anesthetized mice were intracerebroventricularly injected with radioactively labeled peptide (1 l of lactated Ringer’s solution containing 125,000 cpm of 125I-labeled peptide) and decapitated 2, 5, 10, or 20 min after injection to determine the half-time disappearance rate of labeled

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peptide over time. At the end of the designated interval, the radioactivity (counts per minute, cpm) remaining in the whole brain (minus the pineal and pituitary glands) was counted with a counter for 3 min. 2. A number of kinetic parameters were determined and used to calculate the brain-to-blood transport rate of the labeled peptide as a function of labeled AVP available in the brain for transport. 3. Because intracerebroventricularly injected materials distribute into the CSF, adjacent brain tissue, and interstitial ﬂuid of the CNS, the amount of material available for transport by the brain is only part of the material injected, and must be determined experimentally. Accordingly, Banks et al. (1987a) used two methods to determine the amount of iodinated peptide available for transport by the brain. One method determined this parameter by extrapolating back to time zero on the curve, plotting the half-time disappearance rate of the labeled peptide. The other method assessing radioactivity in brains of mice killed by an overdose of anesthetic before ventricular injections (see original study for more details). 4. In additional tests, unlabeled VP, OT, or one of a number of selected unlabeled peptides or peptide fragments was added to the injectate solution and tested for its ability to inhibit transport of the labeled peptide. These tests helped to characterize the PTS for saturability and for its involvement in the transport of peptides other than VP or OT. The results of these general procedures, and of those speciﬁc to the individual study, are described below. 2. Relevant Studies a. Brain-to-Blood Transport of AVP: Banks et al. (1987a) The results of the Banks et al. (1987a) study were as follows. 1. The half-time disappearance of the labeled AVP from brain, graphed in Fig. 11A, was found to be 12.4 min. The transport rate (T) of labeled AVP [nmol/g (brain) min] as a function of AVP available for transport (Cb) (nmol/g) is plotted in Fig. 11B. The two methods used to determine the amount of AVP available for transport by the brain gave almost identical results. These ﬁndings indicated that the brain-to-blood transport of labeled AVP is much more rapid than can be accounted for by bulk ﬂow (i.e., reabsorption with CSF). 2. Of the unlabeled substances tested for competitive inhibition, seven of them [AVP, arginine vasotocin (AVT), lysine vasotocin (LVT), mesotocin, tocinoic acid, pressinoic acid, and pressinoic amide] signiﬁcantly inhibited this transport, whereas the remaining six (OT, LVP, tocinoic amide, AVP free acid, Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1), and cyclo[Leu-Gly]) did not do so. Noteworthy was the observation that at the same dose level of the coinjected ineffective OT, unlabeled AVP inhibited the brain-to-blood transport of iodinated AVP by 47%. The self-inhibition by coinjected unlabeled AVP demonstrates that the carrier system is saturable, and the failure of the

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FIGURE 11 (A) Disappearance of iodinated AVP from brain; half-time was found to be 12.4 min. Source: Banks et al., 1987a (Fig. 1, p. 328). Copyright ß 1987 by Alan R. Liss, Inc. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. (B) Transport rate of AVP out of brain (T) as a function of AVP available for transport (Cb). Vmax was 1.41 0.0279 nmol/gmin; Km was 28.7 1.23 nmol/g. Source: Banks et al., 1987a (Fig. 2, p. 328). Copyright ß 1987 by Alan R. Liss, Inc. Reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

system to transport OT and several other structurally related peptides indicates that it is highly speciﬁc for AVP and certain closely related compounds. 3. Two types of ﬁndings strongly suggest that the intact molecule of the labeled AVP, not products of its metabolic degradation, was transported across the BBB. First, a 30-nmol dose of unlabeled AVP added to the injectate containing either iodide (125I) or iodotyrosine ([125I]tyrosine) (i.e.,

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the two nonpeptides that could have been generated from metabolic degradation of labeled AVP) did not interfere with the brain-to-blood transport of either nonpeptide. Second, HPLC performed on blood collected from mice decapitated 2 min after an intracerebroventricular injection, or an intravenous, 2 min-long infusion of labeled AVP, indicated that (a) most of the radioactivity found in the blood eluted at the position of labeled AVP, and (b) comparison between HPLC results for intracerebroventricularly and intravenously injected labeled AVP indicated that most of the degradation of the peptide occurred during peripheral circulation and not during transport across the BBB. 4. The ﬁndings of two types of kinetic parameters, assessed in mice intravenously injected with labeled AVP, were as follows: (a) determination of the half-time disappearance rate and volume of distribution (Vd) of labeled AVP in the peripheral blood collected from the carotid artery 2, 5, 10, and 20 min postinjection indicated values of 10.9 min and 6.99 ml, respectively; (b) the blood-to-brain transport rate in these mice, determined in part by a graphic method used by Patlak et al. (1983), yielded a slow blood-to-brain movement of the intravenously injected label (i.e., 0.00247 ml/g min). 5. The effect of hydration status on the brain-to-blood transport of labeled AVP was tested. Three groups of mice were either water deprived for 24 h or water loaded 2 h before testing by an intraperitoneal injection of 5 ml of 0.45% NaCl, or served as nontreated controls. The failure of dehydration to affect transport shows that the PTS-2 is separate from, and operates independently of, the classic VP system (i.e., the hypothalamic– neurohypophysial system that is primarily concerned with the maintenance of normal body ﬂuid osmolality). This independence of the two systems is consistent with the improbability that AVP, injected into the lateral ventricle of the brain, could be taken up by the hypothalamus or posterior pituitary so rapidly. The ﬁnding that, unlike water deprivation, water loading did affect AVP transport (i.e., reduced it by about 40% in comparison with controls) was interpreted as a possible stress effect associated with abdominal distention induced by water loading. This stress interpretation is consistent with evidence of a role for AVP in stress (Ermisch and Landgraf, 1984; Kendler et al., 1978 and see Chapter 1). The authors concluded that these ﬁndings support the presence of a saturable carrier-mediated efﬂux system that transports intact AVP and certain structurally related peptides out of the brain. The presence of this efﬂux system on the brain side of the BBB is consistent with the thesis that the BBB is not totally impermeable to blood-borne AVP. b. Brain-to-Blood Transport of OT: Durham et al. (1991) Durham et al. (1991) determined a number of kinetic parameters of brain-to-blood transport in anesthetized mice, intracerebroventricularly injected with iodinated OT. Their procedures and results were as follows.

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1. The brains of these mice were removed and assayed for radioactivity at various intervals after injection. The small dose of labeled OT available for transport (6.72 fmol/g brain) was chosen to approximate physiological levels of OT expected in CSF (Robinson, 1983). The rate of brain-to-blood disappearance of labeled OT over time is graphed in Fig. 12A and half-time disappearance was 19.1 min. The amount of the radioactive label available for transport was 13,888 cpm and that remaining in the brain 10 min later was 9267 cpm. 2. Unlabeled peptides or peptide fragments, coinjected with iodinated OT into the lateral ventricle, were tested for their ability to inhibit brain-toblood transport of the labeled peptide. It was found that the 10-nmol dose of unlabeled OT signiﬁcantly inhibited this transport whereas the 1-nmol dose produced a similar trend but failed to reach statistical signiﬁcance. Of the remaining substances tested (each at a 10 nmol/mouse dose level), Tyr-MIF-1 and pressinamide signiﬁcantly inhibited this transport; tocinamide produced a nonsigniﬁcant trend in the same direction; but MIF-1, AVP, tyrosine, and iodotyrosine were without effect. These ﬁndings suggest that the peptide transport system for OT has a saturable component (i.e., is inhibited by an excess amount of OT), is speciﬁc for OT and certain other peptides (e.g., competitively inhibited by Tyr-MIF-1 and pressinamide), and transports intact OT rather than a metabolically degraded product (i.e., transport not inﬂuenced by unlabeled iodotyrosine). 3. Determination of whether Tyr-MIF-1 and OT shared a single transport system, or whether two closely related systems existed, was made by simultaneously evaluating their brain-to-blood transport. This was achieved by measuring radioactivity remaining in the brain 10 min after an intracerebroventricular injection of both 131 I-labeled Tyr-MIF-1 and 125 I-labeled OT with or without the addition of unlabeled OT or Tyr-MIF-1. The addition of each of these inhibited transport of both labeled peptides proportionately (see Fig. 12B). Because the regression lines describing the simultaneous transport of the two labeled peptides did not differ, it was concluded that they shared a single transport system, the PTS-1 system. The ﬁndings that unlabeled OT, pressinamide, and tocinamide signiﬁcantly inhibited transport of labeled Tyr-MIF-1 were consistent with this conclusion. 4. Pretreatment with aluminum chloride (intraperitoneal injection of a solution delivering 100 mg of elemental aluminum per kilogram, 60 to 90 min before an intracerebroventricular injection of labeled OT) indicated that the administered aluminum inhibited brain-to-blood transport of OT, as it has been observed to do for iodinated Tyr-MIF-1 (Banks and Kastin, 1989; Banks et al., 1988) and methionine enkephalin (Banks et al., 1987b). Because both of these latter peptides are transported by PTS-1, this ﬁnding further supports the idea that all three peptides share a single peptide efﬂux system.

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FIGURE 12 (A) Disappearance of 125I-oxytocin from brain; half-time was 19.11 1.3 min [y ¼ 4.11 (0.0158 0.0010) , r ¼ ()0.994]. Source: Durham et al., 1991 (Fig. 1, p. 449). Copyright ß 1991 by S. Karger. Reprinted with permission. (B) Transport after simultaneous injection of labeled oxytocin and Tyr-MIF-1 with or without unlabeled oxytocin or Tyr-MIF-1. Controls (d, 125I-oxytocin and 131I-Tyr-MIF-1) [y ¼ 0.1510 þ 0.7982x, r ¼ 0.785 (n ¼ 7)], with unlabeled oxytocin [&, y ¼ 0.1837 þ 0.6637x, r ¼ 0.535 (n ¼ 6)], and with unlabeled Tyr-MIF-1 [s, y ¼ 0.1778 þ 0.6801x, r ¼ 0.806 (n ¼ 6)]. Lines are not signiﬁcantly different. Source: Durham et al., 1991 (Fig. 2, p. 449). Copyright ß 1991 by S. Karger. Reprinted with permission.

5. The radioactivity that crossed from brain to blood was characterized by HPLC, and blood collected from the jugular vein of control mice that had received an intravenous infusion of 125I-labeled OT over a 2-min interval was used to determine the amount of degradation due to circulation in the

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vascular system. Two minutes after intracerebroventricular injection, radioactive material was found in the blood, and of this material 39% eluted as intact OT. By contrast, 33% of material infused directly into the blood over 2 min eluted as intact material. These ﬁndings indicated that most of the radioactivity transported across the BBB appeared to represent intact, labeled OT with some of the degradation occurring after transport. If a large amount of the material being transported had been degraded in the CNS, then a smaller percentage of intact peptide and a larger amount of degraded peptide should have been recovered from the blood in comparison with the control infusion. On the basis of these and related ﬁndings, the researchers concluded that (1) OT is transported out of the brain in an intact form by a carrier transport system (PTS-1) that appears also to serve the enkephalins and Tyr-MIF-I (Banks and Kastin, 1990); (2) although the rate of transport of OT is slower than that of Tyr-MIF-1, it is still faster than bulk ﬂow, which represents the rate of reuptake of CSF into the blood; and (3) the inhibition of iodinated OT brain-to-blood transport by unlabeled OT (self-inhibition) is consistent with a saturable component of the PTS-1. 3. Efflux Systems in the Brain for VP and OT: Relevance for Transport of VP and OT Across Brain–Fluid Barriers When Banks and colleagues (1987a) undertook their investigation of brain-to-blood transport of AVP, they noted that the prevailing attitude at the time was that AVP levels in the CSF and brain were independent of that in the periphery because of the impermeability of the BBB to AVP (Ermisch et al., 1982, 1985b; Robinson, 1983). A number of lines of evidence seemed to support this view, including the ﬁndings that (1) peripherally administered AVP or AVP analogs, sufﬁcient to increase plasma levels up to 100-fold, generally did not produce changes in CSF levels (Stegner et al., 1983; Vorherr et al., 1968); (2) only a small amount of radioactivity was produced in the brain after peripherally injected radioactive labeled AVP. This was often attached to fragments rather than to intact peptide (Ang and Jenkins, 1982; Zaidi and Heller, 1974); and (3) some stimuli increased plasma, but not CSF levels of the peptide (e.g., Mens and Van Wimersma Greidanus, 1982; Sorensen and Hammer, 1985; Wang et al., 1984), suggesting independent regulation of AVP in the two compartments. Similarly, Durham et al. (1991) noted that the corresponding assumption of BBB impermeability to OT received support from observations indicating that (1) the OT level in the brain is independent of that in the periphery (Amico et al., 1983; Robinson and Jones, 1982; Takagi et al., 1985), and (2) uptake of peripherally administered OT by the brain is 30-fold less in brain regions containing a BBB than in those with fenestrated capillaries (Ermisch et al., 1985a; Landgraf, Ermisch, and Heb, 1979; Zaidi and Heller, 1974).

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Several additional ﬁndings reviewed in Section V.A.1. of this chapter reinforced the view that the BBB is impermeable to these neurohypophysial peptides. However, Banks et al. (1987a) and Durham et al. (1991) also pointed out several anomalies in this research literature that are unexplainable under the assumption of BBB impermeability to these neurohypophysial peptides. These include the demonstration of (1) BBB permeability to AVP after peripheral administration of an extremely large dose of the peptide (Mens et al., 1983); (2) the ability of unlabeled AVP, coinjected with radioactively labeled AVP, to inﬂuence BBB penetration of the labeled AVP (Ermisch et al., 1982); (3) the ability of labeled AVP to exit from the CSF more rapidly than predicted on the basis of bulk ﬂow (Jones and Robinson, 1982; Mens et al., 1983); (4) the appearance of AVP-like material in the urine after centrally administered AVP (e.g., Clark et al., 1983); (5) low levels of OT in the brain after a peripheral injection of radiolabeled OT (Ermisch et al., 1985a; Landgraf et al., 1979; Zaidi and Heller, 1974); and (6) intracerebroventricularly administered OT exiting the CSF faster than predicted by reabsorption of CSF (Jones and Robinson, 1982; Mens et al., 1983). Moreover, the subsequent demonstration that AVP seems to readily cross from blood to brain when studied by the VBP method used by Zlokovic and colleagues (Zlokovic et al., 1990, 1991, 1993) can be added to this list of anomalies. The ﬁndings of Banks et al. (1987a) and Durham et al. (1991), which strongly supported the presence of saturable carrier-mediated brain-toblood transport for AVP (PTS-2) and for OT (PTS-1), can account for the anomalies listed above, and for many of the controversial ﬁndings described in Section V.A of this Chapter. Thus, depending on factors such as the dose level of the peripherally injected peptide and the saturable status of the relevant peptide transport systems, this efﬂux system may immediately remove any peripherally administered AVP or OT that does enter the brain before it can sufﬁciently accumulate to be detected and/or to exert an effect in the brain. This could account for the failure of Deyo et al. (1986) to obtain evidence of AVP after peripheral administration of a relatively low dose of the peptide (see Section V.A.1). On the other hand, the high dose of peripherally administered AVP by Mens et al. (1983) may have saturated PTS-2 so that sufﬁcient amounts of the labeled peptide entered the brain and were detected. This pattern of low brain uptake from blood after a peripheral injection of a relatively small amount of unlabeled peptide (Deyo et al., 1986) or with only radioactive labeled compound (Banks and Kastin, 1990), but higher uptake after an injection of a larger amount of the unlabeled peptide (Mens et al., 1983) or with unlabeled plus radioactive compound (Banks and Kastin, 1990), characterizes efﬂux transport systems in addition to PTS-1 and PTS-2 (Chikhale et al., 1995). Thus, the efﬁciency of a saturable peptide efﬂux system that resists blood-to-brain peptide transport is reduced as it becomes increasingly

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saturated by increased concentration of plasma levels of its peptide substrate, as well as by introduction of a potential inhibitor of the efﬂux system (Chikhale et al., 1995). Much remains to be learned about the operation, speciﬁc locations, and speciﬁc roles played by these efﬂux systems with respect to VP and OT distribution between brain and periphery. For example, the speciﬁc site(s) at which these efﬂux systems transport VP or OT out of the brain was not determined in the studies by Banks and colleagues (Banks et al., 1987a; Durham et al., 1991). They could be localized in the endothelium within the brain parenchyma, or at the endothelium near the ventricles or at the choroid plexus. A location at the endothelium within the brain parenchyma is consistent with the observations of AVP-binding sites at brain capillaries (Ermisch et al., 1982; Kretzschmar and Ermisch, 1985). However, the ﬁnding cited by Banks and Kastin (1995), that saturable efﬂux of Tyr-MIF-1 occurs after injection into the lateral ventricle but not after injection in brain parenchyma, suggests that PTS-1 may be located at the choroid plexus but not at the brain endothelium.

VI. AVP Influences Permeability of Brain–Fluid Barrier to Nutrient Transport

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A. Introduction Following an early study (Landgraf et al., 1978) that found a VPinduced enhancement of blood–brain barrier uptake of the RNA precursor, orotic acid, Ermisch and colleagues investigated the potential interaction of VP with the LNAA transporter, the mechanism by which large neutral amino acids (including precursors for catecholamine and serotonin biosynthesis) enter the brain interstitial ﬂuid (ISF) from the blood. These studies used either the single-pass technique of Oldendorf, which exposes the nutrient only once to the blood–brain barrier, or the integral technique in which the nutrient remains in circulation for varying amounts of time before decapitation. An advantage of the integral technique in studies of amino acid transport is that it permits observation of BBB transport under in vivo competition with the normal physiological concentrations of amino acids sharing the same transporter (Ermisch et al., 1992). Kinetic transport values are determined by data analysis (e.g., see data analysis section in Brust, 1986). In studies of amino acid transport, the analysis provides information concerning regional accumulation of the tested amino acid, a measure of its afﬁnity for the amino acid transporter [e.g., the large neutral amino acid (LNAA) transporter]. Regional accumulation values are in turn used to calculate transport kinetic constants such as rate of transport of the amino acid when the transporter is at half-saturation (Km), nonsaturated transport (Kd), and maximal velocity (Vmax) of transport.

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Because cerebral blood ﬂow (CBF) can inﬂuence the rate of transport, the potential inﬂuence of vasopressin on this variable is assessed. The rate of CBF in a sampled brain region is assessed by regional accumulation of radioactively labeled iodoantipyrine, a substance that readily diffuses across the BBB. A lack of difference between AVP-treated and nontreated subjects in this measure is interpreted as a lack of AVP inﬂuence on CBF in the study. A second consideration in transport kinetic studies is to correct for residual vascular activity—the possibility that the tested amino acid remains in residual plasma in the capillary network of the brain and is not taken up by the BBB transporter. Ermisch and colleagues typically correct for residual vascular activity by subtracting a quantity signifying regional accumulation of inulin (a substance that is not transported across the BBB) from the total accumulation of the tested amino acid (At). The net result is the corrected accumulation value (Ac) for the amino acid, a value signifying its afﬁnity for the amino acid transporter localized on the blood or brain side of the capillary endothelium. The purpose of these studies has been to determine whether AVP and/or OT interacts with the blood–brain barrier to inﬂuence the transport of the studied nutrients, and if so, the nature of that inﬂuence and its functional signiﬁcance, including a putative contribution to memory processing.

B. Relevant Research 1. AVP and Brain–Fluid Barrier Permeability to the RNA Precursor, Orotic Acid a. Selected Study: Landgraf et al. (1978) Landgraf et al. (1978) studied the effect of LVP (1, 10, 100, 1000, or 10,000 U per rat, intracarotid injection) on BBB uptake of the RNA precursor, [3H]orotic acid, using the single-pass technique. Regional amount of tracer radioactivity was investigated in each of 19 dissected brain regions (olfactory lobe and ﬁve cortical regions, striatum, hippocampus, thalamus, hypothalamus, colliculi, cerebellum, tegmentum, pons, medulla, and four regions lacking a blood–brain barrier). For each sampled region, tracer uptake (concentration of radioactivity) was correlated with dose of injected AVP. Thus, a signiﬁcant positive correlation between tracer uptake and LVP dose level was interpreted as a peptide-induced increase in barrier uptake of the tracer, whereas a signiﬁcant negative correlation between the two variables indicated the opposite. Control experiments were conducted to determine whether (1) the orotic acid may have lodged within capillary blood instead of being transported into the adjacent brain tissue; this was done by testing the effect of LVP on residual vascular activity for the nondiffusible tracer [3H]inulin, and (2) an LVP-induced alteration in CBF altered brain uptake of orotic acid rather than a peptide-induced inﬂuence on barrier permeability to the tracer;

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this was done by comparing the effect of LVP on the highly diffusible tracer tritiated water (dependent on CBF) with that on the much more slowly diffusing orotic acid (independent of CBF). The results and interpretations were as follows: (1) brain uptake of orotic acid was changed by LVP in a regionally differentiated manner. Some regions were uninﬂuenced (frontal, motor, and most of the sensory cortical regions, striatum, thalamus, colliculi, pons, medulla, cerebellum), whereas in others orotic acid uptake was increased (olfactory cortex, hippocampus, hypothalamus, and all the CVOs) or reduced (tegmentum); (2) in the control test for residual vascular activity for injected orotic acid, there was no signiﬁcant difference between LVP-treated and nontreated subjects in amount of residual vascular radioactivity, indicating no inﬂuence of LVP and thus that the tracer was taken up by a BBB transporter and was not lodged in residual capillary blood (the measure was therefore disregarded in the other experiments); and (3) correlational data indicated that tritiated water uptake varied reciprocally, and orotic acid uptake varied directly, with LVP dose level. This was interpreted as indicating that whereas tritiated water uptake in the hippocampus was dependent on CBF, that for orotic acid was not. In discussing these results, the authors cited evidence that an enhanced supply of orotic acid to the brain affects memory processing (Matthies, 1974) and speculated that systemic administration of vasopressin in behavioral studies may exert its inﬂuence on memory processing by facilitating the BBB transport to the brain of nutrients that act as precursors for chemicals (e.g., mRNA) having a role in memory processing (e.g., protein synthesis). 2. AVP and Brain–Fluid Barrier Permeability to Large Neutral Amino Acids a. Selected Studies i. Brust (1986) Brust (1986) studied the effect of peripherally injected AVP on leucine transport in ether-anesthetized rats, using the single-pass technique. l-Leucine is a large neutral amino acid that, along with eight other amino acids, is transported across the capillary from blood to brain by the large neutral amino acid (LNAA) transporter present on the luminal side of the capillary wall. Each rat received an intracarotid injection of l-[3H]leucine (0.4 mM) and unlabeled l-leucine (0.0, 0.02, 0.10, 0.20, 0.80, or 6.4 mM) either in the absence (controls) or presence of AVP, at physiological concentration (41.2 9.8 pM). Cardiovascular changes in heart rate (HR) and BP were not expected because AVP, at physiological concentration, does not pass the BBB in signiﬁcant amounts (Ermisch et al., 1983), nor does it result in an increase in arterial blood pressure (McNeill, 1983). Moreover, cerebral blood ﬂow was measured to monitor possible cardiovascular effects of AVP.

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The animal was killed 15 s later, the brain was removed, and the hemisphere ipsilateral to the injection was dissected into nine brain regions (olfactory bulb, frontal cortex, visual cortex, septum, hippocampus, striatum, hypothalamus, thalamus, and colliculi). Regional radioactivities [disintegrations per minute (dpm) per gram] were determined by liquid scintillation counting. The results indicated the following. 1. AVP did not signiﬁcantly inﬂuence regional CBF in this study; thus a plot of [14C]iodoantipyrine accumulation values versus time after carotid injection showed no difference between AVP-treated and nontreated animals. Nor did these groups differ in their regional accumulation values of this tracer, although both groups showed regional differences in rate of CBF, which was higher in certain brain regions (visual cortex, frontal cortex, colliculi) than in others (olfactory bulb, septum, hippocampus). 2. Accumulation of [3H]leucine decreased once nonlabeled leucine was added to the injectate, indicating the presence of competitors for the LNAA transporter; AVP did not differentially affect this outcome. 3. Regional differences in tracer afﬁnity for the LNAA transporter were indicated by the observed regional differences in [3H]leucine accumulation when the tracer was administered alone (i.e., in the absence of competition for the LNAA transporter from unlabeled leucine added to the injectate). Tracer accumulation was signiﬁcantly higher in the visual cortex than in all brain regions tested except for the colliculi, and that for the colliculi was signiﬁcantly higher than that in the hippocampus, olfactory bulb, and hypothalamus. 4. When only the tracer concentration of leucine was injected, AVP treatment signiﬁcantly increased [3H]leucine accumulation relative to control values in all brain regions tested except the septum and the hypothalamus. When 0.2 mM unlabeled leucine was added to the injectate, AVP treatment signiﬁcantly decreased tracer accumulation in all brain regions except the frontal cortex, septum, and striatum. These VP-induced effects on leucine accumulation were interpreted as suggesting that the peptide may increase the concentration sensitivity of leucine transport under circumstances associated with low amino acid blood levels. 5. The regional values of the leucine transport constants, maximum velocity of transport (Vmax), and nonsaturable transport (Kd), calculated from nonlinear regression analysis of the tracer accumulation data, also showed regional differences. An increase in velocity of leucine transport may partially reﬂect an increase in CBF due to capillary recruitment, thereby providing a greater capillary surface area for transport of the amino acid. AVP treatment signiﬁcantly reduced the transport kinetic constants Vmax and Km to about one-third of control values in all brain regions. The authors acknowledged that the mechanism underlying this peptide action was not

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clear from this study. Direct or indirect interaction with the transporter at the luminal membrane of the BBB was possible. ii. Reith et al. (1987) Reith et al. (1987) used the integral technique to test the hypothesis that peripherally circulating AVP inﬂuences the transfer of LNAAs across the BBB by an interaction with capillary endothelial receptors rather than by AVP entry into brain tissue through the BBB. The hypothesis was tested by measuring (1) capillary permeability to [3H]AVP relative to the impermeable tracer [14C]mannitol in several brain regions to determine whether AVP crosses the capillary to a signiﬁcant degree in regions protected by the BBB; and (2) the immediate effect of AVP on transfer of [3H]leucine from blood to brain in the presence of normal plasma components and the other eight LNAAs, which normally compete with leucine for the transporter. Two experiments studied the BBB permeability to leucine (group I) and AVP (group II), using a single time measurement (20 s after injection), and to AVP and mannitol (group III), using a multiple time measurement (10–120 s after injection). The experiment with group I compared the accumulation of test tracer [3H]leucine with reference tracer [14C]iodoantipyrine. The experiments with groups II and III compared the accumulation of test tracer [3H]AVP with that of the nonpermeable reference tracer, [14C]mannitol. In the control groups, each test tracer was injected alone: [3H]leucine in group Ia, and [3H]AVP in group IIIa, whereas in the experimental groups (Ib and IIb), each of the tracers was coinjected with 50 nmol of unlabeled AVP. The transport of [3H]leucine across the BBB in the absence and presence of AVP was demonstrated for two non-BBB regions (pineal and pituitary glands), and for eight BBB regions (olfactory bulb, colliculi, hypothalamus, cortical regions [visual, frontal], thalamus, striatum, and hippocampus). The permeability value for each brain region was calculated from the clearances of test tracers, as the ratio between tracer content in the brain and the time– concentration integral in arterial plasma (determined mechanically by continuous withdrawal of arterial blood at a known and constant rate from the femoral artery). Arterial BP was continuously monitored to ensure that BP did not signiﬁcantly alter autoregulated CBF. The results were as follows. 1. In group I, brain uptake of [3H]leucine, measured 20 s after intravenous injection, showed a marked accumulation in the non-BBB regions tested, but was much lower in the BBB-protected regions. Peripherally injected LVP further reduced this transport, especially in the hippocampus, thalamus, and hypothalamus. 2. In group II it was assumed that accumulation of tracer [14C]mannitol in the BBB regions reﬂects the blood plasma space of the brain, whereas in non-BBB regions the tracer can diffuse beyond the vascular bed to

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the interstitial space. [3H]AVP was retained in the non-BBB regions (pituitary and pineal glands) and in the hippocampus to a greater degree than was [14C]mannitol, and this retention effect declined once unlabeled AVP was added to the injectate, indicating saturable retention in both types of region. This result did not identify the site of AVP retention in the hippocampus, which may have been intra- or extravascular (i.e., beyond the BBB); the retention of AVP was likely to have been extravascular only if the BBB was substantially more permeable to AVP than to mannitol. 3. To conﬁrm that permeability of the BBB to AVP in these experiments was not substantially greater than that to mannitol in BBB-protected regions (e.g., hippocampus), and to determine whether AVP binds saturably to the endothelium of hippocampal vessels rather than penetrates into the hippocampal tissue, the authors followed the time course of accumulation of [14C]mannitol and [3H]AVP in the hippocampus and in non-BBB regions of the brain. The results indicated that the accumulation of [3H]AVP in the hippocampus, as a function of time, was not substantially greater than that of [14C]mannitol (i.e., the BBB permeabilities to the two tracers were not substantially different). In the non-BBB regions, both AVP and mannitol showed unlimited diffusion from blood to brain, as expected. In addition, whereas the steady state distribution of mannitol corresponded to the volume of the extracellular space, the 3-fold larger steady state distribution of AVP suggested binding or trapping in the extracellular space, transport into cells, or a combination of these two possibilities, for example, an internalization of the receptor-bound ligand. On the basis of these ﬁndings the authors suggested that (a) AVP interacts with the LNAA transporter to reduce the transport of leucine, and likely other amino acids carried by the transporter, across the BBB from blood to brain; (b) this AVP inﬂuence is unrelated to changes in CBF; and (c) because there was negligible transcellular transport of AVP across the capillary in BBB-protected regions, it is likely that AVP does not enter the brain in these regions but is retained in the endothelium itself, where it may bind to the LNAA transporter to mediate its effect on leucine transport. iii. Brust and Diemer (1990) Brust and Diemer (1990) investigated whether the previously reported effect of AVP on blood-to-brain transfer of leucine (Brust, 1986; Reith et al., 1987) extended to other LNAAs, such as l-phenylalanine. Speciﬁcally, the study was designed to (1) measure the regional blood to brain transfer of l-phenylalanine after peripheral administration of AVP and (2) help localize the AVP receptors on the BBB that mediate this inﬂuence by determining whether centrally as well as peripherally applied VP elicits changes in BBB transport of LNAAs. The integral technique was used, which permitted simultaneous measurement of cerebral blood ﬂow (CBF) and barrier permeability to

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phenylalanine (permeability–surface area product, PaS) and allowed assessment of the relative independence of these two measures. [14C]Iodoantipyrine was used to measure CBF as in previous studies (Brust, 1986; Reith et al., 1987). Two groups of ether-anesthetized Wistar rats were used to determine the effect of peripherally (group I) and centrally (group II) applied AVP on CBF and on barrier permeability to phenylalanine. The rats in group I were intravenously injected via an implanted cannula in the femoral vein with [3H]phenylalanine, [14C]iodoantipyrine, and different amounts of AVP (0, 10 pmol, 500 pmol, 30 nmol). The rats in group II were perfused with AVP in the dorsal hippocampus of the right hemisphere or with Ringer’s solution in the left hemisphere by means of previously implanted microdialysis probes. After 90 min of perfusion (AVP added to the perfusate of the right hemisphere probe during the last 30 min of perfusion), these rats received the intravenously injected tracers used for group I rats. Both groups were monitored for blood pressure and blood was sampled for several physiological variables (blood levels of oxygen, carbon dioxide, and glucose, and pH) during experimental testing. Radioisotope content was assessed in withdrawn blood and in brain tissue samples from group I (olfactory bulb and lobe, frontal cortex), group II (dorsal, medial, and ventral parts of the hippocampus) or from both groups (septum, striatum, hypothalamus, thalamus, visual cortex, and colliculi). The results were as follows. 1. Most of the monitored physiological variables (blood levels of glucose, oxygen, and carbon dioxide; hematocrit, and pH) were within the normal range and did not differ between the two groups. 2. All three dose levels of peripherally administered AVP (group I) increased mean arterial blood pressure approximately 150% over that of control values, and dose dependently decreased cardiac output to 32–66% of control values. No change in either of these measures occurred after intracerebral dialysis of AVP (group II). 3. Compared with control values, intravenously administered AVP reduced CBF in all sampled brain regions. Depending on the brain region, this was a reduction of 24 to 56% for the two highest doses of AVP (500 pmol and 30 nmol) and of 11 to 33% for the lowest VP dose (10 pmol). The authors argued against an AVP-induced vasoconstrictor action on cerebral vessels as a direct cause of the decrease in CBF because a previous study (Reith et al., 1987) demonstrated no change in cerebral plasma volume (mannitol space) after an intravenous injection of large amounts of AVP. 4. VP perfusion into the dorsal hippocampus signiﬁcantly reduced CBF in this region relative to all other brain regions sampled. In addition, the CBF of sampled regions in both hemispheres was less than that in the

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control subjects (effect signiﬁcant for the striatum, and for the dorsal, medial, and ventral parts of the hippocampus). 5. Compared with controls, intravenously administered AVP reduced phenylalanine transport across the BBB in most of the sampled brain regions by 16 to 39% at the two highest dose levels (30 nmol and 500 pmol), and by 11 to 25% at the lowest dose level (10 pmol). Microdialysis of AVP into the dorsal hippocampus had no inﬂuence on this variable. In summary, the results were interpreted as indicating that (1) AVP reduced permeability to phenylalanine transport across the BBB as it had done for leucine (Reith et al., 1987), supporting the theory that this peptide interacts with the LNAA transporter; (2) the AVP-induced inﬂuence on the LNAA transporter occurs at the blood and not the brain side of the BBB because peripherally, but not centrally, applied AVP reduced phenylalanine transport from blood to brain; and (3) the AVP-induced inﬂuence on the LNAA transporter is independent of its effect on CBF because both centrally and peripherally administered AVP decrease CBF, but only peripherally administered AVP reduced LNAA transport from blood to brain. iv. Ermisch et al. (1992) Ermisch et al. (1992) tested the hypothesis that vasopressin interacts with V1 receptors on the luminal surface of the endothelial capillary membrane to affect the transport of LNAAs. This hypothesis was tested by (1) ﬁrst excluding an effect of the applied AVP on CBF and (2) comparing the effect of AVP on the transport kinetics (Vmax and Km) of leucine and phenylalanine. Because Brust (1986) determined the effects of AVP on these transport kinetics for leucine, this study used the same methodological steps in investigating phenylalanine transport to ensure comparable data for comparison. [14C]iodoantipyrine (used as a measure of CBF), the test tracer [3H] phenylalanine, and various concentrations of unlabeled phenylalanine (0 to 6.4 mM) were injected in the presence or absence (controls) of a physiological concentration of AVP (100 pM). The bolus was rapidly injected into the right common carotid artery of ether-anesthetized rats, allowing a single passage of the bolus through the brain microcirculation (single-pass technique). The animals were decapitated 15 s after the injection and the brain hemisphere ipsilateral to the side of injection was dissected into nine regions (olfactory bulb, frontal cortex, visual cortex, hippocampus, septum, striatum, thalamus, hypothalamus, and colliculi). The results were as follows. 1. The addition of AVP (100 pM) to the intracarotid injection of [14C]iodoantipyrine did not signiﬁcantly alter accumulation of this tracer, relative to non-AVP-treated subjects, in any of the nine brain regions studied, indicating that a possible AVP effect on CBF can be excluded from consideration in this study.

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2. The regional accumulation of nine different concentrations of l[3H]phenylalanine, with and without a coinjection of AVP, was determined for the whole brain hemisphere. The amount and direction of the inﬂuence of AVP on this accumulation depended on the concentration of the amino acid and the brain region sampled. With low amino acid concentrations (0.4 M), AVP increased phenylalanine accumulation in all brain regions, but this was signiﬁcant only for the hemisphere itself and for three of the nine brain regions sampled (i.e., hippocampus, hypothalamus, and visual cortex). With somewhat higher amino acid concentrations (0.05 to 0.2 mM), AVP decreased accumulation in all brain regions, and at an amino acid concentration of 0.2 mM the decreased accumulation was signiﬁcant for three brain regions (olfactory bulb, frontal cortex, and hippocampus). At still higher phenylalanine concentrations (0.4 to 0.8 mM, i.e., when the transporter was nearly saturated), AVP had no signiﬁcant effect on accumulation of the amino acid. 3. The kinetic constants of phenylalanine transfer across the BBB were signiﬁcantly decreased after AVP injection in nearly all regions studied (Km, eight of nine; Vmax, seven of nine) and in the hemisphere by about 30% for Km, and 25% for Vmax. The reduction was most pronounced in the hippocampus, frontal cortex, and hypothalamus. 4. Comparisons between the data obtained for phenylalanine (this study) and for leucine (Brust, 1986) indicated that although both transport parameters [Km (half-saturation constant for transport) and Vmax (maximum velocity of transport)] were signiﬁcantly decreased in AVP-treated animals relative to nontreated controls in most of the brain regions studied, leucine transport was altered more strongly than was phenylalanine. This study extended the ﬁndings by Brust (1986), and indicated an AVP effect on BBB transport of phenylalanine as well as leucine. Together, the two studies support the proposal that, at a physiological concentration level, peripheral AVP interacts with the LNAA transporter and thereby decreases barrier transport of all the amino acids carried by this transporter. This study also suggested speciﬁcity of the VP-induced altered transport with respect to both the amino acid and the brain region. Comparisons of these data with those obtained by Brust (1986) suggest that AVP induces a preferred transport of phenylalanine versus leucine across the BBB. v. Reichel et al. (1996) Reichel et al. (1996) tested the hypothesis that the interaction of vasopressin with the LNAA transporter leads to preferential blood-to-brain transport of some LNAAs relative to others. The ﬁnding that AVP decreased blood-to-brain transport more effectively for leucine than for phenylalanine was interpreted as support for this hypothesis (Ermisch et al., 1992). This study used the single-pass technique with ether-anesthetized rats to compare the effect of peripherally administered

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AVP on the blood-to-brain transport of two other LNAAs, l-tyrosine and l-valine. The rats received an intracarotid injection of a 200-l bolus of Ringer’s solution containing either the test tracer (l-[3H]tyrosine or l-[3H]valine), together with a given concentration of its respective, unlabeled test agent [l-tyrosine (0.0, 0.05, 0.1, 0.2. 0.8, or 6.4 mM) or l-valine (0.0, 0.05, 0.1, 0.2, 0.8, or 6.4 mM)], or [14C]iodoantipyrine (to measure CBF). The injectate for the experimental subjects also contained a physiological dose of AVP (0.1 nM). The animals were killed 15 s after the injection and the ipsilateral hemisphere was dissected into nine regions: colliculi, hypothalamus, thalamus, striatum, hippocampus, septum, visual cortex, frontal cortex, and olfactory bulb. The results were as follows. 1. Although CBF values differed by a factor of 3.4 among the nine brain regions, peripherally administered AVP did not affect this variable. This was indicated by the lack of peptide inﬂuence on the accumulation of [14C]iodoantipyrine, used to calculate regional CBF. This ﬁnding replicated that of previous studies (Brust, 1986; Ermisch et al., 1992). 2. Assessment of the effect of AVP on accumulation of labeled tyrosine and valine in the whole brain hemisphere and in each of the nine dissected brain regions depended on the concentration of the corresponding unlabeled amino acid that was included in the injectate. In the absence of unlabeled tyrosine, accumulation of this amino acid was increased by AVP treatment (statistically signiﬁcant in all brain regions except the septum). However, the peptide decreased this accumulation at a higher concentration level of coinjected tyrosine (i.e., 0.2 mM) and leveled off at still higher concentrations. Accumulation of l-[3H]valine was similarly affected by AVP treatment, although statistical signiﬁcance occurred only when unlabeled valine was coinjected at a concentration level of 0.2 mM, and this effect was signiﬁcant for all brain regions except the frontal cortex. 3. VP decreased saturable transport (Km) and maximum velocity of transport (Vmax) in all brain regions, and with one exception (Vmax for tyrosine in the thalamus) these effects were statistically signiﬁcant. For the whole hemisphere saturable transport declined by 72%, and velocity of transport by 55%, for tyrosine, and by 91 and 89% for valine, respectively, indicating that the peptide induced a comparatively larger reduction of transport for valine than for tyrosine. The ﬁndings that AVP increased or decreased valine and tyrosine transport, depending on the concentration of the unlabeled amino acid in the injectate, suggests that the LNAA transporter exhibits enhanced afﬁnity for a given LNAA in the presence of relatively low plasma concentrations of competing amino acids, whereas at higher concentrations this afﬁnity lessens as the transporter approaches carrier saturation.

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The investigators interpreted these results, and their similar previous ﬁndings indicating peripherally but not centrally induced AVP effects on blood-to-brain transport of other LNAAs (Brust and Diemer, 1990; Reith et al., 1987), as supporting the thesis that VP receptors associated with the LNAA transporter are located on the blood side of the BBB, and exhibit differences in concentration between various brain regions. vi. Reichel et al. (1995) Reichel et al. (1995), noting the evidence of a peripherally circulating AVP-induced decrease on blood-to-brain uptake of LNAAs (see Brust, 1986; Brust and Diemer, 1990; Brust et al., 1992; Reichel et al., 1996) investigated whether this peptide inﬂuence is reﬂected in a corresponding reduction of AA content in the CSF and brain. In addition, they assessed the ability of the peptide to inﬂuence CSF and brain concentrations of AAs other than those belonging to the LNAA group. They measured concentrations of 15 AAs in plasma (sampled from the femoral artery), CSF (sampled from the cisterna magna), and brain ISF (sampled from hippocampal tissue) of male and female Wistar rats before and after intravenous infusion into the right jugular vein of AVP at two rates of infusion: 34 and 68 ng/min kg. The AAs studied belonged to each of four classes (large neutral AAs, small neutral AAs, acidic AAs, and basic AAs). At the end of the infusion and postinfusion sampling of CSF and plasma, the subjects were killed and the hippocampus was dissected from the brain for further study. The relative changes in AA levels in plasma and CSF as a percentage of the preinfusion (baseline) level were determined and statistically evaluated. The results were as follows. 1. Both rates of AVP infusion signiﬁcantly raised basal plasma levels of the peptide (1.7 0.2 pg/ml) by approximately 2.5 and 4.4 times, respectively. Control subjects similarly infused with isotonic saline showed no signiﬁcant change in baseline level of AVP. Neither rate of AVP infusion increased blood pressure. 2. Saline infusion mainly increased plasma and CSF levels of AAs, but only a small number of these changes were signiﬁcant. These plasma alterations were paralleled in CSF and brain tissue, as indicated by a signiﬁcant correlation between changes in plasma and CSF AA levels. These altered AA levels were attributed to the effects of anesthesia, surgery, and sampling procedure per se. 3. AVP infusion produced AA alterations in plasma, CSF, and hippocampal tissue that did not correspond to those caused by the general procedure (anesthesia, surgery, etc.). 4. Plasma levels of about half the tested AAs were signiﬁcantly altered relative to initial baseline values by both doses of AVP. Although there was considerable overlap, the individual AAs affected were not entirely the same

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for both dose levels. With two exceptions these alterations involved an increase in plasma AA concentration. 5. The high correlation between plasma and CSF AA levels observed after saline treatment was lost after AVP treatment. In the CSF, the majority of the AAs were signiﬁcantly decreased, compared with baseline levels, that is, 12 of 15 AAs after the low dose, and 8 of 15 AAs after the high dose, of infused AVP. One AA (arginine) was increased after the high dose of AVP. 6. For hippocampal tissue, AVP treatment resulted in signiﬁcant changes in four AAs: LNAAs (methionine after the low AVP dose, phenylalanine and tyrosine after the high dose), and one acidic AA (aspartate after the low AVP dose). All three of the LNAAs were increased, and the acidic AA level was decreased. The ﬁnding that elevated levels of circulating AVP over a 60-min period reduced LNAA concentration in the CSF was interpreted as support for the theory that an AVP interaction with LNAA transporter not only inﬂuences AA supply to the brain endothelium but also inﬂuences their levels in brain extracellular ﬂuid, and hence their availability for brain metabolism. The further ﬁnding that, in addition to reducing CSF levels of LNAAs, the increased circulating AVP reduced concentrations of most of the other AAs tested needs explanation. The basic AAs are known to be more readily taken up by the BBB than others (small neutral and acidic AAs) that appear to be readily transported, along with other AAs, at the blood side of the choroid plexus (Preston and Segal, 1990; Segal and Zlokovic, 1990). The authors therefore suggested the need to consider movement of AAs across the choroid plexus epithelium. That AVP may affect these choroidal transport processes was considered a distinct possibility given the evidence of V1 receptors at the blood side of the choroid plexus. An alternative explanation offered for the reduced levels of AAs in the CSF was that CSF may act as a reservoir from which the brain obtains AAs for its metabolism during periods when AVP reduces AA transport from blood to brain in preserving volume regulation in the brain. Thus, during stress (e.g., disturbances to osmotic homeostasis) hormonal AVP released in the circulation is associated with increased levels of AAs (Milakofsky et al., 1985), which are potent osmolytes (Lang et al., 1990). The AVP-induced reduction of blood-to-brain AA transport could serve as a protective brain volume regulation mechanism. At this time the metabolic needs of the brain for amino acids could be met by the CSF AA reservoir. In contrast to the VP-induced AA decline in CSF, three LNAAs were signiﬁcantly increased in the hippocampus. Of these three, phenylalanine and tyrosine are precursors in the synthesis of catecholamine neurotransmitters (Pardridge, 1983b), and methionine is a precursor in taurine synthesis (Bender, 1975). Because catecholamine neurotransmitters have been implicated in memory processing, as noted in earlier chapters (see Chapters 4 and 10), elevated levels of phenylalanine and tyrosine are in accord with

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the putative role of AVP in memory processing. Taurine serves an osmoprotective role in the brain (Law, 1991), which may be a means by which AVP exerts its role in brain osmoregulation.

C. Vasopressin Influence on Nutrient Transport Across the BBB: A Role in Memory Processing? The ﬁnding by Ermisch, Landgraf, and colleagues (Landgraf et al., 1978) that vasopressin increased permeability of the blood–brain barrier to orotic acid especially interested these researchers, given the observation that availability of this RNA precursor affects cerebral protein synthesis and enhances memory processing (Matthies, 1974; Matthies et al., 1976). Additionally intriguing was the ﬁnding that this effect on BBB permeability was regionally differentiated and especially prominent in the hippocampus (Landgraf et al., 1978), given the 1970s proposal by De Wied and colleagues that peripherally administered vasopressin improved memory consolidation in active and passive avoidance conditioning tasks (see Chapter 2). Ermisch, Landgraf, and colleagues perceived a connection between the inﬂuence of AVP on avoidance conditioning tasks and its inﬂuence on BBB uptake of a precursor for protein synthesis in a region (hippocampus) considered to play an important role in memory consolidation (Ermisch et al., 1982; Landgraf et al., 1978). Indeed, these researchers observed that retention in a brightness-discrimination shock-avoidance learning paradigm was more greatly facilitated after a combined injection of orotic acid and vasopressin than after either treatment given alone (Ermisch et al., 1982). Ermisch, Landgraf, and colleagues (Landgraf et al., 1979) proposed the existence of vasopressin receptors on the blood-facing (lumenal) side of the BBB, which would interact with vasopressin to inﬂuence barrier uptake of orotic acid and other nutrients that might play a role in memory processing. The results of several studies with laboratory rats, cited in Ermisch et al. (1988), led these researchers to conclude that speciﬁc VP-binding sites are present on hippocampal microvessels and perhaps that nonspeciﬁc binding also occurs in other brain regions such as the striatum and neocortex. These studies did not localize the binding sites on the capillary wall. The authors suggested, however, that because VP cannot pass through the BBB in effective amounts (Ermisch et al., 1985b), and VP can inﬂuence substrate transport across the BBB when coinjected with a number of nutrients (e.g., Landgraf et al., 1978; Reichel et al., 1995), the binding sites mediating the inﬂuence of VP on blood-to-brain nutrient transport probably occur on the lumenal side of the capillary endothelium. The ﬁnding that peripherally injected, but not brain-applied, AVP inﬂuenced phenylalanine BBB transport kinetics (Brust and Diemer, 1990) supported this proposal. Subsequent studies examined a role for vasopressin inﬂuence on BBB transport of other nutrients that could play a role in memory processing.

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Because large neutral amino acids (LNAAs) are selectively transported across the BBB by the LNAA transporter, and several of these are precursors in the biosynthesis of catecholamines (phenylalanine and tyrosine) and serotonin (tryptophan) neurotransmitters, these researchers extensively studied the inﬂuence of AVP on LNAA transport across the BBB (see Section VI.B). They have demonstrated that peripheral AVP, given at physiological dose levels, increases arterial blood pressure but does not disturb the autoregulatory mechanisms governing cerebral blood ﬂow (Reith et al., 1987). This treatment has signiﬁcantly inﬂuenced barrier uptake of each of the LNAAs tested to date: leucine (Brust, 1986; Reith et al., 1987), phenylalanine (Brust and Diemer, 1990; Ermisch et al., 1992), valine (Reichel et al., 1996), tyrosine (Reichel et al., 1996), and methionine (Brust et al., 1992). Regional differences in uptake may reﬂect corresponding differences in vasopressin receptor distribution in the brain. In all these studies, calculation of transport kinetic values has indicated that vasopressin decreases blood-tobrain transport of these LNAAs, and this is matched by corresponding reductions in amino acid levels in brain interstitial ﬂuid (Reichel et al., 1995). The observation that vasopressin reduced phenylalanine BBB transport to a lesser degree than leucine transport (Ermisch et al., 1992) suggests that, although vasopressin generally reduces LNAA blood-to-brain transport, differential reduction effects among these competing amino acids may favor transport of certain LNAAs over others. Although it is probable that the AVP-induced reduction of LNAA transport from blood to brain is primarily an adaptive mechanism that maintains osmotic regulation in the brain, as discussed in Reichel et al. (1995), this VP– LNAA transporter interaction may have the additional effect of selectively favoring transport of those amino acids important to memory processing [e.g., phenylalanine relative to leucine (Ermisch et al., 1992) and tyrosine relative to valine (Reichel et al., 1996)].

D. Ermisch–Landgraf Proposal on the Functional Significance of Interaction of VP with the BBB In 1985 Ermisch, Landgraf, and colleagues (Ermisch et al., 1985b) expressed their long-held view that endogenous, physiological levels of circulating AVP or OT do not cross the BBB to inﬂuence memory processing. However, they did suggest the possibility that peripherally circulating VP may interact with the BBB to inﬂuence memory processing. That is, they cited supportive evidence that blood-borne VP binds to endothelial receptors and thereby alters BBB permeability to substances such as orotic acid, which in turn contribute to memory processing (Landgraf et al., 1978). Five years later, Ermisch and Landgraf (1990) formulated a model that emphasized the synergistic relationship between centrally and peripherally acting vasopressin in memory processing. A putative peripheral–central

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synergism in neurohypophysial peptide functioning under certain circumstances is in accord with experimental observations that have indicated a simultaneous release of peripheral and central VP and/or OT in laboratory rats monitored under various stimulus conditions. These have included strong osmotic stimulation (intraperitoneally injected hypertonic saline) (Demotes-Mainard et al., 1986; Landgraf et al., 1988); experimentally induced hypovolemia (removal of blood from the right atrium) (DemotesMainard et al., 1986); electrical stimulation of the hypothalamic SON (Demotes-Mainard et al., 1986) or PVN (Landgraf et al., 1990); and during controlled observations of suckling in lactating rats (Neumann and Landgraf, 1989) and of rats near term and during parturition (Landgraf et al., 1991b). The Ermisch–Landgraf model proposing a peripheral–central synergistic action of VP in information (memory) processing is diagrammed in Fig. 13. The central component of the inﬂuence of VP on memory processing is the

FIGURE 13 Vasopressin and neuronal performance. Signaling: AVP molecules (closed triangles) are produced by distinct hypothalamic neurons and transported through the axons to different terminals. There, the peptidic signal will be released. Information processing: The AVP molecules addressed to other neurons of the brain occupy AVP receptors and induce alterations in the electrophysiologically detectable postsynaptic events, which lastly alter the information processing of the neuron. Microcirculation: There is some evidence that AVP molecules are released from terminals contacting the wall of blood vessels within the brain. The effect of released AVP molecules occupying receptors at, e.g., the smooth muscles might be an alteration of the brain microcirculation. Transport: AVP molecules released into the bloodstream occupy receptors at the luminal surface of the endothelial cells of the brain vessels and induce an alteration of the transport of essential substances (closed circles) from blood to brain. The altered transport of essential substances across the BBB is necessary for the metabolic supply of activated neurons. Lastly, identical peptidic molecules, AVP, induce different reactions at different targets that serve synergistically for neuronal performance. Source: Ermisch and Landgraf, 1990 (Fig. 1, p. 72). Copyright ß 1990 by Plenum Press. Reprinted with permission.

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VP-containing pathways that project to limbic system structures, especially the septal–hippocampus system, where VP performs transmitter/neuromodulator actions (see Chapter 5). The peripheral component is the release of AVP from the posterior pituitary gland into the systemic circulation where, within a few seconds, the peptide is distributed to all regions of the vertebrate body including the vessels of the brain microvasculature. In memory-processing sites, circulating VP interacts with V1 receptors on the lumenal surface of the capillary endothelium, and initiates a cascade of biochemical reactions. This activity results in the modulation of BBB permeability to substances (e.g., orotic acid and/or neutral amino acids) involved in memory processing. Although Ermisch and Landgraf (1990) do not propose the entry of peripheral VP or metabolites into the brain as suggested by De Wied and colleagues (see Chapters 2–5), they offer an alternative mechanism by which circulating hormones can contribute to memory processing.

VII. Chapter Summary and Commentary

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Initial discussion pointed out the important role played by the brain– ﬂuid barriers in regulating nutrient transport into and out of the brain, and in maintaining a brain microenvironment conducive to normal neuronal functioning. Because elaborate and complex barrier mechanisms mediate this regulation, it has been difﬁcult to envision BBB penetration by peripherally circulating VP or OT. Nevertheless, there are several views of how behavioral effects could result from peripheral administration of these peptides. Two of these have been extensively studied: (1) peptide entry into the brain after penetration of the brain–ﬂuid barriers; and (2) peptide inﬂuence on the barrier transport of substances that contribute to memory processing. Peptide entry into the brain after penetration of the brain–ﬂuid barriers: Studies relevant to penetration of the brain–ﬂuid barriers have been of two types. One type used bioassay or radioactive labeling to learn whether increases in circulating VP and/or OT can penetrate the blood–CSF or BBB. Studies of this type increased plasma peptide levels by applying stimuli that released AVP or by peripheral injection of AVP or OT, but had a variety of drawbacks that compromised interpretation of their results. First, although CSF levels of VP were increased under conditions known to release AVP (e.g., general anesthesia, vagal stimulation) it was not clear to what degree this effect was due to the release of central AVP by these conditions (Heller et al., 1968). Second, increases in AVP levels in the CSF or brain after peripherally administered AVP could have resulted from a temporary opening of the BBB due to hypertensive effects associated only with high dose levels of the injected peptide (Heller et al., 1968; Mens et al., 1983; Zaidi and Heller, 1974). Third, in studies using radioactive labeling, there were problems in assessing how much of the radioactive label was indicative of

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metabolically altered rather than intact peptide (Ang and Jenkins, 1982), and whether the radioactively labeled tracer remained within the brain microvasculature rather than entering neural tissue (Deyo et al., 1986). Studies of the second type were designed to learn whether AVP or OT may be taken up by barrier transport systems, and if so, to characterize the transport kinetics of these systems. These studies have shown that BBB uptake of vasopressin and/or oxytocin is greater than that for the relatively impermeable tracers such as inulin (Ermisch et al., 1992; Landgraf et al., 1979) and mannitol (Zlokovic et al., 1990, 1992). The studies by Zlokovic and colleagues (1990, 1992) have identiﬁed, characterized, and localized an AVP inﬂux system on the luminal side of the BBB that transports intact blood-borne AVP into the brain. Once in the brain this peptide appears to undergo rapid metabolism possibly forming metabolites relevant to memory processing as proposed by De Wied et al. (see Chapter 5). To this author’s knowledge an inﬂux system for OT has not yet been demonstrated, and remains an important topic to investigate. Speciﬁc efﬂux transport systems for AVP (PTS-2) and OT (PTS-1), demonstrated by Banks and colleagues (Banks et al., 1987a; Durham et al., 1991), provide important mechanisms for regulating levels of these peptides in the ﬂuid compartments of the brain. These efﬂux transport systems, together with the complementary AVP inﬂux transport system demonstrated by Zlokovic and colleagues (1990, 1992), are consistent with a revised picture of the BBB. Rather than the old view of the BBB as a static, walllike structure barring passage of many substances from entering the brain, it is becoming increasingly apparent that the BBB is a ﬂexible and dynamic system regulating blood–brain interaction for many substances including AVP, OT, and a variety of other peptides. AVP and OT, rather than being independently regulated in the brain and periphery, may be involved in a variety of speciﬁc functions requiring interaction between their central and peripheral levels. Peptide inﬂuence on the barrier transport of substances that contribute to memory processing: Ermisch, Landgraf, and colleagues have carried out studies of a vasopressin interaction with nutrient transporters in the BBB. Aside from an earlier demonstration of an AVP enhancement of BBB transport of a precursor of mRNA, this line of investigation has been primarily concerned with determining the inﬂuence of the peptide on transport of large neutral amino acids. The Ermisch et al. (1992) interpretation is that the interaction of VP with the LNAA transporter produces preferential transport of amino acids that are precursors for catecholaminergic and serotoninergic neurotransmitter systems. This has potential relevance for the De Wied et al. ‘‘VP/OT Central Memory Theory,’’ especially given the evidence suggesting a central VP-ergic interaction with these classic transmitter systems in memory processing (see Chapter 4). Although this is an intriguing line of research, the evidence does not yet strongly support a connection

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between the role of VP or OT in nutrient transport or other dynamic activities of the brain–ﬂuid barriers and its role in memory processing. The problem that the brain–ﬂuid barriers have posed for the theoretical views of De Wied and colleagues remains unresolved at this time, although promising leads have been suggested by the research reviewed in this chapter.

Barbara B. McEwen

Part VIII

Closing Remarks: Review and Commentary on Selected Aspects of the Roles of Vasopressin and Oxytocin in Memory Processing

I. Chapter Overview

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After this overview (Section I), Chapter 15 is divided into the following major areas: Section II, a retrospective summary highlighting the progress made in vasopressin/oxytocin (VP/OT) memory-processing research from its inception in the mid-1960s to the present; Sections III.A–III.C, and Sections IV.A and IV.B, a discussion of a number of major themes, issues, and study areas that have emerged in the course of the research presented in this text; Section III.A pertains to the role of VP, and Section III.B to the role of OT, in memory processing tested in appetitive and avoidance paradigms especially relevant to self-preservation. Viewpoints and relevant evidence relating to a number of issues raised in the course of these investigations are reviewed and summarized, followed by relevant commentaries. Section III.C reviews and updates evidence relevant to the neuropeptide concept as it applies to the effects of VP and OT on memory processing (i.e., that these peptides are Advances in Pharmacology, Volume 50 Copyright 2004, Elsevier Inc. All rights reserved. 1054-3589/04 $35.00

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precursors of metabolic fragments that exert highly potent effects on memory processing via central and not peripheral hormonal actions). The discussion in Section IV reviews major theoretical positions and issues, summarizes relevant evidence, and provides follow-up commentaries about the roles of VP (Section IV.A) and OT (Section IV.B) in olfactory-based social recognition memory (SRM). Species-typical conspeciﬁc recognition behavior is necessary for social interactions directly involved in reproductive behavior and thus species preservation. Section V comments on the future course of research in this ﬁeld of inquiry.

II. In Retrospect: Historical Highlights in the Study of the Roles of VP and OT in Memory Processing

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More than 30 years ago De Wied and colleagues carried out the initial studies that led to the conclusion that these neurohypophysial peptides have a role in learning and memory in addition to their well-known endocrine effects. Between 1965 and the mid-1980s, they investigated the effects of these peptides on the acquisition, storage, and retrieval phases of memory processing, mainly in avoidance learning paradigms. The single-trial stepthrough passive avoidance (PA) learning paradigm was frequently used because of its value in dissociating treatment effects on memory consolidation from those on memory retrieval. Within this time frame, their experimental protocols permitted study of (1) the effect on memory processing of increasing VP or OT levels in systemic circulation, in the brain as a whole, or within speciﬁc brain structures implicated in memory processing; (2) the physiological role of endogenous hormonal and neuronal VP and OT in memory processing; (3) the inﬂuence on memory processing of interactional effects between these neuropeptides and the catecholamine neurotransmitter systems that project to speciﬁc brain sites involved in memory processing; (4) a putative role for VP and OT in the memory consolidation that is theorized to occur during the rapid eye movement (REM) sleep state; (5) the inﬂuence on memory processing exerted by VP and OT peptide fragments formed by proteolytic breakdown of the parent peptides within the brain; and (6) the role of the neuromodulatory action of VP on glutamate neurotransmission within the septal and hippocampal circuitry, which may be directly involved in mediating certain types of VP-dependent memory. Thus, by the mid-1980s the experimental ﬁndings were in place that provided basic support for the De Wied et al. ‘‘VP/OT Central Memory Theory’’ (see Chapters 2–5). During the early 1980s, several other research groups—Koob et al. (see Chapter 6), Sahgal et al. (see Chapter 7), and Beckwith et al. (see Chapter 8)—focused on the role of VP in learning and memory. At the outset these groups challenged the ‘‘VP/OT Central Memory Theory,’’ particularly

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objecting to the proposal that VP exerts a direct neuronal action at brain sites involved in long-term memory storage and retrieval. Instead, they agreed that the arousal system plays a key role in the inﬂuence of VP on memory processing. Speciﬁcally, Koob et al. formulated the the ‘‘VP Dual Action Theory’’ (see Chapter 6), and Sahgal et al. presented the ‘‘VP Central Arousal Theory’’ (see Chapter 7), which explained the memory-processing effects of VP as a result of a primary inﬂuence on the arousal system, which secondarily inﬂuences the various phases of memory processing. Beckwith et al. (see Chapter 8) proposed that the retention effects of VP are attributable to a primary inﬂuence on attention. The ensuing debate among these research ‘‘rivals’’ did much to stimulate and shape research throughout the 1980s. Numerous other researchers interested in the role of VP in memory processing then further expanded knowledge in this ﬁeld of study (see Chapters 9–11). In the late 1980s and early 1990s, Dantzer, Bluthe, and colleagues (the ‘‘Paris contingent’’ of the Koob et al. research team) played a major role in launching a new subﬁeld of VP memory research: the role of VP in a speciespredictable type of memory necessary for conspeciﬁc recognition, rodent olfactory-based social recognition memory (SRM) (see Chapter 12). Subsequent study of SRM by other research groups included OT in many research paradigms, perhaps in part because of its demonstrated role in reproduction-related social behavior (see Chapter 13). With the exception of the research by De Wied and colleagues, and these more recent studies of SRM, the role of OT in memory processing has had scant research attention. It is interesting that although OT has been dubbed a natural amnestic by De Wied and colleagues (Bohus et al., 1978a) because of its attenuation of memory consolidation and retrieval in aversive research paradigms (see Chapters 2, 4, and 5), low dose levels (i.e., closer to physiological values) have also been shown to facilitate memory processing in olfactory-based SRM paradigms (see Chapter 13).

III. VP and OT and Memory Processing: Avoidance and Appetitive Learning Paradigms

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A. VP and Memory Processing 1. Central versus Peripheral Locus of Action for the Memory-Processing Effects of Peripherally Administered VP a. Contrasting Views of De Wied et al. and Koob et al. A major question debated by these two research groups is whether the memory-modulating effects of peripherally administered arginine vasopressin (AVP) is attributable to a peripheral or a central action of the peptide. Both groups agree that a supraphysiological dose of the peptide produces pressor and aversive effects that increase the subject’s arousal level and thereby affect one or more

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stages of memory processing. In contrast to the Koob et al. proposal that this event sequence is the mechanism by which the peptide achieves its effects on learning and memory, De Wied and colleagues argue that it is the central, not the peripheral, actions of the peptide that are essential for its effects on the memory storage and retrieval stages of memory processing. The De Wied et al. position on this issue requires an explanation of how the peripherally administered peptide gains access to central VP receptor sites. One possibility is that a sufﬁcient fraction of the injected peptide, or of one of its behaviorally active AVP metabolites, crosses the blood–brain ﬂuid barriers and activates central VP receptors. Koob and colleagues, as well as numerous other researchers (Pardridge, 1983a; Reppert et al., 1981; Wood, 1982; Zaidi and Heller, 1974) consider this possibility highly improbable. Evidence relevant to their respective positions has been obtained in studies by each of these research teams. The following lines of evidence support the De Wied et al. position: (1) nanogram and picogram doses of centrally administered VP produced effects on memory processing that are comparable to those produced by microgram dose levels of the peripherally administered peptide [e.g., Bohus et al., 1978b (see Chapter 2); De Wied et al., 1984a (see Chapter 3)]; (2) peripheral administration of behaviorally active VP metabolic fragments, which lack the peripheral endocrine effects of the parent peptide, are highly effective in their ability to enhance memory processing in appetitive (Vawter et al., 1997; see Chapter 2) and avoidance (Burbach et al., 1983b; see Chapter 5) learning tasks; (3) De Wied et al. (1984a; see Chapter 3) dissociated the peripheral and central effects of peripherally administered AVP in a study that examined the interactive effects between peripherally administered AVP and peripheral/central administration of a VP V1 receptor antagonist, capable of crossing the BBB. The results indicated that a subcutaneously injected dose of the receptor antagonist (sufﬁcient to reach central receptor sites) blocked both the peripheral physiological (pressor)and central behavioral (retention) effects of the peripherally administered peptide. However, an intracerebroventricular injection of the antagonist (insufﬁcient to reach peripheral AVP receptor sites) blocked the memory but not the pressor effects induced by peripherally administered AVP; and (4) in a study using radioimmunoassay technology, Mens et al. (1983; see Chapter 14) reported ﬁndings indicating that tiny amounts (nanogram to picogram quantities) of a large dose of subcutaneously injected radiolabeled AVP (2.0 or 5.0 g/rat) were able to penetrate blood–brain ﬂuid barriers and enter the cerebrospinal ﬂuid (CSF). This evidence supported the hypothesis that a supraphysiological dose of peripherally administered peptide could access the CNS in behaviorally effective quantities. However, the dose levels used in this study were far greater than the 1-g/rat dose level typically used for peripheral administration in the behavioral studies cited in this text.

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Two lines of evidence support the position held by Koob and colleagues: (1) Lebrun et al. (1984) tested the effects of a centrally administered VP receptor antagonist on the pressor and retention effects induced by peripherally injected AVP. Although the VP antagonist used in this study was different from that used by De Wied et al. (1984a) it was equally capable of crossing the blood–brain barrier (BBB) (Koob et al., 1986). This study did not dissociate the peripheral and central actions of peripherally administered AVP. Instead, the V1 receptor antagonist blocked the retention effect induced by peripherally injected VP, but only at a dose level that also blocked the agonist pressor effect (i.e., a dose level sufﬁcient to diffuse across the BBB and reach the peripheral V1 receptor sites). The explanation for the opposing ﬁndings of Lebrun et al. (1984) and those of De Wied et al. (1984a) is as yet unknown. However, it does not appear to be due to the use of a different VP receptor antagonist, because Koob et al. (1989) replicated the results of Lebrun et al. (1984) in a follow-up experiment with the same V1 antagonist used by De Wied et al. (1984a); and (2) although Deyo et al. (1986; see Chapter 14) found traces of immunoreactive (ir) AVP in BBB-protected brain sites after a subcutaneous injection of a dose of the peptide approximating that typically used in behavioral experiments, a brain perfusion control procedure indicated that the irAVP had remained lodged within the brain microvasculature and had not penetrated the BBB. b. Commentary Despite a considerable amount of research (see Chapter 14), the question of whether peripherally administered AVP or related peptides can penetrate the blood–brain ﬂuid barriers and activate central VP receptors has not as yet been resolved. Although there is deﬁnitive support for carrier-mediated transport of AVP across the BBB, both in the blood-tobrain (Zlokovic et al., 1990, 1992) and brain-to-blood (Banks et al., 1987a) directions, this demonstration of the ability of the peptide to penetrate the BBB offers no assurance that it can reach and activate central receptor sites involved in memory processing for the following reasons: (1) the functions served by these transport systems have yet to be clariﬁed; (2) Zlokovic’s ﬁndings (Zlokovic et al., 1990, 1992, 1993) indicate that the intact VP taken upon the blood side of the BBB might be quickly metabolized at some point on the brain side of the BBB, and if so, it is not known whether these VP metabolites are behaviorally active ones; and (3) the presence of the VP efﬂux system demonstrated by Banks et al. (1987a) suggests that even if behaviorally active VP is transported into the brain, it may not accumulate to a sufﬁcient degree to produce a behavioral effect of the kind speciﬁed by the ‘‘VP/OT Central Memory Theory.’’ It is noteworthy that the problem of explaining behavioral effects after peripheral administration is not speciﬁc to the neurohypophysial hormones. Numerous stress-activated anterior pituitary and adrenal hormones [e.g., adrenocorticotropic hormone (ACTH), endorphins, enkephalins, and

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adrenaline do not readily cross the BBB, but also modulate memory processing after peripheral administration. McGaugh and colleagues (McGaugh, 1989; McGaugh and Gold, 1989) have consistently demonstrated that peripherally administered adrenaline dose dependently modulates memory processing in the passive (inhibitory) avoidance paradigm. Their more recent studies suggest that this memory modulation effect involves noradrenaline release in the basolateral amygdala via a catecholaminergic pathway initiated by adrenaline activation of peripheral receptors of the vagus nerve (Cahill and McGaugh, 1996; Williams et al., 1998). To this author’s knowledge, the question of whether vagal nerve endings contain receptors for AVP has not as yet been raised or explored. If they do, it would provide another route by which the neurohypophysial peptide could access central brain sites involved in memory processing. Peripherally administered adrenaline can also facilitate memory processing by virtue of its ability to increase glucose availability to the brain (Gold, 1995). This comprises a parallel but independent mechanism by which the amine improves memory processing, because it results in direct glucose actions at several brain areas (e.g., hippocampus) that release acetycholine (Korol and Gold, 1998; Messier et al., 1990) rather than catecholamines as occurs in the epinephrine-activated pathway to the basolateral amygdala. The evidence, cited in Chapter 1, suggesting that stress-associated increases in hormonal AVP can increase glucose availability to the brain is consistent with the speculation that this provides yet another mechanism by which peripherally administered AVP may contribute to memory processing. In this context, it is noteworthy that the inﬂuence of AVP on memory processing is closely afﬁliated with adrenergic catecholamines at both peripheral (Borrell et al., 1983a,b) and central (Kovacs et al., 1977, 1979a,b, 1980) levels. This afﬁliation is more fully reviewed and discussed in a later section of this chapter. 2. Position Statements on the Role of Peripherally Circulating VP in Memory Processing a. De Wied and Colleagues Numerous lines of evidence obtained by De Wied and associates led them to conclude that, in contrast to centrally released VP, release of the peptide into systemic circulation does not play any essential role in stress-associated learning encounters (see Chapter 3). These studies, reviewed in Chapter 3, showed that (1) neutralization of central, but not peripheral, VP severely impaired PA retention whereas the converse treatment regimen was without effect, even though the amount of antiserum injected peripherally was 100 times greater than that used for central application (Van Wimersma Greidanus et al., 1975a); (2) plasma levels of VP were not increased during footshock (FS) training in either active (Van Wimersma Greidanus et al., 1979b) or passive (Laczi et al., 1983c; Mens et al., 1982a) avoidance paradigms; (3) PA retention scores (reentry

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latency) increased in correspondence with increases in intensity of the training footshock but in the absence of an increase in plasma levels of VP (Laczi et al., 1983c; Thompson and De Wied, 1973); and (4) the increase in the plasma level of VP that occurs after the conclusion of the PA retention trial was interpreted as a hormonal response to the conditioned fear cues present in the test (Laczi et al., 1983c; Thompson and De Wied, 1973); hence it was a memory retrieval-mediated and not a learning-mediated effect. Other investigators have noted that footshock and a number of other types of emotional stress fail to release hormonal VP from the posterior pituitary gland (Onaka, 2000; see Chapter 1), but do induce VP release in the portal circulation (e.g., Bartanusz et al., 1993; De Goeij et al., 1992a,b; and see Chapter 1) and at central brain sites (Engelmann et al., 2000; Landgraf et al., 1998; Nishioka et al., 1998; Wotjak et al., 1998). Taken together, these results support the proposal that during a stress-associated learning encounter, VP released at central brain sites, and perhaps also in the portal but not the systemic circulation, has an essential role to play in the memory processing occurring at this time. b. Koob and Colleagues The writings of the Koob et al. research team make clear their position that VP actions at both peripheral and central receptor sites contribute to memory processing, albeit indirectly by a VP interaction with the arousal system. Speciﬁcally, they theorize that VP enhancement of behavioral arousal is accomplished through different mechanisms by peripherally and centrally localized VP; thus the dual action nature of the effect of VP in memory processing. Moreover, these independent but homologous inﬂuences on the arousal system may act in parallel and in response to the same stimulus, or at different times to independently occurring events. An example of the former is the simultaneous release of VP at central brain sites and into the systemic circulation in response to an osmotic stimulus (e.g., an injection of hypertonic saline) (see Chapter 6). It is clear that the Koob et al. position on the dual actions of hormonal and neuronal VP in memory processing is consistent with their view that VP does not readily penetrate the BBB (noted above), and that interaction of the peptide with the arousal system is of primary importance in mediating the effects of VP on memory processing (discussed in Section III.A.3). c. Commentary The suggestion that endogenous VP localized at nonneural tissue sites might be implicated in memory processing was previously presented in Chapters 1 and 14. In general, VP may inﬂuence the delivery of glucose (energy) and other nutrients to brain sites actively engaged in processes supporting memory processing (e.g., selective attention) or to one or more phases of memory processing itself. Relevant observations consistent with this suggestion include the following: (1) VP may regulate cerebral blood ﬂow and thereby inﬂuence the rate of delivery of these nutrients to

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metabolically active brain sites including those involved in memory processing; (2) VP localized on the blood side of the BBB appears to inﬂuence transport of orotic acid and certain large neutral amino acids into the brain, where these substances, in turn, may contribute to memory processing, for example, by acting as precursors for neurotransmitters engaged in memory processing (Ermisch and Landgraf, 1990; see Chapter 14); (3) VP enhances glucose release from storage depots in the liver, thereby making this nutrient available to the brain, where it has been demonstrated to enhance memory processing (Cahill and McGaugh, 1996; Gold, 1986; Messier and White, 1987); and (4) VP, acting at the level of the anterior pituitary and adrenal medulla during stress, contributes to the release of a number of stress hormones, which, according to behavioral pharmacological study, have a role in memory processing (see Chapter 1). These nonneural actions of VP may thus provide a parallel and independent mechanism complementing the memory modulation provided by centrally acting VP. With the exception of the research by Koob et al. (see Chapter 6), and by Ermisch, Landgraf, and colleagues (see Chapter 14), little research attention has been given to potential supplementary mechanisms by which VP actions at nonneural tissue sites contribute to memory processing. 3. Importance of the Arousal System in Mediating the Influence of VP on Memory Processing a. Viewpoints of De Wied and Colleagues and of the Arousal-Centered Theorists i. De Wied and colleagues As noted earlier, De Wied and colleagues acknowledge that VP can modulate behavioral arousal but argue that the inﬂuence of the peptide on the memory storage and retrieval stages of memory processing is independent of this arousal effect. More speciﬁcally, their position on this issue may be characterized as follows: (1) VP can modulate the subject’s baseline arousal level during learning encounters; (2) the VP-induced shifts in arousal level have prominent effects on both attention processing and on the short-term memory processing that occurs during learning, but these arousal shifts are neither necessary nor sufﬁcient for the effect of the peptide on memory consolidation and retrieval; and (3) the inﬂuences of VP on these latter phases of memory processing involve a neuronal action at brain sites engaged in either memory modulation (facilitate or inhibit the formation of numerous types of memory stored elsewhere in the brain) or in memory storage per se. Mechanisms by which these mnemonic actions of VP are accomplished are reviewed and discussed in follow-up commentaries in later sections (Sections III.A.5 and III.A.7) of this chapter. ii. Arousal-centered theorists According to Koob and associates (see Chapter 6), a pressor/aversive effect of peripherally administered AVP activates the central arousal system via noxious signals carried by the

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peripheral nervous system. Although pressor activity is not associated with the centrally administered peptide, a homologous arousal state is achieved by a direct effect on the pathways comprising the central arousal system. Sahgal et al. (see Chapter 7) also theorized that a VP-induced arousal effect mediated the inﬂuence of the peptide on behavioral performance in learning/memory paradigms. Their research was focused on demonstrating that, whether peripherally or centrally administered, the effect of VP in these paradigms was best explained in accordance with an arousal as opposed to a direct mnemonic action of the peptide. In accordance with the postulated inverted U-shaped functional relationship between arousal level and performance efﬁciency, VP administration may or may not enhance any of the stages of memory processing, an outcome that is not expected if the direct action of the peptide was on enhancing memory storage and retrieval. Although initially reluctant to accept the ‘‘VP Dual Action Theory’’ in its entirety, Sahgal et al. subsequently agreed that the cardiovascular action of peripherally circulating VP could generate signals that increased the arousal level, based in part on continued accumulation of supportive evidence and acceptance of the view that the neurohypophysial peptides were unlikely to cross the BBB (Sahgal, 1986). This essentially led to rapprochement between the ‘‘VP Dual Action Theory’’ and the ‘‘VP Central Arousal Theory.’’ b. Relevant Research i. De Wied and colleagues Three types of experimental ﬁndings by De Wied and colleagues support their position that a VP-induced arousal effect cannot fully account for the effect of the peptide on performance in tests of memory storage and retrieval: (1) in contrast to the short-term inﬂuence of ACTH-like peptides [-melanocyte-stimulating hormone (-MSH); ACTH(4–10)] in maintaining a previously learned avoidance response (lasts the length of time that the peptide treatment remains in the body), vasopressin exerts a long-term effect that lasts hours, days, and even weeks after treatment is discontinued (Ader and De Wied, 1972; Bohus, 1977; De Wied, 1971; De Wied and Bohus, 1966; see Chapter 2). The short-term effect suggests activation of a mechanism (arousal, attention to the stimulus trace) that can sustain memory of recent learning for a brief time, whereas the long-term effect indicates an action on memory consolidation that constitutes a more permanent change in the CNS (De Wied and Bohus, 1966, and see discussion in De Wied et al., 1974; De Wied and Gispen, 1977); (2) peripherally administered VP metabolites, which lack the pressor effects of the parent peptide [e.g., desglycinamide-arginine vasopressin (DG-AVP) and C-terminal fragments such as AVP(4–8), AVP(5–8), and AVP(5–9)] facilitate PA memory consolidation and retrieval (Gaffori and De Wied, 1986), retention of a conditioned FS avoidance response (De Wied et al., 1972, 1987), and sexually rewarded learned spatial discrimination (Bohus,

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1977), food-rewarded reference memory tested in a hole board search task (Vawter et al., 1997), as well as retrieval tested by the ability of the peptide to prevent experimentally induced retrograde amnesia (Rigter et al., 1974); and (3) Skopkova et al. (1987, 1991) showed that a DG-AVPassociated arousal effect inﬂuenced the rate of acquisition of a shuttlebox avoidance response in accordance with the postulated inverted U-shaped arousal–performance efﬁciency curve; however, the effect of the peptide on the long-term retention of the learned response was independent of this arousal effect on the short-term memory processing involved in response acquisition. ii. Arousal-centered theorists The strongest support for the Koob et al. position regarding peripherally circulating VP was obtained from VP receptor antagonist treatment protocols that indicated that the memoryprocessing effects induced by high levels of peripheral VP were dependent on pressor/aversive effects of the hormone: (1) treatments that increased peripheral levels of AVP, sufﬁcient to produce a pressor effect, enhanced retention in active avoidance (Koob et al., 1985a; Le Moal et al., 1981), passive avoidance (Lebrun et al., 1984), and water-ﬁnding appetitive (Ettenberg et al., 1983a) learning paradigms. These retention effects were prevented by pretreatment with a V1 receptor antagonist that also blocked the pressor effect (e.g., Koob et al., 1985a; Lebrun et al., 1984; Le Moal et al., 1981); and (2) treatment that increased the plasma level of VP sufﬁciently to raise blood pressure (BP) was also accompanied by aversive properties (Dantzer et al., 1982; Ettenberg et al., 1983a), and this pressor/aversive effect was necessary for the VP-induced facilitated retention effect in both avoidance (Le Moal et al., 1981) and appetitive (Ettenberg et al., 1983a; Packard and Ettenberg, 1985) learning tasks. Two sets of experimental ﬁndings support the thesis that different mechanisms mediate the arousal effect induced by peripherally and centrally localized VP in memory processing: (1) treatment that induces small increases in central levels of VP enhances long-term memory in an active avoidance paradigm, without exerting pressor or aversive effects (Koob et al., 1986); and (2) peripherally injected VP at a dose that increased BP and enhanced memory processing, and centrally injected VP at a dose level that enhanced memory processing without inﬂuencing BP, both increased the subject’s arousal level. This was indicated by the neocortical electroencephalographic (EEG) proﬁle and degree of alert behavior observed in the VP-treated rats compared with the controls (Ehlers et al., 1985). In comparison with Koob et al., the Sahgal research group carried out relatively few research studies on the role of VP in memory processing. Using heterogeneous samples of laboratory rats, their studies (see Chapter 7) yielded a number of ﬁndings consistent with their thesis that an increase in behavioral arousal, rather than a direct mnemonic action, accounted for the

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effects of VP in tests of learning as well as long-term memory: (1) VP did not unequivocally enhance retention tested in the step-through PA task (e.g., Sahgal and Wright, 1983; Sahgal et al., 1982) or in the combined active/ passive avoidance test paradigm (e.g., Sahgal, 1986; Sahgal and Wright, 1984); (2) Centrally (Sahgal et al., 1982) as well as peripherally (Sahgal and Wright, 1983, 1984) administered VP produced a bimodal distribution of PA retention scores, an outcome in accordance with the postulated Ushaped arousal–performance efﬁciency relationship; (3) peripherally administered VP disrupted acquisition of an autoshaped lever touch response in moderately aroused female homozygous diabetes insipidus (HODI) rats (Sahgal, 1983), and produced a bimodal effect (tendency) on acquisition of this response in normal female rats (Andrews et al., 1983); and (4) a VPinduced performance effect observed in delayed matching (Sahgal, 1987a; Sahgal et al., 1990) and nonmatching (Sahgal et al., 1990) to position tasks suggested an enhancement of short-term memory. However, further examination of the improved performance scores indicated that they resulted from a position bias produced in large part by a VP-induced increase in behavioral arousal. c. Commentary As noted above, the ‘‘arousal-centered theorists’’ provide convincing support for their position that (1) sufﬁcient increases in peripheral and/or central levels of VP increase behavioral arousal, which in turn modulates performance in learning and memory tasks, and (2) the nature of this performance effect is in accordance with the inverted U-shaped arousal–performance efﬁciency curve. Moreover, VP interacts with both central noradrenergic (Kovacs et al., 1979b) and cholinergic (Baratti et al., 1989; Faiman et al., 1987, 1988, 1991) transmitters in memory processing. These transmitter systems are well known for their effects on cortical activation and correlated behavioral arousal during the waking state (Aston-Jones, 1985; Aston-Jones et al., 2001; Berridge and Foote, 1991, 1996; Cape and Jones, 1998; Jones, 1993). De Wied and colleagues, although not denying a VP-induced arousal effect, have provided equally strong evidence (see above) that this peptide inﬂuences long-term memory (LTM) formation and retrieval independent of its arousal effects. Of relevance to the De Wied et al. position is the fact that central monoaminergic and cholinergic transmitter projection systems that contribute to cortical activation and behavioral arousal also terminate in limbic brain structures, where they serve as memory modulators (systems, extrinsic to the neural circuitry that stores the memory, that nevertheless contributes to the strength of the memory formed therein) [see Martinez et al. (1991) and Cahill and McGaugh (1996) for discussion of the concept of memory modulation]. In their studies of emotional memory, McGaugh and colleagues (e.g., Cahill and McGaugh, 1996; McGaugh, 2000) have provided evidence that

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has traced the linkage between activation of the stress hormone adrenaline, and the release of noradrenaline (NA) into the basolateral amygdala (BLA), where it interacts with glucocorticoids, and numerous neurotransmitters [e.g., acetylcholine, opiate peptides, and -aminobutyric acid (GABA)] in modulating the formation of memories consolidated elsewhere in the brain. Aside from the BLA the septohippocampal system may be an additional brain region involved in the modulation of memory consolidation (Cahill and McGaugh, 1996). This latter brain site may be important for mediating the modulatory effects of VP on memory consolidation via its interaction with central NA-ergic and/or cholinergic systems (Section III.A.5). It is possible that VP interacts with septohippocampal cholinergic circuitry to modulate memory consolidation during the REM sleep state. Consistent with this hypothesis are anatomical, electrophysiological, and behavioral observations that suggest that (1) forebrain cholinergic projection systems contribute to both the neocortical EEG activation pattern and to the hippocampal EEG theta pattern that accompany the REM sleep state (Apartis et al., 1998; Szymusiak, 1995); and (2) hippocampal theta is a neural correlate of memory consolidation during REM sleep, and VP is involved in the regulation of this theta activity during REM sleep (see Chapter 5) and has been implicated in memory consolidation within the hippocampal system (see Chapters 4 and 5). Moreover, the postulated VP modulation of memory consolidation during REM sleep occurs at a point in the sleep–wake cycle when cortical activation is high but behavioral arousal is negligible (see Chapter 5). Taken together, these data lead this author to conclude that (1) VP does inﬂuence memory processing as well as other types of cognitive processing by virtue of its enhancement of behavioral arousal, and (2) in addition, VP exerts a mnemonic role in memory processing that is independent of its role in behavioral arousal. Thus, in agreement with De Wied and colleagues, the VP-induced arousal effect is herein considered neither necessary nor sufﬁcient to explain the mnemonic role of the neuropeptides in memory processing. Further discussion of this mnemonic role of VP in memory processing is presented in Sections III.A.5, III.A.6, and III.C. 4. Influence of VP on Attentional Processing and Its Relationship to Memory Processing a. Views of Beckwith and Colleagues and of Strupp, Bunsey, and Colleagues On the basis of their experimental ﬁndings and those of others, Beckwith and colleagues have concluded that VP enhances selective and other types of attention during various stages of cognitive processing, including those in which memory encoding and retrieval occur. In the latter cases, VP enhancement of attention is a primary contributor to the effect of the peptide on memory processing. Moreover, the interaction of VP with the locus coeruleus noradrenergic (LC–NA-ergic) system may be an important

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mechanism by which the peptide exerts its inﬂuence on attention and memory processing. On the other hand, their studies with intranasally applied 1-desamino-8-d-arginine vasopressin (DDAVP) in human subjects have dissociated the role of the peptide in attentionally mediated memory processing from any postulated pressor/aversive effect of the peptide. Strupp, Bunsey, and colleagues hold a similar viewpoint that can be characterized as follows: (1) VP inﬂuences a variety of cognitive operations, which include attentional mechanisms (vigilance, selective attention, divided attention), and all three stages of memory processing [memory encoding (acquisition), memory consolidation, and memory retrieval] (Bunsey and Strupp, 1990; Bunsey et al., 1990; Strupp, 1989; Strupp et al., 1990); (2) VP modulates arousal level, either on its own or more probably by means of its interaction with one of the transmitter systems comprising the central arousal system, especially the LC–NA transmitter system (Bunsey et al., 1990; Strupp et al., 1984); and (3) a VP-induced increase in arousal level has been shown to increase vigilance (heightened surveillance of the environment) as well as selective attention (narrowing the range of cues under perceptual focus) (Bunsey et al., 1990; Strupp et al., 1984), which, in turn, may contribute to the acquisition phase of memory processing. In addition, VP-enhanced arousal may also increase concentration and thereby facilitate memory retrieval (Strupp et al., 1984). b. Supportive Evidence of a VP Role in Attentional Processing A vasopressininduced enhancement of attentional processing has been demonstrated for human subjects in the behavioral and electrographic studies cited in Chapter 8. The behavioral studies of Beckwith and associates showed that intranasally applied DDAVP facilitated (1) selective attention on a conceptual shift discrimination task (Beckwith et al., 1982); (2) attention (alertness, digit encoding, and response selection), but not memory as tested and operationally deﬁned in the Sternberg item recognition task (Beckwith et al., 1983); (3) immediate and/or delayed recall of taped presentations of implicational sentences (Beckwith et al., 1984; Till and Beckwith, 1985), where VP presumably facilitated attentional processing that was active during both the encoding and retrieval stages of memory processing (Till and Beckwith, 1985); and (4) immediate recall of each of a number of tape-recorded passages of prose where recall was dependent on the selective and divided attentional processing that occurred during memory encoding (Beckwith et al., 1987a). The electrographic ﬁndings of Fehm-Wolfsdorf and colleagues (Born et al., 1986; Dodt et al., 1994; Fehm-Wolfsdorf et al., 1988; Pietrowsky et al., 1989, 1996) were interpreted as a VP-induced enhancement of attentional mechanisms present at many stages of stimulus processing. Given this, they adopted the view that, in humans, VP inﬂuences many cognitive functions, primarily by ‘‘an action on basic mechanisms involved in the general

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regulation of central nervous activation, i.e., on arousal systems that could also alter affective aspects of stimulus processing’’ (Fehm-Wolfsdorf and Born, 1991, p. 1399). Animal research reported in Chapter 8 also supported a VP inﬂuence on attentional processing that was relevant to learning and memory: (1) Beckwith and colleagues (Beckwith and Tinius, 1985; Beckwith et al., 1987b; Tinius et al., 1989) concluded that chronic treatment with peripherally administered AVP during original learning in a Wþ/B discrimination reversal paradigm enhanced selective attention to the relevant stimulus dimension, because this treatment facilitated learning the reversed discrimination as predicted by the attentional model developed by Mackintosh and Sutherland (Mackintosh, 1965; Sutherland and Mackintosh, 1971); (2) Bunsey et al. (1990) interpreted the results of two separate experiments as indicating that peripherally administered AVP(4–9) enhances selective attention to highly salient cues in the stimulus environment. In experiment 1 salience was determined by dominant perceptual attributes, and in experiment 2, by previous experience with the stimulus cues; and (3) Meck (1987) found that peripherally administered AVP(4–9) enhanced performance in a task that required dividing attention among three simultaneously presented stimulus cues to efﬁciently obtain a food reward under the FI schedule in effect during any speciﬁc trial. c. Commentary: Relationship between the Effects of VP on Activation/Arousal, Attention, and Memory Processing i. Relationship between the role of VP in attention processing and its effects on memory processing The Fehm-Wolfsdorf et al. human studies using EEG recordings provide consistent evidence in support of the view that VP can facilitate attentional mechanisms during various stages of stimulus processing. The behavioral studies with human and animal subjects carried out by Beckwith and colleagues, and the animal studies of Bunsey et al. and Meck, implicate a VP-induced inﬂuence on both selective and divided attentional processing present during at least the learning and retrieval stages of memory processing. Given this evidence, it would seem plausible to infer that a VP facilitation of attentional mechanisms that presumably operate during the postlearning period will in turn facilitate the consolidation process underlying the formation of long-term memory. However, other ﬁndings cited in Chapter 8 were inconsistent with this inference, suggesting instead that the VP facilitation of attentional processing can be dissociated from its effect on retention tested in the same task paradigm: for example, Beckwith et al. (1987b) found that chronic posttraining AVP treatment during original learning on the Wþ/B discrimination reversal problem facilitated selective attention to the relevant stimulus dimension (i.e., to the memory traces), but did not affect long-term memory of this learned discrimination. A dissociation between the effect of VP on attentional

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processing and on retention in the same test paradigm was also observed in the Sternberg item recognition task (Beckwith et al., 1983). Taken together, the studies reported in Chapter 8 supported the argument that a VP-induced enhancement of one or more forms of attention may be causally related to enhancement by the peptide of memory encoding and retrieval, but there is no substantial experimental evidence that a VPfacilitated attentional effect is necessary to its role in memory consolidation. ii. Activation/arousal effect of VP and its putative role in attentiondependent memory processing Two sets of ﬁndings contraindicated a pressor-associated arousal effect as a causal agent in the VP-induced enhancement of attentional processing reported in Chapter 8. First, in several of their studies, Beckwith et al. (i.e., Beckwith et al., 1982, 1983) and Fehm-Wolfsdorf and colleagues (e.g., Born et al., 1986; Fehm-Wolfsdorf et al., 1988) monitored heart rate and blood pressure in their human subjects at set times during the test period, and found that intranasally administered DDAVP or other tested AVP analogs did not affect either of these parameters, although they did enhance attentional processing. Second, in their studies with laboratory rats, both Bunsey et al. (1990) and Meck (1987) showed that peripherally administered AVP(4–9) enhanced learningdependent attentional processing in the absence of a pressor-induced increase in arousal level, because this peptide lacks the endocrinological effects of its parent peptide [AVP(1–9)]. On the other hand, the studies cited in Chapter 8 do not contradict the possibility that a cortical activation/behavioral arousal effect of centrally acting VP is causally linked to the ability of the peptide to facilitate attentional processing, and more speciﬁcally attention-dependent memory encoding and retrieval. As noted earlier, on the basis of their electrographic ﬁndings, Fehm-Wolfsdorf and colleagues (Fehm-Wolfsdorf and Born, 1991) favor an arousal explanation for the VP-facilitative effect on attentional mechanisms presumed to be present in various stages of stimulus processing and contributory to a variety of cognitive activities. The VP–NA-ergic interaction (see Chapter 4, and see below, Section III.A.5) exempliﬁes one mechanism by which the VP arousal, attention, and memory-processing effects may be interlinked. Strupp, Bunsey, and colleagues (Bunsey et al., 1990; Strupp and Levitsky, 1985) cited evidence indirectly or directly supportive of a VP interaction with the LC–NA-ergic system in memory processing, cortical activation, and selective attention (and see discussion below, Section III.A.5). The speculation that the activation/arousal action that results from the VP–NA-ergic interaction is importantly involved in the modulation of memory for an emotionally arousing experience is close to a view original expressed by Kety (1972) and subsequently explicated by Harley (1987). According to this perspective, ‘‘the level of arousal at the time of learning may provide an adaptive means by

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which important events are selectively remembered and less relevant ones, forgotten’’ (Strupp et al., 1984, p. 273). More speciﬁcally, this activation/ arousal effect can assist memory processing by enhancing attentional processing during memory encoding as well as memory retrieval (Bunsey et al., 1990; Strupp and Levitsky, 1985; Strupp et al., 1984). On the other hand, observations cited by Strupp and Levitsky (1985) challenge the thesis that the arousal effects of VP fully account for its ability to enhance memory processing. For example, in contrast to VP treatment (Bartus et al., 1982), catecholaminergic drugs or other CNS stimulants were unable to improve memory functions in aged monkeys (Bartus, 1979; Bartus et al., 1983) or to reverse anisomycin-induced amnesia (Kastin et al., 1981). 5. Interactions between VP and Catecholaminergic and Cholinergic Systems: Relevance for Memory Processing a. VP–Catecholamine Interactions in Memory Processing: Relevant Evidence De Wied and colleagues initiated VP–catecholaminergic interactional studies in the late 1970s in response to a number of lines of evidence. This evidence (1) implicated a role for both hormonal and central adrenergic systems in memory processing (Crow, 1968; Kety, 1970, 1972; McGaugh et al., 1975); and (2) indicated that central adrenergic systems (NA) project to brain sites that contain VP ﬁber terminals and/or receptor-binding sites (see Chapter 1) and are involved in mediating the inﬂuence of VP in memory processing (see Chapter 4, Section III). Their ﬁndings supported a VP–catecholamine (CA) interaction in memory processing and further provided insights into the nature of the interaction, its role in memory processing, and the brain structures in which these interactional effects occurred. In addition to this research by De Wied and colleagues, Hamburger-Bar et al. (1984; and see Chapter 11) investigated a potential role for DA involvement in the VP inﬂuence on memory processing in avoidance paradigms. The relevant evidence is presented below. i. De Wied and colleagues Findings from a number of studies presented in Chapter 4 support the thesis that depending on the brain site and type of memory involved, VP inﬂuences transmission and release in one of the central CA transmitter systems [NA or dopamine (DA)], which then mediates the action of the peptide on retention and/or retrieval in the learning paradigm. Behavioral, coordinated behavioral/biochemical, and biochemical studies have provided supportive evidence of this thesis. Moreover, it appears that the central VP–NA interactional inﬂuence on memory processing requires the presence of a normally functioning adrenergic hormonal system (adrenaline). Behavioral ﬁndings in support of this thesis included the following: (1) a VP-induced enhancement of PA retention after peripherally or centrally administered VP was blocked by (a) coinjection of an enzyme that inhibited

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CA synthesis (Kovacs et al., 1977), (b) surgical removal of the adrenal medulla (source of peripheral adrenaline) (Borrell et al., 1983a,b), and (c) chemical lesions in the central LC–NA transmitter pathway to the forebrain (Kovacs et al., 1979b); and (2) posttraining AVP facilitated PA retention when locally injected into the dorsal raphe nucleus (DRN), but not when injected into the LC, indicating that AVP interacts with the LC–NA projection system at its target sites (e.g., DRN) and not at its site of origin (LC) (Kovacs et al., 1979b). In addition, prior lesioning of the DRN prevented the facilitated PA retention induced by DRN-injected AVP into the DRN, suggesting that serotonin has a secondary role in the inﬂuence of VP on noradrenergic transmission in memory processing (Kovacs et al., 1979b). Studies that obtained coordinated behavioral/biochemical ﬁndings in favor of this thesis were as follows: (1) Kovacs et al. (1979a) showed that VP microinjected into the dentate gyrus of the hippocampus facilitated both PA memory consolidation and NA neurotransmission in that structure; and (2) Veldhuis et al. (1987) demonstrated that VP antiserum attenuated PA memory consolidation and retrieval, and decreased NA release when microinjected into either the dorsal or ventral hippocampus. Supportive evidence cited in Chapter 4 was also obtained from biochemical studies that examined the inﬂuence of centrally applied VP or its antiserum on CA neurotransmission in brain sites previously demonstrated to mediate a VP-induced facilitation of retention in avoidance paradigms. These ﬁndings were as follows: (1) intracerebroventricularly injected anti-VP serum attenuated NA neurotransmission in the dorsal septal nucleus and parafascicular thalamus, and DA neurotransmission in the caudate nucleus (Versteeg et al., 1979); and (2) AVP signiﬁcantly increased DA neurotransmission when microinjected into a terminal region of the DA nigrostriatal pathway (caudate nucleus) but not into its site of origin (substantia nigra) (Van Heuven-Nolsen and Versteeg, 1985). ii. Hamburger-Bar and associates Hamburger-Bar et al. (1984; see Chapter 10) investigated the potential for a VP–DA interactional effect in memory processing, using DA-lesioned and nonlesioned rats in a shuttlebox avoidance paradigm. Either saline, LVP, or DDAVP (a nonpressor VP analog) was peripherally administered 1 h before each day of acquisition training but not during retention (resistance to extinction) testing. The following ﬁndings indicated that DA was not critical for memory processing in this task, nor was it an essential mediator in the VP-induced memory-processing effect observed in this study: (1) the saline-treated lesioned rats showed a learning deﬁcit only during the early stages of acquisition and did not differ from the nonlesioned saline-treated controls in either the later stages of acquisition training or in the maintenance of the learned response during the subsequent period; (2) LVP-but not DDAVP-treated nonlesioned rats demonstrated enhanced learning on the last day of training, and enhanced

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retention throughout extinction testing; (3) both LVP and DDAVP treatment facilitated learning and retention in the DA-lesioned rats, indicating the increased sensitivity of the animals to the memory-processing effects of VP and analogs; and (4) histological assessment indicated that VP content was affected (reduced) by the DA lesion only in the anterior pituitary, but was normal in the caudate nucleus, where the VP level was correlated with learning proﬁciency. A correlation between caudate level of VP and strength of retention (resistance to extinction) was not tested in this study. The ﬁnding that DA did not inﬂuence the release of VP into the caudate nucleus in no way contradicts the converse effect, that is, that VP inﬂuences the release of DA into this brain site (Van Heuven-Nolsen and Versteeg, 1985; Versteeg et al., 1978, 1979). The observation that DA was not necessary for the VP-facilitative memory effects observed in this study does not preclude the possibility that DA normally interacts with VP in memory processing in instrumental avoidance paradigms, because VP–NA and/or VP–acetylcholine (ACh) interactions may have substituted for the loss of DA produced by the lesion method used in this study. b. VP–Cholinergic Interactional Effects in Memory Processing: Studies of Faiman and Associates A decade after De Wied and colleagues initiated their investigation of a VP–CA interactional effect in memory processing, Faiman and associates began a series of studies to investigate the possibility that VP might interact with central cholinergic systems in mediating its inﬂuence in memory processing. Similar to the De Wied et al. proposal for the catecholamines, Faiman and colleagues theorized that ACh release in response to VP-ergic activation of central nicotinic cholinergic receptor sites mediates, at least in part, the inﬂuence of VP in memory processing. Experimental ﬁndings supporting this view, discussed in Chapter 10, were as follows: (1) peripheral injections of LVP in combination with each of four cholinergic blockers, acting at central or peripheral nicotinic or muscarinic cholinergic receptor sites, resulted in only the central nicotinic receptor antagonist blocking enhancement by VP of memory consolidation and retrieval in a step-through PA learning paradigm (Faiman et al., 1987, 1988); (2) after peripherally administered injections of each of the four cholinergic receptor antagonists, only pretreatment with the centrally acting nicotinic receptor antagonist prevented the facilitated PA retention effect induced by endogenous VP released by an osmotic stimulus (intraperitoneally injected hypertonic saline) (Baratti et al., 1989); (3) on their own, peripherally administered LVP and nicotine (activates central cholinergic nicotinic receptor sites) produced similar U-shaped dose–response curves and time gradient effects on PA retention. Moreover, when coinjected, each at a subthreshold dose level having no behavioral effect on its own, PA retention was enhanced. These observations indicate synergistic effects between nicotinic cholinergic mechanisms and VP in memory processing (Faiman

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et al., 1991); and (4) a peripheral injection of a nicotinic receptor antagonist prevented nicotinically induced as well as LVP-induced enhancement of PA retention, whereas an injection of a V1 receptor antagonist prevented the LVP-induced, but not the nicotinically induced, effect on PA retention. These latter ﬁndings were consistent with the thesis that the inﬂuence of VP on PA retention depends, at least in part, on its activation of central nicotinic cholinergic mechanisms implicated in LTM formation (Faiman et al., 1991). c. Commentary: Mechanisms by Which These VP–Transmitter Interactions Might Influence Memory Processing VP-released monoaminergic and cholinergic transmitters might mediate a VP-induced inﬂuence on acquisition, storage, and/or retrieval stages of memory processing by one or more of the following functional activities in which these central transmitters systems have been purported to play a signiﬁcant role. First, by virtue of their putative roles in regulating cerebrovascular activity, they may increase blood supply to brain regions engaged in memory processing. In support of this speculation are the neuroanatomical observations and physiological lines of evidence indicating that, in a manner similar to that of VP (see Chapter 1), centrally projecting monoaminergic (Cohen et al., 1997; Raichle et al., 1975) and cholinergic (Itakura et al., 1977; Sato and Sato, 1992) neurotransmitters terminate on brain microvasculature and participate in the regulation of regional cerebral blood ﬂow. Second, they may modulate learning, memory, and other types of cognitive processing by their ability to increase cortical activation/behavioral arousal in response to emotionally and/or motivationally arousing stimulation. The inverted U-shaped arousal–performance curve describes the functional relationship theorized to relate the two sets of parameters. Consistent with this proposed mechanism is evidence that these transmitter systems operate in a coordinated manner to regulate levels of cortical activation and behavioral arousal during various stages of the sleep–wake cycle. For example the LC–NA projection system is neuronally quiescent during sleep, but active during waking, when it helps maintain levels of cortical activation and behavioral arousal sufﬁcient to sustain conscious wakefulness and produces ﬂuctuations in these levels in response to varied stimulus input (Aston-Jones and Bloom, 1981). The forebrain cholinergic system (FB–ACh system) contributes to cortical activation and behavioral arousal during waking, and also to cortical activation during the REM sleep state (Jones, 1993; Szymusiak, 1995). The latter effect is especially relevant to the memory consolidation that is theorized to occur during the REM sleep state (discussed in Chapter 5 and see Section III.A.6, below). Third, these transmitter projection systems may inﬂuence memory processing by means of their contribution to attentional mechanisms present during one or more stages of memory processing. The LC–NA projection system is activated by unexpected novel stimuli, at which time it increases

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cortical activation and behavioral arousal, and widens the span of the attention type known as ‘‘vigilance.’’ This system also narrows attentional focus by enhancing selective attention to relevant stimuli present during stimulus encounters involving memory processing and/or other types of adaptive processes (Aston-Jones, 1985; Aston-Jones et al., 2001; Sara, 1985). The FB–ACh system also facilitates attentional processing during the waking state (McGaughy et al., 2002; Muir et al., 1994; Szymusiak, 1995; Wenk, 1997). A ﬁnal mechanism by which these aminergic and cholinergic transmitter systems may mediate a VP-induced facilitation of performance in tests of memory processing is by virtue of their ability to modulate the processes of associative learning, long-term memory storage, and memory retrieval. In support of this suggestion are numerous ﬁndings indicating that both the LC–NA system (Devauges and Sara, 1991; Harley, 1987, 1991; Kobayashi and Yasoshima, 2001) and the FB–ACh system (Berger-Sweeney et al., 2000; Everitt and Robbins, 1997; Miranda and Bermudez-Rattoni, 1999) play an important role in one or more stages of at least certain types of memory processing. However, the difﬁculty of dissociating the effects of these two classic transmitter systems on memory processing per se from their effects on cortical activation/behavioral arousal and/or attentional processing (Dunnett et al., 1991; Sara et al., 1994; Wenk, 1997) has been frequently noted. In conclusion, to the extent that the classic transmitters with which VP interacts mediate the effect of the peptide on memory processing, then the interpretative problems encountered in explaining the mechanisms by which the classic transmitters modulate memory processing also apply to VP. 6. Influence of VP on Hippocampal Theta Rhythm During REM Sleep and Memory Processing Electrophysiological studies carried out by Urban, De Wied, and Bohus, presented in Chapter 5, have provided evidence of a VP role in the regulation of hippocampal theta activity during the REM sleep state. It has been theorized that during this sleep state the memory consolidation initiated during that day’s learning encounters continues to occur, thereby permitting these memories to be integrated with those already stored in relevant brain circuitry. Speciﬁcally, experimental ﬁndings cited in Chapter 5 (Section IV.B), and in an updated review of this research (Urban 1998), support the following postulates: (1) the REM hippocampal theta rhythm is a neural correlate of memory consolidation; (2) endogenous VP helps maintain the normal frequency of this theta rhythm (Urban and De Wied, 1975, 1978); and (3) the inﬂuence of septal VP on this hippocampal theta rhythm appears to involve a long-acting neuromodulatory action [i.e., a picogram quantity (0.01 pg) of

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VP, microinjected slowly into the septum, produced a slight acceleration of the sleep theta rhythm that lasted for several hours] (Urban, 1981, 1998). This latter ﬁnding has been interpreted as indicating that ‘‘minute amounts of VP could alter the functioning of the theta rhythm-producing network in the septum for a number of hours, suggesting that this, or a similar effect of VP could be related to the memory-enhancing, long lasting action of the peptide’’ (Urban, 1998, p. 287). 7. Neuromodulatory Action of VP in the Septum and Hippocampus: Relevance for Memory Processing In Chapter 1, the concept of neuromodulation was brieﬂy discussed, and the neuromodulatory action of VP was distinguished from its neurotransmitter action. As a neuromodulator, VP inﬂuences (enhances or diminishes) the effect of another transmitter with which it interacts; moreover, this neuromodulatory action results in a relatively long-term effect on processes regulated by that transmitter. The ability of VP to modulate neurotransmission in monoaminergic and cholinergic systems and its signiﬁcance for the role of the peptide in mnemonic processing were discussed in a preceding section (Section III.A.5). The neuromodulator action of VP on glutamate neurotransmission in the septohippocampal system, and its relevance for memory processing, are the subject of discussion in this section. Brieﬂy, it results in a long-lasting enhancement of glutamate-mediated neurotransmission, theorized as having an important role in reinforcing synaptic connections in this circuitry. Relevant electrophysiological evidence obtained in the electrophysiological studies of Joels, Urban, De Wied, and colleagues (see Chapter 5) is brieﬂy reviewed below. a. Neurotransmitter and Neuromodulator Actions of VP in the Septum and Hippocampus: In Vivo Studies with Exogenous AVP and Related Peptides In these studies, AVP was iontophoretically administered to single cells in the septal region or in the hippocampus, or topically applied to the exposed dorsal surface of the lateral septum (LS) or ventral hippocampus (VH) for recording responses from single cells or from cell populations (ﬁeld potentials), respectively. Iontophoretically applied VP to single neurons produced both neurotransmitter and neuromodulatory actions in the septal–hippocampal system. An excitatory neurotransmitter action (i.e., a discernible increase in the spontaneous discharge rate of the cell, which began promptly after onset of VP application and ceased abruptly after its withdrawal) was observed in the LS, medial septum (MS), dorsal hippocampus (DH) (Joels and Urban, 1982, 1984a), and VH (Urban and Killian, 1990). Joels and Urban (1982) noted that in the septum, the LS, which receives hippocampal input, appears

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to be the preferential target for this neurotransmitter action, because far fewer of the MS septal cells, which project to the hippocampus, were thus inﬂuenced. The neuromodulatory action of VP in these single-cell studies was initially observed when Joels and Urban (1984a) noted that iontophoretic application of AVP had no noticeable effect on the spontaneous activity in some tested LS neurons, but enhanced cellular excitatory responses to similarly iontophoretically applied glutamate (Joels and Urban, 1982, 1984a) and other tested excitatory amino acid (EAA) transmitters (Joels and Urban, 1984a). In contrast to the rapid onset/offset that characterized the neurotransmitter effect of VP, this modulatory inﬂuence on LS neuronal responsivity was relatively long lasting (persisted 3–15 min after termination of VP treatment in about half the responsive neurons) and EAA speciﬁc [did not alter the inhibitory responses elicited by GABA (Joels and Urban, 1984a) or monoaminergic input (Joels and Urban, 1985)]. In addition, LS neurons treated with a similar amount of AVP also showed a large increase in the number of action potentials in response to electrical stimulation of the ﬁmbria–fornix (ﬁ–fx) pathway (contains the ﬁbers that transmit glutamate and other EAA neural signals from the hippocampus to the lateral septum) (Joels and Urban, 1984a). In the experiments that recorded ﬁeld potential (FP) responses in the LS, the neuromodulatory action of VP was observed as a long-lasting enhanced amplitude of the FP negative wave component (theorized as an excitatory population response to glutamatergic transynaptic input) elicited by electrical stimulation of the ﬁ–fx pathway. The positive wave component (theorized as an inhibitory population response to GABA-ergic transynaptic input) was not inﬂuenced by this stimulation (Joels and Urban, 1984a; Urban and De Wied, 1986). As expected, these ﬁndings were consistent with the results obtained in the single-cell experiments by Joels and Urban (1984a) cited above. The neuromodulatory action of VP in an FP test was tested for VP(1–9) and a number of its highly potent behaviorally active fragments [VP(4–9), VP(4–8), and VP(5–9)]. Contrary to expectation, given the highly effective actions of these metabolites in learning studies, the neuromodulatory action of these metabolites was far less potent than that for the parent peptide (Urban and De Wied, 1986). This could be because the receptors for VP(1–9) and these metabolites are located in different areas of the hippocampus (see commentary in Section III.A.7.a of this Chapter). In single-cell experiments performed in the VH (Urban and Killian, 1990), iontophoretically applied AVP elicited a neurotransmitter action (excitatory response of rapid onset and brief duration) in only 30% of the tested neurons. In contrast, most of the VH neurons so tested showed a VP-induced neuromodulatory action, had no effect on cellular spontaneous discharge activity, but enhanced glutamate-evoked activity that was of long duration (lasted for tens of minutes after VP application) and glutamate

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speciﬁc (no inﬂuence on ACh-induced excitation). Moreover, pretreatment with V1 and N-methyl-d-aspartate (NMDA) receptor antagonists, each of which prevented VH excitatory responses elicited by either VP or glutamate application, had no inﬂuence on VP neuromodulatory action on glutamate neurotransmission. Altogether, these ﬁndings suggest that factors responsible for the neuromodulatory action of VP differ from those affecting its neurotransmitter action. A VP neuromodulatory action has also been demonstrated in the dorsal hippocampus in single-cell studies conducted by Joels and Urban (1982; see Chapter 5). b. Neuromodulatory Action of VP in the LS after Stimulation of VP-ergic cells in the Diagonal Band of Broca and the Bed Nucleus of the Stria Terminalis: An In Vivo Study In a progress report on the research topic reviewed in this Section, Urban (1998) reported ﬁndings supporting the view that endogenous VP released into the LS exerts a neuromodulatory action that enhances glutamatergic neurotransmission at hippocampal–lateral septal synaptic sites. Noting that VP-ergic cells originating in the diagonal band of Broca (DBB) and bed nucleus of the stria terminalis (BNST) project to the LS, Urban and colleagues examined the effect of stimulated release of VP from these projection systems on the negative and positive waves in FPs generated in the LS by stimulation of a band of ﬁmbrial ﬁbers in the ﬁ–fx pathway. Long-Evans (LE) rats, genetically normal for VP synthesis, and Brattleboro HEDI and HODI rats were subjects in these experiments. The major ﬁndings indicated that stimulation of speciﬁc areas in the DBB or in the BNST of LE normal rats for 3 min (stimulation pattern simulated phasic ﬁring of VP-producing neurons) signiﬁcantly increased the amplitude of the FP negative wave elicited by ﬁ–fx stimulation in contrast to controls that were not stimulated in the DBB or BNST. The data obtained for the HEDI rats did not signiﬁcantly differ from that found for the LE rats, but none of the HODI rats showed a change in the ﬁmbria-evoked FPs after DBB or BNST stimulation. The long-lasting nature of the effect of VP on this response was demonstrated by the fact that the increased amplitude of FP negative waves, induced by DBB and BNST stimulation, was maintained for the duration of the 180-min period of ﬁmbrial stimulation. Taken together, these results support the thesis that endogenous VP released into the LS exerts a neuromodulatory action that results in a long-lasting enhancement of glutamatergic neurotransmission at hippocampal–lateral septal synapses. c. Significance of the Neuromodulatory Action of VP for Memory Processing Experimental evidence obtained by Van den Hooff et al. (1989) and Chepkova et al. (1995) provided support for the suggestion that a neuromodulatory action of VP and related peptide fragments may facilitate glutamate neurotransmission in LS and VH circuitry involved in LTM formation.

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Van den Hooff et al. (1989) demonstrated a role for endogenous VP in the maintenance of long-term potentiation (LTP) in the LS. In this in vitro study, septal brain slices (included almost the entire LS and a long bundle of ﬁmbrial ﬁbers) from normal LE and Brattleboro HODI rats were prepared for LTP experiments. LTP was manifested as an enhanced amplitude of the negative wave component of the FP in response to standard stimulation of the ﬁmbrial ﬁber bundle after tetanic stimulation of this bundle. A number of experimental manipulations were used to explore the putative role of endogenous VP in the induction and/or maintenance of LTP. The results indicated (1) normal induction and maintenance of LTP in the brain slices from the Wistar rats, (2) the importance of endogenous AVP for the maintenance but not the induction of LTP, and (3) the necessity of NMDA glutamate receptors for establishing LTP in this preparation. Chepkova et al. (1995) carried out an in vitro study with hippocampal brain slices from male Wistar rats and tested for a neuromodulatory action of AVP and its behaviorally active metabolite, AVP(4–8). This study used picomolar amounts of these drugs, which are sufﬁcient to enhance retention in PA avoidance (Kovacs et al., 1979a) and discrimination learning tasks (Metzger et al., 1993), whereas larger quantities (nanomolar to micromolar range) of AVP are required to demonstrate the fast-acting neurotransmitter actions of VP (Raggenbass et al., 1988, 1989). The various experimental tests produced the following major ﬁndings: (1) the application of low concentrations of AVP produced long-lasting enhancement of glutamatemediated neurotransmission in pyramidal neurons in the VH/subiculum, and AVP(4–8) produced a similar, but more pronounced, effect than that of the parent peptide; (2) the neuropeptide effects on various tested parameters indicated an action different from a transmitter effect; and (3) pretreatment of the brain slice with a GABAA receptor antagonist, on its own or in combination with an NMDA receptor antagonist, indicated that neither GABA-mediated inhibition nor the NMDA subtype of glutamate receptor were necessary for the peptide-induced potentiation of glutamate neurotransmission observed in this study. d. Commentary: Neuromodulatory Action of VP and Long-Term Memory Formation in the LS and VH A number of observations, presented below, are consistent with the theoretical viewpoint that a VP neuromodulatory action within septal–hippocampal circuitry is responsible, at least in part, for the facilitative role of the peptide in memory consolidation. First, are a number of correspondences between electrophysiological ﬁndings relevant to the neuromodulatory actions of VP on glutamate neurotransmission in septal–hippocampal circuitry and behavioral ﬁndings pertaining to the effects of VP on retention performance in various learning paradigms: (1) AVP in the picomolar dose range is most effective for demonstrating the neuromodulatory action of VP in the LS and VH (Chepkova

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et al., 1995; Joels and Urban, 1982, 1984a), and similarly for producing the retention effects in avoidance (Kovacs et al., 1979a; see Chapter 4) and discrimination (Metzger et al., 1993) learning tasks when injected into these same brain sites; and (2) the VP metabolite AVP(4–8) is far more potent than its parent peptide in producing a neuromodulatory effect on glutamatergic neurotransmission involving LTP-like phenomena (Chepkova et al., 1995), and in facilitating LTM when centrally administered to animals tested in learning paradigms (e.g., Burbach et al., 1983a; see Chapter 5). A second type of correspondence pertains to features characterizing both the neuromodulatory action of VP on glutamate neurotransmission in the LS and VH and LTP phenomena observed in these brain structures: (1) the experimental induction of LTP and the neural induction of the VP neuromodulatory action both result in a long-lasting enhancement of neuronal responsivity to afferent stimulation in the LS (Joels and Urban, 1984a) and in the VH (Urban and Killian, 1990); (2) glutamate neurotransmission is theorized to be importantly involved in mediating LTP (Teyler and DiScenna, 1987), and glutamatergically mediated neurotransmission is enhanced by a VP neuromodulatory action in both the LS (Joels and Urban, 1982, 1984a; Urban, 1998; Urban and De Wied, 1986) and VH (Urban and Killian, 1990); (3) exogenous VP produces a long-lasting enhancement of excitatory responses in LS neurons elicited by stimulation of the ﬁ–fx pathway (Joels and Urban, 1984a), and endogenous VP released in LS neurons by tetanic stimulation of the ﬁmbria bundle is important for the maintenance, but not the induction, of LTP (Van den Hooff et al., 1989); and (4) microinjection of small amounts of AVP, which induced a VP neuromodulatory action, also produced an LTP-like effect in the VH (Chepkova et al., 1995), and a similar effect (termed ‘‘long-term vasopressin potentiation’’) in the dentate gyrus of the hippocampal formation (Chen et al., 1993). Moreover, LVP has been reported to maintain LTP induced in the hippocampus (Chepkova and Skrebitski, 1982). Finally, the structures in which VP-induced neuromodulatory action has resulted in a long-lasting enhancement of glutamatergic neurotransmission, or has exhibited an LTP-like phenomenon, are also those implicated in the inﬂuence of VP on retention assessed in avoidance learning paradigms (see Chapter 4).

B. OT and Memory Processing It is clear from the various studies reviewed in this text that memoryprocessing research involving VP and OT has focused primarily on the former. This is especially true for the studies using stress-associated appetitive and avoidance learning paradigms. De Wied and colleagues have been the leading investigators of the role of OT in memory processing and with rare exceptions they have relied on the use of avoidance learning paradigms, especially the step-through PA task.

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1. OT as an Amnestic Neuropeptide in Stress-Associated Learning Paradigms In their initial studies, De Wied and colleagues concluded that OT was not a major factor in learning and memory. In contrast to VP, peripherally administered OT failed to normalize the rapid extinction of a learned avoidance response shown by posterior lobectomized rats (De Wied and Bohus, 1966; see Chapter 2). Schultz et al. (1974) were the ﬁrst researchers to demonstrate an OT-induced retention impairment in an aversive learning situation. They found that in contrast to the facilitative memory effect of AVP, chronic treatment with physiological doses of OT attenuated retention of a learned active avoidance (AA) response in a platform-jump shock avoidance paradigm. Subsequent research by De Wied and colleagues (see Chapters 2–5) reinforced the notion that VP and OT exerted opposing effects on memory processing in aversive learning encounters. As discussed below, their ﬁndings generally indicated that under favorable experimental conditions (e.g., appropriate dose levels), OT attenuated memory consolidation and retrieval, and that this OT effect on memory processing was of physiological signiﬁcance. Moreover, the opponent effects of the two peptides appeared not to be due to a simple competitive or functional antagonism, because pretreatment with either peptide was unable to reverse the PA retention effects exerted by posttraining administration of the other peptide (Drago et al., 1981). Experimental ﬁndings pertaining to the postulated ‘‘amnestic’’ action of OT, a key proposition in the ‘‘VP/OT Central Memory Theory,’’ have been reviewed throughout the course of this text (see Chapters 2–5, 7, and 9) and the major ﬁndings are summarized and interpreted in the discussion that follows. 2. Effects on Memory Processing of Treatments That Modulate Peripheral and Central Levels of OT a. Evidence Obtained with Treatments That Increase Peripheral and/or Central Levels of OT Experiments using peripheral (subcutaneous injections) as well as central (intracerebroventricular or brain site-speciﬁc) administration of OT and related peptides (OT fragments) have demonstrated an OT amnestic effect on memory processing, but these results have been far from consistent. The variable effects of OT on memory processing are summarized separately for the peripherally and centrally administered peptide. An interpretative discussion, related to the dose-associated bimodal effect observed after both routes of drug delivery, and the site-speciﬁc differences observed after locally injected OT into different brain structures, then follows. i. Peripherally administered OT and OT fragments An OT(1–9) amnestic action has been observed in an active avoidance (Schultz et al., 1974) and a passive avoidance (PA) (Kovacs et al., 1978) version of the

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bench-jump shock avoidance task in rats, and a step-through PA task in mice (Boccia et al., 1998) as well as in rats (De Wied et al., 1987; Gaffori and De Wied, 1988). Moreover, subcutaneously injected OT C-terminal fragments dose dependently attenuated PA memory consolidation and retrieval, as did OT(1–9), and were far more potent in these effects than the parent peptide (Gaffori and De Wied, 1988). On the other hand, OT failed to inﬂuence longterm memory tested in an AA (Bohus et al., 1978b; De Wied, 1971), PA (Bigl et al., 1977 as cited in Kovacs and Telegdy, 1982; Schultz et al., 1974), and combined active/passive avoidance (Sahgal and Wright, 1984) paradigm, or the rate of acquiring a conditioned taste aversion (Verbalis et al., 1986). Bohus et al. (1978b) found highly equivocal effects of OT on retention tested in a pole-jump AA and in a step-through PA task, depending on the dose level and the treatment regimen. For example, a high dose of OT given before the ﬁrst 3 days of a 5-day extinction session produced VP-like facilitated maintenance of the learned AA response, whereas lower dose levels given in the same manner, or chronically administered before testing throughout acquisition and extinction, had no effect. A medium-sized dose of OT given immediately after the PA learning trial produced a bimodal effect, with the majority of the OT-treated subjects having higher (longer) or lower (shorter) retention (reentry latency) scores than the placebo controls. A different type of bimodal dose effect, a bimodal ‘‘dose-variable’’ action, was observed in pole-jump AA task by Gaffori and De Wied (1988). In this case, doses of OT(1–9) and its glycinamide-containing fragments [e.g., OT(4–9) and OT(5–9)] at the high end of the tested dose range mimicked AVP and facilitated retention of the AA response, whereas doses at the low end tended to exert an amnestic action, which was especially prominent for the OT fragments. The results also indicated that the Cterminal amino acid, glycinamide, was necessary for the observed bimodal dose-variable action. Dose factors and/or duration of treatment may have contributed to the contrasting OT effects on retention observed in the few human studies reported in Chapter 9. Thus, after 4 and 8 h of a high dose of intravenously infused OT treatment, female patient volunteers were impaired in delayed recall (retention), but not in immediate recall (learning) of verbal paired associates or photographs of faces (Ferrier et al., 1980; Kennett et al., 1982). In contrast, acute treatment with a lower dose of intranasally administered OT impaired immediate recall of recently presented lists of words, but did not inﬂuence delayed recall of these word lists (Bruins et al., 1992; Fehm-Wolfsdorf et al., 1984). ii. Centrally administered OT and OT fragments Relatively few of the studies on the memory-modulating actions of OT used the intracerebroventricular route of drug delivery. Experimental ﬁndings by Bohus et al. (1978b) supported an OT-induced attenuation of memory consolidation in a

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step-through PA paradigm, when the peptide was intracerebroventricularly injected within 3 h but not 6 h after the PA learning trial. This ﬁnding is in accord with the view that memory consolidation is a time-dependent process (McGaugh, 1966). Intracerebroventricularly administered OT has also resulted in dosevariable effects. Bohus et al. (1978b) tested the effect of various doses (0.05, 0.10, 1.0, and 10.0 ng/rat) of OT on PA retention (memory consolidation). When centrally (intracerebroventricularly) injected immediately after the footshock trial, OT exerted dose-dependent effects on retention, in which the lowest dose was without inﬂuence, the two middose levels attenuated retention, and the high dose produced a bimodal distribution of retention scores (reentry latencies) suggesting some tendency for the high dose to produce a VP-like facilitative retention effect. In a similar manner, Burbach et al. (1983b) observed that OT(1–9) and its glycinamide-containing fragment, OT(4–9), attenuated PA consolidation with intracerebroventricularly injected low doses, whereas doses at the high end of the tested dose range did not differ from controls [OT(1–9)] or showed a tendency to produce a VP-like memory-facilitative effect [OT(4–9)]. The desglycinamide version of the OT fragment [OT(4–8)] attenuated memory consolidation at all the tested dose levels, indicating that the terminal glycinamide amino acid in the OT molecule was required for the bimodal dose-variable trend exhibited by OT(4–9). Site-speciﬁc variability of OT effects on memory processing was observed in treatments that microinjected OT directly into a given brain structure. An amnestic action of OT has been observed in (1) a step-through PA task performed by rats microinjected with OT in the hippocampal dentate gyrus or in the dorsal raphe nucleus (Kovacs et al., 1979a; see Chapter 5), and (2) a pole-jump shock avoidance task performed by rats chronically microinjected with OT in the ventral hippocampus before daily testing during response acquisition and extinction (Ibragimov, 1990; see Chapter 10). On the other hand, OT had no inﬂuence on PA memory consolidation when injected into the central amygdala, and facilitated it when injected into the lateral septum (Kovacs et al., 1979a). Ibragimov (1990) also found that chronic intrahippocampal injection of a relatively high dose of the C-terminal OT metabolite, prolyl-leucyl-glycinamide (PLG), produced an amnestic action in the pole-jump shock avoidance paradigm, a ﬁnding in stark contrast to the VP-like effect reported on the same task by Gaffori and De Wied (1988; see Chapter 2). iii. Interpreting some of the inconsistent results Drug dose level is one factor that clearly appears to be contributing to these discrepant ﬁndings. Dose-related variation in OT effects on memory processing has been observed in studies using different task paradigms, treatment regimens, and peripheral as well as central (intracerebroventricular) routes of drug

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delivery. A dose-dependent effect is one type of dose-related variation and involves the ﬁnding that one or a few dose levels within a given dose range produce a statistically signiﬁcant effect of the ligand whereas doses outside this narrow range have no signiﬁcant effect. In another type of dose-related variation high and low doses of a ligand exert opposing effects on a given function, each of which is statistically signiﬁcant. Both types appear to be implicated in the effects on memory processing exerted by OT(1–9) and its C-terminal derivatives (e.g., Bohus et al., 1978b; Burbach et al., 1983b; Gaffori and De Wied, 1988). A dose-dependent effect of OT and fragments, together with the ﬁnding that a single dose level of these peptides produces a bimodal distribution of reentry latencies in a PA task (Bohus et al., 1978b), could reﬂect an OT interaction with the central arousal system, as proposed for AVP by Sahgal and colleagues (see Chapter 7). Taken together, the observations indicating that OT acts centrally and hormonally (i.e., on the pituitary–adrenocortical axis) in response to a variety of emotional stressors (see Chapter 1), interacts with central catecholamine neurotransmitters (see Chapter 4), and induces EEG cortical arousal effects (this chapter, Section III.B.3) are consistent with a proposal that OT arousal effects may explain, at least in part, the dose-associated variable effects of OT in avoidance learning tasks. De Wied and colleagues have proposed an alternative explanation, especially relevant for the bimodal dose-variable action of OT on memory processing in an AA task observed by Gaffori and De Wied (1988) for OT(1–9) and the glycinamide-containing OT fragments [e.g., OT(4–9) and OT(5–9)]. These ﬁndings together with structure–activity studies (e.g., Walter et al., 1975) suggested to De Wied and colleagues that the OT molecule contains two opponent messages, and that metabolic degradation of OT in the blood and in the brain produces OT metabolites that attenuate memory processing at all dose levels tested [i.e., desglycinamide fragments of OT such as DG-OT, OT(4–8) and OT(5–8)], whereas those at the Cterminal end of the molecule (LG and PLG) facilitate memory processing at all dose levels. The parent peptide and other glycinamide-containing OT fragments display the dose-variable action and facilitate memory processing at high dose levels and attenuate it at low dose levels. It has been suggested that a more rapid or more complete degradation of OT occurs in the septal nucleus to form PLG and LG, and that this is responsible for the VP-like behavioral effect that occurs in this nucleus, but not in other limbic brain sites, after site-speciﬁc injections of OT (Kovacs et al., 1979a). The experimental evidence obtained by Gaffori and De Wied (1988), and the observation that brain regions differ in their rate of degradation of OT (Burbach et al., 1980a) are consistent with this suggestion. However, in this connection the physiological signiﬁcance of a PLG effect on memory processing is questionable, because unlike [pGlu4,Cyt6]OT(4–9) and its desglycinamide derivative OT(4–8), there is no evidence that PLG

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is produced (Burbach and Lebouille, 1983; Burbach et al., 1980a,b) or present (Manberg et al., 1982) in the brain. Van Wimersma Greidanus and Baars (1988) have offered an alternative, although not mutually exclusive, explanation for this OT–septal effect. Noting that the VP-like effects of OT in the septal nucleus have been observed in peptide–catecholamine interactional studies (Van HeuvenNolsen et al., 1984a) and in EEG studies (Urban, 1981), they suggest that this effect could result from the ability of the microinjected OT to stimulate VP receptors, which in contrast to the sparse supply of OT receptors are in abundant supply in the lateral septal nucleus (Barberis and Tribollet, 1996). b. Evidence Obtained with Treatments That Reduce Endogenous OT or Interfere with Its Receptor Transmission i. Behavioral studies Treatments that severely reduce brain levels of OT indicate both the physiological signiﬁcance of the peptide in memory processing and provide supportive evidence for its postulated ‘‘amnestic’’ action in avoidance paradigms. Relevant ﬁndings include the following: (1) intracerebroventricularly injected OT antiserum facilitated retention in a pole-jump FS avoidance paradigm, and memory consolidation in a PA paradigm (Bohus et al., 1978b); and (2) OT antiserum microinjected into the dorsolateral septum or in the ventral hippocampus facilitated PA memory consolidation and retrieval (Van Wimersma Greidanus and Baars, 1988). This ﬁnding is consistent with the opposing effects induced by VP and OT on PA memory consolidation when they are microinjected into the hippocampus (Kovacs et al., 1979a). ii. Hippocampal theta activity during paradoxical sleep, and memory processing The thesis that hippocampal theta activity during REM sleep is correlated with ongoing memory consolidation of learning experiences initiated during the prior waking state was discussed in Chapter 5. Electrophysiological ﬁndings pertinent to this thesis and to the physiological role of OT in LTM formation are as follows: (1) intracerebroventricularly injected OT and DG-AVP exerted opposite effects on the hippocampal theta frequency spectrum during REM sleep (Urban and De Wied, 1978) as they do on retention in avoidance learning tasks (Bohus et al., 1978b); (2) intracerebroventricularly injected OT antiserum inﬂuenced the hippocampal theta band frequency spectrum during REM sleep in a manner opposite to that produced by intracerebroventricularly injected OT (Bohus et al., 1978b), but similar to that produced by intracerebroventricularly injected DG-AVP (Urban and De Wied, 1978); and (3) HODI rats, which De Wied and colleagues have shown are impaired in PA retention (see Chapter 3), exhibited a hippocampal theta frequency pattern during REM sleep that resembled that of OT-treated normal rats (Urban and De Wied, 1975). Taken together, these ﬁndings suggest a direct relation between the depressing

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action of OT on the electrophysiologically recorded hippocampal theta frequency spectrum and its ‘‘amnestic action’’ in avoidance behavioral studies. Moreover, it is possible that the opposing effects of VP and OT on hippocampal EEG activity during REM sleep are related to the effects of these peptides on the level of excitability in the neural substrate contributing to hippocampal theta activity during REM sleep. 3. Means by Which Peripherally and/or Centrally Circulating OT Might Influence Memory Processing a. Roles of OT in Cerebral Circulation, Glucose Metabolism, and Release of Stress Hormones: Relevance for Memory Processing As noted in Chapter 1, OT, like VP, activates nonneural receptor sites located in: (1) cerebral blood vessels, where it participates in cerebral vascular regulation; (2) the liver, where it inﬂuences glucose metabolic activity; and (3) the adrenal gland where it contributes to the release of the stress hormone, adrenaline. Further discussion in Chapter 1 cited evidence that these activities, in turn, may contribute to one or more phases of memory processing. It may be speculated that this nonneuronal OT contribution to memory processing, especially its roles in supplying glucose and blood to brain sites engaged in memory processing, promotes the metabolic underpinnings of memory processing without determining its speciﬁcity (whether it results in an enhancement or attenuation of memory formation and retrieval). The speciﬁcity of the OT effect on memory processing would be determined by its neural signaling activity. For example, a nonneural OT-induced increase in brain glucose level during a stress-associated learning encounter may promote memory-processing activity via a glucose-induced release of ACh in the hippocampus (see Cahill and McGaugh, 1996). Simultaneously, an OT neuronally induced OT–cholinergic interaction in that brain site may impair consolidation and thereby weaken the memory for the stress experience (see Boccia and Baratti, 2000; see Chapter 10) (and see below). b. Role of the Arousal System in Mediating OT Effects on Memory Processing Involvement of OT in stress responding (see Chapter 1) makes it plausible that an OT-induced arousal effect may explain, in part, the effect of the peptide on memory processing during stress-associated learning encounters. Observations consistent with this possibility are as follows: (1) peripherally administered OT desynchronizes cortical EEG activity, and enhances the intensity of startle reactions to appropriate stimulation (Arletti et al., 1995); and (2) Morris et al. (1995) and Nishioka et al. (1998) reported ﬁndings indicating that shaker stress stimulation of an OT cell system in the paraventricular nucleus (PVN) mediated an autonomic nervous system (ANS)-induced increase in heart rate and blood pressure. The pressor response itself could have induced an arousal effect consistent with the proposals and ﬁndings of Koob and associates (see Chapter 6). In addition,

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the earlier reported variable effects of exogenous OT on avoidance memory processing, as well as the bimodal effects induced by central injection of a high dose of the peptide (Bohus et al., 1978b), are consistent with an OTinduced arousal effect on memory processing in accordance with the proposed inverted U-shaped functional relationship between arousal level and performance efﬁciency (see Chapter 7). However, the variable effects observed with peripherally and centrally administered OT on memory processing could reﬂect pharmacological effects of exogenous OT, as opposed to a physiological action of the peptide. In fact, treatments that evaluate the role of endogenous OT in stress-associated memory processing suggest a consistent ‘‘amnestic’’ action of the neuropeptide rather than the variability that would be expected if arousal level mediated the effect of the peptide on memory processing. It appears more likely that, during stressful learning encounters, endogenous OT is directly released into memory-processing brain sites, where it attenuates memory storage and retrieval by neuronal actions at speciﬁc brain sites involved in memory processing, via mechanisms such as those proposed by De Wied and colleagues for VP (Sections III.A.5 and III.A.7). This is discussed in more detail below. c. OT–Catecholaminergic Interactions in Memory Processing Chapter 4 discusses evidence that indirectly supports the proposition that, similar to VP, OT interacts with catecholamines in mediating its inﬂuence on memory storage and retrieval in avoidance learning paradigms. Speciﬁcally, these studies demonstrated the ability of OT to inﬂuence neurotransmitter turnover (i.e., synthesis and release) in monoaminergic neuronal projections terminating in a number of brainstem and forebrain limbic areas where OT exerts a variety of physiological and behavioral effects. Thus, peripherally, centrally (intracerebroventricularly), and locally injected OT inﬂuenced steady state levels of monoamines (Schwarzberg et al., 1981), and modulated the release of catecholamines (Kovacs and Telegdy, 1983; Van Heuven-Nolsen et al., 1984a,b) in midbrain, hypothalamic, striatal, and forebrain limbic structures (e.g., the hippocampal dentate gyrus, several nuclei of the amygdala, and the lateral, medial, and dorsal septal areas). However, in addition to memory processing, OT is involved in many sexual, parental, and social afﬁliative as well as self-maintenance activities mediated by neuronal circuitry within these structures. Because these studies were not designed to ascertain the functional signiﬁcance of the interactional effects they observed, it was not possible to relate the ﬁndings to the postulated amnestic action of OT in avoidance paradigms. d. OT–Cholinergic Interactions in Memory Processing In contrast to the absence of direct evidence relating OT–monoaminergic interactional effects to the role of the peptide in memory processing, the study by Boccia and Baratti

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(2000; see Chapter 10) provided evidence supporting involvement of an OT–ACh interactional effect in memory processing. In addition, it helped to clarify the nature of that interaction and its relation to the amnestic action of OT in aversive learning situations. Brieﬂy, their ﬁndings showed that (1) pharmacological treatment, which prevented the metabolic degradation of ACh at its central synaptic sites (functional ACh agonist), blocked the amnestic action of OT in a PA task; and (2) centrally acting cholinergic antagonists, which interfere with cholinergic receptor-induced transmission, impaired the facilitative PA retention effect induced by a highly potent OT receptor antagonist. These ﬁndings strongly imply that the peripherally administered OT and OT receptor antagonist acted at central cholinergic sites, because the amnestic action of the former, and the memory-facilitative action of the latter, were prevented by pretreatment with a centrally acting cholinergic functional agonist and antagonist, respectively. This interpretation was the basis for the proposal by the investigators that the amnestic action of OT in avoidance paradigms might be due, at least in part, to its ‘‘negative modulatory effect’’ (i.e., its inhibitory effect) on cholinergic neurotransmission at memory-processing sites during the posttraining period when recently acquired information is undergoing memory consolidation. An unanswered question suggested by the ﬁndings of this study concerns how the peripherally administered OT treatments inﬂuence cholinergic neurotransmission at central brain sites. As these authors noted, peripherally circulating OT, like VP, crosses the BBB with difﬁculty (Zaidi and Heller, 1974). Therefore the means by which OT exerted its effect on central cholinergic mechanisms has yet to be clariﬁed. 4. Commentary Although there is convincing evidence that OT exerts an amnestic action in aversive learning situations, there are numerous reports indicating its failure to do so, or conversely, its production of a VP-like facilitative action. A dose-dependent effect could potentially explain these disparate ﬁndings, suggesting that high doses facilitate and low doses (close to physiological levels) impair retention. However, this explanation cannot completely account for these discrepancies, because Schultz et al. (1974) found that the low (physiological) dose level of OT that attenuated retention in an AA paradigm had no inﬂuence on retention in a PA paradigm, suggesting that dose-dependent behavioral effects are not independent of the paradigm used in the study. In a similar manner, Gaffori and De Wied (1988) found that two doses of OT, representing the high end of the tested dose range, facilitated retention of a learned pole-jump AA response, but attenuated memory for a footshock experience in the step-through PA paradigm. Findings by Gaffori and De Wied (1988) were consistent with the alternative, although not mutually exclusive, explanation that OT(1–9) is

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a precursor to the generation of bioactive metabolites, some of which (e.g., PLG) oppose the amnestic action of the parent peptide. Findings from structure–activity studies (e.g., Walter et al., 1975) provided support for this proposal. However, ﬁndings by Ibragimov (1990) demonstrated that PLG was an effective amnestic in an AA task when injected into the ventral hippocampus. Further discussion of the role of OT in memory processing is given in a later section of this chapter. Compared with VP, there is relatively little speciﬁc information concerning the mechanisms by which OT exerts its amnestic action in memory storage and retrieval. However, the evidence that is available suggests that this peptide, like VP, interacts with central catecholaminergic and cholinergic transmitter systems that project to and modulate activity in limbic, striatal, and cortical systems involved in memory processing. Although, OT, like VP, exerts a neuromodulatory as well as a neurotransmitter action in various brain sites (Joels and Urban, 1982; Raggenbass et al., 1998; Urban and De Wied, 1986), no concerted effort has been made to link the neuromodulatory action of OT to its role in memory processing as has been done for VP. The ﬁnding by Joels and Urban (1982) that the neuromodulatory action of OT enhances septal neuronal responsivity to glutamatergic neurotransmission is another instance of an OT-induced VP-like action in the septum and consistent with behavioral data obtained by Kovacs et al. (1979a; see Chapter 4). This anomalous action of OT in the septum has yet to be satisfactorily clariﬁed. Findings that OT appears to interact with GABA-ergic interneurons in the hippocampus to enhance tonic inhibition of CA1 pyramidal neurons (Zaninetti and Raggenbass, 2000) suggests the feasibility of pursuing a search relating the neuromodulatory actions of OT in this structure with its amnestic behavioral effects in avoidance learning paradigms.

C. The Neuropeptide Concept 1. Relation of the Neuropeptide Concept to the ‘‘ VP/OT Central Memory Theory’’ The basic premise of the neuropeptide concept is that neuropeptides (peptides with neurogenic properties) are synthesized in neurons from larger precursor proteins and peptides (see Chapter 5; and De Wied, 1969, 1987). In its application to the neurohypophysial peptides, it proposes that in addition to producing peripheral hormonal and central behavioral effects, VP(1–9) and OT(1–9) are precursors of smaller neuropeptides that exhibit highly potent centrally mediated actions, but lack the peripheral endocrine effects of their parent peptides (for additional discussion see De Wied, 1987, Burbach et al., 1998; Reijmers et al., 1998). In the context of the ‘‘VP/OT Central Memory Theory,’’ the concept is paraphrased as proposition 9:

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‘‘VP(1–9) and OT(1–9) are precursors of metabolic fragments that modulate memory processing in the brain’’ (see Chapter 5). 2. Relevant Observations and Experimental Findings a. Biochemical/Anatomical Evidence i. Formation of bioactive C-terminal VP and OT metabolites Bioactive C-terminal VP and OT fragments derived by proteolytic metabolism from the parent peptides in the rat brain have been studied in vitro and in vivo. The in vitro research (Burbach and Lebouille, 1983; Burbach et al., 1980b, 1983a; Wang and Burbach, 1986) demonstrated that when AVP or OT was exposed to synaptic plasma membranes (SPMs), derived from limbic brain areas of the rat brain, these parent peptides were converted to pyroglutamic acid fragments ([pGlu4,Cyt6]VP(4–9), [pGlu4,Cyt6]VP(5–9), [pGlu4,Cyt6]OT(4–9), and [Glu4,Cyt6]OT(5–9)) and to similar fragments lacking pyroglutamic acid ([Cyt6]AVP(5–9), [Cyt6]AVP(5–8), [Cyt6]OT(5–9), and [Cyt6]AVP(5–8)). These fragments were sequentially generated by the action of aminopeptidase enzymes present in these SPMs, initiated at the N terminus of the parent peptide. Subsequently, in vivo studies provided immunological and chromatographical evidence that C-terminal VP and OT metabolites, similar to those observed in vitro, are present in the brain, and that aminopeptidase activity is involved in their formation (Burbach et al., 1984; Stark et al., 1989; Wang et al., 1986). Speciﬁcally, this evidence suggests that C-terminal VP fragments are formed from the endogenously released peptide in the medulla and in a number of extrahypothalmic brain sites such as the hippocampus, amygdala, septum, and thalamus but are absent from the pituitary gland, consistent with ﬁndings that these peptide fragments produce central but not peripheral hormonal activity (for reviews see Burbach, 1987; De Wied, 1991; Burbach et al., 1998). ii. Specific binding sites for C-terminal VP and OT fragments? One major question as yet resolved is whether the receptors that mediate the behavioral actions of these C-terminal VP and OT fragments are shared by the parent peptides, or are separate and distinct. A study by De Wied et al. (1991), which used agonist–antagonist interactional effects to designate the properties of CNS VP and OT receptor sites, found evidence of a limbic system receptor complex responsive to AVP(1–9) and OT(1–9) as well as their respective C-terminal fragments, VP(4–8) and OT(4–8). This ﬁnding suggested that, in the case of this receptor, these neurohypophysial peptides as well as their metabolites share a common receptor, although the VP-associated and OT-associated effects on the receptor complex are of an opposing nature. On the other hand, ﬁndings from a number of in vitro studies with 35S-labeled C-terminal AVP fragments support the view that the central receptors for the parent peptides are separate and distinct from

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those for their metabolites (Brinton et al., 1986; De Kloet et al., 1985b; Du et al., 1998). Thus De Kloet et al. (1985a,b) localized binding sites in the rat brain for AVP(4–9) that were highly concentrated in certain regions of the brainstem (nucleus of the solitary tract) and diencephalon (region of the arcuate nucleus) and in two circumventricular organs (pineal gland; OVLT) whereas binding sites for both VP(1–9) and OT(1–9) were highly concentrated in extrahypothalamic limbic regions (ventral subiculum, central amygdala, and posterior olfactory nucleus); binding sites for VP(1–9) alone were found in the lateral septum. More recent conﬁrmatory evidence of this view has been obtained by Du and colleagues, and is discussed below in the updated commentary relevant to this topic. b. Behavioral and Electrophysiological Experimental Evidence i. Behavioral studies Three of lines of behavioral evidence are relevant to the neuropeptide concept. One line pertains to research with the desglycinamide derivatives of AVP (DG-AVP) and OT (DG-OT). It is noteworthy that endogenous DG-AVP is present in the peripheral circulation but is absent in the brain of the laboratory rat (Laczi et al., 1991). Nevertheless, numerous studies cited in this text have reported that these derivatives retain the behavioral but not the endocrine effects of their parent peptides. Speciﬁcally, De Wied and colleagues have repeatedly observed that, whether peripherally or centrally administered, DG-AVP facilitates, and DG-OT attenuates, memory consolidation and retrieval in both aversive (De Wied et al., 1972; Lande et al., 1971; Rigter et al., 1974; Skopkova et al., 1991; Walter et al., 1975) and appetitive (Bohus, 1977; Vawter and Van Ree, 1995) paradigms. Other laboratories reported ﬁndings that are consistent with the aforementioned studies cited for De Wied and colleagues: DG-LVP facilitated memory consolidation, and memory retrieval for a LiCl-induced conditioned taste aversion (Vawter and Green, 1980), enhanced retention of a conditioned shuttlebox avoidance response when microinjected into the ventral hippocampus (Ibragimov, 1990), and facilitated retention for previously learned word lists in human subjects (Bruins et al., 1992). On the other hand, there were replication failures: DG-AVP failed to inﬂuence memory consolidation assessed in a combined active/passive avoidance task (Sahgal, 1986), a water-ﬁnding task (Ettenberg et al., 1983a), as well as retention of a changed response–reward contingency experienced in an appetitive radial maze task (Packard and Ettenberg, 1985). A second line of behavioral evidence came from structure–activity studies. The ﬁnding that removal of the terminal amino acid residue (glycinamide) from the AVP and OT molecule dissociated the endocrine from the memory-processing effects of the parent peptides led De Wied and colleagues to search for those sites in the molecule that were essential for the effects of the peptide on memory processing. The modiﬁed VP and OT analogs and fragments tested in these studies had substitutions or deletions

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of amino acid residues at speciﬁc loci within the original molecule. Several general conclusions were drawn from these studies (see De Wied et al., 1988). First, the covalent ring structure of VP appears to be the essential site for its effect on memory consolidation assessed in an active avoidance paradigm; thus modiﬁcations of the covalent ring were more effective than those of the linear tail in interfering with the normal ability of VP to retard extinction of a learned pole-jump active avoidance response (De Wied, 1976; Walter et al., 1978). Second, the tripeptide linear tail, but not the covalent ring of the VP and OT molecules, is highly effective in inﬂuencing memory retrieval as assessed in a retrograde amnesia paradigm (Walter et al., 1975). Third, structure–activity studies (see Kovacs et al., 1982b) led to the conclusions that for the passive avoidance task: (1) the covalent ring structure of OT [tocinamide, OT(1–6)] as well as that of VP [pressinamide, VP(1–6)] facilitates PA memory consolidation; (2) the tripeptide tail structure of OT (PLG) is even more effective than that of VP (prolylarginyl-glycinamide, PAG) in facilitating PA memory retrieval; and (3) in contrast to VP, approximately the entire OT molecule [i.e., OT(1–9) or OT(1–8)] is required for the amnestic effect of the peptide on PA memory consolidation. In addition, an OT-like amnestic effect on retrieval was observed with fragments containing the proline amino acid residue attached to the ring structure of OT [i.e., OT(1–7)]. Behavioral studies using synthesized VP and OT C-terminal fragments in a variety of paradigms provide a third line of behavioral support for the neuropeptide concept. These neurohypophysial metabolites are both more potent (effective at much lower dose levels) and selective (behavioral but no peripheral hormonal effects) than their parent peptides. Studies by De Wied and colleagues have consistently demonstrated these two features whether peripherally or centrally administered, or tested in aversive or appetitive paradigms. More speciﬁcally, various VP C-terminal fragments, with or without the pyroglutamic acid residue, have facilitated PA memory consolidation and retrieval after subcutaneous, intracerebroventricular, or local microinjection into speciﬁc limbic brain sites (Burbach et al., 1983a; De Jong et al., 1985; De Wied et al., 1987; Kovacs et al., 1986), enhanced retention in an active avoidance paradigm after peripheral administration (De Wied et al., 1987), reversed pentylenetetrazole-induced retrograde amnesia after subcutaneous administration of the peptide fragment 1 h before the PA retention test (De Wied et al., 1987), and improved long-term or short-term memory tested in the holeboard search task (Vawter et al., 1997). After subcutaneous or intracerebroventricular injections, variously tested C-terminal OT fragments attenuated PA memory consolidation and retrieval (De Wied et al., 1987, 1991; Gaffori and De Wied, 1988) and reduced conditioned freezing behavior (Stoehr et al., 1992). When subcutaneously injected after polejump active avoidance training, OT fragments containing the glycinamide

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residue [OT(4–9) and OT(5–9)] produced a dose-related bimodal action (low doses retarded retention, high doses facilitated it). Those metabolites that lacked this residue [OT(4–8) and OT(5–8)] produced a straightforward amnestic effect. Findings reported by other research groups using appetitive paradigms indicated that subcutaneously injected C-terminal VP metabolites enhanced selective (Bunsey et al., 1990) and divided (Meck, 1987) attention, and provided that baseline learning proﬁciency and memory accessibility were sufﬁciently low, enhanced memory consolidation in a spatial maze task (Strupp, 1989) and memory retrieval in a socially transmitted food preference test (Bunsey and Strupp, 1990; Strupp et al., 1990), respectively. In addition to the self-preservative types of aversive and appetitive learning paradigms described above, there is also evidence that these VP and OT metabolites modulate rodent olfactory-based SRM, considered herein as closely related to reproductively related social behavior. Several studies reviewed in Chapter 13 demonstrated that peripherally administered C-terminal VP and OT fragments facilitated and attenuated SRM, respectively (Popik and Van Ree, 1992; Popik et al., 1991, 1996; Sekiguchi et al., 1991a). ii. Electrophysiological studies Findings from electrophysiological studies with DG-AVP and C-terminal fragments also support the neuropeptide concept. First, it was shown that DG-AVP normalized hippocampal theta rhythm during paradoxical sleep in HODI rats (Urban and De Wied, 1975, 1978), and this EEG activity is considered to be associated with memory consolidation (see Chapter 5). Second, studies carried out with rat hippocampal slice preparations showed that, similar to VP(1–9), VP(4–8) enhanced glutamate-mediated synaptic transmission and increased the duration of long-term potentiation of synaptic transmission at hippocampal CA1–subiculum synapses and did so without requiring NMDA receptor participation (Chepkova et al., 1995). As with PA behavioral studies, the enhancement of LTP induced by the application of VP(4–8) was signiﬁcantly more potent than that for the parent peptide (Chepkova et al., 1995). 3. Commentary: Research Update a. Research Support for the Neuropeptide Concept : VP(4–8) as a New MemoryEnhancing Molecule The evidence summarized above is based on studies reviewed in earlier chapters of this text. A more recent line of support for the neuropeptide concept is illustrated by the pharmacological and biochemical research with the C-terminal vasopressin metabolite VP(4–8), carried out by Du and associates at the Shanghai Institute of Biochemistry (see Du et al., 1998, for review of this research). The experimental protocols used in their study of the role of this pentapeptide in memory processing have

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ranged from the molecular to the behavioral level of analysis. Their ﬁndings are consistent with the postulate that this pentapeptide can qualify as a ‘‘new memory-enhancing peptide’’ functionally independent of the parent peptide, and characterized by its own distinctive receptor system. To this end they have renamed this oligopeptide ZNC(C)PR. However, for purposes of this discussion, it continues to be referred to as VP(4–8) in recognition of its derivation from the parent peptide, VP(1–9). Two of their studies conﬁrmed earlier cited ﬁndings of De Wied and colleagues. Thus, VP(4–8) was far more effective than the parent peptide in its ability to (1) facilitate retention in a PA task (Lin et al., 1990) and (2) enhance NMDA-independent long-term potentiation of synaptic transmission in CA1–Schaeffer collateral and CA3–mossy ﬁber synapses in rat hippocampal slices (Rong et al., 1993). Of particular interest for this discussion are their research ﬁndings, discussed below, which have helped to (1) verify the independence of the VP(4–8) receptor system from that subserving VP(1–9), with respect to both receptor distribution and ligand– receptor-binding properties; and (2) clarify the nature of the intracellular signaling system by which the pentapeptide mediates its effects on short-term and long-term memory formation. b. VP(4–8) Receptor Localization and Characterization of Its Ligand–Receptor Interactions in the Rat Brain Using chemically synthesized 35S-labeled VP(4–8) with high speciﬁc activity, Du et al. (1994b) provided evidence of speciﬁc binding to synaptic membranes obtained from various structures within the rat brain. These included structures in the hindbrain (nucleus of the solitary tact, cerebellum, and medulla), midbrain (superior and inferior colliculus), diencephalon (hypothalamus), and telencephalon (amygdala, anterior cerebral cortex, caudate nucleus, and hippocampus), and indicated a regional distribution similar to that reported for AVP(1–9)-binding sites (see Table III in Chapter 1). However, comparison of ﬁndings from studies that mapped receptor sites for the two peptides within the hippocampal area suggested that their intraregional receptor distributions might be separate and nonoverlapping. Thus, a developmental study by Du et al. (1994b) used selective lesioning and autoradiography to localize VP(4–8) binding within the hippocampal complex, and showed them to be present on the whole hippocampal pyramidal cell layer and granular cell layer of the dentate gyrus. This hippocampal distribution markedly differed from that reported for VP(1–9) (Petracca et al., 1986; and see Table III in Chapter 1) and for VP(4–9) (Brinton et al., 1986; De Kloet et al., 1985b). Neurochemical testing by Du et al. (1994b) characterized 35S-labeled VP(4–8) speciﬁc binding to synaptic membranes isolated from the anterior cortex. Comparisons between their results and those obtained for

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[3H]AVP(1–9)-binding sites by Junig et al. (1985) and Pearlmutter et al. (1983) indicated notable differences between the two peptides regarding the nature and speciﬁc properties of their respective ligand–receptor interactions (see Du et al., 1994b, for details). In addition, Du et al. (1994a) showed that AVP(1–9) did not compete detectably with VP(4–8) binding to anterior cortical synaptosomal membrane receptor sites, indicating that the parent peptide does not bind to these VP(4–8)-binding sites. Taken together, these ﬁndings support the proposal by Du and colleagues that VP(4–8) is subserved by a central receptor system separate and distinct from the central vasopressin V1 receptor with respect to both its speciﬁc distribution within the brain, and the deﬁning properties that characterize its ligand–receptor interactions (Du et al., 1994a,b, 1998). c. The VP(4–8) Receptor Intracellular Signaling System That Mediates the Effects of the Peptide on Memory Processing A number of the studies carried out by Du and colleagues (reviewed in Du et al., 1998) have yielded valuable information about the nature of the VP(4–8) receptor-coupled intracellular signaling pathway that mediates the effects of the peptide on memory processing. Their studies have indicated that like the VP V1 receptor, the VP(4–8) receptor is a G protein-coupled receptor (GPCR), which when activated by the receptor–ligand complex leads to the breakdown of the membrane phospholipid, phosphatidylinositol-4,5-biphosphate (PIP2) and the formation of inositol triphosphate (IP3) and diacylglycerol (DAG), the second messengers. Thus, an in vitro study with rat hippocampal slices showed that, in the presence of GTP, VP(4–8) markedly stimulated inositol phospholipid metabolism, with a potency 100 times greater than that of VP(1–9) (Gu and Du, 1991a). However, in contrast to the VP(1–9) V1 GPCR, the G protein to which VP(4–8) is coupled is sensitive to pertussis toxin, and the signaling pathway for the VP(4–8) GPCR is blocked at the receptor level by a selective VP(4–8) receptor antagonist, ZDC(C)PR (Qiao and Du, 1996a). Additional investigations carried out by this research group, and by others, further characterized the nature of the VP(4–8) GPCR transduction pathway. 1. Studies by Chen et al. (1993), and Qiao et al. (1996, 1998) provided evidence consistent with the following: (a) intracellular calcium (Ca2þ) released by the second messenger IP3 binds with calmodulin (CaM), an intracellular calcium receptor, to form the Ca2þ–CaM complex, which activates (phosphorylates) a protein kinase, calmodulin-dependent protein kinase II (CaMKII). This enzyme subsequently undergoes autophosphorylation (self-phosphorylation), in which form it plays an essential role in the induction of LTP (Liu et al., 1999); and (b) DAG activates protein kinase C (PKC), which phosphorylates other proteins and enzymes in the

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intracellular pathway, including the GAP43–CaM complex. This complex is formed by an interaction between growth-associated protein 43 (GAP43) and CaM, and when activated by PKC releases CaM, which is then available to reform CaMKII. In this way GAP43 helps regulate the intracytoplasmic supply of CaM necessary for activation of CaMKII. As noted, the latter is necessary for induction of LTP. Du et al. (1998) postulate that this molecular effect may be, at least in part, the mechanism by which AVP(4–8) facilitates NMDA-independent LTP formation. 2. Qiao and Du (1996b) reported that the intracellular kinase, mitogenactivated protein kinase (MAPK), also participates in the VP(4–8) GPCR transduction pathway, and subsequent evidence suggested that this transduction pathway divides into two independent branches: one involving CaMKII activation and the related PKC activation of the GAP43–CaM complex, and the other involving PKC phosphorylation of MAPK (Qiao and Du, 1998; Yan et al., 1999). MAPK signaling cascades can inﬂuence transcription factors that lead to the genetic expression of proteins involved in synaptic structural modiﬁcations that underlie long-lasting LTP (a few hours to a few weeks in duration) and long-term memory storage (Adams and Sweatt, 2002; Adams et al., 2000). 3. Du and colleagues (Gu and Du, 1991b; Zhou et al., 1995) have also obtained evidence that this AVP(4–8) GPCR mediates the transcription of immediate-early genes (IEGs) and late-onset genes. As noted in Feldman et al. (1997; and see Chapter 1), the protein products of IEGs are transcription factors that bind to the target gene and regulate its transcription. Two known target genes inﬂuenced by AVP(4–8) receptor activation in the rat hippocampus and cerebral cortex are those that encode two neurotrophic factors (NTFs): nerve growth factor (NGF) (Zhou et al., 1995, 1996) and brain-derived neurotrophic factor (BDNF) (Zhou et al., 1997). Aside from their roles in the central and peripheral nervous systems during early development (critical for neuronal differentiation, growth, axonal guidance to target structures, and survival) (e.g., Levi-Montalcini, 1987), and in the adult (normal maintenance and protection from trauma in various neuronal cell populations) (Lindsay et al., 1994; Lindvall et al., 1994), NTFs have also been implicated in synaptic plasticity relevant to long-lasting LTP and memory formation (Poo, 2001; Thoenen, 1995). Therefore, it is quite probable that these NTFs mediate, at least in part, AVP(4–8) facilitation of long-term memory formation. As Du et al. (1998) proposed, this may occur via an indirect effect, such as the ability of NGF to maintain forebrain cholinergic systems, which in turn exert an important role in memory processing (Jerusalinsky et al., 1997). However, it may also occur more directly as a result of the contribution of BDNF to functional and morphological synaptic alterations involved in long-lasting LTP and long-term memory storage (Poo, 2001).

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IV. VP, OT, and Rodent Olfactory-Based Social Recognition Memory

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A. VP and SRM 1. Views and Research of Dantzer, Bluthe, and Colleagues These researchers propose that two functionally distinct VP-ergic systems mediate olfactory-based SRM in the rodent brain: (1) an androgenindependent VP-ergic system that enhances performance in a conspeciﬁc SRM paradigm by virtue of its interaction with the central arousal system, as is theorized to occur in non-SRM tests of retention; and (2) an androgendependent VP-ergic system that mediates SRM independently of the arousal system, but relies heavily on chemosensory processing in the vomeronasal (accessory) olfactory system. The ‘‘VP Dual Action Theory’’ is applicable to the former, but not the latter, VP-ergic system (see Dantzer, 1998; and Chapter 12). a. Role for the Androgen-Independent VP-ergic System in SRM Experimental ﬁndings supportive of the putative arousal-dependent and androgenindependent VP-ergic system are as follows: (1) a dose of peripherally administered AVP that had been shown to produce a pressor response (Le Moal et al., 1981; see Chapter 6), and pressor-associated aversive properties (Bluthe et al., 1985a,b; Dantzer et al., 1982; see Chapter 6) facilitated retention in the SRM test paradigm (Dantzer et al., 1987); (2) doses of peripherally as well as centrally administered AVP that produced an EEG proﬁle associated with behavioral arousal (Ehlers et al., 1985; see Chapter 6) also facilitated recognition memory in this paradigm (Le Moal et al., 1987); and (3) peripherally injected AVP enhanced SRM in male castrates, and this enhancement was blocked by pretreatment with a peripherally administered VP V1 pressor antagonist (Bluthe et al., 1990), indicating that the androgen-independent VP-ergic system continues to inﬂuence SRM by an arousal effect in the absence of the androgen-dependent VP-ergic system. b. Role for the Androgen-Dependent VP-ergic System in SRM i. Defining characteristics and linkage to the accessory olfactory system As noted in Chapter 1 (and see De Vries and Miller, 1998) a number of extrahypothalamic VP-ergic systems (e.g., within the medial amygdala and bed nucleus of the stria terminalis) are gonad dependent (rely on circulating gonadal hormones during prenatal life for their development, and during postnatal life for their sustained ability to synthesize VP) and sexually dimorphic (exhibit greater production of VP and denser VP ﬁber projections to target sites in males than in females). The androgendependent VP-ergic system involved in SRM processing is anatomically and

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functionally linked to the vomeronasal organ (VNO), which receives the chemosensory input that identiﬁes the conspeciﬁc, and the accessory olfactory bulb (AOB), which processes and perhaps stores the information received from the VNO. The accessory olfactory components of the medial amygdala (MA) and BNST relay the chemosensory information from the AOB to brain sites involved in social reproduction-related activities (e.g., the medial preoptic nucleus, which inﬂuences reproductive functions via neuroendocrine, autonomic, and somatomotor pathways; Simerly, 1990) and to limbic system structures involved in SRM processing (e.g., the lateral septal nucleus; Dantzer et al., 1988). ii. Evidence in support of a role for androgen-dependent VP in SRM Experimental ﬁndings in support of an important physiological role for the androgen-dependent VP system in rodent olfactory-based conspeciﬁc SRM are as follows: (1) given alone, a peripherally injected lipophilic V1 antagonist, which can cross the BBB (Lebrun et al., 1985), blocked normal SRM in sexually intact male rats (Dantzer et al., 1987); (2) castration, which depletes androgen-dependent VP, temporarily impaired SRM (Bluthe et al., 1990, 1993) whereas testosterone replacement therapy restored it (Bluthe et al., 1990). The temporary nature of the memory impairment suggested that another (as yet unidentiﬁed) system can compensate for the loss of the androgen-dependent VP-ergic system (Bluthe and Dantzer, 1990, 1993); (3) SRM in female rats, as in males, was facilitated by peripherally administered AVP, but unlike in males peripheral administration of the lipophilic V1 antagonist on its own did not block SRM. These ﬁndings indicate that in female rats the role of VP in conspeciﬁc SRM is mediated only by the ‘‘androgen-independent VP-ergic system’’; (4) androgen-deprived (untreated castrated) male rats (Bluthe et al., 1990) and mice (Bluthe et al., 1993) are as insensitive to the memory-blocking effects of the peripherally administered lipophilic V1 antagonist as are their female counterparts; and (5) surgical removal of the VNO mimicks the effects of androgen-dependent VP deletion by castration. That is, these vomerectomized rats are temporarily impaired in SRM and are no longer sensitive to the SRM-blocking effects of the peripherally administered lipophilic V1 antagonist (Bluthe and Dantzer, 1993). 2. Contributions to This Line of Inquiry from Other Research Groups a. Introductory Remarks The VP/SRM research initiated by Dantzer, Bluthe, and colleagues (the Paris group) was subsequently continued and expanded by other research groups as described in Chapter 13. Several of these studies conﬁrmed the earlier ﬁndings of the effect of the androgendependent VP-ergic system on SRM (Section IV.A.2.b). Other studies supported a role for androgen-independent VP in this form of memory

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processing, although without the necessary implication that this was accomplished via an associated VP-induced increase in behavioral arousal (Section IV.A.2.c). Still other studies yielded ﬁndings consistent with the proposal that SRM, like other types of memory, consists of both short-term memory (STM) and long-term memory (LTM) components, and that structurally differentiated VP analogs differentially interact with these two SRM components (Section IV.A.2.d). b. Androgen-Dependent VP and SRM Several lines of evidence are consistent with the Paris group conclusion that septal VP, a component of the postulated androgen-dependent VP-ergic system, has a physiological role in mediating SRM (Dantzer et al., 1988; see Chapter 12). Speciﬁcally, SRM is impaired (1) in Brattleboro (HODI) rats, and subsequently improved by microdialysis administration of VP into the mediolateral septum (MLS) of these rats (Engelmann and Landgraf, 1994); (2) by infusion of VP antiserum into the dorsal septal region (Van Wimersma Greidanus and Maigret, 1996); (3) by intraseptal infusion of a highly selective V1 receptor knockout treatment that markedly reduced AVP receptor density in the septal area (Landgraf et al., 1995); and (4) by infusion of a VP V1 (Everts and Koolhaas, 1997) or V1/V2 receptor antagonist (Everts and Koolhaas, 1999) into the lateral septum (LS), or of a V1 receptor antagonist into the MLS (Engelmann and Landgraf, 1994). Moreover, the SRM impairments induced by the two types of VP antagonists noted in point 4 were speciﬁc to SRM because neither VP receptor antagonist blocked object recognition (Everts and Koolhaas, 1997) or acquisition/retention in a spatial memory task (Everts and Koolhaas, 1999). Given that androgen-dependent VP-ergic cells in the MA also project to the hippocampus (Caffe et al., 1987), the observation that anti-VP serum microinjected into the ventral hippocampus prevented expression of normal SRM (Van Wimersma Greidanus and Maigret, 1996) might signify involvement of the hippocampus as well as the LS in the androgen-dependent VP-ergic circuitry postulated in the mediation of SRM. c. Androgen-Independent VP and SRM i. Olfactory system Findings by Dluzen et al. (1998a,b) were relevant to the role of androgen-independent VP in SRM processing. They demonstrated that infusion of VP into the main olfactory bulb (MOB) extended the duration of SRM from 30 to 120 min, and attributed this preservation of SRM to an androgen-independent VP-ergic inﬂuence for the following reasons: (1) the MOB is a component of the main, not the accessory, olfactory pathway; (2) the infused VP remained localized in the MOB and did not activate the septal VP system; and (3) infusion of a V1 receptor antagonist into the MOB did not impair normal SRM as expected if it were part of the androgen-dependent circuitry involved in SRM processing. This is especially

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so, because Bluthe and Dantzer (1993) had demonstrated the disruptive effect on SRM produced by removal of the VNO. The additional observation of Dluzen et al. (1998a,b) that the preservation of SRM induced by VP infusion into the MOB depended on simultaneous input from the locus coeruleus NA-ergic system, indicated that a VP–NA interaction was necessary for this SRM effect. Given the role of the LC–NAergic system in cortical activation/behavioral arousal, this latter observation can be interpreted as support for the theory of Dantzer and colleagues that behavioral arousal is the means by which androgen-independent VP inﬂuences SRM. However, as noted in previous discussion, the LC–NA cortical projection system also contributes to memory consolidation. Hence, this VP–NA interactional effect in the MOB might signify a mnemonic action justifying adding SRM to the other types of memory processing that fall under the explanatory framework of the ‘‘VP/OT Central Memory Theory.’’ ii. Hippocampus One may speculate that the SRM impairment observed by Van Wimersma Greidanus and Maigret (1996) after infusion of VP antiserum into the hippocampus was due to depletion of an androgen-independent VP system of hippocampal origin rather than an androgen-dependent VP system of MA origin. If so, it may be further speculated that this androgenindependent VP system mediates the LTM component of SRM proposed by Popik and Van Ree (1992, 1998). The following observations suggest the merit of pursuing future study of this speculation: (1) Hallbeck et al. (1999), using a sensitive in situ hybridization assay capable of localizing small numbers of cells that contain mRNA for a given neurotransmitter, found VP mRNA expression suggesting the presence of VP cells in the CA1–CA3 ﬁelds and dentate gyrus of the hippocampus. It is hoped that follow-up study will conﬁrm the presence of these cells, and determine their androgendependency status; (2) Kogan et al. (2000) found that experimental lesions induced in the hippocampus of male mice impaired SRM 30 min but not 30 s after the initial investigative encounter with the juvenile conspeciﬁc. This indicated that the hippocampus has a physiological role in mediating recognition memory, but not in learning the standard SRM task paradigm; and (3) Kogan et al. (2000) also found support for an LTM component in olfactory-based social recognition. This included both behavioral observations (described in the next section) and physiological ﬁndings (a subcutaneous injection of a protein synthesis inhibitor blocked SRM tested 24 h but not 30 min after the initial 2-min encounter with the juvenile), indicating blocking of the protein synthesis-dependent LTM, but not the protein synthesis-independent STM component of SRM. d. Postulated Short-Term Memory and Long-Term-Memory Components in SRM, and the Relevance of VP for These Components It has been consistently reported that when tested for SRM under laboratory conditions, normal adult male

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rats and mice recognize a reencountered conspeciﬁc juvenile for between 30 min and 1 h, after which memory dissipates and is no longer present when tested 2 h after the initial encounter (e.g., Bluthe et al., 1993; Dantzer et al., 1987; Sekiguchi et al., 1991a). However, it has also been noted that this duration of SRM-mediated conspeciﬁc recognition is much shorter than that observed for wild rats tested under similar circumstances (Thor, 1979), and has led to the commonly held assumption that SRM in laboratory rats qualiﬁes as a form of short-term memory (STM) (Dantzer et al., 1987) as deﬁned in accordance with McGaugh (2000). Citing unpublished observations, Popik and Van Ree (1998) questioned this assumption, noting that according to certain behavioral criteria, SRM in these laboratory tested rats lasts well beyond a 30-min interexposure interval (IEI). They observed, for example, that 3 months after the initial encounter, resident laboratory rats engaged in similar amounts of anogenital snifﬁng of the preencountered and the stranger juvenile, but directed fewer aggressive behaviors toward the preencountered juvenile. Such observations led these researchers to propose that rodent olfactory-based SRM is considerably longer in duration than the 30-min IEI typically observed, but is too weak to be expressed in the SRM test paradigm (Popik and Van Ree, 1992). Although they did not explain why this should be so, they nevertheless proposed that as in other types of memory processing, SRM involves both an STM and an LTM component. A study by Kogan et al. (2000) also provides evidence in favor of the postulated LTM component in SRM. Their experimental ﬁndings can be interpreted as support for the view that traditional laboratory housing practices are mainly responsible for the short-term nature of SRM typically observed in the SRM test paradigm, and that the LTM component of SRM will be expressed provided the animal housing arrangements permit normal species-typical social interaction up to the day of behavioral testing. Speciﬁcally, they found that group-housed mice showed social memory for a familiar juvenile when tested immediately, 30 min, 24 h, 3 days, and 7 days after a single 2-min-long interaction. These researchers further showed that social isolation (housing mice in individual cages for 3 weeks or even 1 day before the start of the experiment) disrupted the LTM, but not the STM, component of SRM (i.e., prevented recognition when tested 24 h, but not 30 min, after the initial encounter). Given these ﬁndings, the typically observed 30-min duration for SRM in laboratory rodents may be an artifact of the housing arrangements, because social contact within the animal colony is typically precluded by the practice of conducting experiments with individually caged laboratory rats and mice. Two sets of experimental ﬁndings by Popik, Sekiguchi, Van Ree, and colleagues indicated that, depending on the VP analog tested, VP interacts with either the LTM or STM components of SRM. First, Sekiguchi et al. (1991a) demonstrated that peripherally administered DG-AVP extended the

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duration of SRM in male rats from 30 min to 24 h, thereby verifying the presence of an LTM component in SRM to which this VP analog contributed. Second, Popik and Van Ree (1992) observed that peripheral administration of AVP metabolites that contained the covalent ring structure [i.e., AVP(1–8), AVP(1–7), or AVP(1–6)] lengthened the duration of SRM from the normally observed 30-min interexposure interval (IEI) to at least 24 h (demonstrating the LTM effect). VP derivatives that lacked this structure [i.e., AVP(4–9) and AVP(4–8)] extended SRM for 2 h but not 24 h (demonstrating only the STM effect). These ﬁndings indicated the critical signiﬁcance of the covalent ring structure for the inﬂuence of VP on the LTM component of SRM. The unpublished observation that on its own, the covalent ring structure of AVP [AVP(1–6)] failed to inﬂuence SRM tested 2 h after the original encounter (an STM effect) was interpreted as further support for the view that this structure is involved in the LTM (not the STM) component of SRM. 3. Commentary Functionally distinctive roles played by ‘‘androgen-dependent’’ and ‘‘androgen-independent’’ VP-ergic systems in SRM, originally noted by Dantzer, Bluthe, and colleagues, have received support from experimental ﬁndings of a number of research groups. However, the view that SRM is a form of STM, and that the androgen-independent VP-ergic system inﬂuences SRM by its effect on the subject’s arousal level, as occurs in tests for LTM, is not unequivocally accepted by other research groups. Questions regarding SRM in general, and the nature of the role of the androgen-independent VP-ergic system in SRM in particular, need research clariﬁcation. These questions are as follows. 1. Is SRM basically similar to other forms of memory processing in having both STM and LTM components? Research by Sekiguchi et al. (1991a), Popik and Van Ree (1992, 1998), and Kogan et al. (2000) provides preliminary evidence suggesting an afﬁrmative response to this question. 2. If so, does androgen-independent VP play a role in the putative LTM component of SRM? Again, preliminary data by Van Wimersma Greidanus and Maigret (1996) suggests that this is so. 3. Are the general mechanisms by which VP appears to inﬂuence other forms of memory processing (e.g., VP interactions with central catecholamine and acetycholine neurotransmitter projection systems) also operative for the androgen-independent VP inﬂuence on SRM? Observations by Dluzen et al. (1998a,b) support an afﬁrmative answer to this question. 4. Of what functional signiﬁcance is the ﬁnding that the covalent ring structure of VP is needed for expression of the LTM, but not the STM, component of SRM? 5. Does the hippocampal VP facilitation of SRM observed by Wimersma Greidanus and Maigret (1996) reﬂect a role for the androgen-dependent or the androgen-independent VP-ergic system? The answer to this depends on

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(a) determining the source of hippocampal VP, which may be released from androgen-dependent VP-ergic cells originating in the MA, or from putative VP cells of hippocampal origin (Hallbeck et al., 1999); and (b) if of hippocampal origin, the androgen-dependent versus androgen-independent status of this VP cell system needs to be determined. 6. Does the androgen-dependent VP-ergic system contribute to both the LTM and STM components of SRM, as has been suggested to occur for androgen-independent VP? At present this author is unaware of any data that is relevant to this question. In conclusion, the pioneering studies of Dantzer, Bluthe, and colleagues have done much to clarify the role of VP in species-predictable rodent olfactory-based SRM. Follow-up research has basically conﬁrmed the androgen-dependent/androgen-independent VP distinction, and has raised intriguing issues that need to be resolved and questions that need to be answered. Of particular interest to this author is the challenge directed to the commonly held assumption that SRM represents a short-term form of memory processing, and the suggestion that, instead, SRM contains both LTM and STM components. Because this intriguing proposal rests on data from only a few studies with the peripheral administered peptide, it is suggested that future studies relevant to this issue include other routes of peptide delivery, and research protocols designed to verify, extend, and further clarify these preliminary ﬁndings.

B. OT and SRM 1. Introductory Remarks As in the case of aversive and appetitive learning paradigms, the amount of research attention given to the study of the role of OT in SRM has been considerably less than that for VP. The studies that have been done have used a variety of protocols examining the effects of peripherally and centrally applied OT and analogs, and also more direct means of manipulating the endogenous OT-ergic systems in the brain. These protocols indicated both facilitative and attenuating effect of OT on SRM and were also consistent with the speculation that two functionally distinct OT-ergic systems may be operating, as has been postulated for VP. This speculation is more fully discussed in the commentary for this section (Section IV.B.3). 2. Relevant Evidence a. Treatments That Increase Peripheral or Central Levels of OT: Dose-Related Attenuation and Facilitation of SRM Dantzer et al. (1987) showed that peripherally administered OT (6 g /rat) attenuated SRM in contrast to the memory-enhancing effect by similar treatment with VP, indicating that the

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two peptides exert opposing actions on this form of short-term memory as they do for long-term memory assessed in avoidance paradigms. In their studies, Popik and colleagues peripherally administered OT over a wide range of dose levels immediately after the initial investigative encounter and demonstrated a dose-dependent effect of the peptide on SRM, whereby high doses impaired, and low dose levels facilitated, SRM. Thus: (1) Popik and Vetulani (1991) found that OT doses at 24 ng/kg and above disrupted normal SRM, an amnestic effect prevented by pretreatment with OT receptor antagonists; (2) Popik et al. (1992) showed that a low dose range of subcutaneously injected OT (0.09 to 6.0 ng/kg) dose dependently facilitated SRM; and (3) Popik et al. (1996) tested the effects on SRM of high (0.6 and 6.0 g/kg, subcutaneous) and low (0.6 and 6.0 ng/kg, subcutaneous) doses of OT(1–9) and of many of its derivative metabolites. They conﬁrmed their previous ﬁndings that high doses of OT(1–9) attenuated, and low doses facilitated, SRM. Moreover, high dose concentrations of most of the OT metabolites produced a similar but somewhat less pronounced amnestic action, whereas, depending on the metabolite, low dose levels either facilitated or had no effect on SRM tested 2 h after the initial encounter. Popik et al. (1992) proposed that the high dose levels of OT that result in an SRM amnestic effect increase plasma OT to the high levels that prevail during restraint stress (Gibbs, 1984), sexual activity (Stoneham et al., 1985), and suckling (Higuchi et al., 1985). In contrast, the low doses that facilitate SRM probably result in mildly elevated plasma levels, slightly above basal values, as might be expected to occur during the low-stress condition of a social encounter tested in the SRM paradigm. Arletti et al. (1995) showed that low doses of intraperitoneally injected OT (3.0 and 6.0 ng/kg) administered 60 min before testing enhanced SRM in 26-month-old male rats. This conﬁrmed ﬁndings by Popik et al. (1992, 1996), and demonstrated that aging does not compromise the memory-improving activities of OT. Centrally administered OT enhanced SRM when intracerebroventricularly injected at low doses including femtogram, picogram, and nanogram dose levels, but this SRM-facilitative effect was not observed when the dose exceeded 10 ng/rat (Benelli et al., 1995). Noteworthy is the ﬁnding by this latter research group that the minimum active doses were in the range of physiological values, thereby helping ensure that endogenous OT was importantly involved in the SRM effects induced by this treatment. b. Treatments That Reduce Peripheral or Central Levels of OT Several types of experimental protocols have been used to determine whether or not endogenous OT exerts an inﬂuence on rodent conspeciﬁc recognition memory, and if so, the nature of this contribution. These protocols (1) assessed the effects on SRM of neutralizing endogenous OT by injecting its antiserum

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peripherally, intraventricularly, or site speciﬁcally; (2) prevented normal OT receptor transmission in selective brain structures by local injections of a selective OT receptor antagonist; and (3) compared conspeciﬁc recognition behavior in wild-type and OT knockout mice. Their ﬁndings indicated that OT-ergic systems localized in certain brain sites, especially those integrated with the main olfactory pathway, were signiﬁcantly involved in sensory/attentional and/or mnemonic mechanisms at some stage of memory processing that contributed to olfactory-based conspeciﬁc recognition. Although limited in number, the studies that used pharmacological techniques to reduce endogenous levels of OT suggested an OT-induced facilitative or amnestic effect on SRM, depending on the dose of the antagonist and the route of its delivery, and the brain site in which endogenous OT was neutralized by the OT antiserum treatment. Comparative studies with OT knockout mice indicated an OT-induced facilitative effect on SRM. The relevant ﬁndings are summarized below. An OT impairment of normal conspeciﬁc recognition memory, reminiscent of the general amnestic action of the peptide in aversive learning paradigms, has been observed in two sets of studies using the standard version of the SRM paradigm. Popik and Vetulani (1991) peripherally injected one of two OT antagonists at one of two dose levels (12 or 24 g/kg) immediately after removal of the juvenile at the end of the initial investigative encounter. The higher dose of each antagonist facilitated SRM, tested 60 min after this encounter. These ﬁndings are consistent with the interpretation that the endogenous OT receptor system affected by these antagonists normally attenuates memory processing, including that tested in the SRM paradigm. Van Wimersma Greidanus and Maigret (1996) found that antiserum neutralization of OT facilitated SRM in male rats when injected into the ventral hippocampus, but was without inﬂuence when injected into the dorsal hippocampus, septal area, or olfactory nucleus. Thus ventral hippocampal OT, as well as that present in brain regions reached by intracerebroventricularly applied antiserum, appears to belong to an OT-ergic system of receptors or circuitry that exerts an attenuating effect on SRM, as it does on LTM for aversive learning experiences. The failure of the antiserum to inﬂuence recognition memory when injected into the other limbic areas suggests that any OT present in these areas at the time of testing was not involved in memory processing relevant to olfactory-based conspeciﬁc recognition. A number of disparate ﬁndings raise the question of whether endogenous OT protects the display of normal SRM, tested 30 min after the initial social encounter, as well as its preservation, tested 120 min after the encounter: (1) an intracerebroventricularly injected OT antagonist blocked only SRM preservation in female rats whereas a similarly injected AVP antagonist was without effect on either variable (Engelmann et al., 1998); (2) in contrast to their OT-normal wild-type counterparts, male and female OT knockout mice were selectively impaired in the display of normal SRM

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and its preservation, but tested normally for other olfactory functions (e.g., odor-based foraging, olfactory habituation, and detection of nonsocial stimuli) and in spatial learning tasks (Ferguson et al., 2000); (3) intracerebroventricularly injected OT fully restored conspeciﬁc recognition in OT knockout mice when injected 10 min before, but not 10 min after, the initial social encounter, indicating that the role of OT in SRM formation requires its presence at the time of processing the relevant olfactory stimulus cues (Ferguson et al., 2001); and (4) a local infusion of an OT antagonist into the MOB of male wistar rats did not interfere with the display of normal SRM, but did prevent its preservation (Dluzen et al., 1998a,b, 2000). This ﬁnding indicated that the presence of OT in the MOB is not a necessary prerequisite for the normal display of this type of recognition memory, but is required for its preservation (i.e., extending its normal duration of action). c. OT-ergic Representation in the Main Olfactory Pathway: Relevance for Conspecific Recognition The main olfactory system, which consists of the main olfactory bulb (MOB) and certain of its branching pathways, processes the volatile odorous stimuli necessary for identifying individual conspeciﬁcs and for determining their socially relevant distinguishing characteristics (e.g., maturational, sexual, emotional, and social rank status). After initial processing in the MOB, olfactory neural signals are relayed along branching pathways making synaptic connections in cortical and subcortical brain sites. Processing in certain of these brain sites may result in memory storage itself or in memory-dependent or memory-independent species-predictable sexual, parental, or other conspeciﬁc social behavioral patterns (Meredith, 1991; and see Ferguson et al., 2001). Olfactory information processed in the medial preoptic area (MPA) ( Macrides, 1976; Pfaff and Pfaffmann, 1969) has an important role in mediating both ‘‘appetitive’’ (proceptive) and ‘‘consummatory’’ (copulatory) phases of rodent sexual behavior (see Caldwell et al., 1986, 1988). Experimental ﬁndings from the studies of Popik and Van Ree (1991) and Dluzen et al. (1998a,b, 2000) (see Chapter 13), and Ferguson et al. (2001) (this chapter) support the suggestion that OT receptor sites along one of the main olfactory branching pathways from the MOB to MPA [MOB ! medial amygdala (MA) ! bed nucleus of the stria terminalis (BNST) ! MPA] have an important role in mediating olfactorybased social recognition behavior in both male and female rats and mice. Relevant ﬁndings are brieﬂy reviewed below. Popik and Van Ree (1991) demonstrated that low doses (0.3 to 1000 pg) of OT, microinjected into the MPA, dose dependently facilitated SRM, with the 0.3-pg dose level being the lowest effective dose level. This was a siteselective effect because these low doses of the peptide failed to inﬂuence SRM when microinjected into the septum. This difference might have been due to the presence of different OT receptors in the two brain sites. The observation by De Wied et al. (1991) that pretreatment with an OT receptor

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antagonist that reduced the amnestic effect of OT on PA behavior when injected intracerebroventricularly had no inﬂuence on OT enhancement of SRM when microinjected into the MPA (Popik and Van Ree, 1991) supports this interpretation. However, Popik and Van Ree (1991) did not investigate the effects on SRM of pretreatment with subcutaneously injected OT receptor antagonists that blocked OT-induced attenuation of SRM and facilitated SRM when injected alone (Popik and Vetulani, 1991). Hence, the type of receptor in the MPA relevant to the OT-induced enhancement of SRM remains to be clariﬁed. Experimental ﬁndings obtained by Dluzen et al. (1998a,b, 2000) led these authors to propose that an OT–NA interactional effect in the main olfactory pathway is an important mechanism by which OT enhances rodent SRM, and might be identical to that mediating the role of OT in the recognition by ewes of newborn lambs (Kendrick et al., 1997). More speciﬁcally, these ﬁndings indicated that an OT-ergic system within the MOB facilitates rodent SRM by enhancing NA release into the MOB via activation of 2-adrenoceptors localized in terminals of the LC–NA-ergic projection pathway. The relevant experimental ﬁndings were as follows: (1) the MOB was a target structure mediating SRM enhancement effects of exogenous OT (Dluzen et al., 1998a); (2) NA released into the MOB was critical for the recognition memory preservation effects of OT (Dluzen et al., 1998b); and (3) an 2-adrenoceptor mediated the OT–NA interaction that was a prerequisite for OT enhancement of SRM (Dluzen et al., 2000). Ferguson et al. (2001) investigated the role of OT in olfactory-based SRM in the various branches of the main olfactory pathways including that projecting from the MOB to the MA. This study compared genotypic differences in this processing in wild-type and OT knockout mice. Their ﬁndings speciﬁed the portions of this olfactory pathway that were involved in processing important to the role of OT in this behavior and speciﬁcally to its contribution to SRM formation. The relevant ﬁndings were as follows: (1) using Fos protein induction as a marker for neuronal activation in a number of brain regions involved in processing olfactory information, genotypic comparisons indicated that neuronal activation during the initial encounter in the SRM paradigm was similar for wild-type and OT knockout mice in the MOB and in several cortical and subcortical brain sites (lateral septum, piriform olfactory cortex, and cortical amygdala), but was signiﬁcantly reduced in others (MA, BNST, and MPA); (2) OT injections into the MA but not the MOB corrected the SRM deﬁcit observed in the OT knockout mice; and (3) in a similar manner, a selective OT antagonist impaired SRM when injected into the MA but not olfactory bulbs of OTnormal mice. These ﬁndings, together with those reported by Dluzen et al. (1998a,b, 2000), indicated that OT activation in the MOB during a social investigative encounter prolongs duration of SRM but is not essential for

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the formation of normal SRM; on the other hand, the presence of OT in the MA at this time is required ‘‘for the proper processing of the olfactory information and the development of the social memory’’ (Ferguson et al., 2001, p. 8282). The ﬁndings cited above are also consistent with the speculation that OT facilitative effects on conspeciﬁc SRM are relevant for the signiﬁcant role of the neuropeptide in reproduction-associated social behavior observed in both male and female rodents. This point is more fully elaborated below (Section IV.B.3). 3. Commentary Concerning the Role of OT in Memory Processing in Avoidance Learning Paradigms, and in Rodent Olfactory-Based Conspecific Recognition a. The Case for Two Functionally Distinct OT-ergic Systems Involved in Memory Processing It seems clear from the evidence reviewed in this chapter (Sections III.B and IV.B) that peripheral or central administration of OT does not induce an ‘‘amnestic’’ effect in every learning situation. Instead, the speciﬁc effect of OT on memory storage and retrieval depends on the dose level injected, the type of learning task given, and, in the case of central administration, the speciﬁc brain site injected. For example, numerous studies reviewed in Section IV.B have demonstrated that whereas low doses enhance SRM, high doses of OT attenuate SRM. Various lines of evidence pertaining to the OT inﬂuence on memory processing, tested in a variety of learning situations, are in general consistent with this author’s proposal that two functionally distinct OT-ergic systems (receptor systems and neuronal circuitry in the brain), herein referred to as types I and II, are differentially activated depending on the learning task and the level of externally applied (or internally released) OT. In certain learning contexts, some of which involve SRM, the type I OT-ergic system is activated and exerts an amnestic effect on memory processing. In contrast, activation of the type II OT-ergic system produces low OT levels and facilitates memory processing. Additional features characterizing these two types of OT-ergic systems are described below. The type I OT-ergic system is activated in emotionally arousing, stressful, and/or energy-demanding situations associated with certain reproductive activities (e.g., sexual activity, parturition, and lactation), or in encounters perceived as threats to the individual’s well-being. Measurement of OT in the CSF and in the plasma at these times indicates that these levels are well above normal basal values (see Chapter 1; and Uvnas-Moberg, 1997). The type II OT-ergic system is activated in social stimulus situations characterized by a mild increase in emotional arousal, and a negligible degree of stress. Activation of this system enhances social afﬁliative behavior and promotes a degree of emotional bonding that is conducive to reproductive success (e.g., Insel, 1992; Nelson and Panksepp, 1998). In such

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situations, small amounts of OT are released, slightly above basal values when measured in the CSF and plasma (Uvnas-Moberg, 1997). According to this model, the SRM paradigm that involves only a mild degree of emotional arousal and negligible stress should activate only the type II OT-ergic system and hence facilitate conspeciﬁc recognition. This prediction from the model is consistent with many of the ﬁndings cited in Chapter 13 and reviewed in this chapter. That is, an SRM-facilitative action was induced by low doses of exogenous OT whereas high doses, expected to engage the type I OT-ergic system, resulted in an SRMattenuating effect as occurs in memory processing tested in aversive learning paradigms. b. Adaptive Value of an OT-ergic Amnestic Action in Stressful Contexts and an OT-ergic Memory-Enhancing Action in Prosocial Encounters From an evolutionary perspective, it is plausible that the OT-ergic systems, postulated above, might have developed in accordance with natural selection because the ability of OT to both attenuate and facilitate memory processing, depending on the circumstance, is functionally adaptive, serving both to protect selfintegrity (self-preservation) and promote reproductive success (species preservation). These points are elaborated and illustrated below. The ability of OT to attenuate memory formation and retrieval of highly emotionally arousing stressful experiences appears to be consistent with its ability to exert a calming emotional effect on behavior and to reduce anxiety and stress (Uvnas-Moberg, 1997). Otherwise stated, a memory-attenuating effect during an extremely physically and/or emotionally painful experience reinforces the anxiolytic effect of the peptide on behavior. Both types of OT actions are protective of self-integrity insofar as they help maintain organized behavior during the stressful encounter, shortly after it, and on future occasions when the stressor is present. The calming and anxiolytic effects of OT on behavior in both social and nonsocial stress contexts are illustrated by the ability of the peptide to reduce (1) ultrasonic distress calling in 6- to 8-day-old rat pups elicited by removing them from their dams and siblings (Insel and Winslow, 1991), (2) anxiety behavior triggered by noise stress (Windle et al., 1997), and (3) the anxiety behavior displayed after placement in the elevated plus maze (McCarthy et al., 1996; Windle et al., 1997). OT enhancement of memory processing is known, or may be expected to be involved, in memory processing associated with promoting (1) parent– offspring bonding in sheep (Kendrick et al., 1997); (2) mate selection, mate bonding, and parental behavior in the prairie vole, especially but not exclusively in the females (Carter et al., 1992; Cho et al., 1999; Insel and Hulihan, 1995) and (3) social afﬁliative behavior in laboratory rats and mice (Nelson and Panksepp, 1998). The demonstration of the role of OT in the SRM

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paradigm tested with laboratory mice and rats provides further support for the latter point.

V. Closing Remarks: Future Considerations

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Suggestions for future research have been made throughout the course of this text and in the commentaries of this last chapter. A number of them are reiterated and elaborated in the topic areas discussed below. It is this author’s opinion that these topic areas should and will receive considerable research attention in the future.

A. Questions and Issues of Long-Standing Interest 1. How Do Peripherally Administered VP and OT Exert Centrally Mediated Behavioral Effects? This topic, a long-standing unresolved issue in memory-processing research with neurohypophysial hormones, is of continued interest on both theoretical and practical grounds. The theoretical debate engaged by this issue was reviewed in this chapter. Its practical signiﬁcance concerns its applicability to research study and clinical treatment with human subjects and patients. Potentially, there are a number of ways in which peripheral administration of supraphysiological doses of neurohypophysial hormones might induce centrally mediated behavioral effects. These were discussed in Chapter 14 and included (1) means by which these hormones might enter the brain, and (2) mechanisms by which they might exert central effects other than by direct entry into the brain. Several of the mechanisms reviewed or elaborated in Chapter 14 (Sections IV.B, VI.C, and VI.D) were initially introduced in Chapter 1 (Sections IV.C and IV.D) discussions that related various nonmnemonic functional activities of these neurohypophysial hormones to their effects on memory processing. These activities involve actions at nonneural receptor sites by which these peptides regulate glucose availability to the brain, cerebral blood ﬂow and nutrient transport across the BBB, as well as stress-associated activation of the anterior pituitary gland. Several lines of evidence support the proposal that these nonneuronal actions of VP and OT might contribute to memory processing either on their own or via an interaction with an aminergic, cholinergic, or peptidergic hormone and/or transmitter. Earlier cited research ﬁndings support a relation between VP enhancement of memory processing and its ability to promote nutrient transport across the BBB (see Chapter 14, Sections VI.C and VI.D). In general, however, there has been negligible research interest in relating the aforementioned functional activities of these neurohypophysial peptides with their roles in memory processing. It is hoped that future researchers may ﬁnd this line of inquiry worthy of study.

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2. Arousal and Attentional Mechanisms as Mediators of the Effects of VP and OT on Memory Processing The effects of VP, and in some cases of OT, on arousal and/or attentional processing continue to crop up as potential explanations for behavioral effects observed in various learning paradigms. The degree to which the effects of these neuropeptides on cortical activation/behavioral arousal and on attentional processing are necessary for their inﬂuences on STM and LTM remains an unresolved question. Evidence presented throughout the course of this text has indicated that peripheral administration of behaviorally active doses of VP, in particular, increases the subject’s level of behavioral arousal. Chapter discussions have also made clear that (1) catecholaminergic and cholinergic neurotransmitter projection systems play important roles in modulating activation/arousal, attentional processing and the various phases of memory processing; (2) the effects of VP and OT on all three of the above-mentioned neuronal/behavioral processes are mediated by their interactions with each of these neurotransmitter systems; and (3) numerous research groups are strongly committed to the view that activation/arousal and attentional processing mediate the memory modulation effected by these interactional effects. This author favors the viewpoint that arousal and attentional factors are more likely to play key roles in mediating STM, as well as the learning and retrieval but not the consolidation phases of LTM processing. Nonetheless, a VP-and/or OT-induced modulation of behavioral arousal continues to be suggested as the prerequisite for the behavioral results obtained in various learning paradigms used in this line of research. For example, an OT-induced arousal effect could explain the dose-dependent effects of OT on conspeciﬁc recognition behavior in the SRM paradigm. Future study relevant to this issue will, and should, continue to occupy research attention until it is satisfactorily resolved.

B. Methodological Issues: Protocols and Paradigms, and the Use of VP and OT Knockout Models in VP/OT Memory Research 1. Protocols Given the interpretative problems associated with peripherally administered VP and OT (noted above), it is puzzling to this author that many present day researchers continue to rely heavily on this route of delivery in studies using both ‘‘self-preservative’’ and ‘‘species-preservative’’ types of paradigms. It is particularly difﬁcult to understand this preference for peripheral administration when the parent peptides are used and the investigators are clearly concerned with investigating centrally mediated actions that are independent of associated peripheral endocrine activity. It is suggested that in those studies in which a peripheral route of administration is

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justiﬁed, a second experiment be included using central application of the same drug, and that in both experiments the drugs be delivered over a dose range that includes, at its lowest end, a dose that approximates the physiological level of the peptide under study. Future research would also do well to make more frequent use of protocols that permit assessment of the physiological signiﬁcance of the role of VP or OT in memory processing tested in a given task paradigm. These include (1) depletion of central VP or OT by intracerebroventricular or site-speciﬁc application of a VP or OT antiserum; (2) reduction of central VP and/or OT receptor activity by intracerebroventricular or local injections of VP and OT receptor antagonists or antisense oligodeoxynucleotides that interfere with synthesis of the VP V1 receptor; and (3) use of the microdialysis technique to measure locally released VP or OT and relating these results to ongoing performance in learning and memory paradigms. 2. Paradigms The studies by De Wied and colleagues have provided a wealth of detailed information in support of (1) the physiological signiﬁcance of central VP and OT participation in memory consolidation and/or retrieval stages of memory processing, (2) the catecholaminergic and glutamatergic neurotransmitter systems with which these peptides (mainly VP) interact in mediating these effects, and (3) the limbic and striatal brain sites involved in these effects. However, their predilection for using active and passive avoidance learning tasks (especially the passive avoidance task) in these studies greatly limits the generality of their ﬁndings. Although a number of other research groups have used a wide variety of other task paradigms, their investigations have been conﬁned to examining the effects of peripherally administered VP on task performance (see Chapter 9). In general, these researchers showed no interest in investigating potential roles played by selected brain structures or VP interactions with classic transmitter systems in the behavioral effects observed in these learning paradigms. Of the seven studies that did investigate the involvement of speciﬁc limbic structures (septal areas, dorsal or ventral hippocampus) in VP and/or OT effects in memory processing (see Chapter 10), two of them used avoidance and ﬁve, appetitive paradigms. Of the ﬁve studies (see Chapter 10) that investigated a potential role for VP– and/or OT–central cholinergic interactive effects in memory processing, all used the passive avoidance paradigm. Taken together, these studies point up the need to include greater representation of appetitive learning paradigms in future studies to extend the generality of the ﬁndings reported by De Wied and colleagues that were tested with the passive avoidance paradigm.

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3. Use of VP and OT Gene Knockout Models in VP/OT Memory Research The Brattleboro rat, derived from Long-Evans hooded rat stock, is an example of a natural VP gene knockout model. It was produced by a spontaneously occurring gene mutation at the VP locus that resulted in a homozygous genotype that is unable to synthesize VP. De Wied and colleagues, and other research groups, have compared retention behavior of homozygous and heterozygous Brattleboro rats with that observed in their normal LongEvans counterparts and in Wistar rats. As discussed in Chapter 3, inconsistent ﬁndings have been observed and alternative explanations have been offered to account for those retention deﬁcits that have been observed in the homozygous variant. More recently, advances in transgenic technology have resulted in the experimental creation of an OT gene knockout mouse model, a mutant mouse whose genotype lacks the gene needed to encode the OT peptide without affecting the gene encoding AVP. As noted earlier, Ferguson and colleagues have used this OT gene knockout mouse to investigate the role of OT in rodent olfactory-based conspeciﬁc SRM. Aside from an experimental test using a spatial learning paradigm, this mutant mouse has not been tested in the conventional stress-associated appetitive and aversive learning tasks used in many of the studies reported in this text. It would be interesting to compare performance of the mutant mouse with that of its wild-type counterpart in learning paradigms in which OT is expected, or has been actually found, to inhibit or attenuate long-term memory consolidation and retrieval. Given the interpretative problems associated with the VP gene knockout Brattleboro rat, it is further suggested that the OT gene knockout mouse mutant receive the same type of research scrutiny given to the former.

C. The Role of OT in Memory Processing As evident in the research discussed in this text, study of the participation of neurohypophysial peptides in memory processing has been focused on VP, with relative neglect of OT. As a result, several questions about the role of OT in this processing remain unanswered. Particular areas in need of future study are listed below. 1. Appetitive Learning Tasks In what is herein referred to as ‘‘self-preservation types’’ of learning tasks, OT/memory processing effects have been tested primarily in passive avoidance (PA), rarely in active avoidance (AA), and not at all in appetitive learning tasks. This was due primarily to the fact that OT/memory processing research was carried out almost exclusively by De Wied and colleagues, who used mainly aversive paradigms in their studies. Given that appetitive learning paradigms typically involve the stress of restricted food or water

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intake to establish behavioral motivation in laboratory animals, one might expect that endogenous OT will exert an amnestic action on memory processing tested in these paradigms. However, to this author’s knowledge such investigations have not as yet been carried out. 2. OT–Classic Transmitter Interactions in Aversive and Appetitive Learning Encounters A number of ﬁndings provide direct or indirect support for the notion that the amnestic action of OT in PA behavior is mediated by OT interactions with central catecholaminergic (CA-ergic) (see Chapter 4) and cholinergic (ACh-ergic) (see Chapter 10) projection systems. However, further study is needed to verify that the reported OT inﬂuence on CA-ergic neurotransmission in the various midbrain and forebrain structures studied relates to its role in memory processing and not to other functional activities in which OT is involved in these brain sites. The results of a PA study (see Chapter 10) that examined interactive effects between peripherally administered OT and centrally and peripherally acting cholinergic antagonists were consistent with the thesis that an OT interaction with central ACh-ergic mechanisms is importantly involved in mediating the effect of the peptide on memory processing. Nonetheless, the means by which peripherally applied OT accessed the central ACh-ergic transmitter system was not addressed by the protocol used in this study. ‘‘It is hoped that future research will succeed in learning (1) the speciﬁc brain structures in which these OT/neurotransmitter interactive effects on memory processing take place, and (2) the speciﬁc role played by, and physiologival signiﬁcance of, OT in these interactions.’’ Microdialysis would be a useful technique in such studies because it can be used to measure OT release at synaptic sites to which the tested neurotransmitter projects, and this release can be related to measures of memory processing performance in appetitive and aversive tasks. 3. Neuromodulatory Actions of OT in the Septum and Hippocampus? Evidence cited in Chapter 5 demonstrates that OT, like VP, exerts a neuromodulatory action in the septal complex and in the ventral hippocampus. However, the concerted research effort that has been made to clarify the relation between the neuromodulatory action of VP in this circuitry and its role in memory processing has not as yet been accomplished for OT. Moreover, ﬁndings suggesting an OT-induced inhibition of neural activity in ventral CA1 pyramidal hippocampal cells, perhaps via an OT interaction with GABA-ergic inhibitory cells in this circuitry, justiﬁes further study of the neuromodulatory action of OT in this circuitry, especially because the ventral hippocampus is a major site where OT has been observed to exert an amnestic action in the avoidance learning paradigm.

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D. VP, OT, and Olfactory-Based SRM 1. VP and Behaviorally Active VP Metabolites: Differential Effects on Short-Term and Long-Term Components of SRM? Several lines of evidence suggest that, like other forms of memory, SRM has both short-term memory (STM) and long-term memory (LTM) components. Moreover, peripherally administered VP and various of its behaviorally active C-terminal fragments appear to differentially affect these short- and long-term SRM components. Given the BBB and attendant interpretative problems associated with the peripheral route of drug delivery, the advantage of follow-up studies using central administration (intracerebroventricular or site-speciﬁc injections into relevant brain structures) is obvious. Should this follow-up research effort support the above-cited ﬁndings, subsequent evaluation of the physiological signiﬁcance of these effects is clearly warranted. And last, because studies investigating the roles of androgen-dependent and androgen-independent VP-ergic systems in olfactory-based conspeciﬁc SRM evaluated only its STM component, it would be of interest to determine whether these two systems were also involved in mediating the putative LTM component of SRM. 2. Dose-Dependent and Site-Dependent Effects of OT on Olfactory-Based SRM Although a number of lines of evidence have been cited that support an OT-induced facilitation of olfactory-based SRM, a species-typical type of recognition memory, the numerous discrepant ﬁndings in this literature attest to the complex nature of the role of OT in this form of memory processing. Both SRM attenuation and facilitation have been observed, depending on the dose of the peptide administered or the brain site in which the level of OT was manipulated. As earlier noted, certain ﬁndings have suggested that the dose-dependent effects reﬂect an OT-induced arousal inﬂuence, and others that the site-dependent effects might indicate involvement of different OT-ergic receptors or circuitries. This author proposed a model to accommodate both the dose- and site-dependent effects of OT in SRM. It is hoped that future follow-up research will help clarify the dose-dependent and site-dependent actions of OT on SRM, and in the process determine the degree to which the proposed model is able to satisfactorily account for these effects.

E. Molecular Aspects of the Roles of VP and OT in Memory Processing As noted in Chapter 1, central vasopressin V1 and oxytocin receptors are connected to a G protein that, when activated by the ligand–receptor complex, leads to the breakdown of phosphatidylinositol from which is

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derived the second messengers that participate in the mediation of both short- and long-term effects of peptide receptor activation. The short-term effects constitute the neurotransmitter action (ionic ﬂux across the neuronal cell membrane). The long-term effects are mediated by gene induction and the formation of proteins necessary to morphological changes that presumably underlie the changes in neuronal circuitry that occur during prenatal and early postnatal development, and the formation of long-term memory in postnatal life. The earlier discussed research studies of Du and associates with the behaviorally active VP metabolite, VP(4–8), have made impressive inroads into linking the role of the peptide in memory processing studied at behavioral (passive avoidance task) and cellular (induction of long-term potentiation) levels with the molecular and biochemical pathways that characterize its receptor-mediated intracellular signaling system. Brinton’s studies with cultured hippocampal and cortical neurons have provided evidence consistent with the thesis that the vasopressin V1 receptor-associated intracellular signaling system produces long-term effects that mediate the neurotrophic actions of the peptide and result in the morphological growth patterns that occur in these neuronal cultures (Brinton, 1998; Chen et al., 2000). Continuation and expansion of these lines of investigation hold much promise for bringing to fruition De Wied’s long-held conceptualization that VP and OT and their bioactive metabolites are fully and directly engaged in inﬂuencing central processing mechanisms concerned with the formation of long-term memory.

F. A Step Toward Integration in VP and OT Memory Research 1. VP and/or OT Roles in Memory Processing: From Stress Stimulation to Memory Modulation Experimental observations cited in Chapter 1 (Section IV.D), and throughout most of the chapters in this text are consistent with the following general outline of the events that take place in the course of stress-associated learning encounters that activate VP and OT cell systems within hypothalamic and extrahypothalamic structures. The aversive and appetitive paradigms used in the study of the roles of VP and OT in memory processing include stimuli of emotional/motivational signiﬁcance, which comprise the ‘‘stressors’’ that activate the transmitter systems comprising the central arousal system as well as hypothalamic and extrahypothalamic VP and OT cell systems. Stress-associated stimulation of VP and OT cell systems, localized within parvocellular areas of the paraventricular hypothalamus, activate the pituitary–adrenocortical system and autonomic brainstem neural centers that mediate the peripheral physiological effects that contribute

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to the so-called stress response. These peripheral effects exert feedback actions that inﬂuence the central arousal system as well as emotional/motivational and cognitive processing within limbic and striatal structures at midbrain and forebrain levels of the brain. At the same time that the stressors associated with these learning paradigms activate the VP and OT cell systems cited above, they also activate hypothalamic and extrahypothalamic VP-ergic and OT-ergic cell systems that exert neuronal effects affecting central circuitry involved in emotional/ motivational and cognitive processing. It is herein postulated that these effects are mediated, in part, by VP and OT interactions with a number of aminergic, peptidergic, and steroidal hormonal systems in addition to those for which supportive evidence has thus far been obtained. It is further proposed that these interactive effects are involved in modulating cortical activation/behavioral arousal levels important to emotional/motivational aspects of central processing, and in cognitive processing mediating stressrelated attentional effects, as well as storage in and retrieval from short-term and long-term memory stores. It is hoped that future VP/OT memory research study will commit to the goal of ﬁlling in the general outline sketched above. This will include the use of a variety of experimental protocols and paradigms in studies designed to yield data that will gradually allow us to trace the consecutive steps, peripheral/central pathways, and interactions with peripheral hormones and central transmitters that intervene between stressor stimulus and memory performance tested in these learning paradigms.

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Index

Abortion, 380–383 Accessory olfactory bulb (AOB), 465 Accurel Systems, 464 Acetylcholine in memory processing, 438 Acquisition, 278 in appetitive paradigms, 427– 429 of lever touch response, 358, 360 trials to, 279 ACTH. See Adrenocorticotropic hormone Active avoidance tasks, 87–88 effects of LVP on, 64 Active/passive avoidance tasks, Combined description of, 280–281 studies using, 281–285 Acute treatment regimens, 447–448 Adrenal hormones, 597–598 Adrenal medulla VP/OT in, 46–47 Adrenalectomies, 170 Adrenocorticotropic hormone (ACTH), 48 amygdala lesions and, 145–146 analogs of, 72 antidiuretic activity and, 119 dosages, 59 effect of, on extinction, 147 fragment, 93 hippocampal theta activity and, 202 in learning and memory, 55–56 LVP and, 354 –356 peptides similar to, 142–143 performance effects of, 601

release of, in rats, 57 treatment with, 57–58 Adrenomedullectomies, 170 ADX rats, 170, 171 retention in, 172 ADXM rats, 170 AEP. See Auditory evoked potential Age differences, 445–446 Agonist-Antagonist interaction, 237–238 Albino CD-1 mice, 354 –356 postdefeat submissive behavior, 356–357 Aminergic transmitter systems, 612 Amino acids concentrations of, 585–586 neurohypophysial hormones and, 179 in peptide synthesis, 1 Aminopeptidase conversion, 187 Amnesia CO2 induced, 92 OT and, 483–490 pentylenetetrazole-induced, 93 puromycin-induced, 79–80 retrograde, 90 Amnestic action glycinamide-containing peptides, 189 of OT, 618 AMP. See Amphetamine AMPH. See D-amphetamine Amphetamine (AMP) effect of, on retention, 283–285

709

710

Index

Amygdala AVP levels in, 127–128 DA utilization in, 163 lesions of, 145–146 Analysis of variance (ANOVA), 234 –235 four-way, 300 for learning rates, 318 on performance accuracy, 288–289 Androgen in olfactory-based social recognition memory, 459–465 VP-ergic system dependent on, 634 VP-ergic system independent of, 636–637 Animal research appetitive paradigms in, 357–380 aversive paradigms in, 340–357 divided attention in, 327 learning phase in, 422–425 overview of, 307–308 socially transmitted information in, 377–380 visual discrimination learning in, 364 –371 ANOVA. See Analysis of variance Anti-AVP serum, 496–498 Antibody-antigen linkage, 10 Antidiuretic activity, 118, 555 ACTH and, 119 Anti-OT serum, 85–86, 496–498 Antisense oligodeoxynucleotide, 504 –506 Anti-VP serum, 85–86 AOB. See Accessory olfactory bulb Apomorphine pretreatment with, 245 Appetitive learning paradigms, 72–78 acquisition/extinction in, 427–429 in animal research, 357–380 memory processing in, 391–397 OT in, 650–651 Arachnoid membrane role of, 539 Arcuate nucleus binding sites at, 192 Arginine vasopressin (AVP) activity levels and, 252 administration routes of, 93–94 arousal effects of, 96 in aversive paradigms, 417–418 aversive properties of, 244 –245 avoidance learning and, 415–417 avoidance retention and, 118–124, 236 behaviorally active doses of, 240–241 bimodal effect induced by, 275–276

brain-fluid barrier penetration of, 554 –575 brain-to-blood transport of, 567–575 CA interaction with, in memory processing, 156 carrier-mediated transport of, 597 conversion of, 181 in CSF, 124 –126 C-terminal antiserum, 186 DG-AVP and, 252 in dialysates, 499 EEG activity and, 254 –255 effect of, in autoshaped lever touch task, 362 effect of, in pole-jump footshock avoidance test, 232 effect of microdialysis on, 502 effect of, on behavioral arousal, 130–132 effect of, on catecholamine transmission, 162–166 effect of, on hippocampal rhythms, 199 effect of, on locomotor activity, 248 effect of, on memory encoding, 301–302 effect of, on memory retention, 75–77, 233, 283–285, 426–427 effect of, on pentylenetetrazole-induced amnesia, 93 effect of, on puromycin induced amnesia, 79–80 effect of, on selective attention, 297–298 effect of, on spontaneous activity, 211 effect of, on ventral hippocampal neurons, 217 effects of, in position task, 286–288 endogenous, 158 EPSP amplitude and, 219 hippocampal theta activity and, 202 HPLC analysis of, 184 immunoreactive, 557–558 intracerebroventricular injection of, 84 –85 iodinated, 569 iontophoretic application of, 614 levels of, in amygdala, 127–128 levels of, in brain structures, 126–129 levels of, in hippocampus, 127–128, 157 in LSC, 206 LSC neuronal responsivity and, 208–209 in memory consolidation, 157 memory processing modulation by, 221–222 memory retrieval and, 171 metabolites of, 596

Index microinjection of, 150, 393 neurotransmitter/neuromodulator actions of, 216–219 nigrostriatal DA system interaction with, 164 –166 in olfactory-based social recognition memory, 467 in original and reversal learned tasks, 310–311 osmotically induced release of, 254 PA avoidance paradigm and, 98 peripherally administered, 524 –525, 595–598 pressor properties of, 240–242, 244 –245, 441–442 proteolytic conversion of, 180 radioimmunoassay analysis of, 253 receptor knockdown strategy, 504 –506 subcutaneous administration of, 83 V1 antagonists and, 249–250 V1 receptor, 242–243 ventral hippocampal, 394 in vivo studies with, 613–615 in Wistar rats, 119–120 Arginine vasotocin (AVT) in original and reversal learned tasks, 310–311 Arousal levels, 96 attention processing and, 326 AVP and, 130–132 in Brattleboro rats, 113–114 learning/memory and, 259 memory processing and, 138–139 memory storage and, 132 neurophysiological substrates of, 259–260 performance-versus-arousal curve, 268 VP and, 607–608 Arousal system attention mechanisms and, 648 behavioral arousal and, 266–267 OT mediation in, 623–624 upper/lower, 270 VP mediation of, 600–604 Yerkes-Dodson, 267–268 Arousal-centered theorists, 600–601 Arousal-performance efficiency relationship peripherally administered VP and, 442–444 Asp, 209 Ataxia, 278 Attention processing arousal levels and, 326

711

arousal mechanisms and, 648 divided, 327 electrophysiological measures of, 327–331 in human research, 327–331 introduction to, 315–316 memory processing and, 604 –608 peptide-induced control of, 323 selective, 297–298 vasopressin fragments in, 316–326 VP activation/arousal effects and, 607–608 VP and, 298–301, 335–337, 605–606 Auditory evoked potential (AEP) in twins, 330 Auditory response to tone pips, 329 Autoradiography, 10, 11 Autoshaped lever touch task acquisition of, 358, 360 in animal research, 357–359 description of, 276–277 extinction of, 358 mean lever touches in, 363, 364 S-D mice in, 361–364 studies using, 277–280 Aversive experiences, 280–281 behavior choice and, 282 Aversive motivation for Holtzman albino rats, 312 Aversive paradigms, 340–357 endogenous AVP/OT in, 417–418 memory consolidation and, 425–426 memory processing in, 390–391 Aversive properties, 238 of AVP, 244 –245 Avoidance conditioning paradigms, 56–72 reliance on, 273 septal area in, 397–399 Avoidance learning endogenous AVP and, 415–417 Avoidance response acquisition of, 148 extinction of, 345 Avoidance retention active, 235 AVP and, 118–124 in brain structures, 126–129 CSF levels and, 124 –126 effect of lesions on, 159–162 neurohypophysial peptides and, 193 VP/OT and, 137 AVP. See Arginine vasopressin

712

Index

AVP-receptor complex, 31–32 AVP-receptor knockdown strategy, 504 –506 AVT. See Arginine vasotocin

BALB/c mice in black/white discrimination task, 369–371 in B/W discrimination tests, 393–394 PA response in, 350–351 Baseline proficiency, 429 individual differences in, 446 Beckwith and colleagues animal research of, 307–315 human research of, 296–307 research summary of, 332–334 Behavioral arousal arousal system and, 266–267 baseline, 291–293 effect of VP on, 278–280 two component model of, 269–271 Bioassay methods, 554 –556 Biochemical paradigm of VP/OT neurotransmission influence, 175 Blood pressure in learning /memory, 258 Blood-brain barrier carrier-mediated transport across, 597 crossing, 256–257 definition of, 533–535 Ermisch-Landgraf proposal on, 588–589 historical development of, 532–533 lumenal side of, 564 –565 LVP and, 576–577 nutrient exchange across, 43, 537 opening of, 549–550 transcellular transfer through, 551 VP in, 253 Blood-CSF barrier historical development of, 532–533 molecule flow across, 538 nutrient exchange across, 537 Body fluid balance, 553 Brain catecholamines in, 160–161 circumventricular organs of, 15 DA utilization in, 168 efflux systems in, 573 –575 MGC/PVC VP/OT in, 20 oxytocin in, 2 perfusion regulation in, 41

peripherally injected hormones entering, 549–552 rat, 12 vasopressin in, 2 VP/OT distribution in, 23–25 Brain capillaries general capillaries and, 534 model of, 535 in nutrient exchange, 537 Brain uptake index technique, 561 Brain-fluid barriers AVP penetration of, 554 –575 defining, 531 historical development of, 532–533 peptide transport across, 566 permeability of, 575–589 regulation of, 542–543 Brain-to-blood transport of AVP/OT, 567–575 hydration status and, 570 of leucine, 580–582 of LNAAs, 585 Brattleboro rats arousal-mediated phenomenon in, 113–114 emotional behavior of, 114 –115 inconsistencies in, 109–110 introduction to, 104 lateral septum of, 214 –215 memory processing and, 135–136 model validity, 273 retention deficits in, 110–113 septal AVP in, 519–521 summary of, 115–116 timidity of, 111 VP deficiency in, 113 Wistar normal rats and, 106–109 Brightness discrimination in S-D rats, 368–369 Broadbent’s two-component model, 269–271 Bunsey, Strupp, and colleagues on attentional processing, 315–316 research summary of, 334 –335

CA4-dentate gyrus region VP fibers in, 396–397 Cannula, 395 Capillaries, 534 Castration social recognition and, 460

Index Catecholaminergic projection system cholinergic systems and, 608–612 selective lesions in, 159–162 Catecholamines, 70 in brain, 160–161 effect of AVP/OT on transmission of, 162–166 in memory processing, 438–440 neurohypophysial peptide interactions with, 174 –176 OT interaction with, 624 transmission of, 162–168 VP interaction with, in memory processing, 169–173 VP/OT antiserum and, 166–169 VP/OT interaction with, 155–168 CBF. See Cerebral blood flow CDP. See Chlordiazepoxide Central arousal theory dual action theory and, 257, 271–272 proposition 1, 290–291 proposition 2, 291–293 two-component model and, 269–271 Central memory theory, 271 neuropeptide concept and, 626–627 VP/OT and, 425 Cerebral blood flow (CBF) measuring, 581 CFY rats, 390–391 Chlordiazepoxide (CDP) bimodal effect induced by, 275–276 Choice alternation reentry latency and, 284 –285 Choice behavior reentry latency and, 282 Cholinergic system catecholiminergic systems and, 608–612 in memory processing, 407–412, 439–440 noradrenic system and, 603 OT interaction with, 624 –625 transmitters in, 611 Choroid plexus, 535–539 location of, 536 in molecule flow, 538 transcellular transfer through, 551 Choroidal epithelial cells, 537–538 Chronic treatment regimens, 447–448 Circadian rhythms, 544 Circumventricular organs (CVOs) in body-brain integration, 553 of brain, 15 role of, 540–541

CNS OT binding sites in, 36 OT localization in, 9–11 VP localization in, 9–11 VP/OT as neurotransmitters in, 37 CNV. See Contingent negative variation CO2-induced amnesia, 92 Cognitive processing human, 380–388 Concentration, 298–301 Conditioned place aversion (CPA) CTA and, 246–247 dose dependent attenuation of, 250 Conditioned response suppression in animal research, 342–343 Conditioned taste aversion (CTA) in animal research, 340–342 CPA and, 246–247 establishing, 241 for milk, 242 OT influence in, 423 V1 antagonist blocking of, 244 in Wistar rats, 243 Contingent negative variation (CNV) in LVP conditions, 332 Corticosterone, 49–50 CPA. See Conditioned place aversion CSF circulation of, 539 formation of, 535–539 secretion of, 536 sink function of, 547–549 VP/OT transport to, 545–546 CSF levels avoidance retention and, 124 –126 CTA. See Conditioned taste aversion C-terminal AVP fragments, 187 hexapeptide fragment, 222 VP/OT fragments, 191–192, 627–628 VP/OT metabolites, 627 CVOs. See Circumventricular organs Cys-1 Cys-6 bridge linking to, 183 Cys-6 Cys-1 bridge linking to, 183

DA. See Dopaminergic activity DA synthesis, 165 DA-lesioned rats, 405–407

713

714

Index

D-amphetamine (AMPH) bimodal effect induced by, 275–276 effects of, in position task, 286–288 Dantzer, Bluthe, and colleagues, 595 on SRM, 634 –635 De Wied and associates evidence presented by, 261–262 groundwork of, 594 position statements of, 598–599 Sahgal on, 272–273 Delayed matching to position tasks description of, 285–286 studies using, 286–290 Delayed matching to sample paradigm, 403–404 Delayed nonmatching to position tasks description of, 285–286 studies using, 286–290 DH. See Dorsal hippocampus Diagonal band of Broca VP-ergic cells in, 615 Divided attention in laboratory rats, 327 DNAB. See Dorsal noradrenergic bundle Dopaminergic activity (DA), 270–271 VP interaction with, 609–610 Dorsal hippocampus (DH), 205 Dorsal noradrenergic bundle (DNAB) fiber system of, 161 lesions of, 159–160 Dorsal raphe nucleus NA-ergic pathways innervating, 176 Dorsal septal region NA-ergic pathways innervating, 176 Dose-dependent effects, 446–447 Dose-response function inverted U-shaped, 70 Druckay hooded rats in radial maze task, 373–374 Drug treatments, 91 Dual action theory central arousal theory and, 257, 271–272 olfactory based social recognition memory and, 468–471 proposition 1 of, 257–258 proposition 2 of, 258 proposition 3 of, 258–259 proposition 4 of, 259 proposition 5 of, 259–260 proposition 6 of, 260

EEG activity. See Electroencephalographic activity EEG records, 198 Efflux systems for VP/OT, 573–575 Electrical stimulation field potential responses to, 212–214 Electroencephalographic (EEG) activity AVP and, 254 –256 relaxed/alert, 267 theta rhythms, 255–256 Electron microscopy, 10 Endocrine activity passive avoidance tasks and, 107–108 -Endorphin, 48–49 Endothelial capillary membranes V1 receptors on, 582 Epinephrine, 70 PA retention and, 170–171 Ermisch-Landgraf model of blood-brain-barrier, 588–589 Ethologically relevant avoidance behavior in animal research, 354 –357 Event-related potentials (ERP) definition of, 327 Fehm-Wolsdorf on, 328 Evolution, 8 Exons in OT, 3 in VP, 3 Experimental treatments learning/memory effects of, 260 Extinction behavior in appetitive paradigms, 427–429 of avoidance responses, 345 of lever touch response, 359 new learning and, 252 pressor antagonists and, 236–237 radial maze tasks and, 251 Extracellular fluid, 533 Extracerebral vasculature intracerebral vasculature and, 40–43

Fehm-Wolsdorf and colleagues electrographic findings of, 605–606 on ERP data, 327–331 Field potential responses (FP) to electrical stimulation, 212–214 of VP neuromodulation, 614

Index Fimbria fibers, 214 –215 neurotransmission from, 222 FLU. See -flupenthixol Fluorescein, 10 -flupenthixol (FLU) behavioral effects of, 275–276 Food approach response, 252 Food deprivation, 75–77 Food preferences, 377–380 Foot shock, 57, 91 FP. See Field potential responses

G proteins, 26 activation, 27–28 GABA receptors, 209 inhibition of, 219 Gene transcription of VP/OT, 4 Glial cells nutrients reaching, 537 Glucose metabolism, 623 Glucose storage VP/OT in, 44 Glutamate excitation induced by, 205 hippocampal response to, 207 iontophoretic application of, 206 neurotransmission of, 220 NMDA subtype receptors, 220 Glutamatergic synaptic sites neuromodulation at, 223–225 Glycinamide, 88 amnestic action induced by, 189 Grooming, 278

Hebbian influence on arousal systems, 266–267 HEDI rats. See Heterozygous diabetes insipidus rats Heterozygous diabetes insipidus (HEDI) rats, 105 emotional behavior of, 114 –115 hippocampal theta activity of, 197–198 paradoxical sleep in, 201 rhythmic slow activity of, 198, 199 VP absence in, 108 High-pressure liquid chromatography (HPLC), 512, 565 Hippocampal theta activity, 612–613 AVP/ACTH and, 202

715

characteristics of, 195–196 of HEDI rats, 197–198 of HODI rats, 197–198 memory consolidation and, 196–204 OT frequency depressing actions, 203 septum in, 203–204 slow activity, 198 VP influence on, in paradoxical sleep, 223 VP/OT and, 197–204 Hippocampus AVP levels in, 127–128, 157 glutamate responses of, 207 lesions in, 144 OT neuromodulation in, 651 role of, in memory processing, 390–397, 434 –436 role of, in retention, 151 in SRM, 637 theta rhythm of, 195–204 VP antiserum injections to, 152 VP neuromodulatory action in, 613–617 Histochemical studies on VP, 42 HODI rats. See Homozygous diabetes insipidus rats Holtzman albino rats aversive motivation for, 312 reversal-learned tasks in, 309–310 Homozygous diabetes insipidus (HODI) rats, 105 DG-AVP treatment of, 199 emotional behavior of, 114 –115 hippocampal theta activity of, 197–198 lateral septum of, 214 –215 LTP induction in, 215 paradoxical sleep in, 201 retention deficit in, 110 rhythmic slow activity of, 198, 199 septal AVP in, 519–521 VP absence in, 108 Horseradish peroxidase (HRP), 550 HPLC analysis, 182–183 fractionation by reversed phase, 186, 187 of immunoreactive AVP peptides, 184 HPLC. See High-pressure liquid chromatography HRP. See Horseradish peroxidase Human research measuring attention processing in, 327–331

716

Index

Human research (continued ) on memory processing, 430–432 overview of, 296–297 Hydration effect of, on brain-to-blood transport, 570 Hypothalamic parvicellular VP-ergic system role of, in memory processing, 437 Hypothalamus mgc cells in, 12

ICC staining, 11 IHC staining, 396 Immunohistochemical analysis of castrated animals, 461 Inhibitory avoidance tasks retention in, 409–410 Interim nose poke response, 360 Intracerebral vasculature extracerebral vasculature and, 40–43 local shifts in, 42–43 Invertebrates hormone-like molecules in, 6–7 Inverted-U performance v. arousal curve, 268

Koob and colleagues evidence presented by, 262–263 position statements of, 599 research of, 594 –595

Large neutral amino acids (LNAA), 542 brain-to-blood uptake of, 585 carrier, 564 transfer of, 579 Latency scores scatter diagram of, 275 Lateral septal complex (LSC) AVP in, 208–209 glutamate in, 206 glutamate-induced excitation in, 207 monoamines in, 210–212 neuronal responses in, 209 neurons in, 205–206 Learning phase in animal research, 422–425 VP/OT in, 100–101 Learning rate ANOVA on, 318 LENO rats. See Long-Evans normal rats

Leucine accumulation of, 578 brain-to-blood transfer of, 580–582 Ligand-receptor interactions of VP receptors, 631 Light microscopy, 10 Limbic system cortical structures in, 412 role of, in memory processing, 173–174 Lithium chloride, 246 LNAA. See Large neutral amino acids Locomotor activity effect of AVP on, 247 facilitating, 278 in radial maze tasks, 251–252 Long-Evans hooded rats autoshaping task in, 359–361 memory retrieval in, 378–379 in Morris water maze, 399–401 Long-Evans normal (LENO) rats, 109 emotional behavior of, 114 –115 lateral septum of, 214 –215 timidity of, 111 Long-term memory, 257 acute /chronic treatment regimens and, 447–448 components of, in SRM, 637–639 OT and, 79 retrieval of, 372–373 short-term memory and, 375 VP neuromodulation and, 616–617 Long-term potentiation (LTP) endogenous VP and, 224 in lateral setpum, 214 –216 Lose-shift object responding, 321 object analysis for, 323 LSC. See Lateral septal complex LTP. See Long-term potentiation LVP. See Lysine vasopressin Lysine vasopressin (LVP), 57 in acquisition training, 65 ACTH and, 354 –356 blood-brain barrier and, 576–577 CNV and, 332 dosages, 59 effect of, after training, 71 effect of five doses of, 69 effect of, on auditory reaction time, 331–332 effect of, on avoidance behavior, 64, 67 effect of, on CO2-induced amnesia, 92

Index effect of, on memory retention, 62–65, 73–74 effect of, on pole jump task, 67–68 effect of, on pole-jumping avoidance response, 146 effect of, on puromycin induced amnesia, 79–80 effect of, on retrograde amnesia, 90–93 exogenously applied, 351 extinction effects induced by, 67, 144 long-term effects of, 65 memory consolidation induced by, 131–132, 408 posttraining treatment with, 69 in shuttlebox avoidance test, 426 synthetic, 63 treatment, 156

Magnicellular neurons VP in, 4 Magnicellular systems cell groups in, 14 VP/OT, 11–13, 20 MANOVA program, 75–76, 77–78 Marsupials neurophysical peptides in, 8 Medial preoptic area (MPOA), 465 definition of, 516 SRM and, 516–518 Memory consolidation bimodal effects on, 274 –276 hippocampal theta activity and, 196–204 LVP induced, 131–132 OT and, 99–101 in paradoxical sleep, 177 at various brain sites, 149–150 VP and, 97–99 Memory encoding effect of AVP on, 301–302 Memory processing acetylcholine in, 438 in appetitive learning paradigms, 391–397 attention processing and, 604 –608 in aversive paradigms, 390–391 AVP/CA interaction in, 156–157 catecholamines in, 155–169, 174 –176, 438 – 440, 608–612 central versus peripheral locus of, 595–598 in cholinergic system, 407– 412 cholinergic system in, 439–440, 608–612 effects of DG-AVP on, 75

717

endogenous VP/OT and, 415– 420, 440 – 441 glucose availability and, 44 – 46 glucose-induced enhancement of, 45–46 hippocampal neuromodulation and, 613–617 history of study of, 594 –595 human research on, 430– 432 hypothalamic parvicellular VP-ergic system in, 437 molecular aspects of, 652– 653 neurohypophysial peptides and, 136–138, 155 nigrostriatal DA system in, 405– 407, 438–439 nutrient transport and, 587–588 OT influence on, 623–625 parvocellular hypothalamic VP-ergic system in, 402–405 passive avoidance tests of, 61 peripheral/central mechanisms of, 116–133 peripherally injected VP/OT and, 133 –134 position statements on, 598– 600 role of hippocampus in, 434 – 436 role of limbic system in, 173 –174 role of septal area in, 436 – 437 septal area neuromodulation and, 613–617 spatial learning tasks and, 399–402 stress hormones and, 48 V1 receptors in, 589 vasopressin transmitters and, 611–612 VP in, 38 VP-CA interactional effects in, 610–611 VP-catecholamine interaction in, 169–173 VP-DA interactional effects in, 609–610 VP/OT direct modulation of, 138–139 VP/OT fragments in, 178–180 Memory retention effects of AMP on, 283–285 effects of AVP on, 233 effects of LVP on, 62–65 effects of VP on, 71–72 effects of VP/OT on, 80 role of hippocampus in, 151–152 sexual gratification and, 73 Memory retrieval, 90 AVP and, 171 in Long-Evans hooded rats, 378 –379 Methionine enkephalin, 571–573 Microdialysis effects of, on AVP release, 502 Microinjection, 164

718

Index

Monoaminergic transmitters, 611 Monoamines in LSC cells, 210–212 Morris water maze, 508 Long-Evans hooded rats in, 399 – 401 spatial navigation in, 420 Wistar rats in, 401–402 Motivational factors, 449 MPOA. See Medial preoptic area -MPT treatment with, 156 -MSH, 59 Multiple-cue tasks, 317 box-relevant, 320 Multitrial one-way active avoidance task, 52 Multitrial two-way active avoidance task, 52

NA. See Noradrenaline activity Narrative prose test, 306–307 Neurohypophysial peptides amino acid sequences of, 179 avoidance retention and, 193 behavioral effects of, 192 catecholiminergic neurotransmitter interactions with, 174 –176 memory processing and, 136–138, 155 Neuropeptide concept definition of, 178 electrophysiological evidence for, 628–630 memory enhancing molecules and, 630–631 VP/OT central memory theory and, 626–627 Neuropeptide fragments in vitro research on, 180–184 in vivo research on, 185–188 Neurophysical peptides in marsupials, 8 in vertebrates, 7 Neurotransmitter pathways in vasopressin action, 161 Neurotransmitters transcription regulation by, 27 Newman-Keuls test, 497 Nicotinic cholinergic mechanisms, 410 Nigrostriatal DA system AVP interaction with, 164 –166 in memory processing, 405–407, 438–439 NMDA receptors, 209 antagonists, 216 glutamate, 220

Nonimmune rabbit serum, 342 Noradrenaline activity (NA), 270–271 depletion of, 511–513 VP interaction with, 438 Noradrenergic pathways activity modulation in, 271 Noradrenergic projections in PVN, 18 Noradrenic system cholinergic system and, 603 Norepinephrine, 70 Nose poke response. See Interim nose poke response Nucleus of the solitary tract (NTS) binding sites at, 192 Nutrient exchange across blood-brain barrier, 43 memory processing and, 587–588

Oldendorf single-pass technique, 560–562 Olfactory system VP/OT in, 509–516 Olfactory-based social recognition memory task androgen in, 459–465 AVP neurotransmission in, 467 definition of, 454 –455 dose dependent OT effects on, 652 dual action theory and, 470–471 endogenous VP in, 469–470 sex differences in, 462–464 sexual dimorphy and, 471–472 vomeronasal organ and, 472–473 vomeronasal system and, 465–467 VP-mediated, 458–459 Organum vasculosum lamina terminalis (OVLT) binding sites at, 192 Original-learned tasks AVP/AVT/PA in, 310–311 Osmotic stimulation of SON, 498–504 OT antagonists, 194 OT genetic knockout models SRM and, 518–524 OT. See Oxytocin OT-Ach interaction in passive avoidance program, 412–415 Ovarectomy, 521–524 OVLT. See Organum vasculosum lamina terminalis

Index Oxytocin (OT) acquisition behavior influenced by, 95 in adrenal medulla, 46–47 as amnestic neuropeptide, 618 amnestic properties of, 483–490 antagonists, 194 antidepressant effects of, 489 anti-OT serum, 85–86 in appetitive learning paradigms, 650 – 651 arousal system and, 623– 624 in aversive paradigms, 417–418 avoidance retention behavior and, 137 bimodal action of, 88, 89, 275–276 binding sites, 36 biosynthesis of, 2–6 in brain blood vessels, 38–44 brain-to-blood transport of, 567–575 catecholamine interaction with, 155–168, 624 central memory theory, 173–176, 228 cerbrovascular activity and, 39–43 cholinergic interactions, 624 –625 CTA influenced by, 423 C-terminal fragments, 191–192, 627–628 C-terminal metabolites, 627 C-terminal peptides, 188–192 definition of, 1, 78–79 desglycinamide, 167 direct memory processing modulation, 138–139 disappearance of, 572 effect of, on catecholamine transmission, 162–166 effect of, on learning phase, 94 –96 effect of, on passive avoidance responses, 80, 81 effect of, on pole-jump shock avoidance task, 81–86 effect of, on puromycin induced amnesia, 79–80 endogenous, in memory processing, 440–441 exogenously administered, 139–140 exon organization of, 3 extinction influenced by, 72–73 extrahypothalamic systems, 17–22 fiber distribution of, 23–25 fiber terminals, 22 fragments in memory processing, 178–189 functionally distinct systems, 645–646 gene transcription of, 4 –5 in glucose storage, 44

719

in hippocampal theta activity, 203 hippocampal theta activity and, 197–204 history of study of, 594 –595 hypothalamic, 13–17 immunoreactive, 557–558 influence of, on stress hormones, 45 intracerebroventricular injection of, 84 –85, 619–620 intranasal administration of, 383–388 intraventricular administration of, 86, 166–169 knockout models, 648–650 in learning phase, 100–101, 136 level modulation of, 618–623 localization of, in CNS, 9–11 long-term memory and, 79 magnicellular/parvicellular systems, 11–13 memory consolidation and, 99–100, 157 memory processing modulation by, 221–222 metabolic degradation of, 6 microinjection of, 149–154 microinjection of antiserum of, 151–154 molecular structure of, 5–6 neuromodulator activities of, 204 –220 neurotransmission influenced by, 175 as neurotransmitter/neuromodulator, 37 neutralizing, 117–118 in olfactory pathway, 643–645 in olfactory system, 509–516 origin of CSF, 543–545 PA behavior and, 153 in pancreas and liver, 44 –45 pathways, 10 peripherally injected, 133–134, 432–434, 647 pituitary-adrenocortical axis and, 47–50 plasma levels of, 341 in PVN, 16 in rat brain, 12 reduction of, 622–623, 641–643 septal-hippocampal, 495–508 septal/hippocampal neuromodulation of, 651 sink function in transport of, 547–549 site-specific variability of effects of, 620 in social recognition memory, 477–490, 526–527 spatial memory and, 418–420 SRM mediation pathways, 527–528 in SRT, 485–487

720

Index

Oxytocin (OT) (continued ) subcutaneous administration of, 82, 83 time course conversion of, 182 transmitter interactions, 651 transportation of, to CSF, 545–546 in vertebrates, 7 in vivo studies using, 204 –205 in Wistar rats, 120–121 Oxytocin fragments peripherally administered, 618–619 Oxytocin receptors classification of, 35–37 interference with, 622–623 ionotropic transmission, 22, 26 metabotropic transmission, 26–28

PA. See Pressinoic acid Paradoxical sleep (PS) in HEDI/HODI rats, 201 hippocampal theta activity in, 195–204 memory consolidation during, 177, 196–204 VP in, 200 VP influence on hippocampal theta activity during, 223 Paraventricular nucleus (PVN) afferent input to, 16–17 cytoarchitectonic/functional subdivisions of, 19 of hypothalamus, 12 noradrenergic projection organization in, 18 parviceullar VP/OT neurons in, 16 as visceral effector integrative center, 17 VP in, 13–16, 20 Parent peptides retention facilitation and, 191 Parvicellular systems cell groups in, 14 VP/OT, 11–13 Parvocellular hypothalamic VP-ergic system definition of, 402 in memory processing, 402–405 Passive avoidance tasks, 55, 87 acquisition and retention from, 121–122 AVP and, 98, 235 behavioral results of, 160 bimodal memory consolidation and, 274 –276 effects of LVP on, 64 effects of VP/OT on, 80, 81

endocrine activity and, 107–108 epinephrine doses and, 170–171 footshock trials and, 149–150 HODI/HEDI rats in, 105–106 memory modulation and, 190–191 memory processing tested through, 61–62 OT antiserum and, 153 OT-ACh interaction in, 412–415 retention of, 158 training for, 62–63 VP levels and, 117–118 VP-ACh interaction in, 407–412 Wistar rats in, 120 Pentylenetetrazole-induced amnesia, 93 Peptide synthesis amino acids in, 1 Peptidergic neurons peptide release in, 3 Perspex box, 280–281 Phagocytic cells role of, 539–540 Pharmacogenetic factors, 445 Phenylalanine, 566, 583 Pineal gland binding sites in, 192 Pitressin tannate, 59, 365 vasopressin and, 60 Pituitary hormones, 597–598 intermediate lobe of, 58 lobectomy, 56–57 retention and, 138 Pituitary-adrenocortical axis VP/OT and, 47–50 Placebo, 56 PLG. See Prolyl-leucyl-glycinamide Pole jump footshock avoidance task, 54 definition of, 229–230 effect of AVP in, 232, 415–416 extinction of, 233 Wistar rats in, 231–232, 238–240 Pole jump task, 52–55, 59–60 acquisition and extinction in, 107 acquisition training for, 62 effect of ACTH on, 147 effect of LVP on, 67–68, 146 effect of VP/OT on, 81–86 extinction of, 144 for lesioned animals, 143 Posterior pituitary lobectomy, 56 Preprooxytocin, 2 Preprovasopressin, 2

Index Pressinoic acid (PA) in original and reversal learned tasks, 310–311 Pressor properties, 238 of AVP, 244 –245, 441–442 Prolyl-leucyl-glycinamide (PLG), 390–391 Proposition 7, 173–174 Proposition 8, 174 –176 Proposition 9, 221–222 Proteolytic conversion of AVP, 180 PS. See Paradoxical sleep PTZ-induced retrograde amnesia, 192 Punishment, 284 Puromycin-induced amnesia preventing, 79–80 Push-pull perfusion, 164 PVG hooded rats in spatial tests, 288–290 PVN. See Paraventricular nucleus Pyroglutamic acid, 189

Quis, 209

Radial maze task in animal research, 371–377 definition of, 231 Druckay hooded rats in, 373–374 eight-arm, 250–253 extinction behavior and, 251 locomotor behavior in, 251–252 spatial memory and, 374 –377 Radioactivity regional distribution of, 562 Radioimmunoassay analysis (RIA), 556–557 of AVP, 253 Ratio of investigation duration (RID), 503 Redundant learning paradigm correct trials in, 325 phases of, 324 Reentry latency choice alternation and, 284 –285 choice behavior and, 282 in non-shock conditions, 409 in retention tests, 351 Reference memory (RM), 75 in Wistar rats, 77–78 Renal tubule cell vasopressin and, 34 Retention behavior, 256–257

721

measuring, 449–450 in Wistar rats, 281–283 Retrograde amnesia effects of LVP on, 90–93 Reversal-learned tasks AVP/AVT/PA in, 310–311, 314 –315 for Holtzman albino rats, 309–310 Rewards, 73 RHA rats AVP deficient, 404 –405 Rhythmic slow activity differences in, 198 of HODI rats, 199 special parameters of, 199 RIA. See Radioimmunoassay analysis RID. See Ratio of investigation duration Ringer’s solution, 584 RM. See Reference memory Roman high-avoidance rats, 402–405

Saccharine in CTA tasks, 341–342 Saccharine-vehicle pairings, 241 Sahgal and colleagues on De Wied, 272 research of, 594 –595 research practices of, 273–274 Saline controls, 87, 88 Sampling ladder, 541 S-D rats. See Sprague-Dawley rats Second-messenger action, 29, 33 Selective attention effects of AVP on, 297–298 effects of VP on, 312–314 Selective lesions in catecholaminergic projection systems, 159–162 Sentence memory task crossover design with, 302–305 delayed recall, 305 immediate recall, 306 sentences recalled in, 304 treatment order groups in, 303 Septal area in hippocampal theta activity, 203–204 long term potentiation in, 214 –216 in memory processing, 397–402 OT neuromodulation in, 651 role of, in memory processing, 436–437 in SRM, 506–508 VP neuromodulatory action in, 613–617

722

Index

Septal-hippocampal system neuropeptides and, 207–208 neurotransmission in, 399–401 SRM and, 495–508 VP neuromodulation action in, 210, 223–225 Serotonin, 161 Sex differences, 301–302 in olfactory based social recognition tasks, 462–464 social recognition memory and, 490–495 Sexual dimorphy, 21 olfactory-based social recognition memory task and, 471–472 Sexual gratification memory retention and, 73–74 Sham operations, 170 Short-term memory components of, in SRM, 637–639 long-term memory and, 375 VP and, 430 Shuttlebox footshock avoidance test, 53, 57 acquisition and extinction in, 106 in animal research, 343–349 learning in, 347 LVP in, 426 S-D rats in, 347–348 Silver grains concentrations of, 541 Single-cue tasks, 317 box-relevant, 319 Single-trial inhibitory avoidance task in animal research, 349–354 definition of, 230 Single-trial water-finding task, 245–250 definition of, 230 Sink function of CSF, 547–549 6-OHDA lesions, 513 Social discrimination test, 476 Social learning paradigm in animal research, 377–380 Social recognition memory (SRM), 418–419 androgen in, 459–465 AVP neurotransmission in, 467–468 castration and, 460 conspecific, 455–458 Dantzer, Bluthe, and colleagues on, 634 –635 definition of, 454 –455 endogenous VP in, 469–470 hippocampus in, 637

long-term /short-term components in, 637–639 medial preoptic area and, 516–518 multitrial, 521–524 OT in, 477–490, 526–527, 640–647 septal VP-ergic systems in, 506–508 sex differences and, 490–495 SRT and, 475–476 vomeronasal system and, 465–467 VP in, 477–490 VP-ergic transmission in, 464 VP-mediated olfactory-based, 458–459 VP/OT genetic knockout models and, 518–524, 528–529 VP/OT pathways and, 527–528 Social recognition test (SRT) dose-dependent memory-facillitation and, 487–489 multitrial, 476–477 OT in, 485–487 SRM testing and, 475–476 SON. See Supraoptic nucleus Spatial tests endogenous OT and, 418–420 male PVG hooded rats in, 288–290 memory processing in, 399–402 performance in, 419 radial maze and, 374 –377 Spontaneous behavior, 87 Sprague-Dawley (S-D) rats in autoshaped lever touch task, 361–364 brightness discrimination in, 368–369 in shuttlebox avoidance tests, 347–348 in Y-mazes, 366–368 SRM. See Social recognition memory SRT. See Social recognition test Stereotypy, 278 Sternberg item recognition task, 298–301 Stress hormones effects of, on memory, 48–50 VP/OT influence on, 45 Stress response VP/OT activation, 47 Stress-associated learning paradigms OT in, 618 Stria terminalis bed nucleus of, 615 Structural encoding task grand means on adjectives during, 331 Subcommisural organ, 15

Index Subfornical organ, 15 Subject variables age differences, 445–446 individual differences, 446 pharmacogenetic factors, 445 Suprachiasmatic nucleus, 17 Supraoptic nucleus (SON) of hypothalamus, 12 osmotic stimulation of, 498–504 VP in, 13 Swiss mice, 352–354

Task difficulty, 448 Task retention AVP in, 246 improving, 247 Task variables training experience, 448 Taste aversion conditioning, 239 Testosterone replacement therapy, 461 Thalamic-limbic lesions, 142–148 Theta activity EEG, 255–256 in paradoxical sleep, 201 Thompson-Bryant discrimination box, 308 Time course experiments, 181–182 Time-dependent effects, 446–447 T-maze task, 110 black/white discrimination, 364 –366, 424 Training experience, 448 Transcellular transfer, 551 Treatment variables acute /chronic treatment regimens, 447–448 dose /time-dependent effects, 446–447 motivational factors, 449 task difficulty, 448 Two-way shuttlebox task, 52 Tyr-MIF-1, 571–572 Tyrosine, 584

V1 antagonists, 194 AVP and, 249–250 CTA blocking, 244 effect of, on castrated rats, 460 lipophilic, 416 pressor, 243–244 V1 receptors, 242–243 on endothelial capillary membranes, 582 in memory processing sites, 589

723

V2 antagonists, 194 V2 receptors nonpressor, 243 Valine, 584 Vascular brain perfusion technique, 562–564 Vasopressin (VP), 278 absence of, in HODI/HEDI rats, 108–109 in adrenal medulla, 46–47 androgen-dependent systems, 634 –635 androgen-independent systems, 636–637 anti-VP serum, 85–86, 117 Arginine, 6 arousal levels and, 96 arousal system mediation of, 600–604 arousal-performance efficiency relationships and, 442–444 attention and, 335–336 avoidance influenced by, 94 avoidance retention behavior and, 137, 159–162 behaviorally active doses of, 240–245 binding-sites, 30–31 biosynthesis of, 2–6 in blood brain barrier, 253, 556 in brain blood vessels, 38–44 CA interaction with, 610–611 catecholamine interaction with, 155–168 catecholamine interaction with, in memory processing, 169–173 central memory theory, 173–176, 228 cerbrovascular activity and, 39–43 cholinergic interaction, 408–409 C-terminal fragments, 191–192, 627–628 C-terminal metabolites, 627 C-terminal peptides, 188–192 DA interaction with, 609–610 DA synthesis and, 165 deficiency of, in Brattleboro rats, 113–114 definition of, 1 depletion of, 95 direct memory processing modulation, 138–139 dual action theory, 238–240, 256–260 EEG theta rhythms and, 255–256 effect of, on acquisition, 83–84 effect of, on attention, 298–301 effect of, on catecholiminergic transmission, 162–168 effect of, on learning phase, 94 –96 effect of, on memory retention, 71–72, 85 effect of, on passive avoidance responses, 80, 81

724

Index

Vasopressin (VP) (continued ) effect of, on pole-jump shock avoidance task, 81–86 effect of, on selective attention, 312 –314 efflux systems for, 573 –575 endogenous, 98–99, 104 endogenous, in memory processing, 440–441 endogenous, in social recognition memory, 469 – 470 exogenously administered, 139–140 exon organization of, 3 extinction influenced by, 72 –73 extrahypothalamic systems, 17– 22 fiber distribution of, 23–25 fiber terminals, 22 fragments in memory processing, 178 –189 gene expression activation and, 33 gene transcription of, 4 –5 in glucose storage, 44 hippocampal theta activity and, 197–204 Histochemical studies on, 42 history of study of, 594 –595 hypothalamic, 13 –17 influence of, on hippocampal theta activity during paradoxical sleep, 223 influence of, on stress hormones, 45 intracerebroventricular injection of, 152–153 intranasal administration of, 383 –388 intraventricular administration of, 86 intraventricular antiserum administration, 166 –169 knockout models, 648–650 learning influenced by, 95–96 in learning phase, 100–101, 136 localization of, in CNS, 9–11 magnicellular/parvicellular systems, 11–13 in Magnocellular neurons, 4 memory consolidation and, 97–99, 135 –136 in memory processing, 38 memory retrieval and, 150 –151 memory storage and, 132 metabolic degradation of, 6 metabolites, 213 microinjection antiserum, 151–154 microinjection of, 149–154 molecular structure of, 5–6 neuromodulator activities of, 204 –220

neuromodulator activities of, in hippocampus, 613 – 617 neuromodulator activities of, in septal area, 613 – 617 neuronal performance and, 590 neurotransmission influenced by, 175 neurotransmitter pathways of, 161 neurotransmitter system interaction of, 405– 415 as neurotransmitter/neuromodulator, 37 neutralizing, 117–118 Noradrenaline interaction with, 438 in olfactory system, 509–516 in olfactory-based social recognition memory, 458–459 origin of CSF, 543–545 PA learning and, 117–118 in pancreas and liver, 44 – 45 in paradoxical sleep, 200 parent peptides and, 191 pathways, 10 peripherally injected, 133–134, 432– 434, 647 Pitressin extracts and, 60 pituitary-adrenocortical axis and, 47–50 position statements on, 598 – 600 pressor effects produced by, 129 –130 primary structure of, 183 proposed structure of, 5 in PVN, 16 in rat brain, 12 renal tubule cell and, 34 in retention behavior, 256–257 in septal-hippocampal system, 223 –225, 495–508 sexually dimorphic circuitry and, 21–22 short-term memory and, 430 sink function in transport of, 547–549 in social recognition memory, 464 – 465, 477–490 SRM mediation pathways, 527–528 tasks used in memory research, 229–231 timidity and, 111 transportation of, to CSF, 545–546 treatment with, 68 V1 antagonist, 194 V1 receptors, 242–243 V2 antagonist, 194 in vertebrates, 7 in vivo studies using, 204 –205 in Wistar rats, 120 –121

Index Vasopressin fibers in CA4-dentate gyrus region, 396 –397 Vasopressin fragments role of, in attentional processing, 316 –326 Vasopressin receptors intracellular signaling system, 632– 633 ionotropic transmission, 22, 26 Ligand-receptor interactions and, 631– 632 metabotropic transmission, 26 –28 in peripheral and neural tissues, 28–35 signaling pathway, 31–33 V2, 34 –35 Via, 28–33 Vasopressin transmitters memory processing and, 611– 612 Vasopressin /Oxytocin central memory theory, 228, 234, 256 Vasotocin, 7 Ventral hippocampus (VH) effect of AVP on, 217 neurons of, 216 Ventricles, 536 Vertebrates neurophysical peptides in, 7 VH. See Ventral hippocampus Virchow-Robin spaces, 550 Visual discrimination learning in animal research, 364 –371 VNO. See Vomeronasal organ Vomeronasal organ (VNO), 465 olfactory based social recognition memory and, 472–473 Vomeronasal system olfactory-based social recognition memory and, 465–467 VP genetic knockout models SRM and, 518–524 VP pressor effects arousal effects produced by, 259 aversive stimulus properties linked to, 258–259 VP receptors direct action on, 260 retention effects mediated by, 262 VP. See Vasopressin VP-ACh interaction in passive avoidance program, 407–412 VP-CA interactional effects in memory processing, 610–611

725

VP-DA interactional effects in memory processing, 609–610 VP/OT knockout models SRM and, 528–529

W1A, 186, 187 W4E, 186, 187 Water deprivation, 253, 553 White/black reversal discrimination task, 308–309, 391–393 BALB/c mice in, 369–371, 393–394 studies using, 309–315 Win-stay object responding, 322 Wistar rats amino acids in, 585 AVP levels in, 119–120 Brattleboro rats and, 106–109 castrated, 468 conspecific social recognition in, 457 CSF levels in, 125 CTA in, 243 EEG activity in, 254 –255 emotional behavior of, 114 –115 iontophoretic application of AVP in, 211 lateral septum of, 214 –215 lesions in, 143–144 in Morris water maze, 401–402 PA acquisition and retention in, 121–122 in PA tasks, 120 in pole jump footshock avoidance task, 231–232, 238 –240 reference memory in, 77–78 retention in, 281–283 in shuttlebox avoidance tasks, 344 VNO-sham, 466 VNO-X, 466 VP-ergic neurotransmission in, 401–402 VP/OT levels in, 120–121 water deprivation of, 253 working memory in, 77–78 Working memory (WM), 75 in Wistar rats, 77–78

Yekers-Dodson postulated arousal/ performance curve, 267–269 Y-maze task, 420 S-D rats in, 366–368

Zinc phosphate, 58

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Contents of Previous Volumes

Volume 37 Opioid and Nonopioid Cardiovascular Effects of Dynorphins Michel Dumont and Simon Lemaire

Cytokines in Neuronal Development G. Miller Jonakait

Heterogeneity and Functional Properties of Subtypes of Sodium-Dependent Glutamate Transporters in the Mammalian Central Nervous System Michael B. Robinson and Lisa A. Dowd

Development and Therapeutic Potential of Calpain Inhibitors Kevin K. W. Wang and Po-wai Yuen

The Pharmacology of ()-Nicotine and Novel Cholinergic Channel Modulators Jorge D. Brioni, Michael W. Decker, James P. Sullivan, and Stephen P. Arneric

Cryptococcosis Judith A. Aberg and William G. Powderly

Antimalarial Activity of Artemisinin (Qinghaosu) and Related Trioxanes: Mechanism(s) of Action Jared N. Cumming, Poonsakdi Ploypradith, and Gary H. Posner

727

728

Contents of Previous Volumes

The Role of Endothelin in the Pathogenesis of Atherosclerosis Mark C. Kowala

The Pharmacology and Molecular Biology of Large-Conductance Calcium-Activated (BK) Potassium Channels Valentin K. Gribkoff, John E. Starrett, Jr., and Steven I. Dworetzky

Update on Invasive Candidiasis Libsen J. Rodriguez, John H. Rex, and Elias J. Anaissie

Volume 38 Antioxidants: The Basics—What They Are and How to Evaluate Them Barry Halliwell

Metabolism of Vitamin C in Health and Disease Ann M. Bode

Regulation of Human Plasma Vitamin E Maret G. Traber

Glutathione and Glutathione Delivery Compounds Mary E. Anderson

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Antioxidant Actions of Melatonin Russel J. Reiter

Antioxidative and Metal-Chelating Effects of Polyamines Erik Løvaas

Antioxidant and Chelating Properties of Flavonoids Ludmila G. Korkina and Igor B. Afanas’ev

Potential Use of Iron Chelators against Oxidative Damage Jean-Baptiste Galey

N-Acetylcysteine: Pharmacological Considerations and Experimental and Clinical Applications Ian A. Cotgreave

Contents of Previous Volumes

729

Ebselen as a Glutathione Peroxidase Mimic and as a Scavenger of Peroxynitrite Helmut Sies and Hiroshi Masumoto

Salen–Manganese Complexes: Combined Superoxide Dismutase/Catalase Mimics with Broad Pharmacological Efficacy Susan R. Doctrow, Karl Huffman, Catherine B. Marcus, Wael Musleh, Annadora Bruce, Michel Baudry, and Bernard Malfroy

Antioxidant Drug Targeting Anthony C. Allison

Antioxidant-Inducible Genes Thomas Primiano, Thomas R. Sutter, and Thomas W. Kensler

Redox Signaling and the Control of Cell Growth and Death Garth Powis, John R. Gasdaska, and Amanda Baker

Protective Action of Nitrone-Based Free Radical Traps against Oxidative Damage to the Central Nervous System Robert A. Floyd

Reactive Oxygen Species and Their Contribution to Pathology in Down Syndrome Judy B. de Haan, Ernst J. Wolvetang, Francesca Cristiano, Rocco Iannello, Cecile Bladier, Michael J. Kelner, and Ismail Kola

Antioxidants, Redox-Regulated Transcription Factors, and Inflammation Paul G. Winyard and David R. Blake

Relationships among Oxidation of Low-Density Lipoprotein, Antioxidant Protection, and Atherosclerosis Hermann Esterbauer, Reinhold Schmidt, and Marianne Hayn

Adult Respiratory Distress Syndrome: A Radical Perspective Samuel Louie, Barry Halliwell, and Carroll Edward Cross

Oxidative Stress in Cystic Fibrosis: Does It Occur and Does It Matter? Albert van der Vliet, Jason P. Eiserich, Gregory P. Marelich, Barry Halliwell, and Carroll E. Cross

730

Contents of Previous Volumes

Oxidative Stress and Antioxidant Function in Relation to Risk for Cataract Allen Taylor and Thomas Nowell

The Macular Pigment: A Possible Role in Protection from Age-Related Macular Degeneration John T. Landrum, Richard A. Bone, and Mark D. Kilburn

Neurological Disease David P. R. Muller

Role of Cysteine and Glutathione in HIV Infection and Cancer Cachexia: Therapeutic Intervention with N-Acetylcysteine Wulf Dro¨ge, Andrea Gross, Volker Hack, Ralf Kinscherf, Michael Schykowski, Michael Bockstette, Sabine Mihm, and Dagmar Galter

Role of Oxidative Stress and Antioxidant Therapy in Alcoholic and Nonalcoholic Liver Diseases Charles S. Lieber

Antioxidant Therapy for the Prevention of Type I Diabetes Birgit Heller, Volker Burkart, Eberhard Lampeter, and Hubert Kolb

Photoaging of the Connective Tissue of Skin: Its Prevention and Therapy Karin Scharffetter-Kochanek

Antioxidant Nutrients and Cancer Incidence and Mortality: An Epidemiologic Perspective Susan T. Mayne

Volume 39 Colorectal Cancer and Nonsteroidal Anti-inflammatory Drugs Walter E. Smalley and Raymond N. DuBois

Mouse Mammary Tumor Virus and the Immune System Susan R. Ross

Sodium Channels and Therapy of Central Nervous System Diseases Charles P. Taylor and Lakshmi S. Narasimhan

Contents of Previous Volumes

731

Anti-adhesion Therapy Carol J. Cornejo, Robert K. Winn, and John M. Harlan

Use of Azoles for Systemic Antifungal Therapy Carol A. Kauffman and Peggy L. Carver

Pharmacology of Neuronal Nicotinic Acetylcholine Receptor Subtypes Lorna M. Colquhoun and James W. Patrick

Structure and Function of Leukocyte Chemoattractant Richard D. Ye and Franois Boulay

Pharmacologic Approaches to Reperfusion Injury James T. Willerson

Restenosis: Is There a Pharmacologic Fix in the Pipeline? Joan A. Keiser and Andrew C. G. Uprichard

Role of Adenosine as a Modulator of Synaptic Activity in the Central Nervous System James M. Brundege and Thomas V. Dunwiddie

Combination Vaccines Ronald W. Ellis and Kenneth R. Brown

Pharmacology of Potassium Channels Maria L. Garcia, Markus Hanner, Hans-Gu¨nther Knaus, Robert Koch, William Schmalhofer, Robert S. Slaughter, and Gregory J. Kaczorowski

Volume 40 Advances in Understanding the Pharmacological Properties of Antisense Oligonucleotides Stanley T. Crooke

Targeted Tumor Cytotoxicity Mediated by Intracellular Single-Chain Anti-oncogene Antibodies David T. Curiel

In Vivo Gene Therapy with Adeno-Associated Virus Vectors for Cystic Fibrosis Terence R. Flotte and Barrie J. Carter

732

Contents of Previous Volumes

Engineering Herpes Simplex Virus Vectors for Human Gene Therapy Joseph C. Glorioso, William F. Goins, Martin C. Schmidt, Tom Oligino, Dave Krisky, Peggy Marconi, James D. Cavalcoli, Ramesh Ramakrishnan, P. Luigi Poliani, and David J. Fink

Human Adenovirus Vectors for Gene Transfer into Mammalian Cells Mary M. Hitt, Christina L. Addison, and Frank L. Graham

Anti-oncogene Ribozymes for Cancer Gene Therapy Akira Irie, Hiroshi Kijima, Tsukasa Ohkawa, David Y. Bouffard, Toshiya Suzuki, Lisa D. Curcio, Per Sonne Holm, Alex Sassani, and Kevin J. Scanlon

Cytokine Gene Transduction in the Immunotherapy of Cancer Giorgio Parmiani, Mario P. Colombo, Cecilia Melani, and Flavio Arienti

Gene Therapy Approaches to Enhance Antitumor Immunity Daniel L. Shawler, Habib Fakhrai, Charles Van Beveren, Dan Mercoa, Daniel P. Gold, Richard M. Bartholomew, Ivor Royston, and Robert E. Sobol

Modified Steroid Receptors and Steroid-Inducible Promoters as Genetic Switches for Gene Therapy John H. White

Strategies for Approaching Retinoblastoma Tumor Suppressor Gene Therapy Hong-Ji Xu

Immunoliposomes for Cancer Treatment John W. Park, Keelung Hong, Dmitri B. Kirpotin, Demetrios Papahadjopoulos, and Christopher C. Benz

Antisense Inhibition of Virus Infection R. E. Kilkuskie and A. K. Field

Volume 41 Apoptosis: An Overview of the Process and Its Relevance in Disease Stephanie Johnson Webb, David J. Harrison, and Andrew H. Wyllie

Genetics of Apoptosis Serge Desnoyers and Michael O. Hengartner

Methods Utilized in the Study of Apoptosis Peter W. Mesner and Scott H. Kaufmann

Contents of Previous Volumes

In Vitro Systems for the Study of Apoptosis Atsushi Takahashi and William C. Earnshaw

The Fas Pathway in Apoptosis Christine M. Eischen and Paul J. Leibson

Ceramide: A Novel Lipid Mediator of Apoptosis Miriam J. Smyth, Lina M. Obeid, and Yusuf A. Hannun

Control of Apoptosis by Proteases Nancy A. Thornberry, Antony Rosen, and Donald W. Nicholson

Death and Dying in the Immune System David S. Ucker

Control of Apoptosis by Cytokines W. Stratford May, Jr.

Glucocorticoid-Induced Apoptosis Clark W. Distelhorst

Apoptosis in AIDS Andrew D. Badley, David Dockrell, and Carlos V. Paya

Virus-Induced Apoptosis J. Marie Hardwick

Apoptosis in Neurodegenerative Diseases Ikuo Nishimoto, Takashi Okamoto, Ugo Giambarella, and Takeshi Iwatsubo

Apoptosis in the Mammalian Kidney: Incidence, Effectors, and Molecular Control in Normal Development and Disease States Ralph E. Buttyan and Glenda Gobe´

Apoptosis in the Heart Samuil R. Umansky and L. David Tomei

Apoptosis and the Gastrointestinal System Florencia Que and Gregory J. Gores

Role of p53 in Apoptosis Christine E. Canman and Michael B. Kastan

733

734

Contents of Previous Volumes

Chemotherapy-Induced Apoptosis Peter W. Mesner, Jr., I. Imawati Budihardjo, and Scott H. Kaufmann

Bcl-2 Family Proteins: Strategies for Overcoming Chemoresistance in Cancer John C. Reed

Role of Bcr-Abl Kinase in Resistance to Apoptosis Afshin Samali, Adrienne M. Gorman, and Thomas G. Cotter

Apoptosis in Hormone-Responsive Malignancies Samuel R. Denmeade, Diane E. McCloskey, Ingrid B. J. K. Joseph, Hillary A. Hahm, John T. Isaacs, and Nancy E. Davidson

Volume 42 Catecholamine: Bridging Basic Science Edited by David S. Goldstein, Graeme Eisenhofer, and Richard McCarty

Part A. Catecholamine Synthesis and Release Part B. Catecholamine Reuptake and Storage Part C. Catecholamine Metabolism Part D. Catecholamine Receptors and Signal Transduction Part E. Catecholamine in the Periphery Part F. Catecholamine in the Central Nervous System Part G. Novel Catecholaminergic Systems Part H. Development and Plasticity Part I. Drug Abuse and Alcoholism

Volume 43 Overview: Pharmacokinetic Drug–Drug Interactions Albert P. Li and Malle Jurima-Romet

Role of Cytochrome P450 Enzymes in Drug–Drug Interactions F. Peter Guengerich

Contents of Previous Volumes

735

The Liver as a Target for Chemical–Chemical Interactions John-Michael Sauer, Eric R. Stine, Lhanoo Gunawardhana, Dwayne A. Hill, and I. Glenn Sipes

Application of Human Liver Microsomes in Metabolism-Based Drug–Drug Interactions: In Vitro–in Vivo Correlations and the Abbott Laboratories Experience A. David Rodrigues and Shekman L. Wong

Primary Hepatocyte Cultures as an in Vitro Experimental Model for the Evaluation of Pharmacokinetic Drug–Drug Interactions Albert P. Li

Liver Slices as a Model in Drug Metabolism James L. Ferrero and Klaus Brendel

Use of cDNA-Expressed Human Cytochrome P450 Enzymes to Study Potential Drug–Drug Interactions Charles L. Crespi and Bruce W. Penman

Pharmacokinetics of Drug Interactions Gregory L. Kedderis

Experimental Models for Evaluating Enzyme Induction Potential of New Drug Candidates in Animals and Humans and a Strategy for Their Use Thomas N. Thompson

Metabolic Drug–Drug Interactions: Perspective from FDA Medical and Clinical Pharmacology Reviewers John Dikran Balian and Atiqur Rahman

Drug Interactions: Perspectives of the Canadian Drugs Directorate Malle Jurima-Romet

Overview of Experimental Approaches for Study of Drug Metabolism and Drug–Drug Interactions Frank J. Gonzalez

Volume 44 Drug Therapy: The Impact of Managed Care Joseph Hopkins, Shirley Siu, Maureen Cawley, and Peter Rudd

736

Contents of Previous Volumes

The Role of Phosphodiesterase Enzymes in Allergy and Asthma D. Spina, L. J. Landells, and C. P. Page

Modulating Protein Kinase C Signal Transduction Daria Mochly-Rosen and Lawrence M. Kauvar

Preventive Role of Renal Kallikrein—Kinin System in the Early Phase of Hypertension and Development of New Antihypertensive Drugs Makoto Kartori and Masataka Majima

The Multienzyme PDE4 Cyclic Adenosine Monophosphate-Specific Phosphodiesterase Family: Intracellular Targeting, Regulation, and Selective Inhibition by Compounds Exerting Anti-inflammatory and Antidepressant Actions Miles D. Houslay, Michael Sullivan, and Graeme B. Bolger

Clinical Pharmacology of Systemic Antifungal Agents: A Comprehensive Review of Agents in Clinical Use, Current Investigational Compounds, and Putative Targets for Antifungal Drug Development Andreas H. Groll, Stephen C. Piscitelli, and Thomas J. Walsh

Volume 45 Cumulative Subject Index Volumes 25–44

Volume 46 Therapeutic Strategies Involving the Multidrug Resistance Phenotype: The MDR1 Gene as Target, Chemoprotectant, and Selectable Marker in Gene Therapy Josep M. Aran, Ira Pastan, and Michael M. Gottesman

The Diversity of Calcium Channels and Their Regulation in Epithelial Cells Min I. N. Zhang and Roger G. O’Neil

Gene Therapy and Vascular Disease Melina Kibbe, Timothy Billiar, and Edith Tzeng

Contents of Previous Volumes

737

Heparin in Inflammation: Potential Therapeutic Applications beyond Anticoagulation David J. Tyrrell, Angela P. Horne, Kevin R. Holme, Janet M. H. Preuss, and Clive P. Page

The Regulation of Epithelial Cell cAMP- and Calcium-Dependent Chloride Channels Andrew P. Morris

Calcium Channel Blockers: Current Controversies and Basic Mechanisms of Action William T. Clusin and Mark E. Anderson

Mechanisms of Antithrombotic Drugs Perumal Thiagarajan and Kenneth K. Wu

Volume 47 Hormones and Signaling Edited by Bert W. O’Malley New Insights into Glucocorticoid and Mineralocorticoid Signaling: Lessons from Gene Targeting Holger M. Reichardt, Franois Tronche, Stefan Berger, Christoph Kellendonk, and Gu¨nther Shu¨tz

Orphan Nuclear Receptors: An Emerging Family of Metabolic Regulators Robert Sladek and Vincent Gigue`re

Nuclear Receptor Coactivators Stefan Westin, Michael G. Rosenfeld, and Christopher K. Glass

Cytokines and STAT Signaling Christian Schindler and Inga Strehlow

Coordination of cAMP Signaling Events through PKA Anchoring John D. Scott, Mark L. Dell’Acqua, Iain D. C. Fraser, Steven J. Tavalin, and Linda B. Lester

G Protein-Coupled Extracellular Ca2þ (Ca2þo)-Sensing Receptor (CaR): Roles in Cell Signaling and Control of Diverse Cellular Functions Toru Yamaguchi, Naibedya Chattopadhyay, and Edward M. Brown

738

Contents of Previous Volumes

Pancreatic Islet Development Debra E. Bramblett, Hsiang-Po Huang, and Ming-Jer Tsai

Genetic Analysis of Androgen Receptors in Development and Disease A. O. Brinkmann and J. Trapman

An Antiprogestin Regulable Gene Switch for Induction of Gene Expression in Vivo Yaolin Wang, Sophia Y. Tsai, and Bert W. O’Malley

Steroid Receptor Knockout Models: Phenotypes and Responses Illustrate Interactions between Receptor Signaling Pathways in Vivo Sylvia Hewitt Curtis and Kenneth S. Korach

Volume 48 HIV: Molecular Biology and Pathogenesis: Viral Mechanisms Edited by Kuan-Teh Jeang Multiple Biological Roles Associated with the Repeat (R) Region of the HIV-I RNA Genome Ben Berkhout

HIV Accessory Proteins: Multifunctional Components of a Complex System Stephan Bour and Klaus Strebel

Role of Chromatin in HIV-I Transcriptional Regulation Carine Van Lint

NF-B and HIV: Linking Viral and Immune Activation Arnold B. Rabson and Hsin-Ching Lin

Tat as a Transcriptional Activator and a Potential Therapeutic Target for HIV-1 Anne Gatignol and Kuan-Teh Jeang

From the Outside In: Extracellular Activities of HIV Tat Douglas Noonan and Andriana Albini

Rev Protein and Its Cellular Partners Jørgen Kjems and Peter Askjaer

Contents of Previous Volumes

739

HIV-I Nef: A Critical Factor in Viral-Induced Pathogenesis A. L. Greenway, G. Holloway, and D. A. McPhee

Nucleocapsid Protein of Human Immunodeficiency Virus as a Model Protein with Chaperoning Functions and as a Target for Antiviral Drugs Jean-Luc Darlix, Gae¨l Cristofari, Michael Rau, Christine Pe´choux, Lionel Berthoux, and Bernard Roques

Bioactive CD4 Ligands as Pre- and/or Postbinding Inhibitors of HIV-I Laurence Briant and Christian Devaux

Coreceptors for Human Immunodeficiency Virus and Simian Immunodeficiency Virus Keith W. C. Peden and Joshua M. Farber

Volume 49 HIV: Molecular Biology and Pathogenesis: Clinical Applications Edited by Kuan-Teh Jeang Inhibitors of HIV-I Reverse Transcriptase Michael A. Parniak and Nicolas Sluis-Cremer

HIV-I Protease: Maturation, Enzyme Specificity, and Drug Resistance John M. Louis, Irene T. Weber, Jo´zsef To¨zse´r, G. Marius Clore, and Angela M. Gronenborn

HIV-I Integrase Inhibitors: Past, Present, and Future Nouri Neamati, Christophe Marchand, and Yves Pommier

Selection of HIV Replication Inhibitors: Chemistry and Biology Seongwoo Hwang, Natarajan Tamilarasu, and Tariq M. Rana

Therapies Directed against the Rev Axis of HIV Autoregulation Andrew I. Dayton and Ming Jie Zhang

HIV-I Gene Therapy: Promise for the Future Ralph Dornburg and Roger J. Pomerantz

Assessment of HIV Vaccine Development: Past, Present, and Future Michael W. Cho

740

Contents of Previous Volumes

HIV-I-Associated Central Nervous System Dysfunction Fred C. Krebs, Heather Ross, John McAllister, and Brian Wigdahl

Molecular Mechanisms of Human Immunodeficiency Virus Type I Mother-Infant Transmission Nafees Ahmad

Molecular Epidemiology of HIV-I: An Example of Asia Mao-Yuan Chen and Chun-Nan Lee

Simian Immunodeficiency Virus Infection of Monkeys as a Model System for the Study of AIDS Pathogenesis, Treatment, and Prevention Vanessa M. Hirsch and Jeffrey D. Lifson

Animal Models for AIDS Pathogenesis John J. Trimble, Janelle R. Salkowitz, and Harry W. Kestler

McEWEN, CHAPTER 14, FIGURE 3 The choroid plexus sits within the ﬂuid-ﬁlled chambers in the brain called ventricles. It continuously secretes cerebrospinal ﬂuid (CSF), which cushions the nervous systems, carries some nutrients to brain tissues, and cleanses the brain of wastes. As new CSF is produced, old CSF is forced to ﬂow (arrows) through the ventricles, around the spinal cord, and into the subarachnoid space around the brain. As the CSF ﬂows, it exchanges substances with the interstitial ﬂuid between brain cells. Eventually it drains into blood in the superior sagittal sinus through structures called arachnoid granulations. Because the choroid plexus and the arachnoid membrane stand between the blood and the CSF, they constitute the blood–CSF barrier. Source: Spector and Johanson, 1989 (p. 69). Published by Scientiﬁc American, Inc. Copyright ß 1989 by Carol Donner and reprinted with her kind permission.

McEWEN, CHAPTER 14, FIGURE 4 Essential nutrients reach the neurons and glial cells in the brain by crossing either the blood–CSF barrier, which is regulated by the choroid plexus, or the blood–brain barrier of the cerebral capillaries. Water-soluble molecules cannot diffuse freely between the blood and the CSF because of impermeable tight-junction seals between the choroid epithelial cells; instead, the epithelial cells transfer certain molecules from one side of the barrier to the other. Once molecules enter the CSF, they can diffuse through the ‘‘leaky’’ ependymal layer and reach the interstitial ﬂuid around the neurons and glial cells. Similarly, wastes in the interstitial ﬂuid can pass into the CSF for disposal. The endothelial cells of the cerebral capillaries, which are also sealed by tight junctions, control the direct exchange of materials between the blood and interstitial ﬂuid. Source: Spector and Johanson, 1989 (p. 70). Published by Scientiﬁc American, Inc. Copyright ß 1989 by Carol Donner and reprinted with her kind permission.

McEWEN, CHAPTER 14, FIGURE 5 Flow of molecules across the blood–CSF barrier is regulated by several mechanisms in the choroid plexus. Some micronutrients, such as vitamin C, are pulled into the epithelial cells at the basolateral surface by an energy-consuming process known as active transport; the micronutrients are released into the CSF at the apical surface by another regulated process, facilitated diffusion, which requires no energy. Essential ions are also controllably exchanged between the CSF and blood plasma. Transport of an ion in one direction is linked to the transport of a different ion in the opposite direction, as in the exchange of sodium (Naþ) ions for potassium (Kþ) ions. Source: Spector and Johanson, 1989 (p. 72). Published by Scientiﬁc American, Inc. Copyright ß 1989 by Carol Donner and reprinted with her kind permission.