Animal Models of Schizophrenia and Related Disorders (Neuromethods 59)

NE U R O M E T H O D S

Series Editor Wolfgang Walz University of Saskatchewan Saskatoon, SK, Canada

For other titles published in this series, go to www.springer.com/series/7657

Animal Models of Schizophrenia and Related Disorders

Edited by

Patricio O’Donnell Departments of Anatomy & Neurobiology and Psychiatry, School of Medicine, University of Maryland, Baltimore, MD, USA

Editor Patricio O’Donnell, MD, Ph.D. Departments of Anatomy & Neurobiology and Psychiatry University of Maryland Baltimore, MD 21201, USA

ISSN 0893-2336 e-ISSN 1940-6045 ISBN 978-1-61779-156-7 e-ISBN 978-1-61779-157-4 DOI 10.1007/978-1-61779-157-4 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011928327 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface to the Series Under the guidance of its founders Alan Boulton and Glen Baker, the Neuromethods series by Humana Press has been very successful since the first volume appeared in 1985. In about 17 years, 37 volumes have been published. In 2006, Springer Science + Business Media made a renewed commitment to this series. The new program will focus on methods that are either unique to the nervous system and excitable cells or need special consideration to be applied to the neurosciences. The program will strike a balance between recent and exciting developments like those concerning new animal models of disease, imaging, in vivo methods, and more established techniques. These include immunocytochemistry and electrophysiological technologies. New trainees in neurosciences still need a sound footing in these older methods in order to apply a critical approach to their results. The careful application of methods is probably the most important step in the process of scientific inquiry. In the past, new methodologies led the way in developing new disciplines in the biological and medical sciences. For example, Physiology emerged out of Anatomy in the 19th century by harnessing new methods based on the newly discovered phenomenon of electricity. Nowadays, the relationships between disciplines and methods are more complex. Methods are now widely shared between disciplines and research areas. New developments in electronic publishing also make it possible for scientists to download chapters or protocols selectively within a very short time of encountering them. This new approach has been taken into account in the design of individual volumes and chapters in this series. Wolfgang Walz

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Preface Animal models of schizophrenia and major psychiatric disorders have been sought for decades. As it is clear that schizophrenia is a uniquely human disease, animal models are not likely to reproduce all facets of this disorder. However, there has been considerable growth in this field in the past several years with models that test possible pathophysiological scenarios, the role of environmental factors, or contributions of gene variants conferring risk for schizophrenia to abnormal function and behavior. As a result, we are now facing new vistas on pathophysiology that could lead to novel therapeutic approaches and even hint at possible preventive strategies. The animal models that yield this advance will be needed to gain deeper insight into biological processes that can yield to behavioral anomalies and, perhaps more importantly, to explore novel treatments. It is critical in this regard that the manipulations used to model schizophrenia-relevant phenomena are used consistently across laboratories. Hence, this book presents an overview of what information can be obtained with several different models and a detailed account of how to generate such models. As the search for animal models of schizophrenia is a decadeslong endeavor, many models have been proposed. Here we cover only some; it would be unrealistic to describe all models proposed. The sample presented in this book includes pharmacological models such as non-competing NMDA antagonists, emphasizing their use in vitro, neurodevelopmental models such as the neonatal ventral hippocampal lesion and the antimitotic MAM, models that reproduce environmental factors such as neonatal hypoxia, vitamin D deficits, and prenatal immune activation, as well as several different genetic model approaches. Although not all models have been studied with the same tools, a remarkable convergence is observed for most: whenever the impact of these manipulations on cortical inhibitory interneurons was tested, it was found abnormal. Of relevance to the disease, these anomalies emerge during adolescence and are consistent with a wealth of human post-mortem and imaging data. Thus, the use of animal models to gain insight into pathophysiological mechanisms of relevance to major psychiatric disorders is paying off and will certainly be expanded to test targets that could restore or ameliorate function. Patricio O’Donnell

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Contents Preface to the Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1.

2.

A Method to the Madness: Producing the Neonatal Ventral Hippocampal Lesion Rat Model of Schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . R. Andrew Chambers and Barbara K. Lipska Gestational MAM (Methylazoxymethanol) Administration: A Promising Animal Model for Psychosis Onset . . . . . . . . . . . . . . . . . . . . . . . . . Gwenaëlle Le Pen, Alfredo Bellon, Marie-Odile Krebs, and Thérèse M. Jay

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Prenatal Infection and Immune Models of Schizophrenia . . . . . . . . . . . . . Alan S. Brown

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The Hypoxic Rat Model for Obstetric Complications in Schizophrenia . . . . . . Andrea Schmitt, Peter Gebicke-Haerter, Ulrich Sommer, Markus Heck, Anja Lex, Mario Herrera-Marschitz, Mathias Zink, Markus Fendt, and Peter Falkai

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The Developmental Vitamin D (DVD) Model of Schizophrenia . . . . . . . . . . 113 Darryl W. Eyles, Thomas H.J. Burne, Suzy Alexander, Xiaoying Cui, and John J. McGrath

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Studying Schizophrenia in a Dish: Use of Primary Neuronal Cultures to Study the Long-Term Effects of NMDA Receptor Antagonists on Parvalbumin-Positive Fast-Spiking Interneurons . . . . . . . . . . . . . . . . . . 127 M. Margarita Behrens

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Glutathione Deficit and Redox Dysregulation in Animal Models of Schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Pascal Steullet, Jan-Harry Cabungcal, Anita Kulak, Michel Cuenod, Françoise Schenk, and Kim Q. Do

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Psychiatric Genetics and the Generation of Mutant Animal Models . . . . . . . . 189 P. Alexander Arguello and Joseph A. Gogos

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DISC1 Mouse Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Yavuz Ayhan, Hanna Jaaro-Peled, Akira Sawa, and Mikhail V. Pletnikov

10. Genetically Engineered Mice for Schizophrenia Research . . . . . . . . . . . . . 231 Juan E. Belforte and Kazu Nakazawa

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11. Epigenetic Animal Models of GABAergic Deficit in Mental Disorders . . . . . . . 243 Patricia Tueting, Erminio Costa, and Alessandro Guidotti 12. Modeling Schizophrenia in Neuregulin 1 and ErbB4 Mutant Mice . . . . . . . . 261 Yisheng Lu, Dong-Min Yin, Wen-Cheng Xiong, and Lin Mei Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

Contributors SUZY ALEXANDER • Queensland Brain Institute, University of Queensland, St. Lucia, Brisbane, QLD, Australia P. ALEXANDER ARGUELLO • Department of Neuroscience, Columbia University, New York, NY, USA YAVUZ AYHAN • Department of Psychiatry, Johns Hopkins University, Baltimore, MD, USA M. MARGARITA BEHRENS • The Computational Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA JUAN E. BELFORTE • Departamento de Fisiologia, Universidad de Buenos Aires, Buenos Aires, Argentina ALFREDO BELLON • Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Paris, France ALAN S. BROWN • Department of Psychiatry, Columbia University, New York, NY, USA THOMAS H.J. BURNE • Queensland Brain Institute, University of Queensland, St. Lucia, Brisbane, QLD, Australia JAN-HARRY CABUNGCAL • Department of Psychiatry, University of Lausanne, Lausanne, Switzerland R. ANDREW CHAMBERS • Laboratory for Translational Neuroscience of Dual Diagnosis and Development, Department of Psychiatry, Indiana University School of Medicine, Indianapolis, IN, USA ERMINIO COSTA • Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA MICHEL CUENOD • Department of Psychiatry, University of Lausanne, Lausanne, Switzerland XIAOYING CUI • Queensland Brain Institute, University of Queensland, St. Lucia, Brisbane, QLD, Australia KIM Q. DO • Department of Psychiatry, University of Lausanne, Lausanne, Switzerland DARRYL W. EYLES • Queensland Brain Institute, University of Queensland, St. Lucia, Brisbane, QLD, Australia PETER FALKAI • Department of Psychiatry, University of Goettingen, Goettingen, Germany MARKUS FENDT • Faculty of Biology, University of Tubingen, Tubingen, Germany PETER GEBICKE-HAERTER • The Central Institute of Mental Health, Mannheim, Germany JOSEPH A. GOGOS • Department of Neuroscience, Columbia University, New York, NY, USA ALESSANDRO GUIDOTTI • Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA MARKUS HECK • The Central Institute of Mental Health, Mannheim, Germany MARIO HERRERA-MARSCHITZ • Facultad de Medicina, Universidad de Chile, Santiago, Chile

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HANNA JAARO-PELED • Department of Neuroscience, Johns Hopkins University, Baltimore, MD, USA THÉRÈSE M. JAY • Centre de Psychiatrie et Neurosciences, Universite Paris Descartes, Paris, France MARIE-ODILE KREBS • Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Paris, France ANITA KULAK • Department of Psychiatry, University of Lausanne, Lausanne, Switzerland GWENAËLLE LE PEN • Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Paris, France ANJA LEX • Faculty of Biology, University of Tubingen, Tubingen, Germany BARBARA K. LIPSKA • Clinical Brain Disorders Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA YISHENG LU • Georgia Health Sciences University, Institute of Molecular Medicine and Genetics, Augusta, GA, USA JOHN J. MCGRATH • Queensland Brain Institute, University of Queensland, St. Lucia, Brisbane, QLD, Australia LIN MEI • Georgia Health Sciences University, Institute of Molecular Medicine and Genetics, Augusta, GA, USA KAZU NAKAZAWA • Unit on Genetics of Cognition and Behavior, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA MIKHAIL V. PLETNIKOV • Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, USA AKIRA SAWA • Department of Psychiatry, Johns Hopkins University, Baltimore, MD, USA FRANÇOISE SCHENK • Department of Psychiatry, University of Lausanne, Lausanne, Switzerland ANDREA SCHMITT • Department of Psychiatry, University of Goettingen, Goettingen, Germany ULRICH SOMMER • The Central Institute of Mental Health, Mannheim, Germany PASCAL STEULLET • Department of Psychiatry, Center for Psychiatric Neuroscience, Centre Hospitalier Universitaire Vaudois, Prilly-Lausanne, Switzerland PATRICIA TUETING • Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA WEN-CHENG XIONG • Georgia Health Sciences University, Institute of Molecular Medicine and Genetics, Augusta, GA, USA DONG-MIN YIN • Georgia Health Sciences University, Institute of Molecular Medicine and Genetics, Augusta, GA, USA MATHIAS ZINK • The Central Institute of Mental Health, Mannheim, Germany

Chapter 1 A Method to the Madness: Producing the Neonatal Ventral Hippocampal Lesion Rat Model of Schizophrenia R. Andrew Chambers and Barbara K. Lipska Abstract The neonatal ventral hippocampal lesion (NVHL) rat model of schizophrenia has demonstrated broad heuristic utility as an investigative platform encompassing many of the behavioral, neurobiological, and developmental aspects of this devastating neuropsychiatric illness affecting 1% of all human beings. This chapter serves as an essential description of materials and methods for generating and verifying the NVHL model in rats, which continues to hold significant potential in helping us understand schizophrenia, comorbid disorders, and their neurodevelopmental dynamics. Many of the approaches described here can be modified or adapted for producing other types of neurodevelopmental models of behavioral disorders. Key words: Ventral hippocampus, neonatal lesions, ibotenic acid, neurodevelopmental, methods.

1. Introduction The neonatal ventral hippocampal lesion (NVHL) rat model, developed at St. Elizabeth’s Hospital in Washington, DC, was first described in 1993 from the intramural program of the NIMH (1). It has become one of the most intensively studied animal models of schizophrenia ever developed, being the topic of over 120 primary research publications by at least a dozen independent research groups worldwide (2). Aside from representing a major advance in schizophrenia research in terms of providing the first significant non-pharmacological model of schizophrenia (i.e., not produced by exposure to psychostimulant or hallucinogenic drugs), this model includes multiple features that make it an attractive investigative platform. As a model of a complex neuropsychiatric syndrome that encompasses many neurobiological, P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_1, © Springer Science+Business Media, LLC 2011

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endophenotypic, and clinical/diagnostic features, which might seem etiologically unrelated, it succeeds in reproducing most of these phenomena in detail, if not in broad strokes, as a result of one experimental intervention: developmental damage of the ventral hippocampus in post-natal rats (see (2) for a comprehensive review). So, for example, instead of representing a paradigm that can test rats for a single schizophrenic trait, such as deficits in pre-pulse inhibition (PPI) of auditory stimuli, the model itself produces deficits in PPI, and many other schizophrenialike features including deficits in latent inhibition (LI), positivelike symptoms (e.g., behavioral hyper-responsiveness to novelty, stress, and psychostimulants that are reducible with neuroleptic treatment), cognitive symptoms (e.g., spatial working memory deficits), and neuroleptic-unresponsive negative symptoms (e.g., abnormalities in grooming and social behavior). Neurobiologically, NVHLs produce a host of structural and functional alterations that are downstream in anatomical space and developmental time from the initial lesion and that also mimic neuroimaging or post-mortem findings in human schizophrenia. Timely to growing interest in the periadolescent neurodevelopmental basis of schizophrenia and many other psychiatric disorders, the positive symptom-like behavioral features of the model show periadolescent emergence, in parallel with progressively abnormal biological or physiological measures of prefrontal cortical dysfunction (3). Moreover, the distributed neurocircuit effects of the lesion also represent mechanisms informative to understanding complex co-morbidities spanning mental illnesses such as addictions (4). Finally, the model itself or many of the techniques for producing it may be used to develop more specific experimental interventions in modeling a wide variety of other mental disorders involving alternative developmental time points, anatomical sites, or bioactive agents of lesion delivery, or combinations of lesions with particular environmental or genetic backgrounds. Successfully producing NVHL rats in sufficient quantities, while similar to approaches commonly used in stereotaxic brain interventions in adult rats, requires more of a nuanced, skilled touch and several special modifications. For the second author, originally developing this model accurately (e.g., achieving >80% lesion accuracy) from scratch required the sacrifice of well over 1000 rat pups, and working though numerous “wrong directions.” Even with the bugs worked out, and as trained directly from the inventor herself, the first author required nearly 4 months of effort and >100 pups to gain >66% proficiency. It is our goal to provide this chapter as a fairly complete and definitive resource for other investigators who are starting out in producing NVHL or similar developmental models so that they may get up and running with sufficient number of rats more quickly. We describe the essential materials and methods for producing the

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NVHL lesion and verifying its accuracy in adulthood. Section 4 describes trouble areas, pitfalls, and miscellaneous advice in this experimental approach.

2. Materials 2.1. Rats

Any rat strain of either gender may be used with the NVHL model, but the majority of papers (>90%) have examined Sprague–Dawleys and probably at least three-quarters of papers have focused on males. Thus if an experiment aims to reference or contextualize experimental results to the bulk of the literature, the best bet is to use Sprague–Dawley males. Alternatively, to explore gender or genetic biases with the lesion, both genders or Spragues with another strain are used in single experiments. The lesions are made in (and stereotaxic coordinates are designed for) 7-day-old rat pups (PD-7). This developmental point was chosen to approximate the second to third trimester equivalent of human fetal brain development, when the hippocampus is undergoing a crucially sensitive period of network formation, thought to make it vulnerable to a host of infectious, hormonal, neuroimmunological, or anoxic risk factors implicated in the early pathogenesis of human schizophrenia. Given this background, and the potential effects of other sources of perinatal stress, it is essential that rats are born in the laboratory from pregnant dams, either bred in or arriving in the laboratory housing facility no more than 17 days into gestation (i.e., 4 or more days before parturition on gestational day 21). Orders for pregnant dams at 13–17 days of gestation usually require 2–4 weeks advanced notice. In our experience, a given pregnant dam will typically provide 4–6 male rats/litter of appropriate weight range for the surgery. With one stereotaxic setup, and performing lesions proficiently at full speed, surgeries may proceed at about 4 rats/h. This rate can be increased with more stereotaxic setups (e.g., 6–8 rats/h with two setups). Depending on the expected rate of surgery, pregnant dams should be ordered with gestational due dates 1–2 days apart to avoid traffic jams of pups available for surgery.

2.2. Ibotenic Acid and Artificial CSF

Ibotenic acid (α-amino-3-hydroxy-5-isoxazoleacetic acid) is an NMDA receptor agonist, naturally produced by the Amanita mushroom species. In low doses delivered generally to the CNS, it has psychedelic effects, and interestingly, a portion of the compound is metabolized to muscimol, a potent and selective GABAA receptor agonist. In concentrated doses delivered to specific brain regions, it causes lethal neuronal calcium influx via the NMDA

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receptor. However, as an advantage among lesion techniques, it predominantly kills cells with cell bodies in the vicinity of the high dose and leaves axonal projections through the “blast zone” intact. Ibotenic acid (IBO) is available in 5 mg quantities (e.g., from Sigma) (>$350) providing enough toxin, theoretically, to lesion up to 800 rats. Ibotenic acid for neonatal lesioning is dissolved in artificial CSF (aCSF) vehicle, which after mixing and storing in Eppendorf tubes may be kept in –80◦ C freezer for >6 months and possibly up to 1 year without losing effectiveness. At room temperature and subjected to ambient light, IBO will lose potency within 6 h. The aCSF is a solution of electrolytes at physiologic pH that is inert when administered intrathecally to the rat. Beyond serving as the vehicle for IBO delivery, the aCSF is (a) delivered alone to the brains of SHAM-operated controls; (b) the general tubing fluid between the IBO or aCSF/SHAM bolus and the injection syringe; and (c) a rinsing solution for the injection cannula/tubing/syringe system after cleaning and storage in alcohol. A 1 l volume of aCSF is made by the following three-part recipe: (1) Into flask A, place 500 ml of sterile distilled H2 O and add the following: NaCl KCl CaCl2 •2H2 O MgCl2 •6H2 O

8.66 g 0.224 g 0.206 g 0.163 g

(2) Into flask B, place 500 ml of sterile distilled H2 O and add the following: 0.214 g or Na2 HPO4 •H2 O (dibasic) 0.358 g Na2 HPO4 •7H2 O NaH2 PO4 •H2 O (monobasic) 0.027 g (3) Add flask A to flask B. This gives a 1 l solution with 150 μM Na, 3.0 μM K, 1.4 μM Ca, 0.8 μM Mg, 1.0 μM P, and 155 μM Cl. This solution has pH 7.4. Artificial CSF may be kept in a regular refrigerator in a covered flask. The somewhat more challenging work of preparing IBO in the aCSF vehicle is described in the following four steps, which require pH litmus paper, micropipettes, and Eppendorf tubes: (1) In the small vial in which the 5 mg IBO arrives, add 500 μl of Millipore sterilized aCSF. Use a button spinner to briefly agitate the solution in the container at this point and after subsequent additions of solution described next. (2) After adding the aCSF to the original container, the IBO does not completely dissolve. Now add 1 μl of 10 N NaOH (MW 40 g/mol) to help ionize the acid and facilitate

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solubility. Check the bottom of the container to see how much IBO remains undissolved; repeat these 1 μl additions of 10 N NaOH until only a few tiny IBO clumps are visible. (3) Now the IBO is nearly, but not totally, in solution, and the pH is still not quite physiologic (e.g., it is <7.0). Check the pH (extract 0.5–1.0 μl from the container and drop onto litmus strips) and add 10 μl of 1 N NaOH. Check for progress in dissolving all IBO and check the pH again. Carefully repeat additions of 5 or 10 μl of 1 N NaOH with pH checks until there are no visible particle and the pH∼7.6. (4) Now you have ∼500 μl solution of IBO with a concentration of 1 μg/0.1 μl aCSF. Using a micropipette, distribute the IBO solution to 10 modified Eppendorf tubes (Fig. 1.1) allocating 50 μl per tube. Without significant waste, each of tubes will contain enough IBO to lesion 50–80 pups. After labeling and dating the tubes, the IBO should be kept in a –70◦ C freezer. Room temperature and ambient light can rapidly degrade IBO; when using IBO for lesioning, bring only one of the 10 tubes to the surgical setting where it may be temporarily stored on ice in a covered bucket prior to cannula loading. 2.3. Surgical Equipment

The surgical setup has three main components: the stereotaxic instrument, the syringe/tubing/injection needle (cannula lines), and the infusion pump. It is recommended that the stereotax be of a standard size (e.g., Kopf) for adult rats. Use of a smaller unit as for mice, even though rat pups are comparable in size, is not advised, as one needs plenty of room to move hands freely around the pup’s body during surgery. The stereotax should also be one armed as the lesion is performed bilaterally with the same cannula while the animal is immobilized. The ear bars of the adult stereotax are entirely removed to clear the mounting space. In this space, a platform should be installed upon which the rat will be

Fig. 1.1. The modified Eppendorf tubes are prepared for use for IBO storage by taking one Eppendorf tube, cutting the lower half off and stuffing it into another Eppendorf.

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Fig. 1.2. Stereotaxic stage set for neonatal lesions with custom insert, marked for pup placement.

mounted prone at a level that approximates where an adult rat’s head would be with the use of ear bars (Fig. 1.2). This platform should be custom designed according to the following guidelines: (1) the rat should rest entirely on the platform without any arms, legs, or tail hanging over the edge (i.e., it should be broad enough to accommodate the rat and tape used to immobilize it); (2) the platform should be unaffected by water damage; (3) it should sit firmly in the stereotax; (4) it should be able to have tape bind tightly to it; and (5) it should be able to be marked upon (so that guide marks perpendicular and parallel to the direction of stereotactic movement may be visualized during initial rat placement). We have found that custom-cut hard Styrofoam can fit these requirements fairly well, but for longevity, we have used custom plastic inserts. The three components of the cannula lines (e.g., syringe, tubing, and injection needle) used to deliver IBO/aCSF to the brain are described below: (1) 10-μl Syringe with thin metal plunger: This component sits in the infusion pump. We use a Hamilton 26S fixed nonblunted needle and a 10-μl syringe (Model #701; Hamilton #80300). The inner diameter of the glass cylinder is 0.485 mm and outer 6.604 mm.

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(2) Tubing: Intramedic, non-radiopaque, polyethylene tubing (PE-20). Inner diameter is 0.38 mm and outer diameter 1.09 mm (e.g., manufactured by Clay Adams). This tubing is cut into 30–50 cm segments (e.g., long enough to reach from pump to stereotax, but not too long), one end fitting snugly (without need for glue or grease) onto the tip of the syringe needle (0.5–1 cm down) and the other onto the blunt short end of the injection needle. For this connection, the tubing is advanced as far over the blunt end of the Hamilton needles as possible (a metal ring on the injection needle stops this advancement). (3) Injection needle: The proximal end has a small metal ring and the distal tip penetrates the rat brain. We have used a Hamilton replacement needle for a model 701 syringe (Hamilton #80427). It has an internal diameter of 0.115 mm and an outer diameter of 0.457 mm. A crucial final modification of the injection needle is the placement of the standardized depth band. This is simply the placement of a small length of the PE tubing (4–6 mm, cut with a scalpel) beyond the sharp tip of the injection needles that serves as a depth guide when the needle is put into the rat’s brain. This eliminates the need for ventral–dorsal (VD) stereotactic measuring and calculation – the needle is simply advanced into the subject’s head until the band contacts the surface of the rat’s skull. For standardized targeting for the NVHL, the piece of tubing should be pushed down from the needle tip to where there is exactly 5 mm between the distal end of the band (tubing) and the middle of the bevel in the sharp end of the needle (Fig. 1.3).

5 mm

Fig. 1.3. Placement of PE tubing section on injection needle serving as 5-mm depth gauge for VD coordinate for NVHL surgery.

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It is advised that a single surgical setup be complimented with four completed cannula lines: line #1 is for NVHL lesioning; line #2 for SHAM lesioning; line #3 for practice lesioning [e.g., with dye (Section 4.2)]; and line #4 as backup. Each of these lines should be appropriately labeled (e.g., with a small band of colored tape on the syringes) according to whether they will deliver aCSF only, IBO, or dye. The pump used to deliver the aCSF, the IBO, or the dye should be able to accommodate the previously described Hamilton needles and deliver 0.3 μl over a 2.25-min (135 s) period. We have used Harvard Apparatus microinfusion pumps, with digital/keypad programmable interfaces for this purpose. Regardless of the pump, it should occasionally be checked for proper calibration, as variability in the amount of toxin delivered can have unwanted effects. A simple calibration check is to see how long it takes to deliver 3 μl as visually observed from the 10-μl Hamilton syringe (e.g., it should be about 22.5 min with pump speed at 0.3 μl/2.25 min). Miscellaneous material needled for the NVHL surgery, including for hypothermic anesthesia, post-operative recovery, and equipment maintenance (Section 3.2), includes: – One or two rubber ice buckets with tops: Used to place rats during induction of hypothermic anesthesia or for shortterm storage of IBO in Eppendorf tubes. – Water ice source: For anesthesia. – Surgical Record Book. – Warming pad/heating lamp: For post-op recovery; should accommodate multiple pups. – Freezer tape (about 1 in. wide): For rat immobilization on the stereotaxic stage. – Surgical tools: Scalpel blades or plain single-edged razor blades, fine tweezers, scissors, and an ear punch. – Stopwatch/timer: Need to time duration of needle placement. – Marking pen. – Veterinary wound closure glue. – Small vials (for alcohol tube cleaning and artificial CSF rinsing). – Tuberculin syringes: Several for cleaning line tubing/injector needles each marked according to cannulation setup (e.g., IBO and aCSF). – Dye (for practice lesioning): Prepare by adding 50 mg of Chicago Sky Blue to 20 ml of aCSF.

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The following equipment and materials are required for animal sacrifice/ brain extraction and cutting and histological processing for lesion accuracy (Section 3.3). Animal sacrifice/brain extraction: – Rat guillotine for decapitation. – Appropriate choice of anesthesia for decapitation (e.g., deep isoflurane/pentobarbital/chloral hydrate). – Surgical kit for brain extraction: Scissors and small bone cutter/rongeurs. – Isopentane in 100–250-ml cylinder: Used to flash freeze extracted whole brains. – Thermometer (reading down to a –50◦ C). – Dry ice (frozen CO2 ) source. – Rubber ice bucket: To contain dry ice. – Aluminum foil: To wrap fresh frozen brain. – Marking pen: To label foil by rat number. – Freezer (–80◦ C) for brain storage. Brain cutting/ histology: – Cryostat. – Superfrost Plus microscope slides. – DPX or equivalent coverslip adhesive with coverslips. – Citrosolv. – Chloroform. – 100% Ethanol. – 90% Ethanol. – Calcite water: This is distilled water with a dash (literally a pinch per liter) of calcium carbonated added to pH balance water to between 7 and 8. It is used in all the water containing solutions used for histological processing except the thionin stain. This preparation may be avoided if the lab’s distilled water is already pretreated in this way. – Thionin (Nissl neuronal stain) (0.05%): The following recipe makes a 1 l solution: (1) Measure out 940 ml of ddH2 O (not calcite H2 O). (2) Add 37 g of sodium acetate and dissolve. (3) Add 500 mg of thionin (baker analyzed) and dissolve. (4) Add 30–35 ml of glacial acetic acid to adjust pH to 4.2 (a pH between 4.0 and 4.5 is acceptable). (5) Filter (takes about 1 h).

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3. Methods 3.1. Animal Selection and Litter Management

For the standard NVHL, rats should undergo surgeries on the 7th day of life which obviously necessitates daily observation of when rats are born. Some grace to this rule is reasonable such that rats may undergo surgeries at any point during a 24-h period centered on the best guess as to when the seventh day of life is timed. Most importantly, however, at surgery, the rat must weigh from 15 to 20 g, preferably close to 17 g. If it does not, there are risks that (a) the stereotactic coordinates will be inaccurate; (b) the rat may possibly not be at the correct stage of neural development; or (c) the rat (or litter) has failure to thrive due to an unknown stressor, medical problem, or other developmental abnormality (any of which could likely interfere with effects of NVHL or SHAM lesions). It should be expected that large, albeit normal litters (>12 rats) will lead to higher frequencies of PD-7 rats below the 15 g weight minimum. To address this possibility, and to anticipate the number of subjects available for surgery, it is recommended that litters be assessed at PD-3 to 5 and gendertyped and culled as needed (more culling will speed growth per animal). Gender-typing pups before PD-3 can be tricky but is quite accurate thereafter with a brief visual exam of pup’s genitalia (Fig. 1.4). Generally, compared to females, males will have a greater distance between anus and phallic prominence, and, no huge surprise, the males have a larger phallic prominence. On the day of surgery, it is important to balance randomized NVHL vs. SHAM assignments across and within available litters. Across litters, this is important for minimizing the effects of genetic heterogeneities between moms. Allowing for expected rates of attrition due to incorrectly placed lesions, this means generating NVHL vs. SHAM ratios (e.g., 5:3 NVHL/SHAM), as consistently as possible across litters. Within litters, even when

female

male

Fig. 1.4. Sketch of gender differences in genitalia in PD-3–7 pups.

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keeping within the 15–20 g weight range, it is important to optimize balance of lesion status by weight (e.g., ensure all the SHAM animals do not tend smaller). 3.2. Performing NVHL and SHAM Control Surgeries 3.2.1. Surgical Setup

On the day of surgery, setting up should include the following: Hypothermic anesthesia bucket: Prepare the anesthesia bucket as a rubber ice bucket filled with small ice chips/balls. The researcher’s fist is used to punch a central crater in the ice field 5–10 cm deep for pup placement. Cannula preparation: Tubing and syringes must be clean and patent with easy gliding motion of the syringe plunger. Between surgical sessions, the lines should be flushed with a tuberculin syringe loaded, first with 90% EtOH (4–5 ml squirted vigorously through a 25-gauge needle into the plunger-less Hamilton syringe end of the cannula) and second with aCSF flush (4–5 ml). During these flushes, one wants to see a thin but vigorous stream of fluid emerging from the injector tip. If the flow is slow, repeated flushing may be necessary, possibly with the use of cleaning wires provided with Hamilton syringes for removal of matter from inside the needles. The functioning cannula is then filled completely with aCSF free of air bubbles in the lines (although air bubbles may initially be used to check for good flow in the cannula as described above). As with the cleaning, this insertion of the “hydraulic” aCSF is accomplished by a more gentle injection of aCSF through the plunger-less Hamilton syringe with a tuberculin syringe. Now, for loading the injection bolus (e.g., IBO, aCSF, or dye), one first pulls back on the Hamilton’s plunger to create an air bubble of 0.2–0.3 μl volume as a mobile boundary between the aCSF hydraulic and the injection solution. This bubble not only prevents dilution of the IBO or dye into the hydraulic aCSF but also provides a visual marker to confirm that flow is occurring within surgeries, and for knowing for how much injection solution is left in the cannula over the course of multiple surgeries. Now with the indicator bubble in place, simply suck up IBO (from an Eppendorf tube), aCSF, or dye as needed. For a run of eight or more NVHL surgeries, we recommend filling the line with >7 μl by gentle backward plunging on the Hamilton syringe. If not anticipating a need for IBO for the rest of the session, return it to –80 freezer; if reloading later in the day, it may be stored on ice and under cover for 2–4 h in the surgical room. Now, mounting the cannula needle onto the stereotax, it is key to have the needle secured to the injector arm as close to the tip of the needle as possible for stability (Fig. 1.5), because the needle itself is somewhat unstably flexible. Also, the bevel of the injection needle should be squarely positioned facing the caudal aspect of the rat. After mounting the Hamilton syringe into the infusion pump, go ahead and manually waste some injection fluid out the injector needle tip

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no

yes

Fig. 1.5. Proper mounting of the injection needle on the stereotaxic arm.

to again confirm patency and ensure that the infusion pump and the syringe are correctly mechanically interfaced. Finally with no pup on stage, make one or two 0.3 μl infusions to verify proper function and volume delivery, and to generate a consistent balancing of hydraulic tension in the system. Wipe away the small infusate bead on the tip of the needle. Pre-op ready chamber: Properly selected pups (about six at a time, at the start of a run) should be on hand in a rat-housing tub. Each rat should be numbered with a marker on their back, with weight references, semi-random lesion assignments, and possibly gender ID noted on paper. Logistically, it is best to perform a series of rats (six or more) as IBO lesions and another series as SHAMS, to prevent from having to change cannula between surgeries. If two setups are used, they may be segregated for NVHL vs. SHAM surgeries. Post-op chamber: This may be a rat-housing tub, containing soft padding overlying a warming pad. 3.2.2. Inducing Hypothermic Anesthesia

Although unfamiliar to some investigators and animal research review committees, generalized hypothermia is safer, easier, and less expensive than chemical anesthesia for infant rats. The pup is placed in the ice crater for 15–20 min, with the cover placed over the ice bucket. During this induction, the pup should be checked every 5 min or so, as initially it will often succeed in crawling out of the crater to the edge where hypothermia occurs unevenly. In the 15–20 min window, the rat should be checked for complete immobilization and lack or respiration. This is achieved as early as 17 min. This induction should be rigorously timed. Leaving pups on ice for longer than 20 min significantly increases risk of death; taking them off too soon increases the risk of premature return of mobility during surgery, which is a major cause of lesion inaccuracies or unintended mechanical lesions in SHAM animals.

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Usually, 17–18 min on ice will provide 12–16 min of immobility, which in experienced hands is a sufficient time to perform the surgery. 3.2.3. Lesioning

The two major issues with the neonatal lesion surgery, as opposed to an adult stereotaxic surgery, are (1) the targets are much smaller and (2) the skull bone is far thinner and highly flexible. The former increases the challenge, while the latter provides both up and downsides. On the upside, there is no need for a bone drilling step; the injection needle tip itself is the drill. On the downside, the pup’s braincase is fragile and flexible, rendering head immobilization virtually impossible with traditional means. Thus, your target is very small, and to some extent, it will be moving, because anything (needles and fingers) that touches the pup will displace it after the stereotaxic coordinates are set. This has two important implications for the attitude of the researcher. First, compared to adult stereotaxic surgery, it has to be accepted that neonatal lesioning is much more of an acquired art or a skill (like bowling, dart throwing, and golf ). Second, like these sports, it takes practice to become reasonably proficient. Next we provide detailed guidelines on how the surgery should be approached, while Section 4.2 outlines approaches in practice lesioning. After induction of anesthesia, lesioning consists of three basic steps: (a) mounting the rat; (b) puncture and infusion; and (c) post-op recovery. Including anesthesia, all of these steps performed for a single rat take 40–55 min. However, when performing serial surgeries in assembly line fashion, one rat is undergoing anesthesia while another is being operated on, while another is in recovery. This shortens the procedure to 15–20 min per rat, or shorter with more than one setup. The remainder of this section assumes only one setup and is performed identically with IBO infusions (NVHL) or aCSF (SHAMs). (1) Mounting the rat: This is a crucial step in the process as mistakes here amplify the chance of inaccurate targeting. After the rat has been adequately anesthetized, place it squarely on its abdomen on the surgical platform with the aid of guide marks drawn on the platform. The rat’s tail should be pulled out straight behind and its four limbs should be spread out to the sides so that they do not sit under the rat’s neck and torso (Fig. 1.6). All this is to ensure that the body and the head sit on the platform in the most stable, central and square position (with respect to the stereotax arm movement). Now, using a scalpel or a sterile razor blade, a single incision is made down the middle of the rat’s head (1.5–2 cm long) so that bregma and the sagittal and coronal cranial sutures are observed. Be careful not to lacerate the veins or the tissue on the exposed skull. Spread out the

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Coronal Suture

Lamboidal Suture

Bregma

Saggital Suture

Fig. 1.6. Proper positioning and taping of pups on the stereotaxic stage.

width of the wound with your fingers to ensure adequate lateral room for insertion of the cannula needle into the skull. Now, using strips of freezer tape, secure the rat’s neck and head to the platform crosswise (Fig. 1.6). The caudal strip is placed first to stabilize the neck. Then, one hand is used to manually re-position the head so that it is flat on the platform and square with the stage (e.g., the sagittal suture is made parallel with the long-axis motion of the stereotaxic arm and non-rotated with respect to the vertical), while the other hand places the rostral strip of tape. Note that either or both the caudal and rostral pieces of tape can be placed just a bit over the open wounds to aid in keeping the wound open. Further, quick adjustments should now be made as needed to the tape and/or the rat’s positioning as necessary to maximize alignment of cranial landmarks with the stereotax (you will notice that the rat’s skull slides around very easily within the skin around the rat’s head). In making all these adjustments, it is important to view the animal from directly above, as a viewing angle off the vertical will result in a misaligned pup. (2) Puncture and infusion: Now using the stereotax, position the needle directly over bregma. In these infant rats, bregma may be quite difficult to see. Confirm that you have bregma

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by very gently taking the needle down to the skull surface. Carefully applying some pressure with the needle (do not puncture the sagittal vein!) will depress the soft tissue between the cranial plates giving contour to bregma and the cranial sutures. Looking at the skull from a sideways view so that the light is reflected from the skull can help reveal these contours. Once bregma is confirmed, write down the anterior–posterior (AP) and medial–lateral (ML) coordinates as given by the stereotax. Remember, ventral–dorsal (VD) coordinates are not needed because of the standardized depth needle band. The coordinates relative to bregma for the NVHL surgery are AP –3.0 mm and ML ± 3.5 mm (VD –5.0 mm). Move the needle using the stereotactic mechanism posteriorly 3.0 mm from bregma. Observe that as you move it back, it traces the sagittal suture. If it does not appear to follow it in this fashion, then you have got an alignment problem that needs to be fixed as described under the previous step (mounting the rat). It does not matter which side of the rat’s head you lesion first as long as you are consistent. Using the stereotactic mechanism and coordinates, move the needle 3.5 mm from the midline toward the side you choose to do first. Now you are ready to penetrate the skull. First use the needle as a punch to make a hole in the targeted spot on the skull. Holding the rat’s head steady with one hand (gentle pressure with thumb and index finger on either side), make a quick and shallow jab with the needle using the stereotax with the other hand. Now, keeping one hand in support of the rat’s head, slowly re-advance the needle into the hole until the standardized depth needle band touches (and somewhat depresses) the rat’s skull. The most likely complication with needle insertion (and a major source of inaccuracy) is rotation of the rat’s head to one side; gentle head support with the fingers and a properly targeted needle should keep this from happening. Once the needle is in, you should notice that the pressure of the needle band has forced blood out of the venous sinuses of the sagittal and lambdoid sutures. Slowly raise the needle just until the blood refills these veins. Now that the needle is in place, the infusion pump may be started. It should pump in 0.3 μl of fluid over the course of 2 min and 15 s (135 s). Once the infusion is complete, let the needle rest in place in the rat’s head for about 3 min to prevent backflow. For this duration of needle placement, it is recommended that a stopwatch set to 5 min be used to time from the start of the infusion to when the needle should be withdrawn. After 5 min of total needle placement, we now lesion the other side of the brain. In our experience, this second infusion is

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more likely to be off target than the first because the effects of pup motion and/or subtle misalignment, if present, are cumulative. Again, gently support the head with thumb and index finger of one hand, as this prevents cranial rotation during withdrawal of the needle. Still holding the head in this way after the needle is out, use the other hand to manipulate the stereotax so that the needle moves over 7.0 mm to the other side of the head (e.g., 3.5 mm to the other side of the sagittal suture). With good head support, the needle is now positioned above a spot that is symmetric with the AP and ML positions of the puncture hole on the contra-lateral side of the head. If when viewing the animal from directly above you do not see this symmetry, you may have to manually adjust the targeting just a bit (i.e., deviate from the exact coordinates) based on your line of sight only. This maneuver can be tricky but is sometimes necessary if inadvertent pup motion has occurred in prior steps, and it is often accurate with the eye of an experienced surgeon. Now, just as for the procedure on the other side of the head, and while providing head support, repeat the steps: punch and needle placement, timed infusion, and 3-min waiting period while the needle rests in the rat’s head. At the end of the second 3-min waiting period, the needle is withdrawn and the surgery is nearly complete. If during the surgery the rat begins to show movement (usually begins with back legs), put more pieces of ice over its back to maintain hypothermia. It is important to maintain immobility as much as possible during all phases of needle placement; movement of the head can make an IBO lesion too large or cause the SHAM surgery to make a mechanical lesion. After withdrawing the needle, carefully pull the tape off the rat. Take advantage of the remaining anesthetized state for using a mouse ear punch as needed to mark animals (one or two punches on either ear as NVHL, SHAM or probable failed attempt). It is best to close the wound after putting holes in the ears to prevent re-opening of the wound. Close the wound with a thin line of veterinary wound closure glue. (3) Post-operative recovery: Pups are placed in the post-operative tub for warming and monitoring. If a post-operative pup has not yet begun to stir, it should within the next 5 min. Animals receiving IBO may be observed to be more mobile compared to SHAM animals within this time frame. Occasionally, an IBO pup will die 5–10 min after becoming mobile, possibly in relation to improper ventricular spread of IBO that may impair respiration after recovery from hypothermia. Animals that die after showing little to no movement most likely had lethal hypothermia (an outcome

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that can be 100% avoided with proper timing of anesthesia induction). Proactive attempts to resuscitate pups with diminished or lost respiration are ill advised, since it may indicate a bad lesion, or extra-lesion damage (e.g., cerebral hypoxia). If after 15 min, the pups are respiring normally, motorically responsive to touch, have normal pink coloration, and dry wound closure cement (if used), they are ready for return to their litters. Ordinarily, rats are returned to their original litters. However if culling or other sources of attrition have greatly reduced litter sizes, adoption to other litters is reasonable, as long as litters remain balanced and equal numbers of NVHLs and SHAMs are adopted. 3.3. Lesion Confirmation

Lesion confirmation is a necessary step for all rats in a given experiment (e.g., including both NVHL and SHAM-operated animals). Bilateral success rates for NVHLs may vary (even for the same surgeon) from 50 to 100% between cohorts, and occasionally a SHAM will have enough needle track damage to warrant exclusion. Rats may be sacrificed at any age for this purpose, but consistent with most experimental designs, we describe methods general to adult rats (>PD-56).

3.3.1. Histology

After sacrifice (by deep anesthesia and guillotine) and brain removal, the brain is placed whole into an isopentane-filled cylinder (kept between –30 and –40◦ C on dry ice), for 20–40 s. The rock-hard brain is then removed with forceps and wrapped in an appropriately sized (∼12 cm × 12 cm) and labeled square of aluminum foil. The brains may then be stored in a –80◦ C freezer until cutting. For sectioning, aim to collect 6–8 coronal sections on a cryostat through most of the rostral–caudal extent of the hippocampus. Generally, sections for slides should begin when the dorsal– rostral blade of the hippocampus comes into view (∼–2.0 mm AP bregma (5) as shown in Fig. 1.7). Sections are collected for slides about every 400 μm. We have used 20-μm-thick sections, in which every 18th–20th section is put on slides, or 40-μm sections, with every 9th–10th kept. Four to six sections are mounted on each appropriately labeled slide. Before staining, mounted slides may be stored in a slide rack for several days in a –20◦ C freezer. For staining, allow slides to warm from cold storage at room temperature for about 5 min. Then, process slides through the following 17 solution steps, while allowing the dipping rack to drain off between emersions. Notably, only the steps marked by (∗ ) require an actual solution dish preparation; steps 4 and 6–9 may reuse solutions 1–3.

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Fig. 1.7. Appearance of dorsal blade of the hippocampus in coronal section; approximate area where first section for histological processing should be taken.

Dehydration (1) 70% ETOH (3 min)∗ (2) 95% ETOH (3 min)∗ (3) 100% ETOH (3 min)∗ (4) 100% ETOH (3 min) Fixing (5) 1:1 chloroform/100% ETOH (1 h to overnight) ∗ Rehydration (6) 100% ETOH (1–3 min) (7) 95% ETOH (1–3 min) (8) 95% ETOH (1–3 min) (9) 70% ETOH (1–3 min) (10) Calcite water (1–3 min)∗ (11) Calcite water (5 min)∗ Staining (12) Thionin solution (0.05%) (5–7 min)∗ (13) Calcite water∗ (14) 80% ETOH (1 min)∗ (15) 95% ETOH (1 min)∗ (16) 100% ETOH (1 min)∗ (17) Citrosolv (2 min)∗ (18) Coverslip with DPX (or equivalent)

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Investigators should examine brains from all animals in a given experiment, blind to the results of the completed study. It is recommended that brains be examined under both very low (4×–10×; magnifying glass) and low magnification (40×; microscope) to allow close visualization of the entire brain and a more detailed exam of the cellular architecture of the hippocampus. Both lesioned and SHAM rats should be examined. Many SHAMs will normally show trace evidence of needle track damage – and that is permissible, unless there is pronounced evidence of tissue distortion or destruction, suggesting a mechanical lesion. The coordinates for the NVHL target the area where the CA3 layers of the dorsal and ventral blades merge [in adult sections, at about −5.0 mm from bregma (5)] (Fig. 1.8). Lesioned animals are typically characterized by one or more of the following: (a) generalized enlargement of the lateral ventricle; (b) tissue atrophy and/or distortion of normally curvaceous cell layers in the ventral hippocampus (involving CA1, CA2, CA3, and subiculum) and/or the CA3 of the ventral extent of the dorsal hippocampus; (c) loss and disarray of individual neurons (e.g., decrease in thionin staining density in cell layers) within cell layers. Between subjects, it is to be expected (and is permissible) that lesion damage within the hippocampus is somewhat heterogeneous in size and exact location, much as histopathological studies of brains of schizophrenia subjects show within-group heterogeneity of morphological findings. However, the following parameters should also be used to disqualify rats and limit this heterogeneity. (1) Lesion size: Lesions can be too small or too large, and either extreme may alter the behavioral phenotype (6). Require evidence for IBO damage (not just needle tracks) in at least two serial sections. Conversely, the lesion should not be a total hippocampectomy or involve significant damage to the dorsal blade of the hippocampus as this structure has differential connectivity patterns and functionality compared to the ventral (more limbic) hippocampus (7). This outcome is easily ruled out by ensur-

Sham (normal)

IBO

Fig. 1.8. Primary target areas (cross hairs) of coordinates for NVHLs (left) and typical zone of tissue loss with a “perfect” bilateral hit in an NVHL (right).

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ing lack of extension of the lesion into the dorsal blade on the first section in the series. (2) Lesion location: The damage should be to the hippocampus proper; significant damage evident to the overlying cortex, underlying thalamus, or ventrally posited amygdala should be inspected for, and warrants disqualification. The NVHL lesion does cause some degree of generalized atrophy to its efferent/afferent structures (e.g., prefrontal cortex, temporal/entorhinal cortex, amygdala, dorsal hippocampus, and likely elsewhere) and so if noted on exam, such trends are not grounds for disqualification unless there is evidence of significant cell loss and/or gliosis in these structures under 40×. (3) Bi-laterality: Small lesions on one side and large on the other are not infrequent and are permissible as long as the lesions on each side meet the above criteria. However, a lack of bilateral lesions is an exclusion, representing either a lack of enough infusion volume on one side or an extrahippocampal infusion that may be hard to find.

SHAM

NVHL

Fig. 1.9. Example of published demonstration of serial maps showing smallest and largest extent of lesion damage in a study (8) and exemplary micrographs of SHAM vs. NVHL, with permission from Springer.

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For purposes of publication, it is typical for researchers to show anatomical maps of largest vs. smallest lesion extents of rats with appropriate lesions (e.g., from among all those not disqualified from a given study) along with a representative micrograph of a SHAM and NVHL brain (e.g., Fig. 1.9).

4. Notes: Problems and Pitfalls 4.1. Making Ibotenic Acid

4.2. Practice Lesioning

This obviously crucial step, if not done carefully and correctly, can cost serious time and money since histological lesion results are typically not assayed until well after rats reach adulthood (PD-56) and experiments are completed. The addition of NaOH for putting IBO into solution is a delicate matter and must proceed in a gradual and deliberate titration with frequent pH checks. The danger is that the pH can suddenly shoot higher than 8.5 from a prior reading of <7.0. In our experience, around or below 8.5 may be OK, but higher than this risks both impotency of the IBO as an excitotoxin and lethality to rat pups in surgery. If in surgery, attrition exceeds >25%, “bad acid” is a likely culprit, and a new batch should be mixed. To obtain a reasonable level of skill and accuracy, it is recommended that surgeons inexperienced with NVHLs initially practice lesions on 50–100 pups. Learning curves are more rapid if this is done in batches of 1–10 rats, with meticulous intra-surgery notes taken on each rat and batch sacrifices for lesion verification conducted within 24 h, if not immediately after a surgery. Keep a record of improving bilateral success rates vs. unilateral hits and total misses. Two major alterations to the general methods are important for practice lesioning: first, use Chicago Sky Blue (50 mg in 20 ml of aCSF) (or equivalent stain) as the infusate. Second, the histological processing involves sacrifice of rat pups [e.g., with lethal (>25 min) hypothermia, brain removal, isopentane-rapid freezing, and cutting on a cryostat only]. The lesion verification is done by visual inspection (with magnifying glass) of dye delivery while the brain is being sectioned on the cryostat – there is no need for slides or histology. Of note, the PD-7 rat brain and hippocampus do differ architecturally somewhat from the adult. After histology, the adult hippocampus appears to have two independent lobes: one dorsal that comes into view first (coronal sections cut caudally) and then a ventral that appears independently, then merges with the dorsal. In the pup, these parts, appearing darker than overlying cortex, seem more contiguous regardless of which section you are looking at. A good bilateral hit vs. common patterns of missed hits are shown in Fig. 1.10. In general, the key will be achieving consistent bilateral

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Bilateral Success

No

No

No

Fig. 1.10. Patterns of success and typical failures in practice lesioning using dye.

symmetry, alignment, and infusion depth. Create small cartoons for each result, compare with written notes, and adjust your technique accordingly. 4.3. SHAM Controls

It is debatable whether SHAM lesions are the best control for all studies. Mechanical damage or other complications of introducing a needle into the brain are likely to have at least some subtle long-term effect consistent with a mild lesion, potentially decreasing effect size in some NVHL vs. SHAM lesion comparisons. Although early piloting studies and some published results have shown little effect of SHAM–NVHL lesions compared to unoperated controls in a limited set of phenotypic measures, SHAM lesions of the neonatal medial prefrontal cortex do show trends approaching significance across several phenotypic measures compared to unoperated controls (9). Therefore, depending on the experiment and experimental justification, one or more alternative controls may also be used (e.g., unoperated pups not removed from litters; pups undergoing hypothermic anesthesia; and superficial wound opening only).

4.4. Cautions About Alternative Approaches

In developing or learning the methods for producing NVHLs, it may be tempting to try alternative modifications to the procedures. Here, we mention a few of these that we would advise caution in pursuing:

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Anesthesia: Although alternative forms of anesthesia (e.g., pharmacological) other than hypothermia have been used for NVHL surgery, we generally advise against these approaches. In our experience, hypothermia, when properly done, is safer, more predictable, cheaper, and cleaner. Using it arguably maintains the model as a more purely non-pharmacological model of schizophrenia, while other anesthetics if used this early in the developmental stage of the animal may have longterm effects and have themselves been used to produce models of schizophrenia (e.g., ketamine). Head stabilization: It may be tempting to create an apparatus for allowing greater head stabilization as an attempt to reduce the amount of practice and skill needed to make accurate lesions. In our experience, such attempts have amounted to wild-goose chases. While some groups appear to have successfully made form-fitting molds for stabilizing the rat’s body and head in the operative platform, we recommend against the use of metal probes, clamps, or ear bar-like instruments. The pup’s skull is simply too soft, flexible, and rotatable under the skin. Wound closure: The wound may of course be closed with wound closure glue, clips, or sutures. However, the latter unnecessarily adds time to the surgery and is susceptible to wound re-opening after return to litters. Mothers may also meddle with sutures and especially clips. Placing clips involves additional risk of injuring the pup’s skull, and they need to be removed a week or two later which unnecessarily distresses the animals. The only caution with glue is to make sure it is dry before returning pups to their litters.

Acknowledgments Compilation of this chapter was supported by NIDA K08 DA019850 (R.A.C.). The authors wish to thank Alena Sentir for her effort in rendering the original drawings into digital form. References 1. Lipska BK, Jaskiw GE, Weinberger DR. (1993) Post-pubertal emergence of hyperresponsiveness to stress and to amphetamine after neonatal excitotoxic hippocampal damage: a potential animal model of schizophrenia. Neuropsychopharmacology 9: 67–75.

2. Tseng KY, Chambers RA, Lipska BK. (2009) The neonatal ventral hippocampal lesion as a heuristic neurodevelopmental animal model of schizophrenia. Behav Brain Res 204: 295–305. 3. O’Donnell P, Lewis BL, Weinberger DR, Lipska B. (2002) Neonatal hippocampal

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damage alters electrophysiological properties of prefrontal cortical neurons in adult rats. Cereb Cortex 12:975–982. 4. Chambers RA. (2007) Animal modeling and neurocircuitry of dual diagnosis. J Dual Diagn 3:19–29. 5. Swanson LW. (2004) Brain maps: structure of the rat brain. 3rd ed. New York, NY: Elsevier. 6. Swerdlow NR, Halim N, Hanlon FM, Platten A, Auerbach PP. (2001) Lesion size and amphetamine hyperlocomotion after neonatal ventral hippocampal lesions: more is less. Brain Res Bull 55: 71–77.

7. Moser MB, Moser EI. (1998) Functional differentiation in the hippocampus. Hippocampus 8:608–619. 8. Chambers RA, Jones RM, Brown S, Taylor JR. (2005) Natural reward related learning in rats with neonatal ventral hippocampal lesions and prior cocaine exposure. Psychopharmacology 179: 470–478. 9. Schneider M, Koch M. (2005) Behavioral and morphological alterations following neonatal excitotoxic lesions of the medial prefrontal cortex in rats. Exp Neurol 195:185–198.

Chapter 2 Gestational MAM (Methylazoxymethanol) Administration: A Promising Animal Model for Psychosis Onset Gwenaëlle Le Pen, Alfredo Bellon, Marie-Odile Krebs, and Thérèse M. Jay Abstract This chapter provides an overview on exposure to methylazoxymethanol (MAM) at embryonic day 17 as a promising animal model for schizophrenia that mimics behavioral abnormalities and deficits in prefrontal cortex networks. This early insult produces in adult offspring from E17 MAM-treated dams the following: (1) behavioral changes including spontaneous hyperactivity and hypersensitivity to psychotomimetic drugs that are reminiscent of positive symptoms of schizophrenia and associated with a temporal pattern of expression; (2) impaired social interaction similar to that observed in schizophrenic patients existing prior to the onset of disease; (3) cognitive deficits in a variety of domain: working and reference memory, behavioral flexibility, attentional functioning, object recognition, and reversal learning; (4) behavioral abnormalities resembling schizophrenia-related endophenotypes like deficient sensorimotor gating and disrupted latent inhibition; (5) anatomical changes with cortical, thalamic, and hippocampal reductions in volume that are associated with an enlargement of the lateral ventricles; and (6) abnormalities in functional connectivity between brain areas that involve deficits in dopamine, glutamate, and GABA systems. The E17 MAM model that incorporates neuropathological and functional manifestations associated with schizophrenia may help forward early preventive interventions that can successfully reduce the risk of developing schizophrenia in exposed individuals. Key words: MAM, animal model, schizophrenia, prefrontal cortex, hippocampus, schizophreniarelated endophenotypes, behavioral and cognitive deficits, anatomical and functional abnormalities.

1. Introduction Schizophrenia is one of the most disabling and emotionally devastating illnesses of the brain. It is characterized by a constellation of distinctive symptoms that can be divided into three groups: (1) positive symptoms, such as delusions, hallucinations, paranoia, P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_2, © Springer Science+Business Media, LLC 2011

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and psychosis, (2) persistent negative symptoms, such as flattened affect, impaired attention, social withdrawal, and (3) cognitive impairments (1). Despite a hundred years’ research, the neuropathology of schizophrenia remains obscure. To better understand its pathogenesis, animal models are important tools in experimental medical science. Schizophrenia can be modeled in animals using standardized procedures that recreate specific pathogenic events and their behavioral outcomes. Availability of suitable animal models could further allow analysis of the mechanisms that underlie the etiology of the disease and may facilitate the development of novel therapeutic strategies. Nevertheless, an animal model that can perfectly replicate all aspects of a human disease such as schizophrenia does not exist. Despite the fact that the etiological origin of schizophrenia is still unclear, there is increasing evidence suggesting that early insults during brain development are associated with an increased risk for schizophrenia (2, 3). In particular, postmortem studies have revealed reduced volume, decreased neuronal size, and loss and/or malpositioning of cells in the hippocampus and prefrontal, entorhinal, and cingulate cortices of schizophrenic patients (2). Epidemiological studies and the observation of minor physical (4) and dermatoglyphic abnormalities (5) also suggest that the second trimester of pregnancy could be a “critical window of vulnerability” during which the fetus may be placed at a higher risk for schizophrenia (4, 5). This period of time in the developing human brain coincides with a massive neuronal migration of cells to the cortex (6). In addition, it has been hypothesized that the interaction between hippocampus and prefrontal cortex might be particularly disturbed in the disorder (7, 8). This so-called disconnection hypothesis (9) is also attractive since the hippocampal formation (HF) is selectively vulnerable to some obstetrical insults (10), and a disturbed interaction with the dorso-lateral prefrontal cortex would thus offer an explanation of epidemiological data linking schizophrenia to early neurodevelopmental disturbances (11). Consistent with this notion, several neuroimaging studies have reported altered functional integration between prefrontal and hippocampal regions in schizophrenia (8, 12–15). Hence, an animal model based on neurodevelopmental defects in the hippocampus and the cortex has been proposed as offering some degree of construct validity for schizophrenia. The use of a mitotoxin administered prenatally and the knowledge of a precise neurogenic timetable (16, 17) has allowed targeting neuronal ablations in specific brain areas of the central nervous system when administered at different gestational days. To this end, brain cellular proliferation can be briefly interrupted with the mitotoxin methylazoxymethanol (MAM), administered

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during late gestation at embryonic day 17 (E17), when neurons that will migrate to the hippocampus and the neocortex are undergoing major cell division (18). Because of its unique characteristics, MAM has long been used to induce developmental brain dysfunction in rodents and to study postnatal behavioral effects of a prenatal administration (17, 19). MAM is an antimitotic agent (20–23) that, in vivo, is rapidly converted to methyldiazonium, damaging DNA through the methylation of guanine nucleic acids at O6 or N7 positions (24). In addition, MAM has specificity for actively dividing neurons, leaving other cell types and differentiated neurons relatively unaltered (25, 26). Indeed, actively dividing neuroepithelial cells during the S phase are affected by MAM, whereas post-mitotic neurons or neuroblasts in the G0 phase are spared (27). Furthermore, MAM is able to readily cross the placental barrier (26, 28). The narrow time window of biological activity of MAM in fetal tissue remains for a limited period of 2–24 h, after intraperitoneal administration to the pregnant dam, with a peak activity at 12 h that allows targeting the proliferation of specific neuronal cell populations (24, 29). While the hypotheses taken into account to develop the E17 MAM model seem to endow the model with construct validity for some aspects of schizophrenia, there is now converging evidence supporting that exposure to MAM at E17 has also reached a certain degree of face validity. Indeed, E17 MAM-exposed animals exhibit behavioral changes that mimic some aspects of the positive, negative, and cognitive symptoms of schizophrenia (30–37). In addition, the diachronic pattern of the phenotype (i.e., peripubertal pattern of behavioral abnormalities), shown in MAMexposed animals, is strikingly similar to that seen in schizophrenia (34–36). Finally, concerning the face validity of the model, E17 MAM-exposed animals also exhibit neurochemical and neurophysiological abnormalities reminiscent of those observed in schizophrenic patients (32, 37–42).

2. Behavioral Changes Observed in E17 MAM-Exposed Rats 2.1. Behavioral Abnormalities Reminiscent of the Positive Symptoms of Schizophrenia

In rodents, both locomotor hyperactivity and stereotyped or perseverative behaviors are thought to reflect hypersensitivity of the subcortical dopamine system (43, 44). In humans, an excess of subcortical dopamine has been associated with delusions and hallucinations (45–47). Then, novelty-induced hyperactivity has been viewed as a preclinical model of positive symptoms of schizophrenia and of psychomotor agitation in particular (48). In the same line, both locomotor hyperactivity and striking stereotyped or perseverative behaviors induced by dopaminergic

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receptor agonists and N-methyl-D-aspartate (NMDA) receptor antagonists, at high doses, were shown to each have various degrees of face validity for behaviors that are reminiscent of changes observed in schizophrenia. In addition, these agents were also shown to exacerbate psychotic symptoms in schizophrenic patients, suggesting that dopaminergic and glutamatergic neurotransmission may be critical in schizophrenia (49–56). Thus, to investigate if MAM-exposed animals exhibit behaviors that could be reminiscent of positive symptoms of schizophrenia, hypersensitivity to novelty, to dopaminergic receptor agonists, and to the locomotor-activating effects of NMDA receptor antagonists as well as the stereotyped or perseverative induced behaviors have been evaluated. Spontaneous locomotor activity measured in MAM-exposed rats revealed discrepancies that could be related to differences in either the strain reactivity or the experimental procedure. In Sprague Dawley rats, MAM (25 mg/kg) induces a robust and persistent spontaneous hyperactivity from postnatal day 63 to 115 in various behavioral tests (open field, social interaction, Y maze) (34, 35). In addition, we showed that the increase in spontaneous locomotion was present only in the adult MAM-exposed rats (Fig. 2.1), suggesting that this behavioral feature reflects the interaction of early developmental disturbances with adolescent brain maturation (34, 35).

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Fig. 2.1. Spontaneous locomotor activity in prenatal E17 MAM- vs. saline-exposed rats. Total distance (±SEM) travelled by sham and MAM-exposed rats during a 60-min testing period in the open field was measured at pre- and post-puberty. ∗∗∗ p < 0.001 vs. sham. (Adapted from Le Pen et al., 2006)

In contrast to these results, data obtained in Wistar rats with 22 mg/kg E17 MAM exposure are more inconsistent. Indeed, in a large open field, Flagstad et al. (32) did not observe any modification of locomotor activity level in MAM-exposed rats in comparison to sham animals, whereas, in the same arena, MAMexposed rats exhibited increased spontaneous locomotor activity when tested in the social interaction test. These discrepancies could be due to a different strain of rats or to the experimental

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conditions (lighting during light (Sprague Dawley rats) vs. dark (Wistar rats) period of the light-dark cycle, MAM dose). 2.1.2. Dopaminergic Receptor Agonist-Induced Hyperactivity

Two different research groups have conducted experiments to investigate amphetamine-induced hyperactivity in animals preexposed to MAM (22 mg/kg) at E17 (32, 36) during the dark period of the light-dark cycle. These two studies performed in two different strains of rats (Wistar and Fisher 344) both revealed that MAM-exposed rats were hypersensitive to the locomotoractivating effects of D-amphetamine (Wistar: 2 mg/kg s.c. measured as salt 90 min and Fisher 344: 0.5 mg/kg i.p. 60 min). In addition, distinct experiments performed before and after puberty (Fig. 2.2) showed that hypersensitivity to amphetamine emerged only in adult animals (36). 600

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Fig. 2.2. Amphetamine-evoked locomotor activity in prenatal E17 MAM- vs. salineexposed rats. Total distance (±SEM) travelled by sham and MAM-exposed rats during a 60-min testing period in the open field after amphetamine (0.5 mg/kg, i.p.) was measured at pre- and post-puberty. ∗ p < 0.05 vs. sham. (Adapted from Moore et al., 2006)

2.1.3. Glutamatergic NMDA Antagonist-Induced Hyperactivity and Stereotyped Behaviors 2.1.3.1. PCP

Hypersensitivity to PCP-induced hyperlocomotion in an open field has been shown at adulthood, in two strains of rats (Wistar and Fisher 344) pre-exposed to MAM (22 mg/kg) (36, 37) after administration of an acute s.c. (120 min) (Fig. 2.3) or i.p. (60 min) dose of 5 mg/kg. In addition, it has been shown that the increase of PCP-induced ataxia in the open field was similar in sham and MAM-exposed rats (Fig. 2.4) (36). Orofacial dyskinesia and stereotypies (vacuous chewing and biting stereotypies) induced either spontaneously or by PCP (2.5 and 5 mg/kg i.p.) were also investigated in adult MAM-exposed rats (36). Oral behaviors were manually scored by direct observation

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Fig. 2.3. Phencyclidine-evoked locomotor activity in prenatal E17 MAM- vs. salineexposed rats. Total distance (±SEM) travelled by sham and MAM-exposed rats during a 120-min testing period in the open field after phencyclidine (5 mg/kg, s.c.) was measured at adulthood. (Adapted from Penschuck et al., 2006)

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Fig. 2.4. Phencyclidine-evoked ataxia in prenatal E17 MAM- vs. saline-exposed rats. Ataxia scores were measured in sham and MAM-exposed adult rats during a 60-min testing period after phencyclidine (5 mg/kg, i.p.) at adulthood. (Adapted from Moore et al., 2006)

(by an observer blind to gestational and drug conditions) and defined as follows: tremor, rapid oscillation of facial muscles progressing from lower jaw through cheek to extra-ocular region, stereotypic mouthing of the tail, and repetitive approaches to the tail with the mouth featuring horizontal and vertical motions of the jaw (follow-through biting rarely occurred). These behaviors were scored every 5 min during a 60-min period as absent [0], present/intermittent [1], or continuous [2]. MAM-exposed animals displayed a significantly higher frequency of spontaneous orofacial dyskinesias and stereotypies; moreover, these

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Fig. 2.5. Phencyclidine-evoked orofacial dyskinesias in prenatal E17 MAM- vs. salineexposed rats. Vacuous chewing (left panel) and biting stereotypy (right panel) were measured following injection of saline or phencyclidine (PCP 2.5, 5.0 mg/kg, i.p.) at adulthood. ∗ p < 0.05 vs. sham. (Adapted from Moore et al., 2006)

behaviors were significantly more sensitive to exacerbation by PCP (Fig. 2.5) (36). 2.1.3.2. MK-801

The sensitivity to (+)-MK-801 (0.05 and 0.1 mg/kg) has been investigated in E17 MAM-exposed (25 mg/kg) Sprague Dawley rats at both pre- and post-puberty in different groups of rats (PD35 and PD115, respectively) to avoid multi-drug exposure (Fig. 2.6). Before MK-801 testing, animals were subjected to a 1-h session of habituation in the locomotor activity apparatus (open field: 40 cm × 40 cm), followed by another 1-h period of locomotor activity after a saline injection. Whereas both sham and MAM-exposed rats exhibited a similar response to the locomotoractivating effects of MK-801 before puberty, MAM-exposed rats developed hypersensitivity to the locomotor-activating effect of MK-801 in adulthood (35).

2.2. Behavioral Anomalies Reminiscent of Negative Symptoms of Schizophrenia

Social withdrawal is a frequent negative symptom of schizophrenia (1, 3). It exists prior to the onset of disease and is predictive of future schizophrenia (57). In rodents, social behavior can be easily evaluated using a social interaction test. This test consists in allowing the experimental subject to freely explore an unfamiliar congener in its home cage or in a neutral environment. Social exploration is measured in time spent by the experimental subject around the congener.

2.2.1. Procedure

The general design of the social interaction test used to evaluate MAM-exposed animals was adapted from the work of SamsDodd (58). The test was performed in an open arena (80 cm × 80 cm, 50 cm high) that was placed in a dimly lit room. Two unfamiliar rats that had approximately the same weight and had received identical prenatal treatment were placed simultaneously

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Fig. 2.6. (+) MK-801-evoked locomotor activity in prenatal E17 MAM- vs. salineexposed rats. The distance (±SEM) travelled by sham and MAM-exposed rats was explored at both (a) pre- and (b) post-puberty in a spontaneous exploration session, after saline injection and after (+)-MK-801 (0.05 or 0.1 mg/kg s.c.) administration. (Adapted from Le pen et al., 2006)

in the opposite corners of the arena and the track of each rat was recorded for a 10-min period with a video tracking system. Social interactions were measured by the time spent and number of contacts at a distance lower than 20 cm from each other. During the

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10-min period of the test, locomotor activity and time spent in the central zone were also recorded. In Wistar and Sprague Dawley rats tested during the dark and light phase of the light-dark cycle, respectively, evaluation of social behavior produced similar results. Indeed, deficits in social interaction revealed by a decreased amount of time spent in contact with the congener were first shown in adult Wistar rats exposed to MAM at E17 (32). We extended these results (Fig. 2.7) by showing that Sprague Dawley MAM-exposed rats display these deficits at both pre-puberty and adulthood (34, 35). The deficits could be dissociated from motor performances, given that hyperactivity emerged only after puberty (34, 35) and were not the result of anxiogenic-like behaviors since the time spent in the central zone was similar in both groups (32, 34, 35). In addition, this abnormal behavior appeared to be the consequence of a lower duration of contacts and not a difference in the number of contacts (32, 34, 35). Sham

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Fig. 2.7. Social interactions in prenatal E17 MAM- vs. saline-exposed rats. The time spent (±SEM) in interaction (a) and the number of contacts (b) between two rats from the same treatment group were explored at pre- and post-puberty. ∗∗∗ p < 0.001 vs. sham. (Adapted from Le pen et al., 2006)

2.3. Abnormalities Reminiscent of the Cognitive Symptoms of Schizophrenia

Cognitive impairments constitute a central feature of schizophrenia (59) that include deficits in abstraction, executive function, verbal memory, language function, vigilance, and attention (60–62). Schizophrenic patients are also commonly described with abnormalities in information processing (63–65). For some authors, sensorimotor gating deficits correlate highly with measures of perceptual and reasoning disturbances. This relationship may then form an important basis for cognitive dysfunction observed among schizophrenic patients (66). Thus, an important future direction of preclinical research relating to schizophrenia is the design of animal models and novel treatments that target cognitive dysfunctions associated with this disorder. It is possible to design behavioral paradigms in rodents that can evaluate cognitive constructs “comparable” with those measured in many human experimental paradigms.

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2.3.1. Working Memory

Working memory may be defined as the capacity to hold information online over short delays while that information is integrated with other ongoing mental operations. A number of studies have shown that schizophrenic patients have deficient working memory (67–69), and imaging studies have confirmed that these deficits in working memory are associated with abnormal activation of the prefrontal cortex (70). Although working memory as measured in rodent might differ from that in human because it is explored for a longer delay, performance in working memory has been investigated in MAM-exposed rats using various paradigms.

2.3.1.1. Radial Arm Maze

In the rat, interactive communication between the hippocampus and the medial prefrontal cortex is necessary to perform a spatial working memory task. Thus, a delay-interposed radial maze learning task, previously shown to be mediated by a distributed neural network linking the hippocampus and the prefrontal cortex (71), has been used to investigate working memory in MAM-exposed rats (33). The radial arm maze task takes advantage of the natural tendency of food-deprived rodents to learn and remember different spatial locations for food in an eight-arm radial maze.

Procedure

The eight-arm radial maze used to test working memory in MAM-exposed rats consisted of a central arena (30 cm diameter) connected to eight equally spaced arms (80 cm × 13 cm) that extended radially. The maze was elevated 60 cm above the floor and surrounded by extra-maze three-dimensional cues in an illuminated room, with music on for background noise. The working memory task was adapted from Packard et al. (72) and Floresco et al. (73). After a 3-day period of habituation to the maze environment, adult food-deprived animals were trained. Each trial consisted of a training and a test phase, separated by a delay. Every day, a random set of four arms (the same for all animals in a given day) was blocked. Food pellets were placed in cups at the end of the remaining four open arms. Each rat was given 5 min to retrieve the pellets and then was replaced in its home cage for the delay period. Then, during the test phase, all arms were open, but food was placed only in the arms previously blocked. Rats were allowed a maximum of 5 min to retrieve the four pellets (Fig. 2.8). The initial delay between training and test phases was 5 min. The learning curves (5-min trials) were compared two by two between groups by means of the log rank test. After achieving criterion performance (i.e., all four pellets retrieved in four or five choices during the test phase for 2 consecutive days), the delay was increased to 30 min. On 30-min delay test days, we recorded the number and order of arm entries. Entries into non-baited arms during the test phase were considered as errors. We distinguished across-phase errors (entries into an arm entered

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Delay

Fig. 2.8. Diagram of the radial arm maze task with delay. Food pellets (plain circle) were placed in a well at the tip of each maze arm. (Adapted from Gourevitch et al., 2004)

previously during the training phase) from within-phase errors (re-entries into an arm entered earlier during the test phase). Results

E17 MAM-exposed rats were able to reach the level of performance of control rats at the initial stages of training as revealed by similar number of 5-min trials necessary to learn the rule in both sham and MAM-exposed animals. Nevertheless, with a 30-min delay, MAM-exposed rats were unable to process and retrieve spatial information when a 30-min delay was interposed, indicating a significant impairment in working memory. Indeed, MAM-exposed rats made more total errors than did sham animals (Fig. 2.9). These errors were mostly related to working memory (entries during the test phase to an arm entered previously during the training phase), while errors related to visual short-term memory (re-entries into an arm entered earlier during the test phase) were similar in MAM-exposed rats compared to sham animals.

2.3.1.2. Spontaneous Alternation

The Y-maze spontaneous alternation procedure used to assess spatial working memory in MAM-exposed rats (34) is based on their natural tendency to explore novel environment. When placed in the Y maze, normal rats prefer to explore the least recently visited arm and thus tend to alternate visits between the three arms. To explore the three arms successively, the rat must maintain an ongoing record of most recently visited arms and continuously update such records. A rodent with impaired working memory cannot remember which arm it just visited and thus shows decreased spontaneous alternation (74, 75).

Procedure

The Y maze used for MAM-exposed rats has three identical arms (40 cm × 9 cm × 15 cm with walls 30 cm high) placed at 120◦ from each other. Numerous visual cues (geometrical forms) were placed on the ceiling and kept constant during the experiments. Rats were placed at the end of one arm of the Y maze and the sequence of arm entries was recorded during 10 min. The introduction arm was randomly assigned for each rat. An arm visit was recorded when a rat moved all four paws into the arm. An alternation was defined as consecutive entries into all three arms (e.g., 1, 2, 3 or 1, 3, 2). The number of maximum alternations was the total number of arm entries minus 2 and the percentage of

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Fig. 2.9. Working memory performance in a delay-interposed, eight-arm radial maze task in prenatal E17 MAM- vs. saline-exposed rats. (a) E17 MAM-exposed animals made more total errors than did controls, and (b) these errors were mostly across-phase errors, whereas within-phase errors were not significantly different in treated and control rats (∗ p < 0.05; ∗∗ p < 0.01). (Adapted from Gourevitch et al., 2004)

alternations was calculated as the ratio of actual to maximum alternations multiplied by 100: (actual alternations/maximum alternations) × 100. Persevering behavior was defined as subjects making significantly fewer alternations than would be expected by chance (50%). Results

Deficits in alternation were present at both pre- and post-puberty in MAM-exposed rats compared to sham animals (Fig. 2.10). There was an improvement across puberty in both groups. Low performances before puberty in both sham and MAMexposed rats were linked to a low exploratory behavior that could reflect anxiety-like behaviors in both sham and MAM-exposed young rats (34). The deficit in alternation indicates a deficit in flexibility and short-term memory, further supporting that E17 MAM exposure impairs the hippocampo-prefrontal networks (76). Nevertheless, spontaneous alternation results also suggest that the working memory skills used in the spatial alternation test were acquired during puberty and that maturation of processes involved in that behavior is not affected by the MAM treatment, since the deficits in MAM-exposed rats are already seen before puberty (34).

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Fig. 2.10. Spontaneous alternation in prenatal E17 MAM-exposed vs. saline-exposed rats. Percentage of alternation (±SEM) at pre- and post-puberty. The dotted line represents the chance level (50%), # different from the chance level p < 0.001. ∗ p < 0.05 vs. sham group.

2.3.1.3. Delayed Non-match to Position

The delayed non-match-to-position task measures the ability of subjects to learn a rule in which they have to associate the position of a stimulus previously presented and an action for getting a reward. Flagstad and colleagues (31) have investigated working memory in MAM-exposed rats using this paradigm.

Procedure

In the procedure used by Flagstad and colleagues (31), in the first step, rats have to learn to press a lever to initiate the delivery of water (here rats were water deprived and water was used as the positive reinforcer). In the second step, the non-match-toposition (NMTP) training schedule, at the start of each trial, one of the two retractable levers is presented to the subject into the operant chamber. The subject has to press the lever for indicating that the sample has been registered. Then both levers are presented and the subject has to press the sample lever at the opposite of the one presented before when the rat received the reward. In the last step, the procedure was as in the NMTP experiment, except that a delay (0, 5, or 10 s, random) was inserted after the initial press on the right or left lever before the reward was given. The animals were initially trained for 25 sessions in this procedure including the delay and the subsequent three sessions were used as the test sessions.

Results

The investigation of working memory using the NMTP paradigm showed no consistent difference between the performances of the sham and MAM-exposed rats, either in the non-delay condition or in the delay condition (Fig. 2.11). The sham animals were superior only in four sessions out of 40 during the learning performance of the NMTP paradigm, and this did not appear to be a consistent effect (31).

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Fig. 2.11. Working memory performance in a delay non-match-to-position (NMTP) task in prenatal E17 MAM- vs. saline-exposed rats. (a) The mean percent correct responses (±SEM) of the groups during the 40 days of acquisition of the NMTP paradigm. ∗ p < 0.05 vs. sham. (b) The mean percent correct responses (±SEM) during three consecutive days of DNMTP. (Adapted from Flagstad et al., 2004)

In the delayed non-match-to-position procedure, a delaydependent decline was apparent, but no differences between groups were observed. The accuracy of the performance in the task is believed to be dependent on prefrontal cortical integrity, as lesions decrease performance in the task, although the published data suggest that the deficits of the prefrontal cortex-lesioned rats are apparent only at the beginning of training (77, 78). In Flagstad’s work (31), the animals were first trained on zero delay and then after an extensive training switched to the delay condition. The authors hypothesized that under the present protocol, the animals use a movement-mediated strategy, that is, the choice of the rat after the delay is mediated from the body position of the animal rather than by information kept online in working memory. Thus, this could possibly explain the lack of effect of the MAM treatment. During development, passively holding information available in working memory as well as recognition and recall from long-term memory is developed before active use

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and manipulations of information (executive functions), and as such not as dependent on later maturation of the prefrontal cortex (79). This might suggest that disturbances in prefrontal functions in MAM-exposed rats are related to a later maturation of this structure, and hence less radical than a dysfunction of all processes governed by the prefrontal cortex. 2.3.1.4. Morris Water Maze

Short-term place memory, or spatial working memory, has been shown to be impaired in schizophrenia (80, 81). In rodents, a typical experimental working memory paradigm is the Morris water maze (MWM) in which rats search for a submerged platform whose position is changed daily in a circular pool filled with water (82). The water maze is a particularly useful tool for assessment of spatial memory ability in rats. Indeed, the motivating stimulus, escape from water, does not require the food or water deprivation that is common in appetitive tasks like the radial arm maze or T maze. Flagstad and colleagues (31) have used the Morris water maze procedure to evaluate spatial working memory in MAMexposed rats.

Procedure

The water maze consisted of a circular, black pool filled with water. The test room contained several permanent extra-maze cues such as the rat housing rack, laboratory table, posters on the walls, etc. In the working memory task, the platform was placed in a different quadrant every day (north-south-west-east) for 4 days. Before each trial, the rat was placed on the hidden platform for 15 s (inter-trial period). The initial placement on the platform each day served to reveal the new location of the platform. If the rat did not locate the platform within 60 s, the rat was gently placed on the platform. Key measures were path length (m), escape latency (s) to find the hidden platform, percent of path length near (10 cm) the sidewall (percent sidewall), and number of animals not finding the platform within 60 s (non-finders). These measures were recorded using a video tracking system.

Results

Flagstad et al. (31) showed that MAM-exposed rats performed significantly worse than controls on days 2 and 3 of testing but only on the basis of the percent sidewall and non-finder parameters. On the last day, however, they performed as well as the controls (Fig. 2.12). They concluded that the MAM treatment does not interfere with spatial working memory performance. Nevertheless, this result has to be confirmed since sham animals strangely performed worse on the last day of the test than on the penultimate day. The MAM treatment seems, however, to affect the search strategy of the rats. When the platform is moved, the rats spend more time swimming along the sidewall, rather than searching in the central part of the maze where the platform is positioned.

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Fig. 2.12. Working memory performance in the reversal learning paradigm of the Morris water maze in prenatal E17 MAM- vs. saline-exposed rats. The mean (±SEM) of latency to reach the hidden platform (a), percent of path length near (10 cm) the sidewall (b), and number of animals not finding the platform within 60 s (c) were presented for four consecutive trials. ∗ p < 0.05 vs. sham. (Adapted from Flagstad et al., 2004)

2.3.2. Attention and Information Processing

Deficits in attention and information processing have been considered a central feature in schizophrenia, which might lead to stimulus overload, cognitive fragmentation, and thought disorder (65, 66, 83–88). Various physiological and neuropsychological techniques related to selective attention and pre-attentional mechanisms have been used to quantify information processing deficits in schizophrenia such as P50 (89, 90), prepulse inhibition (PPI) (83), latent inhibition (LI) (91, 92), continuous performance test (93, 94), span of apprehension (95, 96), visual backward masking (97, 98), and dichotic listening (99, 100). The advantage of PPI (101) and LI is that they can be used in both a human and an animal experimental setup. Whereas PPI assesses early attentional gating mechanisms, often referred to as pre-attentional deficits, LI assesses later stages of information processing related to attentional filtering. In addition, in rats, the fivechoice serial reaction time task (5-CSRTT) developed by Carli and colleagues (102) exhibits closed analogies with the continuous performance test and Leonard’s 5-CSRTT used in humans to assess the processes involved in sustained attention (103, 104). Finally, attentional set-shifting task is considered as a rodent analog of the Wisconsin card sort task. PPI, LI, 5-CSRTT as well as attentional set-shifting tasks have been used to clarify attention and information processing in MAM-exposed rats (30, 31, 34, 36).

2.3.2.1. Prepulse Inhibition (PPI) of Startle Reflex

A well-established method for evaluating sensory filtering is the paradigm of PPI which refers to the inhibition of a startle reflex by presentation of a weak intensity prepulse immediately before the startle stimulus. The impact of MAM exposure on PPI has been investigated using two different protocols (34, 36).

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Acoustic startle reactivity and prepulse inhibition of startle reflex are assessed in a single session using standard startle chambers (SR-Lab, San Diego Instruments). (A) In Sprague Dawley rats (34, 35), after a 5-min acclimatization period (68 dB background noise), 10 startle pulses (120 dB, 40 ms duration) were presented with an average inter-trial interval of 15 s. During the next 20 min, no stimulus (background noise, 68 dB), prepulses alone (72, 76, 80, or 84 dB, 20 ms duration), startle pulses alone, six times for each condition, were randomly distributed. (B) In Fischer 344 rats (36), after a 5-min acclimatization period (55 dB background noise), in a first experiment, the startle threshold of both sham and MAM-exposed rats was determined with a repeated ascending, then randomized, series of 40-ms noise bursts ranging from 70 to 105 dB and in a second experiment, for PPI evaluation, rats were exposed to 100 dB startle tone (white noise burst), and 62 and 70 dB prepulses (6 or 12 ms duration). Prepulses were presented on 75% of the startle trials. PPI was defined as the percent reduction in startle amplitude in the presence of the prepulse compared with the amplitude in the absence of the prepulse [100 × (startle amplitude on prepulse−pulse trials/startle amplitude on pulse-alone trials) × 100] (Fig. 2.13).

Fig. 2.13. Scheme of prepulse inhibition of startle reflex response.

Results

PPI experiments in both Sprague Dawley and Fisher 344 rats revealed that E17 MAM-exposed animals at adulthood exhibited sensorimotor gating deficits (34, 36). In addition, we showed that these deficits emerge only after puberty (Fig. 2.14) (35). Besides, Moore and colleagues (36) showed that the PPI deficits were neither due to a decrease in sensory processing, since MAM-exposed

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Fig. 2.14. Effect of prenatal (E17) MAM vs. saline exposure on prepulse inhibition (PPI) of startle reflex. PPI of the acoustic startle reflex (±SEM) was measured at pre- (a) and post-puberty (b). The insets show acoustic startle amplitude. ∗∗ p < 0.01 vs. sham group. (Adapted from Le Pen et al., 2006)

rats showed a lower threshold for responding to startle stimuli than did sham animals, nor due to a change in motor function, since the maximal startle response of MAM-exposed rats did not differ from that of sham. 2.3.2.2. Latent Inhibition (LI)

The LI paradigm is based on the phenomenon of reduced conditioning after stimulus pre-exposure. It is used to measure a form of selective attention, namely the ability to ignore irrelevant stimuli.

Procedure

The experiment performed in MAM-exposed rats by Flagstad and colleagues (32) was conducted using an automated shuttle box (42 cm × 16 cm × 20 cm) divided into two compartments by a partition with one opening. On the first day, the animals were left undisturbed in the shuttle box for 50 min with the houselights turned on. On the second day, the animals were divided into groups of pre-exposed (PE) and non-pre-exposed (NPE). The PE rats were subjected to 50 presentations (10–60 s randomized inter-trial interval) of the to-be-conditioned stimulus (cue light on for 10 s). The NPE rats were left undisturbed in the test box with only the houselights on for a corresponding duration. On the third day (test session), the animals were subjected to 100 trials of avoidance learning (10–60 s randomized inter-trial interval). Upon presentation of a light signal, the rat had 10 s to avoid a 0.5-mA scrambled foot shock by moving to the other compartment (avoidance). If the rat did not move to the other compartment within 10 s, a foot shock of a maximum duration of 10 s was delivered. The shock was terminated if the rat moved to

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the other compartment. The position of the animal and crossings from one compartment to the other were detected by two photocells placed on either side of the dividing wall. The outcome measure was the number of avoidances (i.e., rats moving to the other compartment of the shuttle box after the presentation of the light but before the shock was delivered). In these experiments (Fig. 2.15), sham animals exhibited a greater number of avoidances in the NPE conditions than in the PE conditions that revealed good latent inhibition processes in those animals. In contrast, MAM-exposed animals exhibited the same number of avoidances in the two conditions, indicating the absence of latent inhibition in those rats (32). The latent inhibition experiment, however, also revealed that under the present experimental setup, MAM-exposed rats had a slower acquisition of the conditioned avoidance.

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Fig. 2.15. Latent inhibition (LI) performances in prenatal E17 MAM- vs. saline-exposed rats. (a) Number of avoidance responses (±SEM) during a 100-trial learning session in a two-way avoidance shuttle box in pre-exposed (PE) and non-pre-exposed (NPE) conditions. ∗ p < 0.05, ∗∗∗ p < 0.001 vs. sham group in NPE conditions. The number of avoidances made during the conditioning in 10 trial bins for sham (b) and MAM-exposed (c) animals. (Adapted from Flagstad et al., 2004)

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2.3.2.3. Attentional Set Shifting

In the set-shifting task, rats are required to solve a series of discriminations by attending a particular perceptual dimension of a multidimensional stimulus. A critical discrimination occurs when rats are required to shift to an alternate perceptual dimension after having acquired an attentional set to the previous dimension. The extradimensional shift (EDS) required to solve this discrimination is analogous to what is required to solve the Wisconsin Card Sorting Task, a test that has repeatedly shown impaired prefrontal cortex-mediated executive function in schizophrenia (105). The rodent set-shifting task also requires the prefrontal cortex (106, 107) and, thus, has been used for assessing attentional processes in MAM-exposed rats (30).

Procedure

For this test, Featherstone and colleagues (30) used a black twocompartment chamber (60 cm × 42 cm × 30 cm). Animals had access to either compartment where a small ceramic bowl (7 cm in diameter and 4 cm in depth) was placed. Each bowl was covered with a texture and filled with bedding material that was scented with an odor. Animals were initially trained to reliably dig and retrieve food from two bowls containing a small amount of bedding and a food reward (Fruit loop cereal). Animals were then trained on a simple discrimination (SD), in which food was paired with one stimulus dimension (either odor or texture) but not the other. Training continued until the animals chose correctly over six consecutive trials. The next phase of training involved the full set of discriminations with only one of the two bowls baited. The location of the correct bowl was randomly determined on every trial. The bowls differed according to odor and texture, with food being consistently associated with a particular odor or texture depending on the current discrimination. Discrimination training began with an SD. For this discrimination, only one stimulus dimension was present. In the second discrimination (CD), another stimulus dimension was added, although food remained paired with the original stimulus. The third discrimination involved a reversal of the second discrimination, in which an alternate exemplar of the relevant stimulus dimension was now paired with food. The fourth discrimination required animals to choose between a new series of stimuli, with the previously relevant stimulus dimension still paired with food (intradimensional shift (IDS)). This discrimination was followed by a reversal in which the alternate exemplar of the relevant stimulus dimension was now paired with food. In the sixth discrimination (EDS) the relevant stimulus dimension was changed such that the animal had to learn to attend to the previously irrelevant dimension. The final discrimination involved a reversal of the EDS discrimination in which the animal had to choose the previously irrelevant exemplar of the new stimulus dimension. In a first part, saline- and MAM-exposed animals received initial training (SD, CD, Rev 1,

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and IDS) with odor as the relevant dimension and were required to make an EDS to texture. In a second part, saline- and MAMexposed animals received initial training with texture as the relevant dimension, followed by a shift to odor during the EDS. A correct trial was considered to have occurred when the animal successfully retrieved food from the bowl. If the animal dug in the incorrect bowl, the trial was scored as an error. Animals were moved onto the next discrimination once they had made six correct responses in a row. The individual who carried out the set-shifting task was unaware of the group identity of any given animal. On the attentional set-shifting task (Fig. 2.16), compared to controls, MAM-exposed animals required a greater number of trials to learn the EDS component of the task, indicative of a deficit in shifting attentional set (30). MAM-exposed rats were able to solve an SD and perform an IDS, suggesting that the deficit in EDS ability was not due to a generalized performance or cognitive impairment (30). Since deficits in performing an EDS are found in animals with a compromised prefrontal cortex, as has been shown on the attentional set-shifting task (106, 107), results obtained in MAM-exposed rats are in good accordance with prefrontal morphology and function abnormalities shown in those animals (30, 33, 35, 36, 39). Nevertheless, it is unclear whether difficulties in shifting attentional set are due to a failure to ignore a previously reinforced

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dimension or to attend to a previously irrelevant dimension. Lesions of the prefrontal cortex have been shown to selectively increase perseveration to a previously learned dimension in non-human primates (108) and humans (109, 110). Since MAM injection at E17 disrupts prefrontal cortex structure and function, Featherstone and colleagues (30) hypothesized that MAMexposed animals would likewise show increases in perseveration and that this might serve as the basis for the deficit observed in the present study. In addition to impairment in EDS learning, MAM-exposed animals were impaired when required to make a simple reversal of a previously acquired discrimination (for two out of three reversals). These results are in good agreement with previous findings. Indeed, difficulties in reversal learning have been reported in E17 MAM-exposed animals within a water maze paradigm (31), as well as in a Y-maze task (36). 2.3.2.4. Five-Choice Serial Reaction Time (5-CSRT) Task

Visual attentional processes in E17 MAM-exposed rats have been evaluated by Featherstone and colleagues (30) using five-choice serial reaction time task (5-CSRTT). The 5-CSRTT provides the possibility to test the effects of E17 MAM exposure on discrete and somewhat independent measures of behavioral control, including accuracy of discrimination, impulsivity, perseverative responses, and response latencies. Several aspects of the animal performance are assessed (accuracy = % correct), anticipatory responses (premature nose pokes which are thought to be analogous to impulsivity in humans) and perseverative responses (additional nose pokes following a correct response before a new trial is initiated, which are thought to be analogous to compulsive behaviors in humans).

Procedure

In their version of the task, the rats are required to attend and respond to visual stimuli displayed at different positions in an array of five niches placed along the wall of an operant chamber to receive a reward (here, 0.06 ml of a 10% sucrose/water solution) distributed in a magazine/liquid dipper located on the opposite wall. The magazine and light apertures contained photodetectors to detect nose poke entries. First, rats were initially trained to respond to signaled presentations of the dipper. Then, they were trained for 40 daily sessions during which they gradually learn to respond in the appropriate aperture within a certain amount of time. If they failed to respond, or respond in the wrong hole or at an inappropriate time, a short period of darkness (time-out) is presented as a punishment and no reward is delivered (Fig. 2.17). Task acquisition: For all subjects, the stimulus duration (SD) was progressively reduced from 30 s until a criterion duration of 1 s was achieved (all other parameters unchanged: ITI 5 s, limited hold 5 s, 100 trials per sessions). At 30 and 20 s, animals were required to perform at least 30 (out of 100) correct responses.

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Fig. 2.17. Schematic illustration of the five-hole box operant chamber. (a) General plan of the 5-choice serial reaction time test apparatus. (b) Flow diagram to illustrate the general task requirements.

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At 10 s, the level was increased to 50 correct trials. For 5, 2.5, 1.25, and 1 s, animals were required to make 50 or more correct responses, have greater than 80% accuracy, and less than 20% omitted trials. Animals were trained for a total of 41 sessions in order to reach criterion at the lowest stimulus duration of 1 s. Behavioral challenges: Once stable performance had been achieved at the 1-s SD, a series of manipulations of the stimulus parameters were employed. First, animals were challenged with different SDs (0.125, 0.25, 0.5, and 1 s). This was performed using a withinsubject design, with each test day separated by a maintenance session using 1 s as SD. The purpose of this manipulation was to increase demand on attentional resources. Second, animals were given an additional session using a prolonged ITI (9 s instead of 5 s). This manipulation typically increases premature responding, since animals must delay responding across a longer duration. Finally, animals were given a session of training with variable ITIs (3.5, 5.5, 7.5, and 9.5 s) occurring within the same session. Results

MAM-exposed rats were able to acquire the task similarly to sham animals since they needed the same amount of time as sham to reach the criterion at each training stage. In addition, at the end of the training, no obvious group differences were observed when comparing sham and MAM-exposed rats in any of the following performances: percent accuracy, percentage of omitted trials, number of perseverative responses, latency (in seconds) to perform the correct response, latency (in seconds) to collect reinforcement, and number of premature responses (30). MAM treatment did not disrupt accurate performance during any of the post-acquisition manipulations (shortened stimulus durations, variable ITI, and lengthened ITI), suggesting that performance in this task is not compromised by MAM treatment. Additionally, the lack of change in omitted trials and response latency suggests that attentional function was spared and, incidentally, indicates that motor and/or motivational functions were unaffected by MAM treatment. Given the effects of MAM treatment on attentional set shifting and reversal learning, as well as on prefrontal morphology, it is surprising that MAM treatment had little effect on the five-choice performance. Indeed, previous work has shown that the prefrontal cortex plays a major role in several aspects of performance in the five-choice task. Hence, prefrontal lesions alter accuracy (111, 112) and can induce changes in perseverative and premature responding (111, 113). MAM-exposed animals did show a trend toward an increase in premature responding during acquisition, suggesting a difficulty in behavioral inhibition (30). However, this change was nowhere near as significant as changes seen in premature responding following other prefrontal manipulations (113). It has to be noticed that rats evaluated in 5-CSRT task by Featherstone and colleagues (30) had

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been previously evaluated in the attentional set-shifting paradigm. Thus, it cannot be excluded that a facilitation of five-choice performance in MAM-exposed rats could result from their attentional set-shifting experience. Therefore the absence of impact of E17 MAM exposure on sustained visual attention should be confirmed in an independent set of experiment. 2.3.3. Behavioral Flexibility

Impairment in different forms of behavioral flexibility such as set shifting and reversal learning has been frequently observed in schizophrenic patients. Indeed, they display great difficulty in shifting between different rules or strategies on tests such as the Wisconsin Card Sorting task (114–117). These impairments appear to be due in part to an inability to shift attentional set from one stimulus dimension to another, as similar impairments have been observed in patients tested on an intradimensional/extradimensional shifting (IDS/EDS) task (118, 119). Furthermore, a subset of these patients also display impairments in reversal learning, a simpler form of behavioral flexibility entailing shifts between different stimulus-reward associations within a particular dimension (120, 121). In both rats and humans, multiple prefrontal cortex regions and subcortical regions connected to the prefrontal cortex contribute to different component processes of behavioral flexibility. The orbital and prelimbic/infralimbic regions of the prefrontal cortex mediate some forms of reversal learning in the rat and human (109, 122–126). Then, it is likely that abnormalities in multiple prefrontal regions contribute to the cognitive inflexibility in the E17 MAM model. Thus, behavioral flexibility have been investigated in MAM-exposed rats through a reversal learning procedure that was tested in a Y maze (36) and a water maze procedure that we previously described (31).

2.3.3.1. Reversal Learning in a Y Maze

The test was performed in a Y maze that consisted of a central chamber with guillotine doors opening into three 36-in. runways, each terminating in a food magazine. Adult control rats and MAM-exposed rats were habituated to the Y maze and then trained in E17 the conditional discrimination task. At the beginning of the task, the rat was placed in an arm and the trial initiated by opening the door to the central chamber. Three seconds later, the doors to the choice arms opened and each food magazine was illuminated. A head poke into the CS+ magazine resulted in the termination of the light and delivery of food pellets. A poke into the CS− magazine resulted in the termination of the light and a 15-s time-out. The next trial was initiated from the choice arm of the previous trial; sessions were 30 trials long. After the rat achieved 67% accuracy for 3 consecutive days, the reward contingency was reversed (i.e., the CS+ became the CS− and vice versa). The number of trials to criterion for acquisition and

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reversal phases was compared between E17 MAM-exposed rats and control rats. Results

First, relative to sham animals, MAM-exposed rats did not show a deficit in learning the novel discrimination. Indeed, these rats learned the discrimination significantly faster than did control rats, indicating that forebrain circuits involved in basic discriminated approach learning were not affected. However, under reversal conditions, MAM-exposed rats required significantly more trials to reach criterion than did sham animals (Fig. 2.18). Thus, MAM-exposed rats exhibited a significant deficit in reversal learning. Regarding the selective deficit in reversal learning, they observed in E17 MAM-exposed rats, Moore and colleagues (36) suggested that (1) cortical circuits involved in sensory and motor processes and association learning are intact; (2) the faster learning may be indicative of a perseverative response, in which the rats fail to test the non-rewarded arm once the association in the rewarded arm has been experienced; and (3) the perseveration is further revealed when the contingencies are reversed. Their learning phenotype is also consistent with the E17 MAM-exposed rats being more sensitive to the negative consequences associated with the original CS−. Each of these abnormalities implicates frontal cortical function.

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Fig. 2.18. Reversal learning performances in a Y maze in prenatal E17 MAM- vs. salineexposed rats. Acquisition of the initial discrimination was significantly faster in MAMexposed rats. +p < 0.05 vs. sham group in acquisition conditions. However, MAMexposed rats required significantly more trials to learn the reversal of the discrimination. ∗ p < 0.05 vs. sham group in reversal conditions. (Adapted from Moore et al., 2006)

2.3.3.2. Reversal Learning in the Morris Water Maze

As previously mentioned, one asset of the water maze tests is that many aspects of performance can be examined, providing several measures of spatial memory. Another asset is its capacity to examine reversal learning as a component of behavioral flexibility processes where rat has to switch from an established strategy to develop a new one.

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Procedure

As previously described (see Section 2.3.1), the water maze consisted of a circular, black pool filled with water. The test room contained several permanent extra-maze cues such as the rat housing rack, laboratory table, and posters on the walls. In the experiment performed by Flagstad and colleagues (32), behavioral flexibility can be evaluated between the first and second days of the working memory version of the task. Indeed, the working memory task takes place after the reference memory version of the task (see below) that involves the acquisition of rules about spatial location of the platform that are constant (north quadrant of the pool) across all trials of the task. On the first day of the working memory task, the platform was located in the same position as for the reference memory task but on the second day the platform was positioned in the opposite quadrant. The rationale underlying this task is that animals displaying behavioral flexibility will rapidly learn to search the platform in its new location, while rats impaired in behavioral flexibility will spend more time around the old location. Key measures were path length (m), escape latency (s) to find the hidden platform, percent of path length near (10 cm) the sidewall (percent sidewall), and number of animals not finding the platform within 60 s (nonfinders). These measures were recorded using a video tracking system.

Results

On the first day of the working memory task, sham and MAM-exposed animals performed similarly to locate the hidden platform. On the second day, when the platform moved to the opposite quadrant, Flagstad and colleagues (32) showed that MAM-exposed rats performed significantly worse than the controls but only on the basis of the percent sidewall and non-finder parameters (Fig. 2.12). These results suggest that MAM-exposed rats exhibit abnormal behavioral flexibility in a reversal learning version of the Morris water maze task.

2.3.4. Episodic Memory

Human episodic memory refers to the recollection of a unique past experience in terms of its details, its locale, and temporal occurrence (127). Episodic memory deficits among individuals with schizophrenia are well established (128–131). Interestingly, studies suggest that non-human mammals have the ability to build higher-order memory for unique events that incorporate information about what, where, and when, lending strong support to the idea that animals are endowed with episodic-like memory (132– 135). Thus, object and spatial recognition tasks as well as spatial reference memory paradigm have been used to evaluate episodic memory in MAM-exposed rats.

2.3.4.1. Object Recognition Task

Previous studies in schizophrenic patients have demonstrated episodic memory deficits reflected by impaired recognition of

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visually presented objects (128, 136, 137). In parallel, neuroimaging studies have provided evidence that schizophrenia is associated with abnormal brain activation within the hippocampus, the thalamus, and/or the prefrontal cortex during the recognition of previously seen or new objects (13, 138, 139). Ennaceur and Delacour (135) presented evidence that rodents are able to form an integrated memory for “what,” “where,” and “when” aspects of single experiences by combining different versions of the novelty-preference paradigm, i.e., object recognition memory, the memory for locations in which objects were explored, and the temporal order memory for objects presented at distinct time points. The novel object recognition task (NORT) was used to assess if MAM-exposed animals are able to form an integrated episodic memory for “what.” This test is based on the natural tendency of rodents to explore a novel object by comparison to a familiar one (135). Naive animals will spend more time exploring a novel object rather than a familiar one. Procedure

The test was performed in an open field (45 cm × 65 cm × 29 cm). On the day before the test, rats were habituated to the test box for 10 min. On the test day, rats were put in the test box, and after 3-min habituation, two similar objects were introduced in the two corners. The time spent investigating the two objects was scored in the next 3-min period (denoted pretest). The rats were then removed and put back in the housing cage. After a delay period of 20 min, the rats were reintroduced to the test box and 3 min later the objects [one similar to that used in the pretest (known) and one the rat had never encountered before (new)] were introduced. The objects were placed in the same position as in the pretest. In the next 3 min, the time spent investigating each of the objects was scored. All scoring was performed using a video tracking system.

Results

The MAM-exposed rats spent significantly less time exploring the objects on the pretest. There was also a main effect of the group in the test session, indicating that the MAM-exposed rats spent less time exploring the objects (Fig. 2.19). There was no main effect of the known/new object factor; however, there was an interaction between the group and the known/new factor. This interaction was caused by a significant difference between the known/new objects in controls, but not in the MAM-exposed rats. Furthermore, there was no difference between the amount of time the animals in each group spent exploring the known object, whereas the controls spent a longer time exploring the new object than the MAM-exposed did. Overall, these results revealed that MAM-exposed rats exhibited object recognition deficits that could be reminiscent of those observed in schizophrenic patients.

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Fig. 2.19. Object recognition in prenatal E17 MAM- vs. saline-exposed rats. (a) Mean investigation time (±SEM) during the pretest of the object recognition paradigm. ∗ p < 0.05 vs. sham. (b) Mean investigation time (±SEM) of the known and the new object during the object recognition test. ∗∗ p < 0.01 known vs. knew. (Adapted from Flagstad et al., 2004)

2.3.4.2. Spatial Recognition Memory in a Y Maze

Memory for spatial context has been shown to be impaired in schizophrenic patients (140–145). Recently, Da Silva and colleagues (132) proposed that spatial recognition memory in the rodent can be likened to human episodic memory. In rodents, various brain areas, including the cerebral cortex and the hippocampus, are known to be pivotal in spatial recognition memory (146–148). A simple two-trial recognition test in a Y maze was performed to investigate spatial recognition memory in MAMexposed rats. This test is based on the innate tendency of rodents to explore novel environments (149–151). The paradigm avoids the effects of punishment (such as electric shock) or reward (such as food) that is commonly used in other paradigms and that may have non-specific effects on the results. In addition, it does not require learning of a rule and is independent of locomotor activity; thus, it is useful for studying memory in rodents (150–153).

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Procedure

This two-trial memory task is based on exploration of novelty (151). Experiments were carried out in a Y maze which consisted of three identical alleys (40 cm long and 15 cm large with walls 30 cm high), diverging at a 120◦ angle from the central point. The experiments were performed in a dimly illuminated room. Numerous visual cues were placed on the walls and were kept constant during the experiments. The test consisted of two trials, separated by a 2-h time interval. During the first trial (acquisition phase), one arm of the Y maze was closed, allowing the rats to explore the remaining two arms for 3 min. The position of the closed arm and the introduction arm was randomized. During the second trial (retrieval phase), the rats had access to the three arms for a 3-min period. During this period, for each rat, the time spent in each arm and the total locomotor activity was measured by a video tracking system. The ability of the animals to remember the two arms already visited during the first phase and to explore preferentially the novel arm opened during the second phase. Then, after a 2-h time interval, the control rats are expected to spend more time in the “novel” arm, which remained closed during the first trial, compared with the two “familiar” ones that were already open during the first phase (151).

Results

Before puberty, both MAM-exposed and sham rats showed poor spatial performances revealed by the absence of clear preference for the novel arm (Fig. 2.20). This is consistent with the literature which suggests that rats younger than 40 days exhibit spatial navigation deficits in relation to the immature hippocampus (154, 155). At adulthood, the same MAM-exposed animals spent approximately the same amount of time in the three arms of the maze, in contrast to sham animals that exhibited a clear preference for the novel arm. The data reflect the physiological brain maturation occurring during puberty in control animals. In contrast, in either pre- or post-puberty, the MAM-exposed rats

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Fig. 2.20. Spatial recognition memory (Y-maze paradigm) in prenatal E17 MAM- vs. saline-exposed rats. Time spent (±SEM) in the novel arm was compared with the time spent in the introduction and third arms at pre-puberty (a) and post-puberty (b). # p < 0.05 vs. novel arm. (Adapted from Le Pen et al., 2006)

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did not exhibit a preference for the novel arm and remained approximately the same amount of time in the three arms of the maze, suggesting that the normal spatial recognition memory skills were not acquired during puberty in the MAM-exposed animals. In addition, MAM-exposed rats spent less time than sham animals in the central zone of the Y maze at both pre-puberty and adulthood, suggesting that memory deficits are unlikely a consequence of anxiety-like behavior. Conversely, when tested in the social interaction test, MAM-exposed rats were not different from controls in the time spent in the central area. So the results in the Y maze could reflect impulsive-like behavior in the MAM-exposed rats. In conclusion, spatial recognition memory, as explored by the Y maze, is thus relevant to follow the late maturation of the hippocampo-prefrontal networks occurring during the rat’s puberty and the related acquisitions of cognitive skills. Y maze results also present interesting similarities with observations of visuo-spatial memory deficits seen in schizophrenic patients (140–145). 2.3.5. Spatial Reference Memory

Reference memory is regarded as a long-term memory for information that remains constant over repeated trials (156). The retrieval of information, based on spatiotemporal context, from long-term memory is affected in schizophrenia. Morris water maze paradigm is frequently used in rodents to assess reference memory in a spatial context (82, 157). In this paradigm, animals have to navigate to a goal (hidden platform) in an allocentric spatial environment and must build a representation of the goal in relation to distal cues (constellations of cues) to form a cognitive map. Then, they must be able to use this information flexibly regardless of where in the environment the subject begins navigating. In animals (158) and humans (159), the hippocampus is involved in this kind of spatial learning, because encoding is based on a flexible knowledge of relationships between environmental cues. Hippocampus-dependent spatial memory in animals is thus phylogenetically viewed as a homologue of human episodic memory (160, 161). Thus, in order to evaluate spatial reference memory in MAM-exposed rats, the reference memory version of the Morris water maze has been used by Hazane and colleagues (34) and Flagstad and colleagues (31).

Procedure

The water maze consisted of a circular, black pool filled with water. The test room contained several permanent extra-maze cues such as the rat housing rack, laboratory table, and posters on the walls. In the reference memory task, the rats were trained to locate a hidden platform positioned 1 cm below the surface of the water. The platform remained in a fixed position throughout the test, although the animal’s starting position varied. Key measures were path length (m), escape latency (s) to find the

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hidden platform, percent of path length near (10 cm) the sidewall (percent sidewall), and number of animals not finding the platform within 60 s (non-finders). These measures were recorded using a video tracking system. Reference memory capacities measured in MAM-exposed rats revealed various results that could result from differences in either the strain reactivity or the experimental procedure. Thus, in the reference memory paradigm of the water maze task, Flagstad and colleagues (31) showed that MAM-exposed rats performed significantly worse than the controls on days 2 and 3 of testing but only on the basis of the percent sidewall and non-finder parameters. On the last day, however, they performed as well as the controls. Thus, it does not appear that the MAM treatment interferes with spatial working memory, as this should also result in a deficit on the last day of testing. The MAM treatment seems, however, to affect the search strategy of the rats. When the platform is moved, rats spend more time swimming along the sidewall, rather than searching in the central part of the maze where the platform is positioned. On the other hand, in Sprague Dawley rats, Hazane and colleagues (34) observed that MAM-exposed rats were significantly impaired in learning to locate the hidden platform compared to control animals (latency to reach the platform) (Fig. 2.21). Analysis of performance in each group revealed that, in contrast to sham rats, MAM-exposed rats did not improve their latency to locate the platform across trials. This contrasts with previous reports showing no effect of prenatal MAM exposure in Wistar rats using a similar spatial reference memory version of the Morris water maze task and a MAM exposure at E17 Sham MAM

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(22 mg/kg) (31). This impaired reference memory is of particular interest since patients with schizophrenia were similarly impaired on the hippocampal-dependent hidden-platform version of a virtual Morris water maze task. Indeed, patients were slower and had longer search paths than did controls to find the hidden platform (162, 163).

3. Neurochemical Changes Observed in E17 MAM-Exposed Rats

3.1. Neurochemical Abnormalities Associated with Hypersensitivity to D-AmphetamineInduced Hyperlocomotion in MAM-EXPOSED Rats

If the behavioral phenotype of E17 MAM-exposed rats is now pretty well characterized, fewer studies have characterized the neurochemical alterations associated with this neurodevelopmental animal model for schizophrenia and its behavioral abnormalities. Structural and/or functional alterations, presumed to reflect abnormal brain development, have consistently been found in schizophrenic patients in several interconnected brain regions such as the prefrontal cortex (164–166), the hippocampus (139, 167–169) and the striatum (170–172). In particular, dysfunctions of glutamatergic and/or dopaminergic neurotransmission have been demonstrated within these structures (164, 169–172) and are thought to play a central role in the pathophysiology of schizophrenia (173, 174). In addition, abnormalities in markers for GABAergic neurons in prefrontal cortex and hippocampus are also well documented in schizophrenia (175–181) with more recent studies suggesting a central role of impairment in the glutamate-GABA interactions, both in the hippocampus and in the prefrontal cortex. In line with these findings, neurochemical abnormalities associated with hypersensitivity to D-amphetamineand MK-801-induced hyperlocomotion in MAM-exposed rats have been investigated using in vivo microdialysis techniques. In addition, possible abnormalities in GABAergic markers have also been explored in MAM-exposed rats using immunohistochemistry. The prefrontal cortex and nucleus accumbens shell regions play a crucial role in mediating the behavioral effects of increased dopaminergic activity in schizophrenia (51, 182–185). In rodents, the nucleus accumbens and prefrontal cortex are strongly implicated in psychostimulant action. Dopamine release in the nucleus accumbens is believed to be the main mediator of the reinforcing and locomotor-activating properties of psychostimulants (186). The prefrontal cortex modulates the nucleus accumbens activity through direct and indirect connections and there is evidence that dopamine release in the prefrontal cortex may be related to the inhibition of reward and locomotor activity (187). Thus, in order to further assess possible corticolimbic dysfunctions associated with D-amphetamine-

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induced hyperlocomotion in MAM-exposed rats, Flagstad and colleagues (32) investigated changes in dopamine release in the nucleus accumbens and the medial prefrontal cortex after stimulation with D-amphetamine in sham and MAM-exposed Wistar rats. 3.1.1. Procedure

At adulthood, intracerebral guide cannulae were stereotaxically implanted in anesthetized Wistar rats. The guide cannula for the nucleus accumbens was implanted in the right hemisphere at AP +1.7 mm, L 0.8 mm, and V –6 mm from the brain surface (188). The guide cannula for the medial prefrontal cortex was implanted in the right hemisphere at an angle of 20 degrees, at AP +3.8 mm, L 0.7 mm, V −0.35 mm. After a 2-day period of recovery, rats were then placed in a hemispheric bowl and the microdialysis probes were inserted in the guide cannulae (0.5 mm diameter, 2 mm probe length for nucleus accumbens, and 4 mm probe length for the medial prefrontal cortex). The probe was connected to a microinfusion pump via a dual-channel liquid swivel, which allows the animals to move freely in the cage during the experiment. The rats had free access to food and water in the test bowls. The dialysis probe was perfused with Ringer solution at a constant flow rate of 1 ml/min. The perfusate was discarded during the initial 100 min and then collected in 20-min intervals. The first four fractions represented the baseline level. Rats were then given D-amphetamine (2 mg/kg, s.c.) and fractions were collected for 160 min. The samples were stored at −80◦ C until analysis. Dopamine was analyzed by HPLC with electrochemical detection. The levels of dopamine in the dialysates were not corrected for probe recovery. Dopamine was separated by reversephase liquid chromatography. Electrochemical detection used a colorimetric detector.

3.1.2. Results

Basal levels of dopamine in the nucleus accumbens and the prefrontal cortex of MAM-exposed rats and sham animals were similar. Flagstad and colleagues (32) demonstrated that acute systemic injection of D-amphetamine induced an exaggerated increase of dopamine release in the nucleus accumbens of MAMexposed rats in line with the striatal dopaminergic hyper-reactivity observed in schizophrenic patients (170–172). In contrast, Damphetamine-induced increase in dopamine concentration in the dialysate from the prefrontal cortex was similar in both sham and MAM-exposed rats.

3.2. Neurochemical Abnormalities Associated with Hypersensitivity to MK-801-Induced Hyperlocomotion in MAM-EXPOSED Rats

In rodents, NMDA antagonists appear to exert their disruptive effects on behavior through the activation of glutamatergic (at non-NMDA receptors), dopaminergic, and noradrenergic transmission (189–195). Thus, acute systemic injection of MK-801, another non-competitive NMDA antagonist, to MAM-exposed rats could exacerbate the neurochemical dysfunctions induced

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during brain development by prenatal exposure to MAM. To test this hypothesis, Lena and colleagues (40), using in vivo microdialysis, examined the effects induced by MK-801 (0.1 mg/kg s.c.) on the extracellular levels of glutamate, dopamine, and noradrenaline in the medial prefrontal cortex and the nucleus accumbens of adult Sprague Dawley rats prenatally exposed to MAM at E17. 3.2.1. Procedure

At adulthood, rats were anaesthetized and mounted on a stereotaxic frame. Two guide cannulae were implanted bilaterally, one in the left medial prefrontal cortex (AP from bregma, +3.0 mm; L, 1.6 mm angled 10◦ toward midline; V, −2.8 mm) and the other in the right nucleus accumbens (AP, +1.4 mm; L, 0.8 mm; V, −6.2 mm) according to the atlas of Paxinos and Watson (188). The two guide cannulae were secured to the skull using dental cement and two stainless steel screws. After 5 days of recovery, rats were placed in a rectangular Plexiglas cage (35 mm × 35 mm × 38 mm), and two concentric microdialysis probes (CMA/12, 500 μm diameter, 20 kDa cut-off) with a membrane length of 3 mm for medial prefrontal cortex or 2 mm for nucleus accumbens were inserted into the guide cannulae. Artificial cerebrospinal fluid was perfused through the probes at a constant rate of 1 μl/min. The probes were connected to the microperfusion syringes via FEP tubing using a dual-channel liquid swivel, allowing free movement of the animal in the experimental cage. After a 4-h stabilization period, dialysates were collected every 10 min over a period of 180 min and immediately stored at −80◦ C before analysis by capillary electrophoresis. Three samples were collected before injection of MK-801 (0.1 mg/kg) or saline to rats prenatally exposed to MAM or saline and were used to determine the basal levels of neurotransmitters. The time delay due to the dead volume of the microdialysis system (probe and output tubing) was taken into account to synchronize the measurement of locomotor activity with sample collection. Catecholamines and glutamate analysis were analyzed by capillary electrophoresis. The concentrations of catecholamines (dopamine and noradrenaline) and glutamate in the dialysate samples were determined using an automatic P/ACETM MDQ system equipped with an external laser-induced fluorescence and ZETALIF detector. Separations were performed using a fused-silica capillary.

3.2.2. Results

The study demonstrates that the neurodevelopmental insults induced by prenatal MAM exposure lead, in adult rats, to dysfunctions of the neurotransmission in the medial prefrontal cortex but not in the nucleus accumbens, in response to a noncompetitive NMDA antagonist. The MK-801-induced increase in cortical glutamate levels was reduced, whereas the cortical

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noradrenaline release was potentiated. These neurochemical alterations were associated with an exaggerated locomotor hyperactivity. Given the implication of the medial prefrontal cortex and the noradrenergic system in the locomotor effects of non-competitive NMDA antagonists, it is conceivable that the potentiation of cortical noradrenaline release might underlie the enhanced locomotor response in MAM-exposed rats. Future studies examining the interactions between the noradrenergic and the glutamatergic systems in the medial prefrontal cortex will be of particular interest in elucidating the mechanisms underlying this abnormal behavioral response. 3.3. Reelin Level in Organotypic Hippocampal Cultures of MAM-EXPOSED Rats

In addition, “postnatal” synthesis of reelin in the hippocampus was investigated in vitro with cultures of hippocampal slices, which allow conservation of cytoarchitectural properties. Hippocampal slices done at 1 week postnatal (P8) and maintained in culture for 3 weeks reach the development of “post-pubertal” hippocampus as it would have in vivo. This method is thus interesting for studying postnatal modifications at the cellular level. Briefly at the age of postnatal day 8 (P8), rats were anesthetized by hypothermia and their brain was aseptically removed. Hippocampal organotypic slice cultures were prepared using the Stoppini method. The hippocampus was dissected out under microscopic control. Tissue pieces were cut perpendicular to the septotemporal axis of the hippocampus with a McIllwain tissue chopper. Hippocampal slices were then transferred into the culture medium, separated, and put onto Millicell-CM membranes. A total of 12 organotypic cultures of adjacent hippocampal slices were obtained per brain. The Millicell membranes were kept in six-well plates above 900 μl of defined medium (neurobasal medium with 20% B27 serum-free supplement and 0.5 mM L-glutamine). Slices were incubated at 36◦ C in 5% CO2 . Cultures were stopped after 1 and 3 weeks and fixed in 4% paraformaldehyde for 1 h. For reelin labeling, the tissue was stained using the diaminobenzidine immunoperoxidase method with anti-reelin G10 monoclonal antibody (Chemicon). Neuronal density was evaluated using the neuronal marker NeuN (neuronal nuclei; Chemicon) coupled to rhodamine fluorophore. Double fluorescence immunohistochemistry to detect expression of both reelin and Calretinin was also performed with anti-calretinin polyclonal antibody (Chemicon) as primary antibody and the secondary antibodies were fluorescent dyes: confocal microscopy and image analysis software were used to analyze the results. Finally, reelin’s expression and methylation levels as well as the number of reelin-positive cells have been investigated in the hippocampal formation of MAM-exposed animals (214).

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Coronal slices from MAM-exposed and control animals were stained with anti-reelin antibody (G10 Chemicon) and 3,39diaminobenzidine used as chromogen. For methylation, hippocampal deoxyribonucleic acid (DNA) was treated with sodium bisulfite using the EpiTect Kit H (Epigenomics; Qiagen, BBBB). Modified DNA was purified by EpiTect H spin columns and used immediately or stored at −20◦ C. Bisulfite-modified genomic DNA was amplified with primers specific for the reelin promoter [59-CGTTTTTTTATTTTGGTTTGGT-39 (forward) and 59-TCATATCATACATAACCACTATCCCTA-39 (reverse)]. The polymerase chain reaction (PCR) conditions were 95◦ C for 15 min, 5 cycles of 95◦ C for 30 s, 56◦ C for 90 s, 72◦ C for 120 s, 25 cycles of 95◦ C for 30 s, 56◦ C for 90 s, 72◦ C for 90 s, and finally 7 min at 72◦ C. The PCR mixture contained 12.5 ml of HotStarTaq Mix (Qiagen), 0.5 ml of each primer at a 20 mM concentration, and 2 ml bisulfite-modified DNA. These first-round PCR products were then used as template (1 ml) and reamplified by tagged primers (59-CCACTCACTCACCCACCC + forward primer-39 and 59-GGGTGGGAGGTGGGAGGG + reverse primer-39). The second-round PCR conditions were 95◦ C for 15 min, 35 cycles of 95◦ C for 30 s, 55◦ C for 90 s, 65◦ C for 120 s, and finally 7 min at 65◦ C. PCR products were then purified with a gel extraction kit (Qiagen) and subjected to direct cycle sequencing on an ABI3100Avant automated DNA sequencer (Applied Biosystems, Foster City, CA, USA), using the primers from the second round of PCR. Sequence chromatographs were analyzed by the software ESMEH (Epigenomics), which performs quality control, normalizes signals, corrects for incomplete bisulfite conversion, and maps positions. 3.4. Abnormal GABAergic Markers in MAM-EXPOSED Rats

Abnormal GABAergic transmission has also been found in several brain regions, including the hippocampus (196, 197) and the prefrontal cortex (198, 199). Decreases in glutamic acid decarboxylase (GAD)-1 mRNA and GAD-67 protein are observed postmortem throughout the cortex of schizophrenic patients (200–202). An increasing number of studies are focusing on GABAergic interneurons. Postmortem studies have consistently reported a significant reduction of GABA interneurons (small, non-pyramidal) in layer 2 of the anterior cingulate and prefrontal cortices (176, 198) and hippocampus (203) of schizophrenia brains. GABAergic interneurons comprise a variety of different subpopulations that can be identified by their neurochemical profile (204). The relative density of subpopulations of GABAergic interneurons is compared using antibodies directed against the calcium-binding proteins, calretinin (CR), parvalbumin (PV), and calbindin (CB). CR-containing neurons have been consistently

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reported to be unchanged in any regions of schizophrenia brains (175, 178–181). CB-containing neurons have been found to be significantly increased in layers 3 and 5/6 of prefrontal cortex (areas 9 and 46) (205) or decreased in these same prefrontal regions (areas 9 and 46) (179). Others have found no significant difference in anterior cingulate (206), prefrontal (area 46), entorhinal (207), or posterior cingulate cortices (areas 30 and 23) (208). Concerning PV-containing neurons, if reduced densities were observed in the hippocampus of schizophrenic patients (181, 209), conflicting results were obtained in the prefrontal cortex. Indeed, most investigators reported lower PV+ cell counts (175, 178, 179, 210), in particular in layers 3 and 4 of the cortex, although others found no change (180, 206, 211). Another marker found in GABAergic neurons is reelin. A marked decrease in reelin in the superficial layer of the cortex has been consistently found in postmortem studies in schizophrenia (reviewed in (2)). In order to investigate the possible GABA neurotransmission abnormalities in MAM-exposed rats, possible changes in the expression of PV, CR, CB, reelin, and GAD-67 have been examined in the prefrontal cortex and/or the hippocampus of MAMexposed rats using immunochemistry (37, 41). 3.4.1. Procedures

Adult male rats were deeply anesthetized and perfused transcardially with PBS or saline followed by paraformaldehyde [4% (w/v) PFA in 0.1 M PBS]. Rats were decapitated and their brains removed, postfixed, and stored in 0.1 M PBS or in 30% sucrose solution until sectioning. In their study, Penschuck and colleagues (37) used 40 μm sections with AP from bregma, 3.7–2.7 mm for the prefrontal cortex and −2.8 to −3.3 mm for the hippocampus The tissue was stained using the diaminobenzidine immunoperoxidase method and primary antibodies for either PV (1:5000), CR (1:2000), or CB (1:10000), according to the manufacturer’s instructions (Swant, Bellinzona, Switzerland). The semi-quantitative analysis was performed blind using stereology software. Lodge and colleagues (41) used a different sampling procedure with sequential sections (50 μm) comprising the prefrontal region (bregma −5.0 mm to −2.0 mm), dorsal (bregma −1.5 mm to −4.5 mm), and ventral (bregma −4.5 mm to −7.5 mm) hippocampus (six slices per region), and double fluorescence immunohistochemistry to detect expression of both GAD67 and PV as described previously (212). Primary antibodies were used for GAD67 (monoclonal 1:2000; Millipore; MAB5406) and PV (1:3000; Swant; PV28) and the secondary antibodies were fluorescent dyes: Alexa Fluor-conjugated goat anti-rabbit (568) or goat anti-mouse (488). Mounted slices were evaluated for fluorescence using confocal microscopy and image analysis software.

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Some of the results remain controversial. Penschuck and colleagues (37) showed that the number of PV-immunoreactive cells/mm2 was significantly decreased in the dorsal part of the hippocampus of MAM-exposed rats in comparison to controls and unchanged in the prefrontal cortex. No changes in the numbers of CR- or CB-immunoreactive cells were found in either structure. In contrast, Lodge and colleagues (41) showed that MAM-exposed rats exhibited a regionally selective reduction in the density of PV-positive neurons throughout the medial prefrontal cortex and in the ventral subiculum of the hippocampus with no significant differences in the number of GAD-67-positive/PV-negative neurons in either the medial prefrontal cortex or the ventral subiculum. These differences in PV cell density were not observed in the dorsal subiculum of the hippocampus, while a small decrease in PV cell density was observed throughout the anterior cingulate cortex. No significant differences in fluorescence intensity for either GAD-67 or PV were found between MAM-exposed and control rats. The negative results concerning GAD-67 obtained by Lodge and colleagues (41) are not in light with those obtained in schizophrenic patients where a decrease was generally observed. Concerning the data on the subset of interneurons that contain the calcium buffer PV, the conflicting findings reported in the two studies (37, 41) could be explained in part by methodological discrepancies. Indeed, the two groups have performed their experiments in two different rat strains (Wistar and Sprague Dawley rats) that have been exposed at E17 to two different MAM doses (22 and 20 mg/kg). In addition, the method of slice sampling could also explain such a difference since the two groups used different brain coordinates to anatomically define the regions being investigated. Thus, results for PV in the E17 MAM model are as much conflicting as those observed in schizophrenic patients for this marker of GABAergic interneurons. Regarding the expression of reelin, the picture is not so clear. In organotypic slices, the number of cells was decreased in the number of neurons that express reelin was increased in the stratum oriens, concomitantly with a decreased number of neurons in the stratum oriens, while no change was seen in the dentate gyrus (a region where neurogenesis is still active in adulthood). In postmortem whole brain studies, the number of reelin-positive cells was not changed in the hippocampus of MAM-exposed rats compared to controls, nor was the methylation or the expression (214). The effect of E17 MAM exposure is thus not so clear and will require further exploration.

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4. Anatomical and Functional Anomalies in E17 MAM-Exposed Rats 4.1. Anatomical Anomalies

Neuroimaging studies in schizophrenic patients have identified morphological and functional anomalies which can be explained by early neurodevelopmental factors. Main findings include enlargement of the lateral ventricles, reduced size of temporal lobe structures, decreased thalamic volume, and enlarged basal ganglia (213). The loss of cortical volume without loss of neurons in frontal and temporal association cortex is associated with the greatest reduction in spine number in schizophrenia. Morphometric data from other brain regions like the hippocampal formation, the entorhinal cortex, and the mediodorsal thalamus coming from brain of patients with schizophrenia is reviewed in Matricon et al. (214).

4.1.1. Prefrontal Cortex and Other Cortices

Brain abnormalities identified postmortem in adult rats prenatally exposed to MAM at E17 point to a size reduction and a distortion in superficial layers of the prefrontal cortex (32, 33, 35–37) (Fig. 2.22). The decrease in cortical thickness appears significant in the prefrontal, occipital, and entorhinal cortices, whereas no significant differences between MAM-exposed rats and controls are noted in the parietal and perirhinal cortices (36, 214). Using complementary staining methods (Bodian-Luxol for myelin and neuronal soma and NeuN, a protein specific for neuron and restricted to the axon hillock), Matricon et al. (214) also evidenced a disruption in columnar organization in the entorhinal cortex (Fig. 2.23). A reduction in the soma size could be observed in both the perirhinal and entorhinal cortices, while this decrease in the entorhinal cortex was associated with a laminar disorganization and the presence of aberrantly clustered neurons in layers 2 and 3. However, no significant difference was observed in the number of neurons in the prefrontal, entorhinal, and perirhinal cortices between MAM-exposed animals and control (36, 214). Conversely, stereological analyses showed increases in neuronal density in the prefrontal, perirhinal, and occipital cortices (36).

4.1.2. Hippocampus and Subcortical Regions

In the MAM-exposed rats, hippocampal and mediodorsal thalamus size reductions have been reported (35, 36, 214). No significant difference between MAM-exposed and control animals could be detected in the size of the ventral tegmental area, the substantia nigra, and the amygdala (214). Within the hippocampal formation, changes in neuronal density appear quite difficult to assess due to the heterogeneity in the different subfields and the

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Fig. 2.22. Representative sections of the hippocampus and adjacent cortices (a–d), the prefrontal cortex (e and f ) from embryonic day 17 (E17) methylazoxymethanol (MAM)-exposed rats (a, c, e) as compared to controls (b, d, f). Dorsal hippocampus sections show heterotopias (white squares) in CA1 in E17 MAM-exposed rats (a). Ventral hippocampus sections illustrate abnormalities in the distribution of CA1/subicular cells (white squares and arrowheads) extended to some cells in E17 MAM-exposed rats (c, CA1/sub). Note alterations in the entorhinal (black square, ent) and retrosplenial cortices (black square, rsp), with dispersed cell and gaps in E17 rats (c). In the same sections, the rhinal fissure (black arrowhead ) points to a deformation of the perirhinal cortex when compared to E17 (c) and control rats (d). Medial prefrontal cortex sections (mpfc, higher magnification) show a distortion in layers II and III, with a decrease in cell density in E17 MAM-exposed rats (e). (Adapted from Gourevich et al., 2004)

structural changes in MAM-exposed rats. Neuronal soma size reductions were mostly present in hippocampal subfields CA3 and CA4 (214). Neuronal density did not differ between MAMexposed and control rats in the mediodorsal thalamus.

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Fig. 2.23. Entorhinal cortex in control and MAM-exposed rats. Coronal sections stained with Bodian-Luxol (a, d) and NeuN immunostaining (b, e and c, f). In controls (a) a well-defined columnar organization and myelin with parallel directions can be observed. Arrows point to myelin pathways. (b) Anti-NeuN immunostaining shows axon hillocks with a parallel orientation and neuronal bodies regularly organized. (c) Neuronal somas and axon hillocks are clearly distinguished in this picture magnified 400×. Arrows point to axon hillock direction. In MAM-exposed animals (d), a less obvious columnar organization is observed, while myelin direction appears random. (e) Neuronal disorganization with clusters of neurons is evident mainly in layers II and III. Circles highlight neuronal clusters. (f) Neuronal disorganization, neuronal size variability, and axon hillocks with random directions can be observed in this picture magnified 400×. Arrows point to axon hillock direction. (Adapted from Matricon et al., 2010)

Brain volume reductions could originate from a variety of causes. Neuronal soma size reduction could be at the origin of the volume reduction reported in some brain regions in both MAM-exposed rats and patients with schizophrenia (198–214). On the other hand, changes in cortical neuropil architecture as revealed by a loss of dendritic spines could explain the loss of cortical volume. Indeed, measurements of spine density and dendrite morphology of pyramidal neurons in schizophrenic patients showed the greatest reduction in spine number in frontal and temporal association cortex where a significant volume loss is reported (215). More detailed investigations on dendrite morphology are necessary to answer this question in MAM-exposed rats. Altogether, these anatomical changes found in E17 MAMexposed animals on top of a significant enlargement of the lateral

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ventricles indicate significant similarities to neuropathological deficiencies encountered in patients with schizophrenia. 4.2. Functional Anomalies

Although the use of neuroimaging techniques has implicated a variety of brain regions in schizophrenia, the emphasis in functional imaging studies has been and continues to be on the frontal lobes. The finding of hypofrontality has been supported by meta-analysis both at rest and under neuropsychological activation (216). Schizophrenic patients perform poorly on working memory tests and these deficits are consistently associated with prefrontal cortex dysfunction, although the basis of this abnormality is unknown. The convergent findings of neuroimaging and pathological studies give strong support to a functional limbic prefrontal network dysfunction in the pathophysiology of schizophrenia, with probably a volumetric alteration of limbic structures more specific to the anterior hippocampus, which would correspond to the ventral hippocampus in animals (8, 139, 217–220). The medial prefrontal cortex in rats is comparable to the ventromedial and subgenual prefrontal cortex in humans, regions which are particularly at risk in schizophrenia. Tamminga and Medoff (219) have shown that regional cerebral blood flow is abnormal in the hippocampal-anterior cingulate circuit in schizophrenic patients who cannot fully activate this circuit with cognitive tasks. Prefrontal cortical activity has been investigated in E17 MAM-exposed animals through in vivo field potential and singleunit recordings in adult anaesthetized rats. The absence of slow and fast oscillations added to an abnormal regular spike firing pattern in the medial prefrontal cortex (38) suggests an alteration in the inputs to the prefrontal cortex. Indeed an increased spike firing in response to ventral tegmental stimulation was observed in the prefrontal cortex of MAM-exposed rats when compared to controls. Using microinjection of either tetrodotoxin (TTX) to inactivate the ventral hippocampus or NMDA in the ventral hippocampus to activate dopamine transmission in the prefrontal cortex, Lodge and Grace have shown an aberrant dopamine response that could result from hippocampal dysfunction (42). Later on, they showed that the deficit in intrinsic GABAergic interneurons within the ventral subiculum may be at the origin of this dopamine dysfunction (41). Knowing the critical control of the mesocortical system on prefrontal circuits by simultaneously activating pyramidal neurons and fast spiking interneurons (221), it remains to be determined which prefrontal neuronal subpopulation is at the origin of the dopamine dysfunction observed in MAM-exposed animals.

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5. Conclusions In summary, data acquired from the last years have emphasized the face validity of the E17 MAM-exposed rat model for schizophrenia by showing its capacity to produce a behavioral phenotype that is reminiscent of positive, negative, and cognitive symptoms observed in schizophrenic patients. Anatomical and functional findings observed in the E17 MAM model indicate that there are significant similarities in neuropathological and imaging data in patients with schizophrenia and MAMexposed rats. However, studies investigating direct deficits in glutamatergic and GABAergic systems or dysfunctions in the interaction of glutamate and dopamine and GABA are still needed. It will also be interesting to directly compare with other models for schizophrenia, in particular genetically based model (such as mice deficient in DISC1 or in neuregulin) or other developmental model (e.g., neonatal lesion of the hippocampus or prenatal infection). Lastly, while most studies have focused on the prefrontal cortex and the hippocampus, subcortical regions in the brain could also participate in the phenotype. The future will determine the precise cellular and molecular mechanisms underlying the anatomical and functional changes observed in the brains of adult MAM-exposed rats. This experimental model also provides an integrative approach to understanding the effects of a developmental insult that induces behavioral anomalies close to those observed in schizophrenia. It may help forward in establishing early preventive interventions that can successfully reduce the risk of developing schizophrenia in exposed individuals.

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Chapter 3 Prenatal Infection and Immune Models of Schizophrenia Alan S. Brown Abstract An increasing number of epidemiologic studies have implicated in utero exposure to infection in the etiopathogenesis of schizophrenia. Recent work has capitalized on the use of prospectively acquired data on infection based on maternal biomarkers. These studies suggest that several maternal infections, including rubella, influenza, toxoplasmosis, herpes simplex virus/other genital-reproductive infections, and elevations in the cytokines interleukin-8 and TNF-α, are associated with increased schizophrenia risk among offspring. Animal models of in utero infection offer the potential to corroborate these findings under controlled conditions and address etiopathogenic mechanisms. Models of maternal immune activation (MIA) and behavioral and brain anomalies in schizophrenia have primarily employed three agents: polyinosinic:polycytidylic acid (poly I:C), lipopolysaccharide, each of which mimic viral and bacterial infections, respectively, and direct maternal inoculation with influenza. Advantages and disadvantages of these approaches are discussed and the findings are reviewed. Each of these MIA models has yielded evidence, reviewed in detail, for neurochemical, neuropathologic, and behavioral anomalies in offspring that are consistent with phenotypes found in patients with schizophrenia. In addition, putative mechanisms by which MIA causes these phenotypes, the role of gestational timing of the insult, and long-term modulation of the immune system following prenatal immune challenge are discussed. Finally, the “back translation” of these findings to the design and implementation of new epidemiologic studies aimed at further refining and testing this hypothesis is discussed. Key words: Infection, immune, schizophrenia, influenza, animal models, cytokines, poly I:C, LPS, epidemiology, risk factors.

1. Introduction An increasing number of epidemiologic studies have implicated in utero exposure to infection in the etiopathogenesis of schizophrenia (1, 2). Although initial studies based on epidemics in populations reported conflicting findings, a new generation of investigations based on birth cohort studies has documented intriguing P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_3, © Springer Science+Business Media, LLC 2011

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associations between prenatal infection and risk of schizophrenia in offspring. These studies capitalized on prospective measures of maternal infection, or indicators of infection, to selected agents during pregnancy, follow-up of offspring, and diagnoses based on direct interviews, psychiatric records, and validated psychiatric registry data. Among other findings, this work revealed that exposure to rubella (3), influenza, elevated toxoplasma IgG antibody (4), periconceptional genital/reproductive infection (5), elevated HSV-2 IgG antibody (6), and increases in the pro-inflammatory cytokines interleukin-8 (7) and TNF-α (8) were associated with increases in risk of adult schizophrenia. The effect sizes in these studies were not trivial, with most ranging from approximately two- to fivefold, significantly higher than effect sizes for most individual susceptibility genes. Although replication of these findings is essential, these promising results suggest that in utero exposure to infection may play an important role in the etiology of schizophrenia. Yet, the biological basis of these findings remains unknown.

2. Rationale for Studies of Animal Models of In Utero Infection

2.1. Maternal Immune Activation Models: Poly I:C and LPS

Animal models of in utero infection offer the potential to provide corroborative evidence that under controlled conditions the associations between infection and schizophrenia-like pathology or behavior can be validated, and address etiopathogenic mechanisms by which in utero exposure to infection can increase liability to schizophrenia. Inspired by the epidemiologic findings reviewed above, a proliferation of such “reverse translational” studies has emerged in recent years. There are two primary types of models of in utero infection that have been tested in paradigms of schizophrenia: noninfectious maternal immune activation and a direct infectious insult. We describe each of these study designs and findings in turn. The first approach relies on induction of a maternal immune response which mimics that of an actual infection. Two agents have been used to stimulate immune activity in animal models of schizophrenia: poly I:C and lipopolysaccharide (LPS). Poly I:C. The first immune-activating agent is the synthetic polymer poly I:C (polyinosinic:polycytidylic acid), which bears structural similarity to double-stranded RNA. Poly I:C acts by binding to the toll-like receptor TLR3, which is expressed intracellularly in B cells and dendritic cells. It is generally thought

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that poly I:C most closely mimics a viral infection. Poly I:C stimulates a potent immune response involving marked increases in cytokines, including interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1-β (IL-1-β), tumor necrosis factor-α (TNFα), interferon-γ (IFN-γ), and interleukin-10 (IL-10). Studies have demonstrated that maternal administration of poly I:C increases several of these cytokines in the placenta, and the fetus, including the fetal brain (9–13). Moreover, IL-6 response genes were demonstrated to be upregulated in fetal brain following maternal poly I:C injection (13, 14). Lipopolysaccharide (LPS). LPS is a large molecule which contains a lipid and a polysaccharide moiety and is found in the outer membranes of gram-negative bacteria, and is therefore believed to most closely simulate a bacterial infection. This agent is an endotoxin, which stimulates the release of pro-inflammatory cytokines. Like poly I:C, maternal administration of LPS increases cytokine secretion in several compartments in the maternal and fetal circulation, and in the fetal brain. Maternal administration of LPS has also been related to changes in brain-derived neurotrophic factors and nerve growth factor expression in the fetal brain (15). 2.2. Advantages of Poly I:C and LPS Models

The use of poly I:C and LPS in animal models of maternal immune activation has several advantages. First, unlike the influenza model, the timing of exposure can be precisely controlled experimentally. The maternal and fetal cytokine response to a single injection of poly I:C appears to be limited to a period of no more than several hours. Consequently, it is possible to narrow the timing of the effect of immune activation to a particular period of gestation. Second, both models allow for testing a nonspecific model of maternal immune activation common to different infections, such as activation of cytokines and other components of the immune response without the confounding influences of varying consequences of different infections. Third, it is not believed that influenza and certain other infections that have been implicated in schizophrenia in epidemiologic studies act by direct effects on the fetus. As noted below, influenza does not appear to be transmitted to the fetal brain. For certain maternal infections that have been associated with schizophrenia in offspring, such as toxoplasma (4) and HSV-2 (8, 16), infection during pregnancy is unlikely to have occurred; hence, it is more likely that the causative factor is the immune response to these agents. MIA models are especially informative for modeling these infections since most cytokines examined in animal model studies have been shown to cross the placenta. Fourth, both poly I:C and LPS circumvent the precautions that need to be taken for studies involving infectious micro-organisms and are therefore safer and more convenient in experimental studies.

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3. Experimental Protocols 3.1. Experimental Protocol: Poly I:C

In order to elaborate the paradigm, we provide an example of the methodologic approach, based on a study by Shi et al. (17). In this study, BALB/c mice that were pregnant on gestational day 9.5 were inoculated by intraperitoneal injections with poly I:C diluted in PBS at dosages ranging from 20 to 0 mg/kg. The offspring of these mice were separated from their mothers after 3 weeks and male and female mice were caged separately. Mice were tested at different ages ranging from adolescence to adulthood, depending upon the protocol. Testing included exploratory behavior and anxiety by open-field and novel object tests, spontaneous locomotor activity by a photobeam activity system, sensorimotor coordination by a Rota-rod Treadmill, social behavior by examining social interaction between pairs of mice, and pre-pulse inhibition to the acoustic startle response using a standard protocol.

3.2. Experimental Protocol: LPS

The methodologic approach for these animal models of schizophrenia is exemplified by a recent publication from Romero et al. (18). In this study, LPS from Escherichia coli was dissolved in saline and subcutaneously administered to pregnant rats at a dosage of 2 mg/kg. Injections were administered daily between days 1 and 21 of pregnancy. Controls were administered saline alone. At 21 days, offspring were weaned and caged in selected groups of animals of the same prenatal treatment and sex. Rats from LPS and control groups were tested in the acoustic startle PPI paradigm at 6 months of age. One day after PPI testing, animals were decapitated, and brains were removed, frozen, and stored for neurochemical assays. Tissue dissections were performed for sampling of the nucleus accumbens, caudate nucleus, putamen, frontal cortex, hippocampus, and amygdala. Samples from nucleus accumbens and neostriatum were homogenized for subsequent high-performance liquid chromatography assays for dopamine and dopamine metabolites. Additional studies of PPI were conducted with and without treatment with haloperidol (see (18) for details).

3.3. Experimental Protocol: Influenza

To exemplify the experimental approach, we discuss the method used in a study by Fatemi et al. (19). In this study, anesthetized pregnant female C57BL/6 mice on 9 days after breeding were inoculated by intranasal instillation of influenza A/NWS/33 (H1N1) virus. Control mice were exposed to sterile virus diluent using the same protocol as the infected animals. Pregnant mice delivered pups and groups of infected and sham-infected neonates were anesthetized. Brains were removed following birth,

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cryopreserved, and coronal sections were cut. Cell counting was conducted for pyramidal and non-pyramidal cell density and pyramidal nuclear size was obtained. In a second study, offspring were allowed to mature to various ages from adolescence to adulthood for testing using the same paradigms as in Shi et al. (17). Of note, loss of pregnancy was not observed to be common so long as the mice were not disturbed. Mothers were noted to have mild lung consolidation with viral titers ranging from 3 to 4 × 103 pfu soon after infection, which became undetectable, and fever was not observed. 3.4. Inoculation with an Infectious Microbe: Influenza

In this model, pregnant animals are inoculated with an infectious microbe, and their offspring are studied for the behaviors or psychophysiologic functions in question. To date, inoculation with influenza is the only known animal model involving a microbial pathogen aimed specifically at investigating schizophrenia. There are two main advantages of this paradigm. First, this model attempts to closely emulate the naturalistic experience involving maternal exposure to the pathogenic microbe in humans. Since it is unclear which of several potential consequences of influenza result in an increased risk of schizophrenia, exposure to the actual infection ensures that the animal is exposed to most or all of these contributory components. This includes, but is not limited to, the respiratory consequences, the full inflammatory response, including elevations of pro-inflammatory cytokines and specific immunoglobulin antibodies (along with the time course by which these antibodies become elevated), and other maternal physiologic changes resulting from these consequences. Second, this model is based on the postulate that if in utero infection plays a role in schizophrenia, not all infectious agents will be implicated. Using this approach, an investigator could theoretically test for a wide array of pathogens to evaluate their specificity for causing brain and behavioral alterations found in schizophrenia. There are three main disadvantages of this approach. First, the infection can last as long as several days after inoculation and the duration of infection can vary between individual animals. This makes it difficult to control the gestational period of susceptibility, a problem that is especially critical in studies of rodents in which the period of gestation is markedly compressed relative to humans. Second, influenza RNA and nucleoprotein corresponding to the serotype of influenza have been identified in fetal brain from pregnancies with maternal inoculation with influenza (20), indicating placental and blood–brain barrier transfer of the virus. This differs from that of the natural influenza serotype in human populations, in which influenza has only rarely been documented in fetal brains of exposed pregnancies in humans. Third, extensive care must be taken to prevent exposure to the infectious agent in

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laboratory personnel, and the methods to prevent exposure and to successfully execute the research paradigm are more complex than alternative approaches discussed below.

4. Review of Study Findings from Animal Models of Schizophrenia

4.1. Findings from Poly I:C Models

Below, we provide a summary of findings that have emerged from animal models of influenza and maternal immune activation. For a more comprehensive review of these findings, we refer the reader to Patterson et al. (10). A plethora of studies have documented phenotypes believed to resemble those observed in schizophrenia following gestational exposure to poly I:C. Patterson and colleagues have demonstrated that impaired PPI, social interaction deficits, and reduced novel object exploration have each been observed in poly I:C-exposed mice (17). Similarities in these outcomes to mice exposed in utero to influenza (see “section 4.3” below) suggest that at least these behaviors may be mediated by the maternal immune response to this infection, in the absence of the virus itself. Several additional findings have been observed among offspring of poly I:C-exposed mice. These include abnormalities in latent inhibition, working memory, and disturbances of dopamine-related functions including enhanced amphetamine-induced locomotion, increased striatal dopamine release, and altered reversal learning (10). Interestingly, in two studies, amphetamine-induced anomalies were not observed in pre-pubertal offspring but emerged during adulthood, reminiscent of the delayed onset of psychotic symptoms in schizophrenia (21, 22). Other findings that relate to a disturbance of the dopamine system include an elevation of tyrosine hydroxylase in the dorsal striatum and the shell of the nucleus accumbens (23), a reduction of dopamine type 1 and type 2 receptors in the medial prefrontal cortex (23), and an elevation of DOPAC in the striatum, suggesting increased dopamine turnover (22). Additional evidence of neurochemical dysfunction has been observed in glutamatergic and GABA-related systems in poly I:C-exposed offspring. An increase in locomotion following administration of MK-801, an NMDA receptor antagonist, has been observed in adult mice in three studies (23–25), and this abnormality was not observed pre-pubertally, similar to the amphetamine-related findings. These results point to NMDA-related dysfunction that appears to be analogous to findings from the extant literature on this receptor in schizophrenia. Moreover, abnormalities in immunohistochemical markers for GABAergic neurons have been demonstrated, including a reduction of reelin and parvalbumin

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in the medial prefrontal cortex, and an elevation of GABA-α-2 receptor subunits in the ventral hippocampus (25). Offspring exposed to poly I:C also evidenced neuropathologic abnormalities. These include disruptions in postnatal neurogenesis in the dentate gyrus, increased apoptosis, and moderate to severe cell loss in the CA1 region of the hippocampus (21). Maternal poly I:C exposure also led to ventricular enlargement, the most well-replicated structural anomaly in schizophrenia (14). Curiously, treatment with antipsychotic medications in pre-pubertal animals exposed to gestational poly I:C prevented ventricular enlargement and other anomalies (26). 4.2. Findings from LPS Models

In utero treatment with LPS appears to produce some of the same behavioral deficits observed with poly I:C. These include PPI deficits (18), which were corrected with administration of antipsychotics (18, 27), and a disruption of social interaction and learning. Dopaminergic abnormalities include amphetamine-induced hyperlocomotion and increases in DOPAC and dopamine in the striatum (18), as well as decreases in tyrosine hydroxylase neurons in the substantia nigra (28). LPS also caused astrogliosis, evidenced by increased glial fibrillary acid protein (GFAP) staining and changes in microglial immunostaining (29).

4.3. Findings from Influenza Models

Fatemi and colleagues (19) have shown that exposure to influenza during mid-pregnancy causes increased packing of pyramidal cells, alterations in reelin, SNAP-25, and nNOS expression. Increased density of pyramidal cells has also been observed. Offspring exposed to influenza in mid-gestation also evidence a decreased number of cerebellar Purkinje cells and a disruption of neuronal migration to layers 2 and 3 of the cortex (30). Patterson and colleagues have demonstrated behavioral abnormalities in influenzaexposed offspring, including social interaction deficits, decreased PPI, and reduced exploration in the open field and in response to novel objects (30).

4.4. Mechanisms of Maternal Immune Activation and In Utero Infection

A key question raised by the MIA and influenza models concerns the mechanisms by which this in utero factor alters fetal brain development in such a way that it leads to the behavioral, histologic, neuroanatomic, and neurochemical abnormalities reviewed above. It is first worth considering the maternal– fetal compartments that are affected by the insult. Studies have demonstrated that maternal serum pro-inflammatory cytokine levels are increased following exposure to poly I:C and LPS (10). Maternal LPS administration has also been demonstrated to increase cytokine levels in the placenta and the amniotic fluid (9, 10). For poly I:C, several cytokines were shown to be increased in the fetal brain (13), and an increase in fetal expression of the IL-6 gene has also been demonstrated. Hence, it is

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conceivable that MIA causes a cascade of events that begin with the maternal immune response and that lead to an elevation in fetal cytokine levels either through placental passage or via activation of a fetal immune response. Another essential question that studies are beginning to address is whether certain cytokines are responsible for the observed effects. Patterson and colleagues have attempted to isolate the role of IL-6 in the brain and behavioral anomalies observed following MIA (31). These investigators found that co-injection of maternal poly I:C and an antibody which neutralized the effect of IL-6 blocked the observed behavioral abnormalities following MIA. This antibody also blocked alterations in gene transcription in the fetal brain that were induced by poly I:C. In a model which utilized intracerebral injection of LPS, ventriculomegaly was diminished and behavioral outcomes were improved, following administration of IL-6 antibody (32). In an extension of this work to IL-6 knockout mice, MIA with poly I:C had no effect on behavior. In a similar vein, genetic overexpression of interleukin-10, and anti-inflammatory cytokine, by macrophages was shown to diminish both behavioral and pharmacological effects of MIA with poly I:C (33). However, when there was no inflammatory stimulus, increased maternal/fetal IL-10 levels were associated with behavioral anomalies in offspring. These intriguing results argue for the importance of a balance between pro- and anti-inflammatory effects of in utero cytokines on behavioral outcomes. 4.5. Gestational Timing of Infection

The identification of windows of vulnerability is of critical importance toward understanding the etiopathogenic effects of MIA and influenza infection, and perhaps of in utero exposures in schizophrenia. Meyer et al. (13) demonstrated that the timing of in utero immune activation in mice may have differential effects on the observed brain and behavioral phenotypes. For example, poly I:C on gestational day 9 (roughly equivalent to midpregnancy in humans), but not gestational day 17 (roughly equivalent to late pregnancy), diminished spatial exploration, while MIA treatment on gestational day 17, but not gestational day 9, resulted in perseverative behavior. Exposure to MIA on GD9, but not GD17, resulted in an impairment in latent inhibition associative learning. MIA in mid-pregnancy caused a more pronounced decrease in hippocampal reelin expression, while exposure during late pregnancy, but not mid-pregnancy, caused apoptosis. These findings suggest an interaction between exposure to MIA and the specific developmental stage of the brain at the time of induction of the exposure. Moreover, these authors also suggest that differences in the fetal brain cytokine response to MIA may contribute to these varying effects.

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4.6. Long-Term Immune Modulation Following MIA and In Utero Infection

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Another particularly exciting avenue of research is addressing the question of whether MIA and in utero infection alter fetal programming such that it produces long-term effects on the expression of cytokines, and of other molecules implicated in the pathogenesis of schizophrenia. An extensive literature has documented abnormalities in peripheral cytokine levels in patients with schizophrenia, though these findings conflict to some degree. Although there are many potential causes for these abnormalities, including infections of childhood or adult onset, the possibility remains that in utero cytokines lead to permanent alterations in gene expression later in life, analogous to fetal programming hypotheses for obesity and hypertension (34). In this regard, the findings of Smith et al. (31), which demonstrated that IL-6 caused alterations in gene expression in adult animals, are particularly intriguing. This group has also demonstrated that MIA results in an upregulation of several IL-6 response genes in fetal brain (14). As previously discussed, it would clearly be of benefit to examine whether this upregulation persists into adulthood and contributes to the observed brain and behavioral anomalies.

Animal models of MIA and in utero infection provide one of the most instructive examples of how epidemiologic research can be translated to basic neuroscience investigations in order to validate observed phenotypes and elucidate causal mechanisms. Given the proliferation of highly informative animal models, it is now worth considering whether the knowledge gained thus far from studies of animal models has implications for the design and implementation of new epidemiologic studies. We suggest three potential avenues of research that may result from such findings. First, as described in detail above, the animal models have documented a variety of brain and behavioral phenotypes that are associated with MIA. For obvious practical reasons, the epidemiologic studies, however, have restricted the phenotypic outcome to the diagnosis of schizophrenia. Research efforts are underway in an attempt to transcend traditional diagnostic classification by assessing neuromorphometric and neuropsychological outcomes in individuals from birth cohorts in which prospective, biomarker-based documentation of in utero infection was obtained. Early studies have demonstrated that in utero exposure to infection is associated with executive function deficits, including anomalies of cognitive set-shifting and working memory (35),

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and with neuromorphometric anomalies, including cavum septum pellucidum (36), which is indicative of in utero brain developmental disruption. One of the caveats of this work is that the samples have been generally small and most of the phenotypes observed in the basic neuroscience literature were not testable, given practical constraints. However, it is conceivable that several of the psychophysiologic and neuromorphometric anomalies currently being examined in the literature on animal models can be directly examined in some of the large birth cohort studies currently underway. Second, as reviewed above, the literature on animal models has revealed differential sensitivity to MIA based on the gestational timing of infection. Although challenging, the potential to investigate this question is plausible in an epidemiologic design. Third, studies of animal models have potential to reveal new candidate cytokines, profiles of cytokine release, or related molecules, which can be tested in maternal or neonatal sera in epidemiologic designs. Fourth, work on alterations in gene expression by MIA may be informative in the selection of candidate genes that can be investigated for interactions between infectious exposures and maternal/infant cytokines. Recent evidence from genome-wide association studies (GWAS) indicating a small but significant association between genetic variants in the extended major histocompatibility complex (MHC) (37–39) suggests that inclusion of data in both in utero infection and immune-related genes in informative samples followed up for schizophrenia may yield significantly larger effect sizes. These samples include prospective birth cohorts with available biological specimens dating back to the in utero period. Presently, efforts are underway to better understand the relationship between in utero exposure to infections and immune disturbances in far larger birth cohorts that have been studied to date.

6. Summary Epidemiologic studies based on in utero biomarkers have revealed tantalizing evidence that prenatal infection may increase the risk of schizophrenia. Inspired by these epidemiologic data, a plethora of studies of animal models of in utero infection have emerged. These studies include maternal challenge with the immuneactivating agents poly I:C and LPS and inoculation with influenza, and assessment of offspring at various stages of development. These studies suggest that prenatal immune activation and infection cause brain and behavioral alterations that are analogous to some phenotypes observed in schizophrenia. Although the causal mechanisms are not well understood, it is clear that MIA

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induces elevated cytokine levels in both the maternal and fetal compartments, including the fetal brain. Among the cytokines investigated to date in these models, it appears that IL-6 may play a particularly important role in the induction of brain and behavioral anomalies in offspring. There appears to be differential vulnerability to certain outcomes of MIA depending upon the gestational period of infection. The potential that these outcomes are transduced by early life programming changes by immune activation and infection is clearly worthy of further exploration. These studies may also enrich epidemiologic studies by a “backtranslational” approach in which cytokines and other molecules shown to be related to immune stimulation in animal models can be examined in prospective studies of informative birth cohorts. This work promises to shed light on the etiologies of schizophrenia, on the pathogenic mechanisms that give rise to this devastating condition, and on new molecular targets for treatment.

Acknowledgments This manuscript was supported by the following grants: National Institutes of Mental Health (NIMH) 1R01MH-60249 (A.S.B), NIMH 2K02MH065422-06 (A.S.B.). References 1. Brown, A. S. (2006) Prenatal infection as a risk factor for schizophrenia, Schizophr Bull 32, 200–202. 2. Penner, J. D., and Brown, A. S. (2007) Prenatal infectious and nutritional factors and risk of adult schizophrenia, Expert Rev Neurother 7, 797–805. 3. Brown, A. S., Cohen, P., Harkavy-Friedman, J., Babulas, V., Malaspina, D., Gorman, J. M., and Susser, E. S. (2001) A.E. Bennett Research Award. Prenatal rubella, premorbid abnormalities, and adult schizophrenia, Biol Psychiatry 49, 473–486. 4. Brown, A. S., Schaefer, C. A., Quesenberry, C. P., Jr., Liu, L., Babulas, V. P., and Susser, E. S. (2005) Maternal exposure to toxoplasmosis and risk of schizophrenia in adult offspring, Am J Psychiatry 162, 767–773. 5. Babulas, V., Factor-Litvak, P., Goetz, R., Schaefer, C. A., and Brown, A. S. (2006) Prenatal exposure to maternal genital and reproductive infections and adult schizophrenia, Am J Psychiatry 163, 927–929.

6. Brown, A. S., Schaefer, C. A., Quesenberry, C. P., Jr., Shen, L., and Susser, E. S. (2006) No evidence of relation between maternal exposure to herpes simplex virus type 2 and risk of schizophrenia, Am J Psychiatry 163, 2178–2180. 7. Brown, A. S., Hooton, J., Schaefer, C. A., Zhang, H., Petkova, E., Babulas, V., Perrin, M., Gorman, J. M., and Susser, E. S. (2004) Elevated maternal interleukin-8 levels and risk of schizophrenia in adult offspring, Am J Psychiatry 161, 889–895. 8. Buka, S. L., Tsuang, M. T., Torrey, E. F., Klebanoff, M. A., Wagner, R. L., and Yolken, R. H. (2001) Maternal cytokine levels during pregnancy and adult psychosis, Brain Behav Immun 15, 411–420. 9. Jonakait, G. M. (2007) The effects of maternal inflammation on neuronal development: possible mechanisms, Int J Dev Neurosci 25, 415–425. 10. Patterson, P. H., Xu, W., Smith, S. E. P., Deverman, B. E. (2008) Maternal immune

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21. Zuckerman, L., Rehavi, M., Nachman, R., and Weiner, I. (2003) Immune activation during pregnancy in rats leads to a postpubertal emergence of disrupted latent inhibition, dopaminergic hyperfunction, and altered limbic morphology in the offspring: a novel neurodevelopmental model of schizophrenia, Neuropsychopharmacology 28, 1778–1789. 22. Ozawa, K., Hashimoto, K., Kishimoto, T., Shimizu, E., Ishikura, H., and Iyo, M. (2006) Immune activation during pregnancy in mice leads to dopaminergic hyperfunction and cognitive impairment in the offspring: a neurodevelopmental animal model of schizophrenia, Biol Psychiatry 59, 546–554. 23. Meyer, U., Nyffeler, M., Schwendener, S., Knuesel, I., Yee, B. K., and Feldon, J. (2008) Relative prenatal and postnatal maternal contributions to schizophrenia related neurochemical dysfunction after in utero immune challenge, Neuropsychopharmacology 33, 441–456. 24. Zuckerman, L., and Weiner, I. (2005) Maternal immune activation leads to behavioral and pharmacological changes in the adult offspring, J Psychiatr Res 39, 311–323. 25. Meyer, U., Nyffeler, M., Yee, B. K., Knuesel, I., and Feldon, J. (2008) Adult brain and behavioral pathological markers of prenatal immune challenge during early/middle and late fetal development in mice, Brain Behav Immun 22, 469–486. 26. Meyer, U., Spoerri, E., Yee, B. K., Schwarz, M. J., and Feldon, J. (2008) Evaluating early preventive antipsychotic and antidepressant drug treatment in an infectionbased neurodevelopmental mouse model of schizophrenia, Schizophr Bull 36, 607–623. 27. Borrell, J., Vela, J. M., Arevalo-Martin, A., Molina-Holgado, E., and Guaza, C. (2002) Prenatal immune challenge disrupts sensorimotor gating in adult rats. Implications for the etiopathogenesis of schizophrenia, Neuropsychopharmacology 26, 204–215. 28. Ling, Z., Chang, Q. A., Tong, C. W., Leurgans, S. E., Lipton, J. W., and Carvey, P. M. (2004) Rotenone potentiates dopamine neuron loss in animals exposed to lipopolysaccharide prenatally, Exp Neurol 190, 373–383. 29. Cai, Z., Pan, Z. L., Pang, Y., Evans, O. B., and Rhodes, P. G. (2000) Cytokine induction in fetal rat brains and brain injury in neonatal rats after maternal lipopolysaccharide administration, Pediatr Res 47, 64–72.

Prenatal Infection and Immune Models of Schizophrenia 30. Shi, L., Smith, S. E., Malkova, N., Tse, D., Su, Y., and Patterson, P. H. (2009) Activation of the maternal immune system alters cerebellar development in the offspring, Brain Behav Immun 23, 116–123. 31. Smith, S. E., Li, J., Garbett, K., Mirnics, K., and Patterson, P. H. (2007) Maternal immune activation alters fetal brain development through interleukin-6, J Neurosci 27, 10695–10702. 32. Pang, Y., Fan, L. W., Zheng, B., Cai, Z., and Rhodes, P. G. (2006) Role of interleukin-6 in lipopolysaccharide-induced brain injury and behavioral dysfunction in neonatal rats, Neuroscience 141, 745–755. 33. Meyer, U., Murray, P. J., Urwyler, A., Yee, B. K., Schedlowski, M., and Feldon, J. (2008) Adult behavioral and pharmacological dysfunctions following disruption of the fetal brain balance between pro-inflammatory and IL-10-mediated anti-inflammatory signaling, Mol Psychiatry 13, 208–221. 34. Godfrey, K. M., and Barker, D. J. (2001) Fetal programming and adult health, Pub Health Nutr 4, 611–624. 35. Brown, A. S., Vinogradov, S., Kremen, W. S., Poole, J. H., Deicken, R. F., Penner, J. D., McKeague, I. W., Kochetkova, A., Kern, D., and Schaefer, C. A. (2009) Prenatal exposure to maternal infection and executive dysfunction in adult schizophrenia, Am J Psychiatry 166, 683–690. 36. Brown, A. S., Deicken, R. F., Vinogradov, S., Kremen, W. S., Poole, J. H., Penner, J. D., Kochetkova, A., Kern, D., and Schaefer, C. A. (2009) Prenatal infection and cavum septum pellucidum in adult schizophrenia, Schizophr Res 108, 285–287. 37. Shi, J., Levinson, D. F., Duan, J., Sanders, A. R., Zheng, Y., Pe’er, I., Dudbridge, F., Holmans, P. A., Whittemore, A. S., Mowry, B. J., Olincy, A., Amin, F., Cloninger, C. R., Silverman, J. M., Buccola, N. G., Byerley, W. F., Black, D. W., Crowe, R. R., Oksenberg, J. R., Mirel, D. B., Kendler, K. S., Freedman, R., and Gejman, P. V. (2009) Common variants on chromosome 6p22.1 are associated with schizophrenia, Nature 460, 753–757. 38. Purcell, S. M., Wray, N. R., Stone, J. L., Visscher, P. M., O’Donovan, M. C., Sullivan, P. F., Sklar, P., Purcell Leader, S. M., Ruderfer, D. M., McQuillin, A., Morris, D. W., O’Dushlaine, C. T., Corvin, A., Holmans, P. A., Macgregor, S., Gurling, H., Blackwood, D. H., Craddock, N. J., Gill, M., Hultman, C. M., Kirov, G. K., Lichtenstein, P., Muir,

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Chapter 4 The Hypoxic Rat Model for Obstetric Complications in Schizophrenia Andrea Schmitt, Peter Gebicke-Haerter, Ulrich Sommer, Markus Heck, Anja Lex, Mario Herrera-Marschitz, Mathias Zink, Markus Fendt, and Peter Falkai Abstract Hypoxia has been discussed as a possible factor of obstetric complications in the pathophysiology of schizophrenia. This study investigated the effects of chronic neonatal hypoxia in rats as an animal model of schizophrenia. Methods: (1) After chronic neonatal hypoxia between postnatal day (PD) 4 and 8, half of the pups were fostered by normally treated nurse animals to control for possible maternal effects and (2) tested on PD 36, 86, 120, and 150 using three different behavioral tests: prepulse inhibition (PPI), social interaction and recognition, and motor activity in an open field. (3) Before the PD 150 test, 50% of the animals had been chronically treated with the antipsychotic drug clozapine (45 mg/kg/day). (4) At PD 155, different brain regions have been used for expression profiling of synaptic genes on cDNA microarrays (“glutamate chip”) with qRT-PCR confirmation. Additionally, at PD 11 and 120, NMDA receptor binding and expression of NMDA receptor subunits have been performed. Rats exposed to hypoxia exhibited deficits in locomotor activity on PD 86, 120, and 150, as well as a PPI deficit on PD 120 and 150 in adulthood, but not before. Chronic treatment with clozapine reversed hypoxia-induced PPI deficits, but not the decreased locomotor activity. In a second experiment, where clozapine was chronically administered before PD 120, development of the PPI deficit in the animals exposed to hypoxia was prevented. In several brain regions, presynaptic genes such as SNAP-25, syntaxin 1A, neurexin, neuropeptide Y, and complexin I were downregulated and the NR1 subunit of the NMDA receptor was upregulated by hypoxia. These differential gene regulations could be partially compensated for by clozapine treatment. NMDA receptor binding was decreased at PD 11 and expression of the NR1 subunit was increased at PD 11 and 120. The time course of hypoxia-induced PPI deficits and their reversal by clozapine support the validity of our animal model and the hypothesis that hypoxia as a factor of obstetric complications plays a role in the pathophysiology of schizophrenia. Differential gene expression in cortical and subcortical brain regions as well as correlations to deficits of PPI support the view of an involvement of synapse-associated gene products and glutamatergic and GABAergic neurotransmission in the pathophysiology of behavioral deficits occurring as delayed responses to neonatal hypoxia in adulthood. Key words: Neonatal hypoxia, schizophrenia, animal model, PPI, gene expression, clozapine, presynaptic genes, NMDA receptor.

P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_4, © Springer Science+Business Media, LLC 2011

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1. Introduction In addition to genetic predisposition, the neurodevelopmental hypothesis of schizophrenia includes a number of environmental factors like maternal infection or obstetric and birth complications with ensuing neuronal damage in the pathophysiology of the disease (Fig. 4.1). Hence, the risk of schizophrenia increases with the number and severity of hypoxia-associated obstetric complications (1–3). Furthermore, twin studies revealed fetal hypoxia as a predictor of reduced cortical gray matter and extended ventricular volume (4), smaller hippocampus sizes (5, 6), and early age of onset (7) in schizophrenia patients (8). In the early postnatal period, the developing rat brain is highly vulnerable to hypoxic damage (9). In contrast to humans, important steps of brain development in rats occur mainly postnatally and the brain growth spurt of early postnatal rats is comparable to the third trimester in humans (10). At this stage, overexpression of NMDA receptors has been observed reaching peak levels at postnatal day 8 (11–13). This resembles the peak in NMDA receptor binding and mRNA expression in gestational stages in the human fetus (14, 15). The tetrameric receptor is composed of several distinct subunits, the obligatory NR1 and several facultative NR2 subunits (NR2A, NR2B, NR2C, and NR2D), responsible for different functional properties (16, 17). During postnatal development, NMDA receptors are composed of more NR2B than NR2A subunits and the NR2D subunit is most prominent around postnatal day 7 (17). NMDA receptors

Fig. 4.1. Risk genes and environmental factors such as hypoxia play a role in the pathophysiology of schizophrenia during the perinatal period, whereas symptoms occur in young adulthood after the synaptic pruning process and wiring of the prefrontal cortex. In some patients with a chronic disease, further neurodegenerative changes with volume loss in several brain regions may play an additional role.

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assembled from NR2B or NR2D subunits show higher affinity for the co-agonist glycine than NMDA receptors with more NR2A subunits in the adult brain. They are also less prone to blockade by magnesium than more mature channels (13). During brain development, NMDA receptors are involved in synaptic plasticity, as well as migration of neurons to their final destination (18–22). In this respect, the early postnatal period of rats is most suitable for investigating the role of hypoxia in the pathophysiology of schizophrenia, as it represents a highly vulnerable time period in brain development. In schizophrenia, NMDA receptor hypofunction has been hypothesized, but the etiology of this dysfunction remains elusive. An early developmental event has been proposed to be involved in the process leading to NMDA receptor hypofunction (23). Hypoxia may be part of this course of events and is investigated in the present animal model. This may be additive with genetic factors, many of which involve the area of glutamatergic transmission (24). We have shown that postnatal, repeated hypoxia can precipitate alterations in NMDA receptor binding and gene expression of subunits early after the event and in adulthood. Indeed, in our animal model of chronic neonatal hypoxia, rats at PD 120 exhibited – along with the PPI deficit – increased gene expression of the N-methyl-D-aspartate (NMDA) receptor subunit NR1 in hippocampal, frontal, and temporal subregions as well as in nucleus accumbens (25), which suggests an involvement of the NMDA receptor in the pathophysiology of hypoxia-induced alterations. The pathogenetic implications of obstetric complications are unclear. One hypothesis suggests that obstetric complications are caused by neurodevelopmental pathology (26–28), another speculates that obstetric complications itself cause developmental neuropathology (26, 29). Either argument is consistent with the neurodevelopmental hypothesis of schizophrenia, which suggests that abnormal brain development and neuropathology occur perinatally, whereas symptoms of the disease appear in early adulthood (8). Thus, if a common factor of obstetric complications such as perinatal hypoxia causes neurodevelopmental disturbances, an animal model of neonatal hypoxia should express schizophrenia-related behavioral alterations in early adulthood of the affected rats. Schizophrenia patients show deficits in attention and processing of sensory information. A reliable model of sensorimotor gating in vertebrates is prepulse inhibition (PPI) of the acoustic startle response, which is disrupted in schizophrenia patients and restored by antipsychotics, mainly clozapine (30). In rats, PPI also reflects sensorimotor gating, and several brain regions such as hippocampus, prefrontal cortex, nucleus accumbens, and striatum are involved in PPI-modulating circuitry (31). In a neurodevelopmental animal model in rats, neonatal hippocampal lesion led to decreased PPI, increased locomotor

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activity after amphetamine challenge, and disturbed social interaction in early adulthood (32), while medial prefrontal lesions led to attenuated locomotor response (33). Additional animal models would be important to further elucidate possible roles of neurodevelopmental factors in the pathogenesis of schizophrenia. Thus, the present approach is meant to mirror the impact of chronic hypoxia during human obstetric and birth complications in more detail. Exposure to anoxia at postnatal day (PD) 9 showed no effects on the PPI paradigm in adulthood (34). However, it must be kept in mind that the duration of the anoxia period of the study be kept relatively short, and long-term hypoxia may indeed affect behavior in adulthood. In a recent study applying chronic neonatal hypoxia, rats at PD 120 exhibited a PPI deficit and increased gene expression of the N-methyl-D-aspartate (NMDA) receptor subunit NR1 at PD 11 and 120 in hippocampal, frontal, and temporal subregions as well as in nucleus accumbens (25). This points to the involvement of the NMDA receptor in the pathophysiology of hypoxia-induced alterations. In another study, Hermans and Longo (35) exposed rat pups to nearly the same oxygen concentrations for 6 h/day and showed increased rearing activity and stereotypies in hypoxia-treated animals. However, in our study, hypoxia-treated rats have been closely examined from the preand postpubertal phase until adulthood and effects of antipsychotic treatment have been examined. Rats exposed to hypoxia exhibited deficits in locomotor activity on PD 86, 120, and 150, as well as a PPI deficit on PD 120 and 150 in adulthood, but not before. Chronic treatment with clozapine reversed hypoxiainduced PPI deficits, but not the decreased locomotor activity. In a second experiment, where clozapine was chronically administered before PD 120, development of the PPI deficit in the animals exposed to hypoxia was prevented (36). Here we show that schizophrenia-related behavioral alterations occur in early adulthood of the affected rats. Schizophrenic patients have deficits in processing of sensory information and attention. A reliable model of sensorimotor gating is prepulse inhibition of acoustic startle response (PPI), which is disrupted in schizophrenic patients and restored by antipsychotics (30, 37). In a neurodevelopmental animal model in rats, postnatal hippocampal lesion led to decreased PPI in early adulthood (38). Additional animal models would be important to further elucidate possible roles of neurodevelopmental factors in the pathogenesis of schizophrenia. Thus, our approach is meant to mirror in more detail the impact of chronic hypoxia during human obstetric and birth complications. In schizophrenia, there is strong support for disturbances of the glutamatergic and GABAergic microcircuitry, mainly affecting

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the expression of presynaptic vesicle proteins including SNAP25 and syntaxin, which form the SNARE complex (39–48). The trimeric SNARE complex consists of the vesicle protein synaptobrevin (VAMP) and the plasma membrane proteins syntaxin and SNAP-25, which drive membrane fusion by guiding the vesicle and plasma membrane proteins in close proximity, thus overcoming the energy barrier for fusion. Complexin I (in inhibitory and excitatory synapses) and complexin II (in excitatory synapses) operate at a post-priming step in synaptic vesicle exocytosis by stabilizing the SNARE complex (49). Neuropeptide Y and neurexins play a role in cell adhesion and are hypothesized to play a role in the pathophysiology of schizophrenia (45, 50–52). Little is known about the influence of chronic hypoxia on the expression of those and other presynaptic proteins. Snails exposed to hypoxia showed a decreased expression of syntaxin 1 (53), but long-term effects of perinatal hypoxia on expression of these genes in mammals and their contribution to behavioral deficits in adulthood are unknown. Additionally, there is only sparse information about the influence of antipsychotic treatment on gene expression, as has been shown for neuropeptide Y and complexins in normal rats (54, 55). In our last study, we showed that in several brain regions, presynaptic genes such as SNAP-25, syntaxin 1A, neurexin, neuropeptide Y, and complexin I were downregulated and the NR1 subunit of the NMDA receptor was upregulated by hypoxia. These differential gene regulations could be partially compensated for by clozapine treatment (56). Differential gene expression in cortical and subcortical brain regions as well as correlations to deficits of PPI support the view of an involvement of synapse-associated gene products and glutamatergic and GABAergic neurotransmission in the pathophysiology of behavioral deficits occurring as delayed responses to neonatal hypoxia in adulthood.

2. Materials 1. Air-tight hypoxia chamber 2. Oxymeter (GMH3690, Conrad, Hirschau, Germany) 3. Startle device (for example, TSE systems, Germany, or San Diego Instruments, USA) 4. Arena (ActiMot, TSE Systems, Bad Homburg, Germany) 5. Rat brain atlas (Paxinos and Watson (63))

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6. Cryostat (Leica) 7. Image analysis system (Inter Active Systems, Interfocus, Germany) R , Amersham, Germany) 8. [3 H]-sensitive film (Hyperfilm

9. [3 H] plastic standards (Amersham, Germany) 10.

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11.

14 C

X-ray films (Biomax MR1)

plastic standards (Amersham, Germany)

12. Clozapine (Novartis, Switzerland) 13. RNA extraction kit (RNeasy, Qiagen, Germany) 14. Brain Punch Set (Föhr Medical Instruments, Germany) 15. Agilent 2100 Bioanalyzer 16. Packard ScanArray 5000 scanner 17. Fast Real-Time PCR cycler (Applied Biosystems, Foster City, CA, USA)

3. Methods 3.1. Animals

A total of 116 Sprague Dawley rats from 12 different litters (bred from 12 females and 5 males; Animal Facility, Department of Animal Physiology, University of Tübingen) were used for the experiments. The rats were housed in groups of three to four animals under a 12 h light/dark cycle (light on at 7 a.m.) with food and drinking water available ad libitum. The experiments were carried out in accordance with the international ethical guidelines for the care and use of laboratory animals for experiments (Declaration of Helsinki, NIH guidelines) and were approved by the local animal care committee (Regierungspräsidium Tübingen, ZP 3/02).

3.1.1. Neonatal Hypoxia

From postnatal day (PD) 4–8, we imposed mild chronic hypoxia (11% O2 , 89% N2 ) on six litters (n = 64) and their mothers by placing them in an air-tight plastic chamber (31 × 40 × 15 cm3 ) for a period of 6 h/day (Fig. 4.2). The six other litters (n = 52) were subjected to identical handling conditions but were placed in identical chambers with regular oxygen concentrations (21% O2 = normoxia). In both chambers, the oxygen concentration was controlled by an oxygen measuring device (GMH3690, Conrad, Hirschau, Germany). During hypoxia, mothers were able to care for their pups. Unsystematic observations revealed no obvious effects of hypoxia on maternal behavior during hypoxia or normoxia.

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Fig. 4.2. Air-tight hypoxia chamber with oxymeter and separate oxygen and nitrogen inflow.

3.1.2. Cross-Fostering

To control for possible effects of hypoxia on maternal behavior, the offspring of the four litters in experiment 1 (n = 62) were cross-fostered on PD 9, meaning that half of the offspring of a hypoxia-treated litter were exchanged with half of the offspring of a normoxia-treated litter. All mothers accepted the unknown offspring without problems. At an age of 4–5 weeks, the young rats were separated from the mother or nurse, respectively, and were housed in groups of the same gender under the conditions described above.

3.2. Testing Procedure

Behavioral tests were carried out at three or four different age levels: between PD 34 and 38, between PD 84 and 88, between PD 118 and 122, and between PD 148 and 152 (only experiment 1). For practical purposes, these test phases are named PD 36, PD 86, PD 120, and PD 150 throughout the manuscript. During each test phase (lasting 1 week), every rat was tested first on motor activity in an open field (day 1 or 2 of the test week), second on social interaction and social recognition (day 3 or 4), and third on baseline startle magnitude and prepulse inhibition (day 4 or 5, for details see below). All behavioral tests were carried out in the light phase between 9 a.m. and 4 p.m. During the last two test phases (experiment 1: between PD 120 and PD 150, experiment 2: between PD 86 and PD 120), half of the rats were being treated over a period of 4 weeks with ca. 45 mg/kg/day clozapine (Novartis Pharma AG, Basel, Switzerland) diluted in some drops of HCl in the drinking water adjusted to pH 6.5. Control rats received some drops of HCl in the drinking water to match the pH levels of the drug solutions. The drug dosage was calculated by the amount of ingested water and body weight, which was measured every day and the dose of 45 mg/kg/day

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is an estimate of the average daily intake over the whole period of administration. This route of administration leads to clozapine plasma levels of ca. 22 ng/ml (57), a level which is shown to be pharmacologically active (57). Furthermore, clozapine, using the same doses and the same route of administration, has been shown to alter the glutamatergic and GABAergic system in rat brain (58–61). 3.2.1. Test on Motor Activity

Spontaneous motor activity was tested in a square arena with walls made from transparent Plexiglass (92 × 92 × 39 cm; ActiMot, TSE Systems, Bad Homburg, Germany) where animals were placed over a 10 min period. Illumination of the arena was between 200 and 250 lux. Rat movements were monitored by infrared detectors (distance between two detectors: 2.5 cm and height: 2.5 cm) and rearings were detected by a second arrangement of infrared detectors (height: 12.5 cm). The ActiMot software automatically calculated motor activity (distance traveled in meters), number of rearings, and total time spent in the middle of the arena (size of the middle: 46 cm × 46 cm).

3.2.2. Test on Social Interaction and Social Recognition

Juvenile rats, ca. 30 days old, were used as a social stimulus. Both the test rats and the social stimulus rats were housed in isolation for 1 h before testing. For testing on social interaction (62), both groups of rats were placed in a cage (38 cm × 22 cm × 15 cm) where their behavior could be observed via camera and monitor. The duration of all social behavior (anogenital sniffing, social sniffing, social grooming, play fights) of our test rats within a 5 min period was recorded by an observer who was not aware of the group (anoxia/normoxia, clozapine treatment) to which the observed animal belonged. The animals were then separated and kept in isolation for a period of 30 min. Thereafter, a second test on social interaction between these two rats was carried out. Test values for social recognition were expressed as the ratio of the time spent on social interaction during the second test divided by the time spent on social interaction during the first test.

3.2.3. Test on Baseline Startle Magnitude and Prepulse Inhibition

The startle magnitude was measured with four identical acoustic startle devices in sound-attenuated chambers. For the tests, rats were placed in wire mesh cages with steel floors (20 × 10 × 12 cm3 ) which were fixed onto piezoelectric accelerometers. Movements of the rats caused changes in the voltage output of the accelerometers. These voltage changes were amplified, digitized, and analyzed by a PC. The startle magnitude was computed as the difference of the accelerometer output 80 ms before and 80 ms after the onset of the startling stimulus. Acoustic stimuli were generated by a function synthesizer (Hortmann, Germany) and delivered through loudspeakers mounted 40 cm away from the test cages. Intensity was measured using a 0.5 in. condenser

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microphone and a measuring amplifier (Brüel and Kjaer, Denmark) after bandpass (0.25–80 kHz) filtering. After a 5 min adaptation period, during which the rats received no stimuli except the background noise, the startle test session began with five initial startle stimuli (100 dB SPL broadband noise pulse, 20 ms duration) followed by four different trial types presented in a pseudorandom order: (1) Startle pulse alone (100 dB sound pressure level (SPL) broadband noise pulse, 20 ms duration), (2) 75 dB prepulse (75 dB SPL, 10 kHz tone pulse, 20 ms duration including 0.4 ms rise/fall times) followed by a startle pulse 100 ms after prepulse onset, (3) 75 dB prepulse alone, and (4) no stimulus. Background noise intensity was 55 dB SPL. A total of five presentations of each trial type was given with an interstimulus interval of 20 s. PPI was measured as the difference between the pulse-alone trials and the prepulse–pulse trials and expressed as percent PPI [100 × (mean ASR amplitude on pulse-alone trials – mean ASR amplitude on prepulse–pulse trials)/mean ASR amplitude on pulse-alone trials]. Experiment 2. The aim of experiment 2 was to test whether chronic clozapine treatment before the onset of the behavioral symptoms induced by neonatal hypoxia (PD 120) prevents the development of these symptoms. To minimize factors, the animals of this experiment (n = 54) were not cross-fostered after neonatal hypoxia or normoxia (experiment 1 showed that this factor has no influence on the hypoxia effect) and only male rats were used. Behavioral testing was identical to experiment 1, except that our tests on motor activity were carried out in a cage (55 cm × 32 cm × 19 cm) with one infrared sensor delivering an arbitrary measure of motor activity (“counts”) being used. Unfortunately, this setup did not allow us to measure rearing behavior. 3.3. Brain Investigations

Brains were removed, frozen in liquid nitrogen-cooled isopentane at –80◦ C, and stored for 3 months. For cryostat sections, brains were processed at –20◦ C. Deep-frozen brains were brought to –12◦ C the day before cutting. Cryostat temperature was maintained at –16◦ C. Adjacent coronal 15 μm sections (receptor autoradiography) and 20 μm sections (in situ hybridization) were thaw-mounted on superfrost plus microscopic slides (Roth, Germany), dried, and stored at –20◦ C for less than 5 days (receptor binding). Sections for in situ hybridization were fixed in 4% paraformaldehyde, dehydrated in ethanol, and stored at –20◦ C for several weeks. For the microarray experiments, slices of 120 μm thickness were used to excise respective brain regions using a micro-punching tool. The resultant cylindrical tissue blocks were collected in –78◦ C test tubes (on dry ice) and stored at –80◦ C until further processing. Brain regions were delineated according to the rat brain atlas of Paxinos and Watson (63). We investigated the frontal

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(motor cortex), infralimbic and medial prefrontal cortex (anterior cingulate cortex) (Bregma 3.20 to 2.20 mm), and temporal cortex; hippocampal subregions CA1, CA2, CA3; and dentate gyrus (Bregma –3.60 to –4.30 mm), nucleus accumbens, as well as striatum (caudate and putamen) (Bregma 1.70 to –0.70 mm). 3.3.1. Receptor Autoradiography

Using quantitative in vitro receptor autoradiography, binding to NMDA receptors was performed with specific ligands such as glycine, spermidine, and MK-801 according to the method of Zilles et al. (64, 65). To remove endogenous ligands such as glutamate and glycine, preincubation of the glass-mounted cryostat sections was carried out with 50 mM Tris–HCl buffer (pH 7.5) for a total of 15 min (3×5 min) at 4◦ C. Binding assays were performed as triplicates in the same magnesium- and zinc-free buffer at 22◦ C for 60 min in the presence of 30 μM glycine and 50 μM spermidine for opening of receptor channels and 5 nM [3 H]-MK801 (dizocilpine maleate). In two adjacent sections per region, non-specific binding was determined by adding 10–4 M (+) MK-801 to the incubation solution. Typically, non-specific binding of less than 5% of total binding was found under these conditions. Non-specific binding was subtracted from total binding to obtain specific binding. Tritium-labeled sections were exposed on [3 H]-sensitive R , Amersham, Germany) for 4 weeks. Autorafilm (Hyperfilm diographs were digitized using an image analysis system (Inter Active Systems, Interfocus, Germany) and a video camera (Nikon, Japan). The gray value images of the coexposed [3 H] plastic standards (Amersham, Germany) were used to compute a calibration curve by non-linear, least-squares fitting, which defined the relationship between gray values and concentration of radioactivity. The plastic standards were calibrated to tissue standards prepared from homogenized brain tissue with known protein content in order to express binding site densities in fmol/mg protein. Concentrations of radioactivity were multiplied by (KD +c)/c to obtain Bmax values (KD = equilibration dissociation constants of the ligand binding kinetics, 5 nM for [3 H]-MK-801; c = incubation concentration of labeled ligand).

3.3.2. In Situ Hybridizations

By in situ hybridization and quantitative real-time PCR, we measured mRNA transcripts (Fig. 4.3). To obtain the probes for in situ hybridization, RNA was prepared from postnatal day 2 rat cerebral cortex using a commercial kit (RNeasy, Qiagen, Germany), and cDNA was synthesized with Superscript II reverse transcriptase (Life Technologies, Germany) and oligo-dT primers (PerkinElmer, Germany). After PCR amplification (Promega, Germany) using specific primer sequences, the PCR product was cloned (Sure Clone Ligation Kit, Pharmacia, Germany) into the

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Fig. 4.3. Base pairs are attached to the DNA strand and this messenger RNA represents the information of a gene. After transport to the cytoplasm it is translated into the protein.

EcoRV site of pBluescript-SK. Sequence identity of the insert was confirmed by sequencing. Asymmetrically digested plasmids served as templates for in vitro transcription with T3 or T7 RNA polymerase (MBI Fermentas, Germany), respectively, to obtain antisense and sense cRNA probes. Efficacy of incorporation of radioactively labeled 35 S-dUTP was measured and hybridizations of duplicate brain sections with antisense and sense probes in concentrations of 107 cpm/ml were performed for 16 h at 55◦ C in the presence of 50% formamide, 20 mM Tris, pH 7.5, 1x Denhardt’s solution, 1.25 mM EDTA pH 8.0, 100 mM DTT, 10% dextran sulfate, 2x SSC, 0.1% SDS, and 1 mg/ml yeast RNA hybridization buffer. After stepwise washings in 2x/1.5x/0.2x SSC dilutions, RNase A digest (10 μg/ml RNase A in 1.5x SSC), and dehydration, three slices (antisense) and one slice (sense) per region and animal were exposed to X-ray films (Biomax MR1) and coexposed with 14 C plastic standards for 4–6 days to obtain radioactivity concentration in Bq/mg brain tissue. Autoradiographs were digitized using the image analysis system mentioned above (Fig. 4.4). 3.3.3. RNA Isolation and Reverse Transcription

One milliliter of Trizol was added to excised tissue and the tissue was homogenized by 30 strokes of trituration through a 27 gauge needle attached to a 1 ml syringe. After addition of 200 μl chloroform and thorough mixing, the solution was transferred to Phase-Lock tubes (Eppendorf) for phase separation. Aqueous supernatants were purified and concentrated by RNeasy mini columns (Qiagen). Quality control was performed by OD measurements in a NanoDrop ND-1000 spectrophotometer (260/280 nm >1.8) and by electrophoretic separation

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Fig. 4.4. 35 S-stained rat brain sections and their analysis after exposure to sensitive films (in situ hybridization) allows the measurement of expression of a single gene (for example, NR1) in several delineated brain regions.

using an Agilent 2100 Bioanalyzer (R.I.N. values >7.0). Reverse transcription of purified total RNA was carried out using oligodT primers with T7 promoter sequence and Superscript II (Invitrogen). 3.3.4. In Vitro Transcription and Labeling

Reverse-transcribed cDNAs were treated with RNase and with DNA polymerase to obtain double-stranded DNA. Purified DNA was used for amplification reactions using RNAMaxx High Yield Transcription Kit (Stratagene) with T7 polymerase. Typically, this in vitro transcription resulted in 40- to 50-fold amplification of mRNA/cDNA. Subsequently, cRNA was reverse-transcribed using aminoallyl dUTP for reaction with cyanine 3 or cyanine 5 NHS esters. Labeled DNA was purified and dried in a speed-vac.

3.3.5. Preparation of DNA (“Glutamate”) Chips

The chips represent a hypothesis-driven selection of 340 cDNA sequences of genes related to glutamatergic and GABAergic neurotransmission and synaptic vesicle proteins (M. Zink, unpublished data), supplemented by clones purchased from RZPD (Berlin) (Fig. 4.5). The cDNA inserts of bacterial clones were PCR-amplified directly from aliquots of bacterial cultures grown overnight (LB medium, containing antibiotic selection). After electrophoretic quality control, PCR mixtures were dried, resuspended in betaine-containing spotting buffer (66), and spotted on aminosilane slides (Nexterion, PeqLab) using a MicroGrid II Arrayer. Each DNA was double spotted, and each array was spotted three times on the same slide. Control DNAs were salmon sperm DNA, human COT-1 DNA, four Arabidopsis thaliana DNAs (for spike-ins), spotting buffer, water, and a series of DNAs derived from artificial genes (library of random external controls, LOREC) (67). Altogether, a chip contained 4608 spots, including markers at the array corners to facilitate finding the correct

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Fig. 4.5. After conversion into cDNA, hybridization of fluorescence-stained samples on the microarray chip allows the simultaneous measurement of the expression of thousands of genes.

orientation after scanning. After spotting, DNAs were UV crosslinked with a radiation of 250 mJ/cm2 . Then they were baked at 80◦ C for 2 h and stored at RT. 3.3.6. Hybridization and Scanning of Slides

Spotted slides were washed in 1% SDS solution and in distilled water. Then they were immersed in 95◦ C distilled water and immediately in 55◦ C prehybridization solution, where they were left for 45 min. Then they were washed again in distilled water and in isopropanol for 20 s each. Finally, they were dried by a stream of nitrogen or air. For hybridizations, SlideHyb Glass Array Hybridization buffer #1 (Ambion) was used throughout. Hybridizations were done overnight at 56◦ C in a water bath with shaking. All subsequent washing steps at increasing stringency were performed in the dark. Scanning was carried out in a Packard ScanArray 5000 scanner. Each slide was scanned twice at laser strengths of 90 and 100% to obtain a larger spectrum of fluorescence intensities.

3.3.7. Data Processing

Data quality was assessed by a number of procedures, including visual inspection of intensities, MA plots, and correlation analysis of replicates. Within-array normalization was performed according to locally weighted scatterplot smoothing (LOESS) method (68). Only rank-invariant probes were used for model generation during this procedure (69). For between-array normalization, scaling (68) was used. After assessing normal distribution of replicate spots using the Kolmogorov–Smirnov test, replicates

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were pooled by their arithmetic means. To identify differentially expressed genes, two-tailed t tests and the RankProduct (RP) (70) method were used. The false discovery rate (FDR) method was employed to avoid accumulation of alpha-error. Corrected p values <0.05 were considered significant. To limit the number of differentially expressed genes, only those that were found by both (t test and RP) methods were used. Finally, to profile overall regulation, only genes that were differentially regulated in at least two regions were selected for further analyses. 3.3.8. Quantitative Real-Time PCR (qRT-PCR)

Quantitative RT-PCR was carried out in 384-well plates in an ABI Prism 7900 HT Fast Real-Time PCR cycler (Applied Biosystems, Foster City, CA, USA). Beta-actin was used as a reference (“house-keeping”) gene. The primer design was such that the annealing temperatures were maintained in a very narrow range for all of them. Pipetting was carried out by a Multi Probe II pipetting robot (PerkinElmer) that reached a reliability and precision far better than by manual pipetting. Data evaluation was performed with the freely available GNU-R statistics software. After normalization across plates through actin-pool DNA values, Ct values were generated by subtraction of the actin value of each sample (delta/delta Ct). After test of Ct values for normal distribution through Shapiro–Wilk test, they were subjected to a multifactorial analysis of variance (ANOVA).

4. Notes Our studies also entail some other limitations. First, our animal model may not mimic the situation of different neurotransmitter systems during neurodevelopment in humans. This has been shown by different brain growth spurts (10). Second, we investigated receptor binding and gene expression only at PD 11 and 120 and did not show the time course of neurobiological parameters. Additionally, our findings of altered gene expression may not directly impact on receptor protein, since altered gene expression is not always reflected in protein expression. Third, although mothers were able to care for their pups, during hypoxia it cannot be excluded that additional malnutrition occurs during the hypoxic periods and future studies should control for maternal behavior. In humans, malnutrition during gestation has been shown to be a risk factor of schizophrenia (71). However, we did not find weight differences in pups between treatment groups. In a subsequent study of effects of postnatal hypoxia on behavior

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where we used cross-fostering of pups we did find the same effects on deficits of prepulse inhibition (36). The aim of the behavioral studies was to further validate the PPI deficit in adult rats induced by neonatal chronic hypoxia as a developmental animal model for schizophrenia (36). Accordingly, in this study we addressed several critical gaps of our previous study (25): First, we cross-fostered the pups after neonatal hypoxia to control for potential hypoxia effects on maternal behavior (experiment 1). Second, we repeatedly tested the same animals pre- and postpubertally as well as in adulthood (PD 36, PD 86, PD 120, PD 150; experiment 1). Third, the effects of chronic, neonatal hypoxia were tested using three different behavioral tests: PPI (as well as baseline startle magnitude), social interaction and recognition, and locomotor activity in an open field (experiment 1). Fourth, for validation of this animal model we subjected subgroups of rats to chronic treatment with the antipsychotic drug clozapine before (experiment 2) and after (experiment 1) onset of behavioral symptoms. Our study shows a PPI deficit and decreased motor activity in adult animals after neonatal hypoxia while their behavior was not affected before onset of puberty compared to controls. This points to an increased vulnerability for psychotic-like symptoms and to the impact of chronic hypoxia as an important factor of obstetric complications playing a role in the pathophysiology of schizophrenia. Further animal studies investigating additional apomorphine or MK-801 challenge and the combination of neonatal hypoxia with adult stress or genetic risk factors may lead to better animal models of schizophrenia comprising several hits during brain development. In brains of the affected animals, we have shown alterations in NMDA receptor binding and gene expression of NMDA receptor subunits after repeated hypoxia in regions involved in the pathophysiology of schizophrenia such as cortex, nucleus accumbens, and hippocampus. Additionally, disruption of PPI has been observed in adult animals and may be related to an NMDA receptor dysfunction. Further animal studies investigating protein levels, neurodegeneration, and treatment effects of other antipsychotics should also improve the validity of this animal model. Presynaptic proteins have been reported to be differentially regulated in schizophrenia. In our animal model of perinatal hypoxia, alterations of presynaptic genes in adulthood during the time period of behavioral deficits have been shown and may be involved in the pathophysiology of environmental factors leading to schizophrenia symptoms. Additionally, chronic clozapine treatment may differentially influence gene expression of these proteins in hypoxic and normoxic animals and in this way contribute to the treatment of symptoms. Since in postmortem studies most of the patients have been treated with antipsychotics,

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Chapter 5 The Developmental Vitamin D (DVD) Model of Schizophrenia Darryl W. Eyles, Thomas H.J. Burne, Suzy Alexander, Xiaoying Cui, and John J. McGrath Abstract It is now widely acknowledged that exposure to adverse environmental factors in utero may not only affect how the brain develops but have long-lasting consequences for later brain function in the adult offspring. This idea has gained particular prominence amongst researchers interested in the etiology of neurodevelopmental disorders such as schizophrenia and autism. Approximately 10 years ago we proposed that developmental vitamin D (DVD) deficiency may explain several epidemiological features of this disease, most noticeably the winter/spring season of birth effect. In 2003 we published results from our first study indicating there were structural changes in how the brain develops in these offspring. Since then we have firmly established that DVD deficiency not only affects brain cell differentiation and gross anatomy but also produces alterations in behavior in these offspring as adults. In this chapter we describe how we came to construct the model we use today. Over the past 7 years the model has proved informative producing both structural brain changes (ventriculomegaly) and behavioral alterations (hyperlocomotion in response to NMDA antagonists) that are thought to be relevant to schizophrenia. Key words: Vitamin D, brain, animal model, development, schizophrenia, behavior.

1. Introduction Despite many decades of concerted, multi-disciplinary research, the etiology of schizophrenia remains poorly understood. In keeping with its clinical heterogeneity, schizophrenia is almost certainly a syndrome with many different etiological factors. The symptoms that contribute to making a clinical diagnosis include hallucinations, delusions, disordered thoughts, and alterations in affect and cognitive impairments – all higher cognitive features. Additionally although the heritability of the disorder is claimed to be as high as 80% (1), the identification P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_5, © Springer Science+Business Media, LLC 2011

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of genetic candidates in all populations has remained elusive (2). For these reasons, it is not surprising that schizophrenia has proved a difficult, some would even suggest impossible, disorder to model in animals. Nevertheless, there remains a stubbornly persistent developmental epidemiology that strongly supports the neurodevelopmental hypothesis of schizophrenia (3, 4). For instance, prenatal exposure to infection, pregnancy and birth complications, and winter/spring birth are all associated with increased risk of developing schizophrenia in later life (5). In general, it is thought that these adverse exposures act against a backdrop of a “vulnerable” genome, which in turn adversely impacts brain development. The neurodevelopmental hypothesis suggests that there are critical early periods of brain development when certain adverse genetic and/or environmental factors contribute to disease susceptibility (6). Despite the inherent difficulties in trying to develop an animal model of a disorder that affects higher cognitive ability (7), results from animal models based on clues from epidemiology have provided important new clues (8). For example, rodent models based on prenatal exposure to virus-like agents (e.g., the synthetic double-stranded RNA and poly I:C), or bacterial membranes, have been used to explore the neurobiological correlates of maternal infection (9). Similarly, pre- or perinatal hypoxia has been used to study obstetric complications (10). Our group has developed an animal model related to prenatal vitamin D deficiency as a plausible explanation for several important epidemiological observations in schizophrenia risk factor biology. Many studies have shown that those born in winter and spring have a significantly increased risk of developing schizophrenia (11). The size of the winter/spring excess increases at higher latitudes (12) and the incidence and prevalence of schizophrenia is also greater in sites at high latitudes (13). Curiously, the incidence of schizophrenia is also significantly higher in dark-skinned migrants to cold countries compared to the native born (14). Given that hypovitaminosis D is more common (a) during winter and spring, (b) at high latitudes, and (c) in dark-skinned individuals (15), low prenatal vitamin D “fits” these key environmental features. It is now 10 years since we first proposed that low prenatal vitamin D may be a risk factor for schizophrenia (16). Initial support for this idea came from studies that established that vitamin D supplementation in the first year of life significantly reduced the risk of schizophrenia in males from a large Finnish birth cohort (17). In addition, a pilot study found that 25-hydroxyvitamin D serum levels in 26 mothers whose children developed schizophrenia were nonsignificantly lower than those of 51 control mothers whose children did not develop the disease, but this group difference was more prominent in mothers with dark skin (18). Larger

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studies investigating a direct link between neonatal vitamin D levels and risk for schizophrenia in later life are ongoing. In order to establish the biological plausibility of this hypothesis, we have developed an animal model to study the impact of developmental vitamin D (DVD) deficiency on a range of brain outcomes (structure, function, neurochemistry, genomics, proteomics, and behavior). We have shown that the brains from DVD-deficient neonates have larger lateral ventricles (19). Brain differentiation also appears delayed with a generalized increased cellular proliferation, increased neurogenesis, and reduced apoptosis (19–21). Ventricular enlargement persists in these animals as adults (22). Behaviorally, adult DVD-deficient rats are more active than controls in novel environments (23, 24). DVDdeficient rats also have enhanced locomotion in response to psychomimetic agents (agents that induce psychosis) such as the NMDA antagonist MK-801(24, 25). The DVD-deficient adult rat is also more sensitive to the widely used antipsychotic haloperidol, a dopamine 2 (D2) receptor antagonist (24). Several features of the DVD-deficient phenotype are therefore informative for schizophrenia research: (a) increased lateral ventricular volume is one of the most consistent neurobiological correlates of schizophrenia (26); (b) behavioral sensitivity to NMDA antagonists is also displayed by patients and is thought to reflect an underlying abnormality in neurotransmission consistent with the hallucinatory or positive symptoms of schizophrenia (27). Intriguingly, our most recent experiments have established that dopamine ontogeny appears to be affected in the developing brains of DVD-deficient animals and that there are also several learning deficits in animals from this model (unpublished observations). In this chapter, we outline how we create a DVD-deficient rat and pitfalls in the process of developing this model.

2. Materials Three diets are used in this model: • The control diet used during gestation and rearing is AIN93G rodent diet custom formula #110700 (Dyets, Inc., Bethlehem, PA, USA) – with #210025 salt mix and #310025 vitamin premix. The amount of pre-vitamin D, cholecalciferol (see Note 1), is 1000 IU/kg, calcium is 5 g/kg, and phosphorous is 1.5 g/kg. • The deplete diet used during gestation and rearing is AIN93G rodent diet custom formula #119266 – (Dyets) with the same salt and vitamin premix – minus cholecalciferol (see Note 2). Calcium and phosphorous contents are

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the same. A casein rather than a cereal-based diet is used as it can be stripped of vitamins and then added back in. This is not possible in non-purified diets such as the grain-based diets. • Weanling diet (all experimental animals) – Rat & Mouse Cubes (Specialty Feeds, Glen Forrest, WA, Australia). Cholecalciferol content is 2000 IU/kg, calcium 8 g/kg, and phosphorous 7 g/kg. The control and deplete diets need to be stored in a dry, cool, dark location, in airtight containers. Shelf life is ∼3 months at 4◦ C (refrigerated) but up to 6 months if stored at less than – 20◦ C (see Note 3). The micronutrient contents of these diets are vulnerable to various sterilization procedures such as irradiation and autoclaving (procedures required in certain animal houses) (see Note 4). Finally we recommend a close inspection of diet constituents if prepared locally (see Note 5). A measure of serum 25-hydroxyvitamin D3 (25OHD3) is the best indication of overall vitamin D3 status (28). We have developed an assay using LC/MS/MS technology that requires a small volume of blood (3 μl) to routinely assess maternal vitamin D status (29). A single drop of blood is drawn routinely from the saphenous vein (30). A commercial radio-immunoassay (Dia Sorin, Inc., Stillwater, MN, USA) can also be used; however, this requires substantially more whole blood to obtain the 50 μl sera required. PTH was measured using a commercial ELISA kit (Immutopics, San Clemente, CA, USA) and calcium and phosphate were analyzed independently with an AutoAnalyser (Hitachi Instruments, Tokyo, Japan).

3. Methods 3.1. Preparation of the Dams

All procedures were performed with the approval of the University of Queensland Animal Ethics Committee, under the guidelines of the National Health and Medical Research Council of Australia. Every 2 weeks, eight-four-week-old female Sprague– Dawley rats (see Note 6) from a specific pathogen-free source are selected. These females come from two litters and four females from each litter are used. Two females from each of the two litters are assigned to a control diet (#110700) and a corresponding two females from the same two litters are assigned to the vitamin D deplete diet (#119266). All breeding animals are housed in groups of four in incandescent light devoid of ultraviolet B radiation on a 12-h light/dark cycle (lights on 0600 h), at a constant temperature of 21 ± 2◦ C and 60% relative humidity, with food

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Time taken to deplete a female Sprague-Dawley rat of 25OHD3

35

30

250HD3 ng/ml

25

20

Control Deplete

15

10

5

0 0

2

4

6

Weeks

Fig. 5.1. Time taken to deplete a female Sprague–Dawley rat of 25OHD3 (•) compared with a control dam (◦) over the same pre-breeding period (mean ± SD). After 6 weeks on a diet deficient in cholecalciferol (119266; Dyets, PA, USA), 25OHD3 is virtually absent.

and water provided ad libitum. These conditions are maintained for 6 weeks prior to mating. Six weeks is sufficient to ensure depletion of 25OHD3 (Fig. 5.1) (see Note 7). 3.2. Preparation of the DVD-Deficient and Control Offspring

At this 10-week period, both vitamin D-deplete females and control females are mated with vitamin D normal males. Males are placed in with the females for 7 days. For experiments in embryos or fetuses where gestational time is required, dams are plug checked and vaginal smears are taken to ensure sperm presence once a day for the first 5 days (or until sufficient dams are confirmed). It is plausible that vitamin D-deficient females could absorb some vitamin D through grooming the vitamin D-replete male during breeding. We cannot rule this possibility out; however, to date we have not observed any increase in vitamin D levels in DVD-deficient females post-mating. This breeding protocol results in approximately 67% pregnancy rates independent of diet. Prospective pregnant dams remain on their respective diets throughout gestation and are housed in their same groups of four, until embryonic day 20 (E20), when they were housed individually and provided with nesting materials. When neonates are required, expectant dams are checked twice a day, beginning with the morning of E21, until litters are born. The first appearance of pups was recorded within 12 h of birth, and this day is referred to

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as postnatal day 0 (P0). On the day dams litter, both control and deplete dams are placed on the casein vitamin D-containing diet (#110700) and they remain under the same lighting conditions until the pups are weaned, at which time they are transferred to a separate animal holding room with standard fluorescent lighting and the dam is culled. Litter sizes of between 8 and 18 are considered normal for Sprague–Dawley rats (31). At birth, litters of size less than 8 or greater than 18 are rejected (see Note 8). Both control and vitamin D-deficient dams and their respective offspring remain on the control diet (diet #110700) till weaning. In pups, 25OHD3 levels return to control levels by weaning (Fig. 5.2). All pups are weaned at 21 days into same sex groups of no specific number and all offspring are placed on a standard cereal-based rat chow that contains vitamin D (diet #119266). At 4 weeks of age, animals are split into groups of 2, 3, or 4, and housed in open-top wire cages, with no additional environmental enrichment. All animals remain under these conditions until behavioral testing (5, 10, or 20 weeks) after which the brain is analyzed for gross or cellular anatomy or protein or mRNA expression.

45

25OHD3 repletion from birth in pups control depletes

40 35 30 250HD3 ng/ml

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25 20 15 10 5 0 1 2 weeks post conception

3

Fig. 5.2. Time taken to replete a vitamin D-deficient dam (•) compared with a control dam (◦) over the same postnatal period (mean ± SD). 25OHD3 levels in vitamin D-deficient dams return to control levels 2 weeks after vitamin D is reintroduced in the diet. Horizontal lines indicate 25OHD3 concentration range (mean ± 1 SD) in control dams over this same postnatal period.

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Vitamin D deficiency had no effect on dam or offspring weight gain either during pregnancy or after conception (Table 5.1). Serum calcium and phosphate measures were also normal in vitamin D-deficient dams despite a slight elevation in PTH across the period of pregnancy (Table 5.2). Rates of pregnancy and fecundity were also unaltered by maternal vitamin D depletion (Table 5.3). These findings conflict with other more drastic models of maternal vitamin D deficiency (see below).

Table 5.1 Vitamin D deficiency had no effect on dam or offspring weight gain either during pregnancy or after conception DVDdeficient offspring weight, male (g)

DVDdeficient offspring weight, female (g)

Time

Control dam weight (g)

Vitamin D–deplete dam weight (g)

Conception

269±12

258±9

E7

281±6

294±16

E14

308±4

328±16

P0

298±5

321±14

6.7±0.8

6.6±0.9

6.4±0.8

6.5±0.7

P7

338±7

337±10

21.5±1.9

21.9±1.4

20.9±1.1

20.8±1.3

P14

335±6

339±11

42.7±2.2

43.7±5.6

42.3±2.9

42.8±4.1

P21

328±6

325±6

61.4±3.0

64.7±8.8

60.4±4.6

61.6±6.0

P35

184.9±12.6

178.4±11.6

147.6±7.6

139.7±8.8

P70

461.1±38.6

475.8±43.2

265±20.9

264.3±30.5

Control offspring weight, male (g)

Control offspring weight, female (g)

Table 5.2 Vitamin D deficiency did not affect serum calcium or phosphate at any stage of pregnancy. PTH levels were however elevated Time

Ca2+ (mM)

PO4 (mM)

PTH (pg/ml)

Control dam

Vitamin D-deficient Control dam dam

Vitamin D-deficient Control dam dam

Vitamin D-deficient dam

Preconception

2.92±0.02

2.88±0.02

2.68±0.18

2.51±0.21

177.4±21.3

350.6±59.5∗

E7

2.86±0.04

3.01±0.03

2.20±0.14

2.47±0.12

98.5±20.8

280.5±55.8∗

E14

2.94±0.06

2.95±0.03

2.52±0.13

2.57±0.15

75.3±22.8

189.5±73.1‡

P0

3.38±0.07

3.25±0.08

2.21±0.29

1.82±0.27

∗ P<0.01; ‡ P<0.1 >0.05 relative to control PTH.

233.7±104.0 725.2±191.2‡

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Table 5.3 Rates of pregnancy and fecundity are unaltered by maternal vitamin D depletion Measure

Control

DVD deficient

%Pregnancies

67.8

67

Average litter size

11.4

11.8

Male/female ratio

1.1

1.0

Litters <8 pups

7.2%

6.4%

Litters >16 pups

3.0%

0.8%

The levels of 25OHD3 and 1,25OH2D3 in pups at birth reflect those seen in dams during pregnancy (25). DVD-deficient newborns are also normocalcemic (i.e., neither the dams nor their offspring have the rickets-like phenotype that would result in more chronic vitamin D depletion) (see Note 9). Observation of the offspring from birth to weaning indicated that maternal vitamin D depletion did not affect the progression of normal development or physical maturity. For instance, there were no significant differences between dietary groups on physical maturity scores (eye and ear opening, ear unfolding, fur development and teeth protrusion, self-righting reflex, and posture or stepping activity) (25). All the above physical and endocrine measures were also normal at the time of behavioral testing at either 10 or 20 weeks.

4. Other Models of Maternal Vitamin D Deficiency and General Measures of Pup Health

We have considered the effects of varying the duration of maternal vitamin D deficiency in the development of this model. We have examined both a shorter period of vitamin D depletion (e.g., gestation only) and two longer periods extending DVD deficiency (e.g., until weaning and throughout adulthood). Although gestational deficiency was also sufficient to produce important behavioral deficits in the offspring (e.g., NMDA antagonist-induced hyperlocomotion) (25), the level of the active form of the hormone 1,25OH2D3 in maternal animals was still well within control levels despite profoundly deficient levels of 25OHD3 (25). Extending maternal vitamin D deficiency until weaning produces changes in gross brain architecture consistent with schizophrenia (22); however, deficiency extended into the period of rearing increases possible associated physiological abnormalities such

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as hypocalcaemia, maternal weight loss, and reduced fertility (32). Lifelong vitamin D depletion apart from being completely non-physiological produced hypocalcaemia and associated cardiovascular and kidney abnormalities (33) and was therefore rejected. We have consistently shown that this model produces no gross abnormalities in birth outcomes, growth rates, or calcium status in either the vitamin D-deficient dams or DVD-deficient pups. However, other models of maternal vitamin D deficiency have not been so benign. Studies in the early 1980s reported that if female rats were kept vitamin D deficient for ≥90 days prior to mating, and during the period of gestation and rearing, then maternal growth and fecundity was reduced (34–36). Hypocalcemia in dams was also prevalent, but not universal (37). Curiously, where assessed, it appeared that in these earlier studies, pups appeared to be calcium normal. It has been suggested that fertility issues in the long-term vitamin D-depleted dams were secondary to low serum calcium and phosphorus rather than vitamin D deficiency per se (38). The duration of maternal vitamin D depletion prior to mating used in our DVD model (42 days) is insufficient to affect maternal calcium and has no adverse effects on fertility, fecundity, or various measures of pup growth. However, this issue would appear to be not completely resolved with a recent study by a Japanese group showing that in female rats depleted in a similar fashion to that used here, both the gravid Sprague–Dawley dams and their offspring were hypocalcemic and fetuses were growth restricted (39). We advise those establishing the DVD-deficient model to first determine that their conditions do not have any adverse effects on either serum calcium levels or the aforementioned indices of maternal or fetal growth.

5. Conclusions Most of our published data have been generated from offspring who experience a transient gestational period of vitamin D deficiency. Since our first studies were published (19), we have refined certain experimental factors such as lighting, litter size, and maternal calcium supplementation: We remain interested in what impact variations in the duration of developmental vitamin D depletion would have for brain development and function. We are also interested in the application of this model in other experimental animals such as wild-type (40) and transgenic mice. Other models could also be employed that, although having less face validity for the environmental nutrient deficiency being studied, could also reveal much about how vitamin D

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affects brain development. For example, studies that allow prolonged but less severe hypovitaminosis D also warrant inspection. Additionally, after birth, maternal vitamin D deficiency could be more rapidly reversed than the simple dietary intervention used here either with injections of the active hormone 1,25(OH)2D3 or by cross-fostering DVD-deficient offspring to control vitamin D normal dams. However, these models would require a substantial amount of preliminary studies to establish the correct drug/dosage/dosing interval prior to any consideration of their suitability for investigation.

6. Notes 1. Cholecalciferol is pre-vitamin D3. This compound is oxidized largely in the liver to form 25OHD3. 25OHD3 undergoes further oxidation in a variety of organs but primarily in the kidney to form the active hormone 1,25(OH)2 vitamin D3. This form of the vitamin is far more labile. 2. When the vitamin D-deficient diet is prepared, making the control and deficient diet of different colors can help reduce mistakes in the animal house. 3. Storage conditions for the control and deplete diets are also particularly critical due to the lack of antioxidants and preservative agents. We have observed that if the diet becomes compromised via storage artifact (i.e., temperatures >4◦ C, high humidity, and continuous exposure to air and light) or if used beyond extended shelf life (more than 6 months at less than –20◦ C or 3 months at less than 4◦ C), pregnancy is severely compromised. If possible, we recommend diets should be vacuum sealed and stored at –20◦ C in the dark to reduce the amount of oxidation. 4. Some countries require strict sterilization procedures prior to importing food. Similarly most animal houses require food to be sterilized. We have found that treatments such as irradiation and autoclaving also interfere with viable animal breeding presumably due to reduced vitamin and nutrient content. The weanling diet is cereal based and can be stored at room temperature prior to autoclaving, for up to 6 months. Once autoclaved, food should be used within 4–6 weeks. The control and vitamin D-deficient diets are casein based and do not withstand autoclaving. We have observed that when sterilized with gamma ionizing irradiation from cobalt-60 at a minimum dose of 2.5 Mrad (25 kGy), successful breeding is also dramatically reduced. If possible we

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recommend another sterilization procedure. To avoid the use of radiation in Australia, we have had to separate our animals during breeding and gestation into separate quarantineapproved premises. 5. We have also had the experience of local manufacturers modifying the original formula to suit locally available raw materials but resulting in compromised pregnancy. Close inspection of each recipe is recommended. The American Institute of Nutrition’s prescribed formula for the AIN93G rodent diet states the requirement of two fatty acids linoleic [18:2(n-6)] and linolenic [18:3(n-3)] as dietary essentials and defines soybean oil as the ideal source of fat to provide these in the right ratio. Substitution of soybean oil for locally available oils, such as canola oil, not only alters the ratio of these essential fatty acids but also often has different biological interactions with vitamin E, hence exacerbating the volatility of vitamin E and possibly increasing the risk of impaired pregnancy. Additionally we have experienced alterations in the prescribed carbohydrates (cornstarch and dextrinized cornstarch) in preparations. This will alter pellet formation and appearance (look/feel/touch), therefore influencing palatability as well as vitamin and mineral dispersal during manufacturing. 6. Almost all published studies on DVD deficiency and behavioral or brain outcomes have been published in rats rather than mice. We have one study outlining DVD-deficient behavioral outcomes in mice (40). An outbred strain such as a Sprague–Dawley rat is possibly subject to greater variation in experimental outcomes compared to say an inbred mouse strain. This strain however is available internationally and has been widely used in behavioral studies modeling schizophrenia. 7. The level of maternal vitamin D depletion in the model is marked; however, these levels have been reported in pregnant women during winter and spring months (41, 42). 8. This has not always been the case. For instance, in the first version of the DVD-deficient model, litters were culled to only two male pups (19). In later versions of the model, only males were tested from litters which were culled to six males and two females at birth (24, 25). A further variation was to restrict litter size to three males and three females (22). In recent years we have abandoned the “cult of culling” in favor of using offspring that reflect the natural variance in litter size from Sprague–Dawley rats. 9. This model has been adopted by collaborators who amongst other minor modifications have included 2 mM Ca2+ in

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the drinking water of both controls and vitamin D-deficient females. We see this as an unnecessary step as serum calcium status is unaffected by this degree of vitamin D depletion. References 1. Sullivan, P. F., Kendler, K. S., and Neale, M. C. (2003) Schizophrenia as a complex trait: evidence from a meta-analysis of twin studies, Arch Gen Psychiatry 60, 1187–1192. 2. Purcell, S. M., Wray, N. R., Stone, J. L., Visscher, P. M., O’Donovan, M. C., Sullivan, P. F., and Sklar, P. (2009) Common polygenic variation contributes to risk of schizophrenia and bipolar disorder, Nature 460, 748–752. 3. Weinberger, D. R. (1987) Implications of normal brain development for the pathogenesis of schizophrenia, Arch Gen Psychiatry 44, 660–669. 4. Murray, R. M., and Lewis, S. W. (1987) Is schizophrenia a neurodevelopmental disorder? Br Med J (Clin Res Ed) 295, 681–682. 5. McGrath, J., Saha, S., Welham, J., El Saadi, O., MacCauley, C., and Chant, D. (2004) A systematic review of the incidence of schizophrenia: the distribution of rates and the influence of sex, urbanicity, migrant status and methodology, BMC Med 2, 13. 6. McGrath, J. J., Feron, F. P., Burne, T. H., Mackay-Sim, A., and Eyles, D. W. (2003) The neurodevelopmental hypothesis of schizophrenia: a review of recent developments, Ann Med 35, 86–93. 7. Arguello, P. A., and Gogos, J. A. (2006) Modeling madness in mice: one piece at a time, Neuron 52, 179–196. 8. McGrath, J. J., and Richards, L. J. (2009) Why schizophrenia epidemiology needs neurobiology – and vice versa, Schizophr Bull 35, 577–581. 9. Meyer, U., Feldon, J., and Fatemi, S. H. (2009) In-vivo rodent models for the experimental investigation of prenatal immune activation effects in neurodevelopmental brain disorders, Neurosci Biobehav Rev 33, 1061–1079. 10. Boksa, P. (2004) Animal models of obstetric complications in relation to schizophrenia, Brain Res Brain Res Rev 45, 1–17. 11. Torrey, E. F., Miller, J., Rawlings, R., and Yolken, R. H. (1997) Seasonality of births in schizophrenia and bipolar disorder: a review of the literature, Schizophr Res 28, 1–38. 12. Davies, G., Welham, J., Chant, D., Torrey, E. F., and McGrath, J. (2003) A systematic review and meta-analysis of Northern Hemi-

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23.

sphere season of birth studies in schizophrenia, Schizophr Bull 29, 587–593. Saha, S., Chant, D. C., Welham, J. L., and McGrath, J. J. (2006) The incidence and prevalence of schizophrenia varies with latitude, Acta Psychiatr Scand 114, 36–39. Cantor-Graae, E., and Selten, J. P. (2005) Schizophrenia and migration: a meta-analysis and review, Am J Psychiatry 162, 12–24. Holick, M. F. (1995) Environmental factors that influence the cutaneous production of vitamin D, Am J Clin Nutr 61, 638S–645S. McGrath, J. (1999) Hypothesis: is low prenatal vitamin D a risk-modifying factor for schizophrenia? Schizophr Res 40, 173–177. McGrath, J., Saari, K., Hakko, H., Jokelainen, J., Jones, P., Jarvelin, M. R., Chant, D., and Isohanni, M. (2004) Vitamin D supplementation during the first year of life and risk of schizophrenia: a Finnish birth cohort study, Schizophr Res 67, 237–245. McGrath, J., Eyles, D., Mowry, B., Yolken, R., and Buka, S. (2003) Low maternal vitamin D as a risk factor for schizophrenia: a pilot study using banked sera, Schizophr Res 63, 73–78. Eyles, D., Brown, J., Mackay-Sim, A., McGrath, J., and Feron, F. (2003) Vitamin D3 and brain development, Neuroscience 118, 641–653. Ko, P., Burkert, R., McGrath, J., and Eyles, D. (2004) Maternal vitamin D3 deprivation and the regulation of apoptosis and cell cycle during rat brain development, Brain Res Dev Brain Res 153, 61–68. Cui, X., McGrath, J. J., Burne, T. H., Mackay-Sim, A., and Eyles, D. W. (2007) Maternal vitamin D depletion alters neurogenesis in the developing rat brain, Int J Dev Neurosci 25, 227–232. Feron, F., Burne, T. H., Brown, J., Smith, E., McGrath, J. J., Mackay-Sim, A., and Eyles, D. W. (2005) Developmental vitamin D3 deficiency alters the adult rat brain, Brain Res Bull 65, 141–148. Burne, T. H., Becker, A., Brown, J., Eyles, D. W., Mackay-Sim, A., and McGrath, J. J. (2004) Transient prenatal vitamin D deficiency is associated with hyperlocomotion in adult rats, Behav Brain Res 154, 549–555.

The Developmental Vitamin D (DVD) Model of Schizophrenia 24. Kesby, J. P., Burne, T. H., McGrath, J. J., and Eyles, D. W. (2006) Developmental vitamin D deficiency alters MK 801-induced hyperlocomotion in the adult rat: An animal model of schizophrenia, Biol Psychiatry 60, 591–596. 25. O’Loan, J., Eyles, D. W., Kesby, J., Ko, P., McGrath, J. J., and Burne, T. H. (2007) Vitamin D deficiency during various stages of pregnancy in the rat; its impact on development and behaviour in adult offspring, Psychoneuroendocrinology 32, 227–234. 26. Harrison, P. J., and Weinberger, D. R. (2005) Schizophrenia genes, gene expression, and neuropathology: on the matter of their convergence, Mol Psychiatry 10, 40–68; image 45. 27. Laruelle, M., Frankle, W. G., Narendran, R., Kegeles, L. S., and Abi-Dargham, A. (2005) Mechanism of action of antipsychotic drugs: from dopamine D(2) receptor antagonism to glutamate NMDA facilitation, Clin Ther 27 Suppl A, S16–S24. 28. Hollis, B. W. (1996) Assessment of vitamin D nutritional and hormonal status: what to measure and how to do it, Calcif Tissue Int 58, 4–5. 29. Eyles, D., Anderson, C., Ko, P., Jones, A., Thomas, A., Burne, T., Mortensen, P. B., Norgaard-Pedersen, B., Hougaard, D. M., and McGrath, J. (2009) A sensitive LC/MS/MS assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots, Clin Chim Acta 403, 145–151. 30. Hem, A., Smith, A. J., and Solberg, P. (1998) Saphenous vein puncture for blood sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret and mink, Lab Anim 32, 364–368. 31. Palmer, A. K., and Ulbrich, B. C. (1997) The cult of culling, Fundam Appl Toxicol 38, 7–22. 32. Halloran, B. P., and DeLuca, H. F. (1979) Vitamin D deficiency and reproduction in rats, Science 204, 73–74. 33. Maka, N., Makrakis, J., Parkington, H. C., Tare, M., Morley, R., and Black, M. J. (2008) Vitamin D deficiency during pregnancy and

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lactation stimulates nephrogenesis in rat offspring, Pediatr Nephrol 23, 55–61. Brommage, R., and DeLuca, H. F. (1984) A maternal defect is responsible for growth failure in vitamin D-deficient rat pups, Am J Physiol 246, E216–E220. Thomas, M. L., and Forte, L. R. (1982) Serum calcium and parathyroid hormone during the reproductive cycle in normal and vitamin D-deficient rats, Endocrinology 110, 703–707. Halloran, B. P., and DeLuca, H. F. (1980) Effect of vitamin D deficiency on fertility and reproductive capacity in the female rat, J Nutr 110, 1573–1580. Hickie, J. P., Lavigne, D. M., and Woodward, W. D. (1983) Reduced fecundity of vitamin D deficient rats, Comp Biochem Physiol A Comp Physiol 74, 923–925. Johnson, L. E., and DeLuca, H. F. (2002) Reproductive defects are corrected in vitamin d-deficient female rats fed a high calcium, phosphorus and lactose diet, J Nutr 132, 2270–2273. Yamagishi, N., Sassa, H., Sato, R., Taniguchi, K., Okura, N., Sato, S., and Naito, Y. (2007) Calcium metabolism of pregnant rats fed a vitamin D-depleted diet, J Vet Med Sci 69, 441–443. Harms, L. R., Eyles, D. W., McGrath, J. J., Mackay-Sim, A., and Burne, T. H. (2007) Developmental vitamin D deficiency alters adult behaviour in 129/SvJ and C57BL/6 J mice, Behav Brain Res 187(2), 343–350. Bodnar, L. M., Simhan, H. N., Powers, R. W., Frank, M. P., Cooperstein, E., and Roberts, J. M. (2007) High prevalence of vitamin D insufficiency in black and white pregnant women residing in the northern United States and their neonates, J Nutr 137, 447–452. Nicolaidou, P., Hatzistamatiou, Z., Papadopoulou, A., Kaleyias, J., Floropoulou, E., Lagona, E., Tsagris, V., Costalos, C., and Antsaklis, A. (2006) Low vitamin D status in mother-newborn pairs in Greece, Calcif Tissue Int 78, 337–342.

Chapter 6 Studying Schizophrenia in a Dish: Use of Primary Neuronal Cultures to Study the Long-Term Effects of NMDA Receptor Antagonists on Parvalbumin-Positive Fast-Spiking Interneurons M. Margarita Behrens Abstract Evidence obtained from schizophrenia post-mortem brain studies have pointed to deficiencies in inhibitory systems, in particular of the fast-spiking parvalbumin (PV)-positive inhibitory interneurons, as responsible for several aspects of schizophrenia pathophysiology. This hypothesis has been confirmed in pharmacological as well as genetic models of the disease, but when and how this dysfunction occurs is still unknown. Exposure to NMDA receptor antagonists is one of the most used pharmacological models for the study of schizophrenia, due to its capacity to produce a psychotic syndrome in humans and to produce an outbreak in schizophrenia patients. Using this model, we and others have shown that dysfunction of the PV-inhibitory system is most probably responsible for the neural network alterations, leading to the schizophrenia-like behavior in primates and rodents. Development of PV-inhibitory neurons occurs postnatally in mammals and follows a predetermined program that occurs also in cultures of cortical neurons. In this chapter we describe in detail the methodology we have used over the last decade to culture these neurons and that led to the discovery of how blockade of NMDA receptors results in the dysfunction of PV interneurons. Key words: Schizophrenia, parvalbumin, fast-spiking, GABA, ketamine, primary neurons.

1. Introduction Because of the nature of its positive symptoms, such as delusions and hallucinations, schizophrenia could be considered an exclusively human disease. Thus, it is difficult to imagine that such features can be studied in animals. However, accumulating evidence shows that particular neural network and behavioral P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_6, © Springer Science+Business Media, LLC 2011

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disruptions observed in schizophrenia patients can be indeed reproduced in animal models. In the last couple of decades, the combination of results obtained from post-mortem studies in humans, together with pharmacological and genetic manipulation of animal model systems, has advanced enormously our knowledge of specific pathophysiological aspects of the disease (1–4). Evidence obtained from schizophrenia post-mortem studies have pointed to deficiencies in inhibitory systems, in particular of the fast-spiking parvalbumin (PV)-positive inhibitory interneurons, as responsible for several aspects of schizophrenia pathophysiology (5–7). Support for this hypothesis comes from mouse reverse-genetic models in which manipulation of different schizophrenia risk genes, such as DISC1, NRG1/ErbB4, Reelin, and GCL (8–11), converges in the dysfunction of this interneuronal system. The time point during the development of the central nervous system when this dysfunction occurs, as well as whether the deficiency in the PV-interneuronal system is a consequence or a cause of the disease, is still a matter of debate. Nevertheless, recent evidence derived from neuroimaging, neuropathologic, neuropsychologic, and epidemiological studies strongly suggest that the dysfunction in the PV-interneuronal system observed in schizophrenia has a neurodevelopmental origin. The shaping of neuronal circuits is essential during postnatal brain development when sensory experiences play critical roles in the refinement of cortical connections. However, both the process of postnatal experience-dependent maturation of neocortical inhibitory networks and its underlying mechanisms remain poorly understood. PV interneurons, which include basket and chandelier cells, represent a unique class of interneurons that elicit powerful control of cortical output through innervation of the soma and axonal initial segment of pyramidal neurons. Furthermore, due to electrical connections with each other, PV interneurons can control ensembles of cortical neurons and thus synchronize cortical activity. Accumulating evidence suggests that PV interneurons are responsible for the generation of gamma oscillations involved in temporal encoding and storage/recall of information required for the correct functioning of working memory processes (12–14). Moreover, adult-level performance in delayed response tasks emerges relatively late in the postnatal development of primates (15), and this functional maturation appears to occur concomitantly with PV interneuron maturation (16, 17). Thus, it is believed that correct development of PV-synaptic inhibition is necessary for the synchronized firing of cortical networks in adulthood and plays a critical role in the development of executive functions associated with frontal brain regions (18–20). Dysfunction of PV-positive fast-spiking interneurons in this brain region is believed to underlie the disruption in evoked gamma-frequency

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oscillations and cognitive deficits observed in schizophrenia (21, 22). The development of pharmacological models that reproduce neurochemical and behavioral features of schizophrenia, together with genetic animal models of high-risk genes found in genome-wide analyses of schizophrenia, has been fundamental to our present understanding of the possible origin of PVinterneuronal disruptions observed in diseased patients. Among the pharmacological animal models developed in the last decades, exposures to NMDA receptor (NMDA-R) antagonists such as phencyclidine (PCP), ketamine, and MK801 are widely used in adult animals to study behavioral and neurochemical disruptions relevant to schizophrenia, principally because similar exposures produce schizophrenia-like behavior in humans. Administration of NMDA-R antagonists in rodents produces deficits in spatial working memory, in reversal learning, and in sustained attention (23–25), and disrupts the ability of humans (26) and non-human primates to switch between tasks (27), something commonly observed in schizophrenia patients. The schizophrenia-like behavioral effects produced by NMDA-R antagonists seem to occur through dysfunction of the PV-interneuronal inhibitory system, as evidenced by results showing that repetitive exposures of adult rodents and primates to NMDA-R antagonists lead to the disruption of the fast-spiking PV-interneuronal system (28–31). Thus, deep understanding of how and when blockade of NMDA-R function leads to the permanent dysfunction of the PV-interneuronal system may allow therapeutic strategies targeted to prevent the development of the disease. Mature PV interneurons are a class of fast-spiking (FS) cells. The ion channels underlying their fast-spiking phenotype include the fast delayed rectifier (containing Kv3.1 and 3.2 subunits), inactivating A-type, and Ca2+ -activated K+ channels (32). PV interneurons receive their glutamatergic excitatory input from thalamocortical projections and the pyramidal cortical network, and are also the target of cholinergic, adrenergic, dopaminergic, and serotonergic modulation (33–35), thus integrating a multitude of limbic inputs to influence cortical control of excitability. The molecular mechanisms initiating and regulating the maturation process of PV-interneuronal circuits are only beginning to be understood. In mice, PV interneurons as well as gamma-band oscillations have a delayed maturation (13). Migration of these interneurons from the median ganglionic eminence to the cortical plate is complete by embryonic day 15, but maturation does not occur until the end of the first postnatal week, coinciding with the emergence of parvalbumin expression. Activity-dependent glutamatergic transmission plays a fundamental role in the maturation of PV interneurons. Among all interneuron subtypes,

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PV interneurons receive the highest number of glutamatergic synapses from thalamic afferents in the adult rodent brain (36). These afferents, by contacting both pyramidal and inhibitory neurons, ensure the correct timing of cortical excitation by local feedforward inhibition (37). During the first week of age, this inhibition is absent, and thalamocortical inputs excite only pyramidal neurons (38). However, between postnatal days 6 and 7, the strength of synaptic contacts from thalamic afferents to layer IV PV interneurons increases substantially, as do the inhibitory synaptic contacts onto principal neurons which now respond to GABA with inhibitory post-synaptic currents. At this same time parvalbumin expression appears in PV interneurons (39). During the following weeks, GAD67-mediated GABA synthesis plays a major role in the development of perisomatic innervation by PV interneurons, regulating interneuron axon branching and synapse formation (40). The maturation phase of perisomatic innervation hereafter seems to be independent of the glutamatergic input from thalamocortical afferents (41) and appears to follow an internal program that is observed even in PV interneurons developing in vitro (Fig. 6.1).

Fig. 6.1. Development of PV-synaptic contacts in primary neurons cultured from cortical tissue. Cultured cortical neurons were immunostained for simultaneous detection of αCaMKII (left panel: glutamatergic neuron) and parvalbumin (right panel: PV interneuron processes) during their fourth week of development in vitro. Note that PV processes surround the soma and primary dendrites of the glutamatergic neuron, making “en passant” synaptic contacts (beaded structures in right panel ).

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1.1. Why Studying PV Interneurons in Culture May Shed Light to Schizophrenia Pathophysiology

The strikingly similar pattern of postnatal development of PV interneurons in vivo and in culture (42) led us to test whether these interneurons were a specific target of prolonged exposure to NMDA-R antagonists. Should this be the case, it would permit the study of the specific effects that blockade of NMDA-Rs have in the development and function of this inhibitory neurons in a simplified system, where only cortical glutamatergic and GABAergic neurons are present.

1.1.1. PV Interneurons in Culture Follow a Preprogrammed Pattern of Development and Respond to NMDA Receptor Blockade Similar to What Is Observed In Vivo

Cortical neuronal cultures give rise to synaptic activity when developed in vitro (43–46). Spontaneous inward currents driven by AMPA-type glutamate receptors have been observed in these cultures during the first week in vitro (47), and expression of inhibitory GABA(A) receptors was observed after 2 weeks in culture (48, 49). This type of cortical culture is widely used for studies of neurodegeneration, excitotoxicity, regulation of ionchannel activity, and neurotrophin or neurotransmitter intracellular signaling (48, 50–55). The primary culture system offers many advantages when studying mechanisms triggered by NMDA-R antagonists, since in this system, all inputs from outside the cortex are severed during the dissection process and the only relevant neurotransmitters are glutamate and GABA. Over the past 5 years we have accumulated evidence demonstrating the advantages of the primary neuronal culture system in studies of PV-inhibitory neuron development and function, as well as in studies of their dysfunction as a possible cause of schizophrenia (28, 42, 56). These interneurons develop in culture in a strikingly similar manner to what has been shown in vivo, with parvalbumin expression appearing at the end of the first week in vitro and the number of PVpositive neurons increasing steadily to reach 50% of the GABAergic population by the fourth week (39, 42, 57). When analyzed during the fourth week in vitro, the characteristic fast-spiking current patterns of PV interneurons are indistinguishable from those observed at a similar postnatal age in vivo (42, 57). By analyzing NMDA-mediated activation of intracellular signaling pathways, we showed that NMDA-Rs are functional in the cultured interneurons. More importantly, we showed that the composition of NMDA-R subunits in these interneurons is different than that present in pyramidal neurons at a similar age (42). These results have recently been confirmed in PV interneurons of rat prefrontal cortex (58), strongly supporting the applicability of the results we obtained in the primary culture system to an in vivo situation. With regard to the effects of NMDA-R antagonists on the GABAergic phenotype of PV interneurons, we showed that prolonged exposure to a non-selective NMDA-R antagonist or to an NR2A-preferring antagonist induced the loss of parvalbumin

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and GAD67 expression exclusively in PV interneurons in culture, without affecting the viability of the neurons. We also showed that these effects were prevented by increasing intracellular calcium concentrations with a calcium channel opener, or by activation of group 1 metabotropic glutamate receptors (42). 1.1.2. NMDA-R Antagonist Exposures Increase Oxidative Stress-Related Mechanism that Leads to the Dysfunction of PV Interneurons In Vitro and In Vivo

In search for the mechanisms by which exposure of NMDA-R antagonists leads to the loss of GABAergic phenotype of PV interneurons, we demonstrated, again using the culture system, that exposures to NMDA-R antagonists lead to disinhibition of excitatory circuits and activation of the superoxide-producing enzyme NADPH oxidase-2 (Nox2) in 3-week-old primary cortical neurons as well as in adult brain. Furthermore, the inhibition of Nox2 with apocynin or the elimination of superoxide with a brain-penetrant SOD mimetic prevented the loss of phenotype of PV interneurons in vitro and in vivo, and ketamine effects were absent in Nox2-deficient animals (28, 56). Exposure to PCP and more selective NMDA-R antagonists such as MK801 and CPP was shown to produce a rapid increase in reactive oxygen and nitrogen species (ROS) in vitro (59) and in vivo (60, 61), and repetitive exposures in vivo led to a substantial elevation of baseline levels of free radicals, suggesting that this treatment results in a persistent change in the oxidative state of the cortex (61). Interestingly, recent results have shown that NMDA receptor activity is required for the expression of antioxidant enzymes (62), further supporting the idea that prolonged blockade of NMDA receptors produces an increased oxidative state in brain.

1.1.3. Inflammatory Mediators Are Involved in the Activation of Oxidative Pathways that Lead to Loss of Function of PV Interneurons

Alterations in cytokine levels have been a consistent finding in schizophrenia patients (63). In particular, elevated plasma levels of IL-6 have been reported in patients and first-degree relatives (64–66), and increased IL-6 levels were found to correlate with exacerbation of psychotic episodes (64–69). To analyze the possible involvement of proinflammatory molecules in the ketaminemediated activation of Nox2, we turned again to the primary culture system. Using this system, we showed that prolonged exposure to ketamine increased the levels of IL-6 mRNA, without affecting the levels of IL-1β or TNF-α mRNAs, and that IL-6 released from neurons was directly involved in the induction and activation of Nox2 by ketamine (56). This result translated to the in vivo situation where we showed that brain IL-6 levels remained elevated 24 h after the last exposure to the NMDA-R antagonist, and was necessary and sufficient to produce the induction and activation of Nox2, and the consequent loss of the GABAergic phenotype of PV interneurons (56). In summary, the use of the primary culture system permitted a detailed study of the mechanisms by which NMDA-R antagonists produce the loss of the GABAergic phenotype of exclusively

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PV interneurons and led to the discovery of the IL-6/Nox2 pathway as intrinsically involved in the enduring effects of NMDA-R antagonists on the PV-inhibitory circuit in vivo (28, 42, 56).

2. Materials For the preparation of primary cultures, the following materials are necessary. 2.1. Equipment

Dissection microscope Horizontal flow clean bench (dissection hood) 70% Ethanol Sterile dissection tools: #3c and #4 forceps Scissors (blunt tips: 9 and 15 cm) Forceps (serrated tip: 12 and 16 cm; 1×2 teeth: 10 cm) Stainless steel tray with lid

2.2. Media

1. Dissection media (DM) Prepare a 10× DM the following way: 280 mM glucose 25 g 200 mM sucrose 4.2 mM NaHCO3 10× HBSS

35 g 1.75 g 500 ml

Dissolve thoroughly, filter sterilize, and store at 4◦ C. 2. Media stock (MS) (1.09 l) H2 O (sterile)

900 ml

10× MEM

100 ml

GB stock (see below)

90 ml

Glucose bicarbonate (GB) stock (for 1 l) NaHCO3 (35.3 mM)

29.66 g

Glucose (246.6 mM)

44.44 g

H2 O

to 1 l

Filter, sterilize, and keep at 4◦ C. The quality of the sterile water is essential for healthy cultures. If in doubt, purchase it from a trusted supplier.

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3. Astrocyte plating media: FBS∗ Horse serum

100 ml (HS)∗

100 ml

Glutamine (200 mM)

10 ml

Pen-Strep (100×)

10 ml

MS

to 1 l

4. Astrocyte growth media: 1× MEM Glutamine (200 mM) Glucose (20%) Pyruvate (100 mM) HS∗

422 ml 5 ml 18 ml 5 ml 50 ml

In general we purchase sterile glutamine and pyruvate already in solution at the indicated concentrations. 5. Neuron plating media: 1× MEM

90 ml

200 mM Glutamine

1 ml

100 mM Pyruvate

1 ml

Glucose (20%)

3 ml

HS

∗

10 ml

6. Neuron growth media: FBS∗

50 ml

HS∗

50 ml

200 mM Glutamine

10 ml

MS

to 1 l

7. Neuron maintenance media: HS∗

10 ml

200 mM Glutamine

10 ml

MS

80 ml

8. MS/N2.1 N-2 supplements

1 ml

100 mM Pyruvate

1 ml

200 mM Glutamine

1 ml

HS∗

2 ml

MS

95 ml

∗ Always

use endotoxin-free, characterized, and heatinactivated sera for preparing neuronal cultures. We always

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check different batches of sera beforehand by preparing one 24-well Primaria plate of one-step cortical cultures as described below. 2.3. Coverslips

Besides the material and media described above, you will need the following: 1. Poly-L-lysine-coated coverslips: For preparing these coverslips, follow exactly the procedure described by Goslin and collaborators (70). The quality of the glass and the preparation of these coverslips are fundamental to the success of the neuronal cultures, principally for the dissociated cultures used in confocal microscopy. For the latter cultures, wax “feet” will need to be added to the coverslips before poly-L-lysine coating as described by Goslin and colleagues. 2. Coat the above coverslips (with or without feet) with neuron plating media in the tissue culture incubator for at least 5 days before using them.

3. Method for Preparing Mouse Primary Neocortical Cultures

3.1. Dissection Procedure and Preparation of Astrocytes

The preparation of mouse cortical neuronal cultures for use in studies of postnatal development of inhibitory interneurons follows well-established protocols that have been optimized by different labs over the last 20 years. Basically, the primary culture system we have used is the one developed for studies of the neurobiology of synaptic development and neurodegeneration, to which we have introduced small modifications to optimize conditions for the correct maturation of the different inhibitory interneuronal types present in cortical tissue. Depending on the type of study, we have used three slightly different ways of culturing cortical neurons which all strictly depend on healthy astrocytes. For all types of cortical neuronal cultures, the preparation and growth of astrocytes, over which embryonic neurons are developed, is fundamental to the success of long-term cortical neuronal cultures. Careful control of the growth of astrocytes has produced, in our hands, cultures lasting for more than 3 months. 1. On the day of astrocyte preparation, place thoroughly washed tools in the tray and fill to cover with 70% ethanol. Sterilize for more than 15 min. Working now in a tissue culture hood, discard or recycle the ethanol and place the tools over sterile pads to dry. Keep the tray in the hood because it will be used to carry the tools to the dissection hood after drying. You can further sterilize by turning

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on the UV lamp in the hood while the tools are drying (about 20 min). Once dried, place them back in the tray and cover it. 2. Place neonatal (P1–3) mouse pups on ice until they are pale and no longer move (see Note 1). 3. Sterilize skin with 70% ethanol. 4. Place pup heads in cold dissection media (DM) in a 100-mm Petri dish. 5. In a dissection hood, remove the brains and transfer them into a clean 100-mm Petri dish with cold DM. 6. Under a dissection microscope, remove the meninges and dissect the cerebral hemispheres. Carefully removing all the meninges reduces the amount of endothelial cells, macrophages, and microglia contaminations in the cultures. 7. Collect the hemispheres in a 60-mm dish containing cold DM and chop the tissue with small scissors. 8. Transfer the tissue into a 50-ml conical tube. 9. Add 5 ml of 0.25% trypsin and 1.5 ml of 1% DNase to the tube, bringing the final volume to 13.5 ml with DM. 10. Incubate at 37◦ C in water bath for 15 min, swirling occasionally. 11. Let tissue settle and transfer the supernatant into a new 50-ml conical tube through sterile cell strainer (70-μm nylon mesh) to remove large chunks of tissue. 12. Add 3 ml horse serum to inhibit trypsin. 13. To the remaining tissue, add another 5 ml of 0.25% trypsin and 1.5 ml of 1% DNase. And again bring the volume to 15 ml with DM. 14. Incubate at 37◦ C as before. 15. Pour supernatant through the nylon mesh, including remaining chunks of tissue. Do not pipette tissue up and down to dissociate! 16. Add 3 ml horse serum through the mesh to inhibit trypsin and to wash the remaining tissue. 17. Centrifuge cells for 5 min at 1000 rpm and resuspend in astrocyte plating media. 18. Plate cells (three pups/flask) in 75-cm Falcon Primaria flask in 15 ml astrocyte plating media with the addition of 15 μl of EGF (10 μg/ml). 19. Replace media with the same astrocyte plating media after 2 days if there is too much debris.

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20. Incubate flasks in 37◦ C/5% CO2 incubator until 80–90% confluency (see Note 2). 21. The day before removing the astrocytes from the flasks, prepare 12-well or 24-well poly-D-lysine-coated plates. If you use Primaria plates, there is no need to coat them. However, Primaria does not have 12-well plates, so these need to be coated in the following way: dissolve 5 mg of poly-D-lysine (Sigma Cat# P7280) in 50 ml sterile water. Add sufficient amount to completely cover the wells and incubate for >5 h in the tissue culture incubator. Wash three times with sterile water and let air-dry in the tissue culture hood. 22. Discard the media from the flask containing the astrocytes and wash in PBS (no Ca2+ no Mg2+ ). If microglia are present, cap the flask tightly and tap it against a hard surface (such as the bench of the tissue culture hood) to release the microglia. Wash again with D-PBS and then incubate at 37◦ C in 3 ml of 0.25% trypsin for approximately 10 min. You may need to bang the flask against your hand to release the astrocytes (do not use a pipette) and transfer to a 15-ml conical tube. Wash the flask with another 5 ml astrocyte plating media, add this to the previous cells, and spin down as before. Gently resuspend cells in 5 ml of astrocyte growth media with a widemouthed, 5-ml pipette and dilute at a ratio of 2 ml of cells into 48 ml of astrocyte growth media. 23. Plate in 12-well poly-D-lysine-coated plates (1 ml/well). 24. Feed by exchanging half of the media once a week only if media become acidic (yellowish). Astrocytes prepared in this manner are used as feeder layers when preparing two-step or dissociated primary neuronal cultures. When used as feeder layers for two-step primary neuronal cultures, astrocytes should be allowed to grow to complete confluency in growth media. In general, we use astrocytes plates that have been confluent for about 3 weeks before seeding primary neurons on top of them (see Note 3). For using as feeder layers for dissociated neuronal cultures, astrocytes should be transferred to MS/N2.1 media when they have reached about 50–60% confluency (see Note 4). These feeder layers should be transferred to MS/N2.1 a week in advance of use. 3.2. Dissection and Preparation of Primary Cortical Neurons

The procedure described below will render two 12-well plates of one-step cultures, and two 12-well plates of dissociated neuronal cultures. If you want to scale up, follow the same procedure up to disaggregating the cells. Then dilute in the corresponding volume

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of neuron plating media such that the final dilution is 1.25 cortices per 10 ml. 3.2.1. One-Step Cultures

These are the cultures of choice when performing electrophysiological recordings or single-neuron transcriptional analysis (42): 1. Dissect cortices of two E15 embryos in DM as described for newborn pups in the preparation of astrocytes, making sure you remove the hippocampus. 2. Collect all four cortices into a 15-ml sterile conical tube and let them settle in the bottom of the tube. Aspirate off the media and add 5 ml of neuron growth media. 3. Using a widemouthed, 5-ml pipette attached to a pipettor, place the mouth of the pipette against the bottom of the conical tube and gently triturate the tissue until no aggregates are observed (be careful to avoid air getting into the pipette and making bubbles). It usually takes around 5–8 times filling the pipette up and down to the 4-ml mark to disaggregate the tissue. Add to 27 ml of neuron plating media in a 50-ml conical tube. 4. Mix and plate 1 ml in each well of two 12-well plates containing poly-L-lysine-coated coverslips that have been treated for 5 days in neuron plating media. Alternatively, for testing of sera, or for biochemical studies, you can plate 0.4 ml in each well of a 24-well Primaria plate. Save the leftover cells for the preparation of dissociated neuronal cultures as described below. 5. Place the plates in a tissue culture incubator (5% CO2 ) and monitor astrocyte growth and neuronal differentiation. The astrocyte monolayer should be confluent in about 12 days. Do not feed the cells during the first 2 weeks after plating. The plating media is sufficiently rich to sustain growth and differentiation of these low-density cultures. When the astrocyte layer is confluent (usually between 10–14 days in vitro), add cytosine arabinoside (Ara-C) to a final concentration of 10 μM. 6. After 48 h of Ara-C addition, exchange 0.5 ml of media with neuron maintenance media. In general, these low-density cultures will need to be fed with neuron maintenance media once a week.

3.2.2. Dissociated Neuronal Cultures

These types of cultures are especially suitable for experiments of intracellular signaling and confocal microscopy analyses: 1. Add neuron growth media to the ∼6 ml leftover cells from the one-step culture preparation to a final volume of 7.5 ml. 2. Add 300 μl of the above neuronal suspension onto each poly-L-lysine-coated coverslip that has been incubated in

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plating media for at least 5 days. Do not exchange media. Incubate overnight in tissue culture incubator. 3. The next day, using sterile forceps and a sterile syringe needle with its tip bent, invert the “feeted” coverslips onto 12-well astrocyte plates that have been grown to ∼50–60% confluency and incubated in MS/N2.1 for 1 week. Do not exchange the MS/N2.1 in the astrocyte plates. 4. Add Ara-C (5 μM final concentration) to prevent astrocyte growth on the coverslip. 5. Monitor the development of neurons once a week and feed with MS/N2.1 only if media becomes yellowish (see Note 5 on monitoring the development of neurons). 3.2.3. Immunocytochemistry Methods to Study Parvalbumin Interneurons in Culture

3.2.3.1. Fixation and Blocking

Due to the low number of PV interneurons, both in vivo and in culture, analysis of protein expression patterns as well as signal transduction mechanisms can be studied only by immunocytochemical methods. Thus, the availability of suitable antibodies is a must when studying these interneurons. Following is the protocol we have used over the last 10 years that results in reliable and reproducible fixation of PV interneurons in culture. 1. Wash coverslips twice with PBS to remove growth or treatment media, by carefully aspirating half of the media and adding 0.5 ml of sterile PBS. Aspiration must be performed with low vacuum and the addition of PBS is best performed with a widemouthed pipette (not automatic) against the wall of the well to prevent the formation of a vortex in the center that will lift up the neurons. Add 1 ml of ice-cold 4% paraformaldehyde in PBS. This is performed sequentially for each coverslip. Each well should be washed and fixed separately. 2. Incubate at room temperature for 30 min. 3. Wash carefully with PBS three times. Once the neurons are fixed, the fixative as well as the washes can be completely aspirated. However, vortexes can still occur, so the same rules apply as for when fixing. 4. Permeabilization, when needed, is performed by incubation in 1 ml of 0.25% Triton X-100 in PBS for exactly 10 min. 5. Aspirate the Triton solution and wash once, again gently, with PBS. 6. Block in 10% normal serum in PBS (see Note 6 on choice of host) for at least 24 h at 4◦ C. If a preservative is added to the blocking solution, such as 0.02% sodium azide, the coverslips can be kept at 4◦ C for prolonged periods.

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Fig. 6.2. Cultured PV interneurons show different morphologies. Primary cortical neuronal cultures were developed in vitro and immunostained for parvalbumin expression during the fourth week in culture. In general, PV interneurons in culture have three or more aspiny primary dendrites and extensive branching.

3.2.3.2. Immunocytochemistry

PV interneurons in culture can be easily identified by immunodetection of parvalbumin expression (Fig. 6.2). In the following, we will describe the immunocytochemistry procedure we have used for simultaneous detection of parvalbumin and GAD67 in cultured neurons, followed by an example where we have quantified the changes in their levels of expression using exposure to NMDA receptor antagonists as an in vitro model to study the molecular effects of these propsychotic drugs on PV interneurons: 1. Aspirate blocking solution and add 1 ml of primary antibody solution containing 2% normal goat serum, anti-parvalbumin antibody (rabbit polyclonal 1:4000 from Swant), and anti-GAD67 (mouse monoclonal from Chemicon). 2. Incubate in a humidified 37◦ C incubator for 2 h. 3. Aspirate antibody solution and wash three times, 10 min each, with PBS using a rocker. 4. Aspirate the last wash and add 1 ml of secondary antibody solution containing 2% normal goat serum, anti-mouse

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Alexa Fluor 488 (1:1000 dilution), and anti-rabbit Alexa Fluor 568 (1:1000 dilution) conjugated secondary antibodies. 5. Incubate protected from light for 45–60 min. 6. Aspirate secondary antibody solution and wash as in step 4 avoiding light exposure. 7. Place a small drop of aqueous fluorescence mount media (such as FluoroMount-G) on a slide. Gently lift a coverslip from its well and place it at an angle (cell layer facing the mounting media) on top of the drop. If the coverslips have wax feet, eliminate these before mounting (see Note 7). 8. Aspirate the excess of mounting media on the edges of the coverslip and seal using nail hardener (not nail polish). 9. Let dry protected from light for 24 h before imaging. 3.2.4. Prolonged Treatment of Three-Week-Old Primary Cortical Cultures with NMDA-R Antagonists Reduces the Expression of Parvalbumin and GAD67 in PV Interneurons

Dissociated or one-step neurons are treated with ketamine (0.5 μM) for 24 h by adding 10 μl of a 100× stock solution of ketamine (Ketaset) made in PBS and returning the plates to the tissue culture incubator. After 24 h, the coverslips containing the neurons are lifted from the wells using a bent syringe needle and forceps, washed by immersion in PBS, and transferred (cells up) into the wells of a 12-well plate containing icecold 4% paraformaldehyde. Immunocytochemistry is performed as described above. After immunostaining, the slides containing the coverslips are imaged with a 40× objective on a confocal microscope set to detect emission of the fluorophores conjugated to the secondary antibodies. We normally use sequential imaging to prevent fluorescence bleed-through and take z-stage images across 3.2 μm in depth every 0.2 μm. The PMTs for the two fluorophores are then set manually such that the fluorescence in control PV interneurons is about 75% of the maximum saturation when the images across the 3.2 μm are collapsed. This prevents the burnout of images when the fluorescence level is too high. The low number of PV interneurons in the primary cultures (∼5%) results in having to scan the coverslip to find them. In our experience, the total number of PV interneurons in one coverslip varies between 40 and 70 for dissociated and one-step cultures, respectively. Each PV interneuron is imaged through 3.2 μm starting from the side of the neurons attached to the coverslip, which allows maintaining consistency in the region that will be analyzed for fluorescence intensity. Because GAD67 tends to aggregate in the somatic region, its co-expression with parvalbumin is imaged in the neuronal soma (Fig. 6.3). PV interneurons in culture have different morphologies (summarized in Fig. 6.2) and their processes tend to spread long distances from their somas. However, PV-synaptic contacts are

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Fig. 6.3. GAD67 immunoreactivity aggregates in the somatic region around the nucleus. This image depicts the typical double immunostaining for GAD67 and parvalbumin observed in PV interneurons. GAD67 aggregation in the somatic region around the nucleus can be observed in the left panel. Parvalbumin immunostaining, on the other hand, is observed throughout the cell including the nucleus (right panel ). For this reason it is important to avoid the nuclear region when demarcating the region of interest (dotted lines in images) to be quantified for the two fluorophores.

always perisomatic (Fig. 6.3). Parvalbumin expression, in difference from that of GAD67, can be nuclear. This is important to take into account when performing the image analysis, as described below. 3.2.4.1. Image Analysis of GAD67 and Parvalbumin Expression

We have used MetaMorph to perform image analysis, but ImageJ (NIH) can also be used. Using either software, the region of interest is delineated manually such that it contains the somatic, but not the nuclear, region of the cell (Fig. 6.3). Each neuron is delineated this way and the fluorescence intensity in the region is measured for both fluorophores. The mean ± standard deviation fluorescence per cell is then calculated across all neurons in the coverslips that were treated in the same manner (we usually perform this in duplicate coverslips obtained from the same dissection and repeat this across three different dissections (six coverslips in total) to take into account differences in culture conditions and gestational age). NMDA-R antagonist exposures produce a pronounced decrease in parvalbumin and GAD67 expression in PV interneurons (Fig. 6.4 and also see (28, 42, 56)).

4. Notes 1. In our experience, dissection of the brain is simplified if we use pups at or near the second postnatal day. At this stage, their skulls are still soft, which makes the extraction of the

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Fig. 6.4. Prolonged treatment with ketamine leads to the loss of GAD67 and parvalbumin immunoreactivity in PV interneurons. Primary cortical-dissociated neurons were treated with ketamine (0.5 mM) for 24 h after which they were fixed in paraformaldehyde and immunostained for the simultaneous detection of GAD67 and parvalbumin expression. The ketamine treatment strongly reduced GAD67 and parvalbumin expression in PV interneurons (arrows in bottom panels) while preserving GAD67 expression in non-PV interneurons (asterisk in left bottom panel ).

brain much easier than in later days. Also, elimination of the meninges is easier at this postnatal age. 2. The use of endotoxin-free sera together with the elimination of meninges helps reduce microglia. It is important to monitor the appearance of these cells in the astrocyte cultures. Should they appear, tap the flask against the bench to make them float and exchange the media completely. 3. Two-step cultures are used in biochemical studies, in studies of neurodegeneration, or when performing nucleic acid analyses. When preparing these cultures it is important that the astrocyte monolayer has reached confluency several weeks in advance of seeding the embryonic neurons. Astrocyte proliferation becomes contact inhibited when they reach confluency and cells acquire a mature phenotype that is not reactive to media changes. If they have not reached this stage when neurons are seeded on top of them, since

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AraC is added to prevent proliferation of astrocyte precursors coming from the neuronal preparation, they become highly reactive, thus preventing the attachment and development of neurons. 4. In the case of dissociated cultures, the proliferative phase is halted by the prolonged incubation in MS/N2.1 and the astrocytes do not react to the addition of AraC. The reason why these types of neuronal cultures require a lower density of astrocytes is probably related to the maintenance of a critical ratio of neurons to astrocytes for optimal development of neurons. 5. It is important that anybody working with primary neurons becomes familiar with the development of processes during in vitro conditions. Just monitoring this will permit correction of media conditions early on. Under the light microscope, you will observe small processes starting to appear about 4 h after plating the neurons. These processes grow to several times the soma size by the end of the first week in vitro. A critical time in the culture process occurs between 9 and 14 days in vitro when NMDA receptors acquire a mature phenotype. It is during this time period when one gets to know whether the astrocytes are healthy and able to transport enough glutamate. If this is not the case, cultures that looked beautiful during the first week will die by necrosis overnight. Should this happen, there is a >95% chance that the problem is in the astrocyte feeder layer. In the following weeks, the chance of death by anoxia increases in the center of coverslips held on wax feet. This is very difficult to avoid, since lifting up the coverslips tends to damage the astrocyte layer underneath, which also produces the death of the neurons. To bypass this problem, we now put the wax feet toward the center of the coverslip such that while keeping the separation with the astrocyte layer, the wax dots will be in the region that becomes more anoxic. 6. Although it may sound redundant, it is not said enough: make sure you block with a serum that is compatible with the host on which your secondary antibodies are made! The most commonly used secondary antibodies are made in goat, thus we normally block in 10% normal goat serum (NGS). However, depending on the hosts of your primary antibodies, you may need a different set of secondary antibodies that are made in a different host. In general, when the primary antibodies are made in rat, mouse, or rabbit, we use NGS as the blocking sera. When one of the primary antibodies is made in goat, we use normal horse or donkey serum. 7. Removing the wax feet from the coverslips before mounting can become an issue, since in the process, the neurons

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can be dislodged. With the aid of a closed #3 forceps, poke gently the center of the wax dot and make a slight movement sideways; the dot will stick to the tip of the forceps. If the dot brakes, using the same forceps, gently pick up the pieces such that they do not “dance around” the coverslip. Yes, you do have to take all the wax out before mounting the coverslip. References 1. Ayhan, Y., Sawa, A., Ross, C.A., and Pletnikov, M.V. (2009). Animal models of gene– environment interactions in schizophrenia. Behav Brain Res 204, 274–281 2. Javitt, D.C. (2007). Glutamate and schizophrenia: phencyclidine, N-methyl-Daspartate receptors, and dopamine–glutamate interactions. Int Rev Neurobiol 78, 69–108 3. Lewis, D.A., Hashimoto, T., and Volk, D.W. (2005). Cortical inhibitory neurons and schizophrenia. Nat Rev Neurosci 6, 312–324 4. Mouri, A., Noda, Y., Enomoto, T., and Nabeshima, T. (2007). Phencyclidine animal models of schizophrenia: approaches from abnormality of glutamatergic neurotransmission and neurodevelopment. Neurochem Int 51, 173–184 5. Beasley, C.L., and Reynolds, G.P. (1997). Parvalbumin-immunoreactive neurons are reduced in the prefrontal cortex of schizophrenics. Schizophr Res 24, 349–355 6. Benes, F.M., and Berretta, S. (2001). GABAergic interneurons: implications for understanding schizophrenia and bipolar disorder. Neuropsychopharmacology 25, 1–27 7. Hashimoto, T., Volk, D.W., Eggan, S.M., Mirnics, K., Pierri, J.N., Sun, Z., Sampson, A.R., and Lewis, D.A. (2003). Gene expression deficits in a subclass of GABA neurons in the prefrontal cortex of subjects with schizophrenia. J Neurosci 23, 6315–6326 8. Ammassari-Teule, M., Sgobio, C., Biamonte, F., Marrone, C., Mercuri, N.B., and Keller, F. (2009). Reelin haploinsufficiency reduces the density of PV+ neurons in circumscribed regions of the striatum and selectively alters striatal-based behaviors. Psychopharmacology (Berl) 204, 511–521 9. Do, K.Q., Cabungcal, J.H., Frank, A., Steullet, P., and Cuenod, M. (2009). Redox dysregulation, neurodevelopment, and schizophrenia. Curr Opin Neurobiol 19, 220–230 10. Fisahn, A., Neddens, J., Yan, L., and Buonanno, A. (2009). Neuregulin-1 modulates hippocampal gamma oscillations: implica-

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Chapter 7 Glutathione Deficit and Redox Dysregulation in Animal Models of Schizophrenia Pascal Steullet, Jan-Harry Cabungcal, Anita Kulak, Michel Cuenod, Françoise Schenk, and Kim Q. Do Abstract Evidence of altered antioxidant systems and signs of elevated oxidative stress are reported in peripheral tissue and brain of schizophrenic patients, including low levels of glutathione (GSH), a major thiol antioxidant and redox buffer. Functional and genetic data indicate that an impaired regulation of GSH synthesis is a vulnerability factor for the disease. Impaired GSH synthesis from a genetic origin combined with environmental risk factors generating oxidative stress (e.g., malnutrition, exposure to toxins, maternal infection and diabetes, obstetrical complications, and psychological stress) could lead to redox dysregulation. This could subsequently perturb normal brain development and maturation with delayed functional consequences emerging in early adulthood. Depending on the nature and the time of occurrence of the environmental insults, the structural and functional delayed consequences could vary, giving rise to various endophenotypes. The use of animal models of GSH deficit represents a valuable approach to investigate how interactions between genetic and environmental factors lead to the emergence of pathologies found in the disease. Moreover, these models of GSH can be useful to investigate links between schizophrenia and comorbid somatic disorders, as dysregulation of the GSH system and elevated oxidative stress are also found in cardiovascular diseases and diabetes. This chapter reviews pharmacological and genetic rodent models of GSH synthesis dysregulation used to address some of the aforementioned issues. Up to date, these models revealed that GSH deficits lead to morphological, physiological, and behavioral alterations that are quite analogous to pathologies observed in patients. This includes hypofunction of NMDA receptors, alteration of dopamine neurotransmission, anomalies in parvalbumin-immunoreactive fast-spiking interneurons, and reduced myelination. In addition, a GSH deficit affects the brain in a region-specific manner, the anterior cingulate cortex and the ventral hippocampus being the most vulnerable regions investigated. Interestingly, a GSH deficit during a limited period of postnatal development is sufficient to have long-lasting consequences on the integrity of PV–IR interneurons in the anterior cingulate cortex and impairs cognitive functions in adulthood. Finally, these animal models of GSH deficit display behavioral impairments that could be related to schizophrenia. Altogether, current data strongly support a contributing role of a redox dysregulation on the development of pathologies associated with the illness and demonstrate the usefulness of these models to better understand the biological mechanisms leading to schizophrenia. Key words: Glutathione, oxidative stress, schizophrenia, parvalbumin, NMDA receptors, dopamine, γ-oscillations, behavior, development, anterior cingulate cortex, ventral hippocampus. P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_7, © Springer Science+Business Media, LLC 2011

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1. Introduction The present review is focused on animal models with a glutathione (GSH) deficit used to investigate the contribution of redox dysregulation to the pathology of schizophrenia. Redox dysregulation is the consequence of an imbalance between an overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) on one side, and a deficiency of enzymatic and non-enzymatic antioxidants on the other side. This leads to oxidative and nitrosative stress-inducing macromolecular damage such as lipid peroxidation, protein oxidation (protein carbonyls), and DNA damages (1). Redox dysregulation can also alter redoxsensitive processes and signaling (2) such as cell cycle regulation and differentiation (3–5), receptor activation (e.g., NMDA receptor) (6), and signal transduction and transcription (e.g., via Nrf-2, NF-κB). ROS include superoxide O2 .− , hydrogen peroxide H2 O2 , hydroxyl radical . OH, and peroxyl radical ROO, while RNS include nitric oxide NO and the highly toxic peroxynitrite ONOO− . The defense systems against oxidative and nitrosative stress are multiple, consisting of enzymes such as superoxide dismutases and catalase, and many other enzymes involved in the redox cycle of GSH, thioredoxin, and glutaredoxin systems. In addition, non-enzymatic antioxidants such as GSH, ascorbic acid (vitamin C), α-tocopherol (vitamin E), carotenoids, and flavonoids act in concert to neutralize ROS and RNS produced in the different cellular compartments. GSH (γ-glutamylcysteine–glycine, Fig. 7.1a) is present in millimolar range in the cytosol, nuclei, and mitochondria. It is the major thiol antioxidant and redox buffer of the cell. It can be found in reduced and oxidized form (glutathione disulphide, GSSG) and constitutes a major storage of cysteine. GSH is a cofactor of several enzymes, which detoxify hydrogen and lipid peroxides through glutathione peroxidases (GPX, Fig. 7.1b, c) and environmental toxins through glutathione transferases (GST, Fig. 7.1a, c) (7). GSH is also able to scavenge directly hydroxyl radicals and singlet oxygen; it participates in the glutaredoxin system and regenerates the antioxidants, ascorbic acid and vitamin E, back to their active forms (Fig. 7.1b). The capacity of GSH to regenerate these antioxidants is linked with the redox state of the glutathione disulphide–glutathione couple (GSSG/GSH) (for recent reviews, see (1, 2, 7, 8)). In addition, the thiol redox state determined by the glutathione disulphide–glutathione couple plays a central role in the modification of cysteine residues of proteins, providing mechanisms to regulate protein function (2) (Fig. 7.1d). In brain, which utilizes 20% of the oxygen

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consumed by the body leading to high amount of ROS during oxidative phosphorylation, GSH plays a key role in protection against oxidative stress. Moreover, in brain, GSH metabolism depends on intact function of glial cells such as astrocytes, which provide neurons with cysteine, the limiting precursor of GSH (9). In peripheral tissues of schizophrenic patients, impaired antioxidant defense systems and increased lipid peroxidation have been reported (10, 11). Reductions of plasma antioxidants such as bilirubin and uric acid were found in both first episode (12) and chronic patients (13). Abnormal antioxidant enzyme activity of superoxide dismutase (SOD), GPX, and catalase (CAT) was observed in plasma and/or blood cells of patients (14– 17). However, large discrepancies were reported (see (18)). Impaired antioxidant systems could lead to oxidative stress and ROS-mediated injury as supported by increased lipid peroxidation products, decreased levels of membrane polyunsaturated fatty acids (PUFAs), and decreased membrane phospholipids in blood cells (19, 20) and fibroblasts (21). It has been suggested that peripheral membrane anomalies correlate with abnormal central phospholipid metabolism in first episode and chronic schizophrenic patients (22–24). Altered redox systems have also been observed in brains of subjects suffering from schizophrenia. Decreased levels of GSH in cerebrospinal fluid (CSF) and in medial prefrontal cortex (PFC) have been measured in schizophrenic patients (25). A microarray and proteomic study showed anomalies of mitochondrial function and oxidative stress pathways in post-mortem brains of patients (26). In addition, lower GPX and glutathione reductase activities (27), elevated Mn-SOD protein levels and activity (28, 29), and elevated Cu- and Zn-SOD activity (29) were found in post-mortem brains of patients. However, it was not clear whether these observations indicated an intrinsic impairment of some antioxidant systems or whether these were the consequences of other dysfunctions and/or environmental insults (i.e., toxic compounds, infections) leading to an excess of ROS production. We recently provided genetic and functional evidence for an intrinsically impaired function of the glutamate cysteine ligase (GCL, Fig. 7.1a), the rate-limiting enzyme of GSH synthesis (30). Polymorphisms of GAG trinucleotide repeat (GAG-TNR) of the catalytic subunit of GCL (GCLC) were associated with the illness in two independent cohorts. Compared to skin fibroblasts of “low-risk” GCLC genotypes, fibroblasts of “high-risk” GCLC genotypes, present in 35–40% of patients, had lower GCL protein expression, GCL activity, and GSH levels under oxidative stress. This demonstrated that GAG-TNR variants are associated with dysfunctional regulation of GSH synthesis in a sub-population of patients. Furthermore, “high-risk” genotype patients had

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Fig. 7.1. Synthesis and role of glutathione (GSH). (a) GSH synthesis requires two ATP-dependent enzymatic steps. The first and rate-limiting enzyme is GCL that binds glutamate (Glu) and cysteine (Cys) to form γ-glutamylcysteine (γ-GluCys). GCL consists of two subunits: a heavy catalytic subunit (GCLC) and a light modifier subunit (GCLM). The second enzyme is GSS, which adds glycine (Gly) onto γ-GluCys to form GSH. Here is also depicted the action site of the pharmacological (BSO) and genetic tool (knockout of GCLM) used to induce a GSH deficit. Roles of GSH include antioxidant action and protection against macromolecular damage (b), detoxification (c), and redox-dependent regulation of protein function (d) and DNA binding (2). (b) Antioxidant action of GSH. Hydrogen peroxide (H2 O2 ) and lipid hydroxyperoxide (LOOH) are reduced by GSH via GPX, producing GSSG, which in turn can be reduced back to GSH via GR. Regeneration of ascorbic acid and vitamin E from their oxidized forms (dehydroascorbate and vitamin E radical, respectively) is achieved via the same GPX-dependent mechanism. Failure of this system may result in irreversible macromolecular damage such as lipid peroxidation, formation of protein carbonyls, and DNA damage. (c) Detoxifying action of GSH. Toxic electrophile compounds (Ep), including diethyl maleate and cyclohexene-1-one, are detoxified by formation of adducts with GSH (GSEp) via the action of GST, and subsequently expelled from the cells via multidrug-resistance proteins (MRPs depicted as circles). This results in GSH depletion. 4-Hydroxynonenal, a product from lipid peroxidation process, can also be rendered

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lower plasma GSH levels and higher oxidized cysteine levels than “low-risk” patients, pointing to a generalized redox dysregulation (31). This is consistent with the decreased GSH levels reported in schizophrenia (25, 27). The drug-naïve status of the patients in the CSF study (25) suggested that the deficit in GSH was not a consequence of treatment. However, GAG-TNR polymorphism was also associated with bipolar patients, but not with major depression (Gysin et al., unpublished), supporting the concept of a psychosis continuum (32) and that various genetic anomalies are common to several forms of psychosis. The implication of redox dysregulation in both schizophrenia and bipolar disease is consistent with the observation that supplementation of N-acetyl-cysteine, a cysteine prodrug, improves both schizophrenic and bipolar patients (33–35). Altogether, this suggests that a dysfunctional regulation of GSH synthesis and a subsequent deficit of GSH in brain and peripheral tissue can be a vulnerability factor to schizophrenia, but also possibly to bipolar disease. Furthermore, other susceptibility genes for schizophrenia may also induce an oxidative state. For instance, functional polymorphisms of PRODH, encoding proline oxidase, that are positively associated with schizophrenia display increased enzyme activity (36), leading to subsequent decreased proline levels. Proline has antioxidant properties and protects the intracellular GSH pool and the GSSG/GSH redox status (37). Consequently, low levels of proline could cause redox dysregulation, which could be particularly severe when combined with impaired GSH synthesis. It is quite striking that many environmental risk factors for schizophrenia (malnutrition, exposure to toxins, maternal infection and diabetes, obstetrical complications, maternal or early life stress, or later insults like brain trauma and stress during childhood, adolescence, and adulthood) result in increased ROS generation, lipid, protein, and DNA oxidation, and decreased GSH levels and antioxidant defense system (see (38) for review). Some of these environmental insults also lead to increased inflammation, emphasizing the tight link between inflammation and oxidative stress (39, 40). Any of the aforementioned environmental insults could worsen the fragile redox equilibrium and, depending on the phase of brain development when

 Fig. 7.1. (continued) into a non-toxic glutathiyl adduct via a GST-dependent reaction. (d) Redox regulation of protein function. The GSSG/GSH redox couple determines in great part the redox state of cysteine residues on proteins. Reversible oxidation and modification of cysteine residues are important mechanisms that control protein function and interaction between proteins. Oxidation of cysteine residues can lead to formation of disulfide bonds (1) within a same protein (P1) or (2) between two proteins (P1 and P2). The GSSG/GSH redox couple also modulates (3) S-glutathionylation and (4) S-nitrosylation that are known to control protein function. NMDA receptor function is for instance modulated by redoxdependent modifications of cysteine residues located on the NR2A subunit (6). GCL, glutamate cysteine ligase; GPX, glutathione peroxidase; GR, glutathione reductase; GSS, glutathione synthetase; GST, glutathione transferase.

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they occur, could perturb normal brain maturation with delayed functional consequences in early adulthood. Thus, a genetic defect that leads to dysregulation of GSH synthesis, combined with environmental insults or other genetic factors generating oxidative stress, could be a critical factor contributing to the emergence of schizophrenia (38). Since the brain is highly vulnerable to oxidative damage because of its high oxygen consumption, its high content of oxidizable polyunsaturated fatty acids, and the presence of redoxactive metals (Cu and Fe), a GSH deficit could be particularly damaging to the neuronal function. Oxidative stress-induced cellular damage is also involved in the pathogenesis of various neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (ALZ), and Huntington’s disease (HD). However, in contrast to schizophrenia where a genetic origin of GSH synthesis could affect brain development, GSH depletion and ROS/RNS increase in neurodegenerative diseases appear later in life and are probably downstream consequences of other primary causes (1). In the following, we review animal models of GSH deficit that have been used to determine the consequences of redox dysregulation and to assess their involvement in the pathology of schizophrenia. The chapter is focused on pharmacological and genetic approaches that induce a GSH deficit in rodents via a downregulation of GCL, the key synthesizing enzyme of GSH. In rodent models, GSH deficit was induced (1) semi-chronically during postnatal development, (2) acutely during adulthood (pharmacological treatment with L-buthionine-SR-sulfoximine, L-BSO), or (3) chronically throughout the life [genetic model GCLM (−/−) mice] (Fig. 7.2). These models were used to address the questions below. First, what are the short- and long-term consequences of a GSH deficit restricted to a period of the development? This can test the contribution of a redox dysregulation in the neurodevelopmental aspect of schizophrenia. Neuroanatomical, neurochemical, neurophysiological, and psychopathological data converge to suggest that interactions between susceptibility genes and environmental insults during pre- and perinatal development cause defects in neuronal integrity and connectivity, setting off a cascade of events that extend into adult life and lead to the emergence of the psychotic symptoms. In this context, a genetic and/or environmentally derived GSH deficit could represent an important susceptibility factor (see (38) for review). Second, what are the effects of an acute and transient GSH deficit during adulthood? GSH deficits have been observed in adult patients (25). This approach is useful to distinguish the consequences of an acute GSH deficit during adulthood from those caused by a GSH deficit during development.

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Finally, what are the effects of a chronic GSH deficit throughout life? This is expected to combine effects of a GSH deficit during development (including during fetal development) and later in life. However, a chronic GSH deficiency may in addition lead to more profound adaptations of the entire system to compensate for the redox dysregulation.

2. Pharmacological Models of GSH Deficit

Two main types of pharmacological agents are available to induce a GSH deficit: electrophile compounds and specific inhibitors of GCL. Electrophile compounds (i.e., diethyl maleate and 2-cyclohexen-1-one) cause very rapid and reversible depletion of GSH through a detoxifying process. These compounds conjugate with GSH directly or via the GSH S-transferases (41). The resulting conjugates are then expelled from the cells through pumps called multidrug-resistance proteins (MRPs), leading to rapid decrease in intracellular GSH (Fig. 7.1c). These GSH-depleting

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agents cross the blood–brain barrier and are able to efficiently decrease brain GSH levels within hours, leading to increased protein carbonyls (42, 43). However, they are quite toxic, not very specific for GSH depletion (at least diethyl maleate), and cannot be used for long-term GSH depletion. In our view, they are not appropriate pharmacological tools to model GSH deficit in the context of schizophrenia but are adequate for toxicological studies. However, two studies describe the effects of these GSH-depleting agents on rodent behavior. Adult rats and mice with acute GSH depletion induced by 2-cyclohexene-1-one (for a few hours) showed impaired short-term spatial memory in a Y maze (44). Rats with an acute GSH deficit induced by diethyl maleate (for a few hours) had impaired acquisition of spatial reference memory in a water maze and displayed normal acquisition in an avoidance test (45). Impaired short-term spatial memory in 2-cyclohexene-1-one-treated animals was also reported for other models of GSH deficit (see following paragraphs). However, impairments in spatial learning and memory in a water maze have not been observed in other models of GSH deficit than diethyl maleate-treated rats, suggesting that some effects of diethyl maleate may be independent of GSH depletion. More specific pharmacological tools are inhibitors of GCL. The most specific inhibitor is DL-buthionine-(SR)-sulfoximine (BSO) (see Fig. 7.1a). It represents a relevant model of GSH deficit, as observed in schizophrenia. Only the L-buthionine(SR)-sulfoximine (L-BSO) is a mechanism-based inhibitor subject to ATP-dependent, enzyme-catalyzed phosphorylation by GCL to form L-buthionine-(SR)-sulfoximine phosphate which tightly binds to the active site of GCL (46–48). L-BSO is only weakly metabolized as 90% is expelled from the body in its original form. L -BSO is considered to be an irreversible inhibitor. But after a washout period, GSH levels in cells/tissue slowly recover via turnover of GCLC and possibly very slow reactivation (48). The main limitation for the use of L-BSO in vivo is its poor ability to cross the blood–brain barrier after weaning age (49, 50). Therefore, only a transitory GSH deficit during postnatal development can be achieved by subcutaneous (s.c.) injections of L-BSO. BSO s.c. injection in pre-weanling animals (Fig. 7.2a) can be used to investigate the effects of a transitory GSH deficit on postnatal development and its long-term effects in adulthood when GSH levels have returned back to normal. Indeed, GSH levels normalize a few days after the last BSO treatment (51, 52). On the other hand, acute GSH deficit in adults, via intracerebroventricular (i.c.v.) administrations of L-BSO (Fig. 7.2c), can help understand the effects of an acute or a semi-chronic GSH deficit in adulthood, independent of any developmental contribution. In rats and mice, a GSH deficit causes upregulation of the synthesis of the antioxidant ascorbic acid (53). Such

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compensation does not exist in humans, as we are not able to synthesize ascorbic acid. In order to use a rodent model that resembles more closely the conditions found in humans, GSH deficit was induced in osteogenic-disorder Shionogi (ODS) rats (54), which are deficient in L-gulonolactone oxidase, the key enzyme in the biosynthesis of ascorbic acid (55). In this rat strain, a GSH deficit does not lead to a compensatory increase in ascorbic acid. Animals with a GSH deficit can be more vulnerable to any insults that cause oxidative stress. For instance, enhanced dopamine release, as observed during the encounter of a stressful situation (56), could be deleterious and exacerbate redox dysregulation during conditions of GSH deficit. Indeed, catecholamines such as dopamine produce ROS (57) that need to be neutralized by antioxidant systems. These catecholamines can also react with cysteine residues of GSH and proteins to form conjugates, leading to decrease of endogenous GSH (58–60). To test the effect of enhanced dopamine release during a transient GSH deficit during development, a specific inhibitor of the dopamine re-uptake transporter 1-(2-(bis-(4-fluorophenyl)methoxy)ethyl)4-(3-phenylpropyl)piperazine dihydrochloride (GBR12909) was used to elevate extracellular dopamine levels locally in brain regions rich in dopamine innervations (61). 2.1. BSO-Induced Transient Postnatal GSH Deficit 2.1.1. Methods 2.1.1.1. Animals

2.1.1.2. BSO Treatment

BSO-induced GSH deficits (Fig. 7.2a, b) were studied in nonmutant rats (OFA, Wistars) and in osteogenic-disorder Shionogi (ODS) rats. A GSH deficit leads to compensatory increase in ascorbic acid synthesis in non-mutant rats, but not in ODS rats. ODS rats were obtained from Clea Japan, Inc. (Tokyo, Japan). Because ODS rats cannot synthesize their own ascorbic acid, they require freshly dissolved ascorbic acid (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) in their drinking water (1 g/l) to prevent vitamin C deficiency (62). ODS rats treated with BSO were in a state of GSH deficiency that was not compensated by ascorbic acid synthesis, but they were not in a state of combined GSH and vitamin C deficiency. A transient postnatal GSH deficit [from postnatal (PN) days 5–24] by about 50% was achieved in brain of rodents through daily subcutaneous (s.c.) injection of L-buthionine(S,R)-sulfoximine (L-BSO; 3.8 mmol/kg animal corresponding to 0.842 mg/g animal) (51, 63). L-BSO (>99% purity) in powdered form can be purchased from Acros Organics (Geel, Belgium) and stored at 4◦ C. BSO was dissolved in a phosphate buffer solution (PBS) consisting of 25 mM NaH2 PO4 , 65 mM Na2 HPO4, and 0.9% NaCl at pH 7.4. Other investigators used 0.9% physiological saline (NaCl alone) to dissolve BSO (64). Solubility of BSO was facilitated by sonication and/or heating the solution to about 45◦ C for 10–15 min. According to the

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producer, BSO solutions can be stored at –20◦ C for up to 3 months. Typically for a rat, 100 μl of a freshly made BSO solution (2–4 mM) was injected daily in the morning. Prior to injection, each animal was weighed in order to adjust the BSO concentration to inject (0.842 mg/g animal). This protocol can be adapted to mice. 2.1.1.3. Enhanced Extracellular Dopamine Levels by GBR12909 Treatment

The specific inhibitor of the dopamine re-uptake transporter, 1-(2-(bis-(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine dihydrochloride (GBR12909), was used to elevate extracellular dopamine levels locally in brain regions rich in dopamine innervations. GBR12909 dihydrochloride (purity >98%) was initially purchased from Tocris Cookson Ltd (Bristol, UK) and stored dessicated at RT. Recently, however, GBR12909 is produced by Biotrend AG (Zurich, Switzerland), on behalf of Tocris. GBR12909 (GBR) was freshly dissolved in PBS (pH 7.4) and subsequently injected s.c. (5 mg/kg animal, in 100 μl) to each rat every other day alone or in combination with BSO (51, 65). Prior to injection, each animal was weighed in order to adjust the GBR12909 concentration to inject (5 mg/kg animal). This protocol can be adapted to mice.

2.1.2. Results

While Slivka et al. (50) reported ∼80% GSH depletion in brain of preweaned mice after four daily injections (s.c.) of BSO, a single daily injection (s.c.) of similar BSO doses in rats from PN days 5–16 resulted in only 40–50% diminution of brain GSH (63). Comparable brain GSH deficit was also observed when BSO was daily administrated to 15-day-old rats for only 1–2 days (66); GSH levels returned back to normal within 2–3 days after the end of the treatment (51). BSO-induced postnatal GSH deficit did not affect the activity of the redox-related enzymes CAT, SOD, GPX, and GR (63). BSO treatment during postnatal development induced similar GSH deficit in ODS and non-mutant rats (63). However, lipid peroxidation was more prominent in ODS rats, particularly in the diencephalon and pons/medulla region (63). Generally, the effects of BSO treatment were stronger in ODS than in any of the non-mutant rats. After 6 days of BSO treatment, ODS rats showed lower body weight than their untreated counterparts (65), but this body weight deficit slowly disappeared after the end of the treatment. Finally, BSO induced more severe cataracts in ODS than in non-mutant rats (65). GSH is indeed an essential antioxidant vital for the maintenance of the transparency of the lenses (67), which are highly vulnerable to oxidative stress. Cataractogenesis due to BSO treatment during postnatal development was largely prevented by melatonin, which has antioxidant properties (68). Altogether, this indicates that the inability to

2.1.2.1. Effects on Brain GSH Levels, Oxidative Stress, and Physical Parameters

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synthesize ascorbic acid (like in humans) is an additional vulnerability factor to a GSH deficiency. In some experiments, rats with a transient GSH deficit during postnatal development were further challenged by a co-treatment with the dopamine uptake inhibitor, GBR12909 (GBR). GBR causes excess extracellular dopamine, which could further disturb the delicate redox balance. Overall, GBR+BSO-treated rats were not notably different from BSO-treated rats in terms of GSH deficit, lipid peroxidation, body weight, and cataract occurrence (51, 65). However, the additional GBR treatment exacerbated some adverse effects of GSH deficit on the development of cortical circuitry and cognitive functions (see below). 2.1.2.2. Effects During the Development of Cortical Circuits

The neuronal circuitry of the PFC is altered in schizophrenic patients (for review, see (69)). This includes reduced spines on pyramidal neurons and reduced expression of parvalbumin (PV), GAD67, and GAT1 in fast-spiking interneurons. ODS rats were used to examine whether a transient BSO-induced GSH deficit during postnatal development (with or without GBR-induced elevated extracellular dopamine) could alter cortical circuits in the PFC. In these experiments, rats were sacrificed after the end of the postnatal treatment (at PN day 16 or 24) and brains were processed for either immunohistochemistry (for interneuron investigations) or Golgi preparations (for dendritic spine quantification). BSO treatment alone affected parvalbumin-immunoreactive (PV-IR) interneurons in the anterior cingulate cortex as demonstrated by a reduced number of PV-IR processes radiating from the somata (70). This effect was more prominent in rats treated with BSO+GBR. Moreover, a GSH deficit combined with elevated extracellular dopamine during postnatal development led to a drastic reduction in the number of small PV-IR profiles (corresponding to PV-IR synaptic boutons and dendritic and/or axonal arborization) in superficial (II–III) and deeper (V–VI) layers of the anterior cingulate cortex, but not in the somatosensory cortex (70). However, there was no loss of PV-IR interneurons as the density of large PV-IR (cell bodies) was not altered. The effect of a GSH deficit on PV-IR interneurons was quite specific seeing that expression of other calcium-binding proteins (i.e., calbindin and calretinin) was not significantly altered by BSO or BSO+GBR treatments. In addition, BSO+GBR treatment during postnatal development (PN days 5–24) also altered the morphology of the dendritic spines on apical and basal dendrites of pyramidal neurons of the layer III of the anterior cingulate cortex (Gheorghita et al., unpublished). Altogether, the use of BSO treatment in rats during their postnatal development, particularly when combined with elevated extracellular dopamine, affects cortical circuitry in the prefrontal cortex (i.e., PV fast-spiking interneurons and dendritic arborization of pyramidal neurons). These observations are

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analogous to anomalies found in post-mortem brains of schizophrenic patients (71–73). 2.1.2.3. Long-Term Effects on Cortical Circuits

Another important question is whether anomalies in PV-IR interneurons caused by a transitory GSH deficit during postnatal development persist throughout adulthood, as GSH levels normalize after the end of the treatment. This has not yet been fully examined. Preliminary observations indicate that BSO or BSO+GBR treatments during postnatal development lead to reduced number of PV-IR interneurons in the anterior cingulate cortex of adult ODS rats (PN day 90) (Cabungcal et al., unpublished). This effect was less severe after BSO treatment alone. Thus, the alteration of PV-IR interneurons by a transitory GSH deficit during development rendered these interneurons more vulnerable during adulthood. This suggests that a transient GSH deficit during postnatal development is sufficient to affect permanently PV-IR interneurons in the anterior cingulate cortex, including a possible cell loss. This long-term effect on cortical circuitry and possibly other regions could be related to the observed long-term cognitive impairments described below.

2.1.2.4. Long-Term Effects on Behavior

A general investigation of the neurological status of the rats up to PN day 50 indicated no obvious effect of BSO treatment. Forelimb muscle strength, novelty reaction, escape, and startle reactions were not affected by BSO treatment with or without GBR (51). A transitory GSH deficit during postnatal development impaired neither spontaneous alternation task performance nor exploratory behavior in an open field (51). The main effect in juvenile ODS rats but not in non-mutant Wistar rats (PN days 25–31) was a significant impaired performance in the spatial water maze task that was observed soon after BSO treatment cessation (74). However, this impairment was no more present in older ODS rats (75). Nevertheless, significant cognitive deficits appeared during the third month of postnatal life. Adult ODS but not non-mutant rats with a transient GSH deficit during postnatal development showed impaired object recognition. This deficit was detected when interval delay was 30 min but not 15 min, suggesting a specific retention impairment. Interestingly, compared to control subjects, the object recognition capacity of schizophrenic patients is also impaired (76–79). The object recognition impairment after BSO treatment emerged earlier in male rats (PN day 65) than in females (PN day 94) and was more severe in males. At PN day 94, males were impaired after BSO treatment (± GBR), while females were impaired only after BSO+GBR treatment (65). This could be due to the protective and antioxidant effects of estrogens (see (80)). Interestingly in humans, first psychotic symptoms occur earlier in men than in women.

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BSO-treated rats trained at a later adult age (above 5 months) in classical spatial tasks expressed a severe impairment in a homing board task, but not in a reference and a working memory paradigm in water maze (74, 75, 81). On a homing board, both adult ODS and Wistar rats treated with BSO during postnatal development were highly inaccurate in discriminating the escape hole position based on distributed visual and olfactory cues (74). Their accuracy was restored only when a single salient cue marked the trained position. In a radial maze, adult BSO-treated rats did more working memory errors and were markedly disturbed by the addition of eight different olfactory cues, a procedure that normally enhances the performance of control rats (74). This suggests that BSO-treated rats have difficulties integrating multisensory cues. The fact, that performance of adult BSO-treated rats was intact in water maze but impaired in homing board, suggests that either the heavy path integration requirement during successive locomotion bouts on a solid floor or the presence of multiple olfactory cues, or a combination of both factors, enhanced task difficulty in the homing board for the BSO-treated rats. Interestingly, schizophrenic patients show impaired multi-sensory processing (82). The data also revealed that the effects of a GSH deficit were more severe in ODS rats unable to synthesize ascorbic acid or when combined with an additional stress factor (GBR treatment). Altogether, these studies demonstrate that a transitory GSH deficit during postnatal development has long-term behavioral consequences, affecting particularly the processing and integration of multi-sensory cues. 2.2. BSO-Induced Acute GSH Deficit in Adulthood 2.2.1. Methods

GSH deficit in adult rodents was achieved via administration of BSO into the lateral ventricle (Fig. 7.2c). Unilateral injection appears to be sufficient to induce similar GSH deficit contraand ipsilaterally to the injection site (52). In adult rats, a dose of 3.2 mg BSO/animal (dissolved in 30 μl of physiological saline, pH 7.4) was routinely administrated every 24 or 48 h for the longest treatments (12 days) (83, 84). A single BSO administration corresponds to approximately 0.01 mg/g rat. In mice, daily doses of about 0.02 mg/g mouse (dissolved in 3–5 μl of physiological saline, pH 7.4) were used (52, 64). Below is the description of the intracerebroventricular injection protocol used by Jacobsen et al. (52) on mice, which is easily adaptable to rats or other rodents. Mice were anesthetized using a saline solution (10 ml/kg; i.p.) of ketamine (100 mg/kg) and xylazine (10 mg/kg) and placed in a stereotaxic frame. A guide cannula (cat. no. C313GS-5; Plastics One, Roanoke, VA, USA) was implanted into the lateral ventricle (AP −0.3 mm; ML 0.9 mm; DV 2.0 mm) and fixed in place with two anchor screws (CMA, North Chelmsford, MA, USA) and dental cement. The guide cannula was protected with a dummy cannula (cat. no. C313DC;

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Plastics One) until used. Mice recovered for at least 1 week before injection and were treated with antibiotics (1.2 mg sulfamethoxazole/ml and 0.24 mg trimethoprim/ml) and analgesics (acetylsalicylic acid; 1 mg/ml) in the drinking water for the first 4 days after surgery and thereafter with only antibiotics. The internal cannula (cat. no. C313I; Plastics One) was inserted into the guide; L -BSO (0.5 mg/mouse; 5 μl) or saline was injected (1 μl/min, 5 min) using a A-99 Razel syringe pump (Stamford, CT, USA). The internal cannula was left in place for 10 min after injection to allow for dispersion away from the injection site. 2.2.2. Results 2.2.2.1. Effects on Brain GSH Levels, Oxidative Stress, and Physical Parameters

2.2.2.2. Effects on Behavior

Intracerebroventricular injections of BSO in adult rodents caused a 40–80% decrease in brain GSH levels. All brain structures, even the most distant from the site of injection, were equally affected (52). However, Shukitt-Hale et al. (84) found that after 12 days of treatment, GSH deficit varied in brain regions, with striatum being the most affected and cerebellum the least. Because of the limited number of studies available, it is difficult to know to what extent GSH deficit varies with the animal model, the dose of BSO, and the duration of the treatment. BSO appears to induce faster GSH depletion in mice (52, 64) than in rats (83), but this difference might well be due to the difference in the BSO concentrations used. Maximum GSH deficiency was reached after 48 h in rats (83, 84) and 24 h in mice (52). The degree of GSH deficit remained stable for 12 days following BSO administration every other day. At the end of BSO treatment, brain GSH levels slowly recovered and normalized almost completely after 72 h (52). This decrease in GSH was accompanied by increased protein oxidation (64). Long-term BSO treatment (12 days) resulted in loss of body weight in rats but did not cause major pathology and did not affect locomotor coordination and performance (84). However, some rats with a GSH deficit exhibited slight and occasional tremors/convulsions, usually in the first few days of BSO treatment (84). Furthermore, Abe et al. (85) showed that BSOinduced GSH deficit in adult mice exacerbated the convulsive action of pentylenetetrazol. This suggests that a GSH deficit can promote epileptic conditions, possibly by depressing the GABAergic system. BSO-induced GSH deficit in adult rodents did not affect locomotor activity (52), spatial reference learning, and spatial reference memory in a water maze (86). However, when GSH deficit was combined with intraventricular injection of dopamine, impairment of locomotion coordination and spatial learning and memory in water maze was observed (84, 86). Since dopamine alone or injected before BSO injection did not have any effect, the authors suggested that reactive compounds resulting from

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dopamine autooxidation led to further redox dysregulation in rats with GSH deficit, thus causing impairment in locomotion coordination and spatial performance. Adult mice with a BSO-induced GSH deficit had reduced capacity in novel object recognition and in a spontaneous alternation task, suggesting impaired short-term memory (64). Interestingly, adult rats which had a transient GSH deficit during postnatal development also showed impaired novel object recognition but not spontaneous alternation (51, 65), suggesting that recognition of objects is particularly vulnerable to a GSH deficit, independent of its time of occurrence. Chronic GSH deficit in GCLM (−/−) mice also led to impaired object recognition (see below). Finally, a GSH deficit in adult mice led to altered psychostimulant-induced locomotor activity (52). Locomotor activity was only transiently increased by amphetamine in BSOtreated mice, while PCP-induced increase in locomotor activity was strongly enhanced in BSO-treated compared to control mice. Dopamine release induced by amphetamine was enhanced in nucleus accumbens but not in PFC of BSO-treated mice, while PCP-induced dopamine release in nucleus accumbens was not affected by a GSH deficit (52). The behavioral responses of the BSO-treated mice to amphetamine and PCP could not however be simply explained by the effects of GSH deficit on dopamine release induced by these psychostimulants. Responses to psychostimulants depend on several neurotransmission systems (i.e., dopaminergic, serotonergic, noradrenergic, and glutamatergic) and interactions between different brain regions (i.e., striatum, nucleus accumbens, PFC, and VTA). A GSH deficit or the resulting increase in ROS could affect many of the above neurotransmission systems in a complex manner. 2.2.2.3. Effects on Neurotransmission

Many proteins are redox sensitive and their functions modulated by the redox state of the cell. This includes NMDA receptors (6), GABAA receptors (87), IP3 receptors (88), ryanodine receptors (89), calcineurin (90), various calcium and potassium channels (91, 92), and dopamine and glutamate transporters (93, 94). Thus, a GSH deficit could potentially alter many aspects of neurotransmission. NMDA receptors. For instance, NMDA receptor-mediated field excitatory postsynaptic potentials (fEPSPs) were weaker in hippocampal slices of BSO-treated rats compared to control rats (66). This hypofunction of NMDA receptors was partly due to an excessive oxidation of their extracellular redoxsensitive sites. In addition, a GSH deficit caused reduced NMDA receptor-dependent, long-term potentiation, decreased pairedpulse facilitation, and increased excitability of pyramidal neurons in CA1 (66).

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Dopamine signaling. A BSO-induced GSH deficit also altered dopamine modulation of NMDA-mediated calcium responses in cultured cortical neurons (95). Dopamine decreased NMDA responses in BSO-treated neurons, while the same dopamine concentration (1 μM) enhanced NMDA responses in neurons with normal GSH. The effect of a GSH deficit on dopamine modulation of calcium responses was due to alteration of dopamine modulation of L-type calcium channels and could be prevented by sulpiride, an antagonist of D2-type receptors. This suggests that antipsychotics with D2 antagonist properties might prevent the alteration of dopamine modulation of calcium responses induced by a GSH deficit. Thus, a GSH deficit can lead to changes in neurotransmission and neuromodulation that are relevant to schizophrenia.

3. Genetic Models of GSH Deficit

3.1. GCLM Knockout (GCLM −/−) Mice

Both the catalytic (GCLC) and the modifier (GCLM) subunits of GCL, the key enzyme of GSH synthesis, have been associated with schizophrenia (30, 96). In particular, some polymorphisms of GCLC that display blunted oxidative stress-induced increase in GCL activity and GSH levels confer a higher risk to schizophrenia. Therefore, genetic animal models targeting GCL to disrupt GSH synthesis (97) are valuable models to investigate the effects of a GSH deficit in the context of schizophrenia. Knockout of the GCLC gene, however, is lethal at an early embryonic stage (5, 98), presumably because GSH synthesis is completely suppressed. By contrast, GCLM (−/−) mice are viable showing a decrease in GSH of 50–80% depending on the organs (99, 100). Association between the GCLM gene and schizophrenia (association with SNPs situated on the 5 and 3 regions) was found in two independent cohorts of patients (Swiss and Danish) and in a family-based study from NIMH cohort (96), but not in the Japanese population (101). In the Swiss and Danish cohorts, no clear causal variant within the coding regions of the GCLM gene was however identified (102). However, reduced expression of GCLM mRNA was observed in cultured fibroblasts of patients compared to control in the Swiss sample (96). Therefore, these findings do not rule out the possibility that DNA change, critical for GCLM function or expression, is located in a region that was not screened (e.g., intronic region). Thus, GCLM (−/−) mice (99) represent a valid genetic animal model to study the effects of a chronic and systemic GSH deficit (Fig. 7.2d). GCLM (−/−) mice show about 60% decrease in GSH across brain regions and throughout life (103). GCLM

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(−/−) mice have no overt phenotype besides a slight reduction in body weight (99) and a reduced female fertility, and cultured cells from these mice are vulnerable to neurotoxicity and ROS (99, 100, 104). However, more detailed investigations revealed that GCLM (−/−) mice display brain morphological, physiological, and behavioral anomalies that could be related to schizophrenia and/or other psychiatric disorders. 3.1.1. Methods

GCLM (−/−) mice were kindly provided by Tim Dalton and Chen Ying from the Department of Environmental Health, Center for Environmental Genetics, University of Cincinnati Medical Center (USA). See (99) for a more detailed description of the generation of these mice. GCLM (−/−) mice were then backcrossed with C57Bl/6 J mice over >10 generations. GCLM (−/−) mice were compared with (+/+) littermates.

3.1.2. Results

GABAergic system. Compared to (+/+) mice, GCLM (−/−) mice showed selective and region-specific anomalies in the GABAergic system. Like in the BSO-treated rats, PV-IR interneurons in GCLM (−/−) mice were particularly affected. The developmental expression of PV was impaired in both the anterior cingulate and the somatosensory cortex at PN day 10, but normalized at PN day 20 (105). Additional stress induced by GBR treatment during postnatal development of GCLM (−/−) mice (from PN days 10–20) led to reduced number of PV-IR interneurons in the anterior cingulate but not somatosensory cortex until PN day 20 (105). PV-IR interneurons in the anterior cingulate cortex of GCLM (−/−) mice remained vulnerable after weaning, as GBR treatment between PN days 30–40 also caused a decrease in PV-IR interneurons in GCLM (−/−) but not (+/+) mice (Cabungcal, unpublished). Interestingly, elevated oxidative stress, as revealed by 8-oxo-dG (marker of DNA oxidation), was present in the anterior cingulate, but not in the somatosensory cortex of GCLM (−/−) mice at PN day 20 (105) (Cabungcal et al., unpublished). This coincides with the higher vulnerability of PVIR interneurons in the anterior cingulate compared to somatosensory cortex. In the hippocampus of GCLM (−/−) mice, PV-IR interneurons also showed a high vulnerability to oxidative stress. The number of PV-IR interneurons was normal in 20-day-old GCLM (−/−) mice, but was strongly reduced in the ventral but not dorsal hippocampus of 4–5-month-old GCLM (−/−) mice (103). The decrease in PV-IR interneurons was particularly selective for the ventral CA3 and dentate gyrus, and this impairment emerged after weaning age as oxidative stress increased or cumulated selectively in the ventral hippocampus (103). These observations confirm that PV-IR interneurons are particularly sensitive to a GSH deficit but their vulnerability depends

3.1.2.1. Morphological Anomalies

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on brain regions and correlates with the level of oxidative stress. This also supports the notion that PV-IR fast-spiking interneurons are highly vulnerable to oxidative stress (106). Thus, signs of DNA oxidation and more severe impairment of PV-IR interneurons were observed in the anterior cingulate cortex and the ventral hippocampus, but not in the somatosensory cortex and the dorsal hippocampus (103; Cabungcal et al., unpublished). Interestingly, the anterior cingulate cortex and the anterior hippocampus (analogous to the ventral hippocampus of rodents) are also two brain regions that are known to be affected in schizophrenia (107, 108). Myelin. Preliminary results suggest less myelination in the anterior cingulate cortex of GCLM (−/−) mice, as revealed by weaker MBP immunolabeling intensity and thinner MBP-IR profiles (105). Further investigations are currently under way to identify anomalies associated with myelin and oligodendrocytes. Indeed, redox state modulates proliferation and differentiation of oligodendrocyte precursors (109). Thus, mice with a chronic deficit in GSH show impaired fast-spiking PV-IR interneurons and altered myelination, both of which occur in schizophrenia (69, 110). 3.1.2.2. Reduction of γ-Oscillations

Fast-spiking PV-IR interneurons control the output of principal neurons and are necessary for the generation of γ neuronal synchrony that facilitates information processing and transfer within and between brain regions during cognitive tasks (111). Such γ-oscillations are reduced in schizophrenic patients during impaired cognitive tasks (112, 113), but also at resting state (114). This suggests that anomalies in synchronized neuronal activity, driven by PV-IR interneurons, are a core feature of the disorder. Therefore, we examined whether the reduced number of PV-IR interneurons in the ventral hippocampus of GCLM (−/−) mice was also associated with reduced γ-oscillations. We found that β/γ-oscillations induced by kainate in CA3 were significantly reduced in ventral but not dorsal hippocampal slices of GCLM (−/−) mice (103). These results indicate that a chronic deficiency in GSH affects PV-IR interneurons and the generation of β/γ-oscillations in the ventral but not dorsal hippocampus. Such anomaly of synchronized neuronal activity in specific brain regions is therefore expected to affect the behavior accordingly.

3.1.2.3. Behavioral Phenotype

A chronic GSH deficit did not affect in an unspecific and broad manner all behaviors investigated to date. Overall, the behavioral differences between GCLM (−/−) and (+/+) mice were quite subtle. Using an array of paradigms that require an intact hippocampus, we found a selective alteration in behaviors that rely on the ventral rather than the dorsal hippocampus. GCLM (−/−) mice showed intact spatial reference learning and memory in a water maze and intact performance

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in a rewarded alteration task (103), indicating normal spatial learning and memory capacity and functionally intact dorsal hippocampus (115). By contrast, tasks that are modulated by the ventral hippocampus (115) were altered in GCLM (−/−) mice. Thus, GCLM (−/−) mice showed a lack of behavioral inhibition in stressful conditions. Compared to wild-type mice, GCLM (−/−) mice entered significantly more often the open arms of an elevated plus maze, performed significantly more transitions between the two compartments of a light/dark box, and spent more time in the anxiogenic light compartment (103). GCLM (−/−) mice also displayed reduced acquisition of delay fear conditioning and expression of contextual and auditory-cued fear, again suggesting a functional anomaly of the ventral but not dorsal hippocampus (103). The ventral hippocampus is indeed implicated in both contextual and auditory-cued delay fear conditioning, while the dorsal hippocampus is involved in trace fear conditioning and contextual delay fear conditioning, but not auditory-cued delay fear conditioning (116–118). Moreover, GCLM (−/−) mice showed novelty-induced hyperactivity and strong exploratory drive toward novel objects. Despite this increased object exploration, however, GCLM (−/−) mice displayed impaired object recognition memory. Interestingly, adult rodents with a BSO-induced GSH deficit during postnatal development or an acute GSH deficit at adulthood also had impaired object recognition abilities (51, 64, 65) but intact spatial reference learning and memory in a water maze ((86); Schenk et al., unpublished). Further characterization of the behavioral phenotype of GCLM (−/−) mice focusing on startle responses and PPI, responses to stress, and specific cognitive tasks requiring functional PFC is under current investigation. 3.2. Other Genetic Models of GSH Deficiency

As genetic and functional evidence of an implication of the GCLC in schizophrenia exists, it would be of interest to study animals with a genetic defect on GCLC. Knockout of GCLC gene is however lethal at early embryonic stage (5, 98). GCLC (+/−) mice, which are viable and display a moderate GSH deficiency (in the liver: 20 and 50% decrease of GSH and GCL activity, respectively (98); but no published data on brain GSH levels), could be another model worth investigating. Similarly, mice knockout for the excitatory amino acid carrier-1 (EAAC1) is also an interesting model of GSH deficiency. Observations of altered expression of EAAC1 (=EAAT3) are reported in post-mortem brains of schizophrenic patients (119, 120). EAAC1, which transports glutamate and cysteine (the limiting precursor for GSH synthesis), is specifically expressed in neurons. EAAC1 (−/−) mice have a neuronal deficiency in GSH, leading to increased susceptibility of neurons to oxidant injury (121). Cerebral cortex

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and hippocampus of these mice show particularly elevated oxidative stress. EAAC1 (−/−) mice have also ventricular enlargement and brain atrophy, which progress with aging (121). The Na+ -independent glutamate–cysteine exchanger (xCT) plays a crucial role in astrocytes and developing neurons to provide them with cysteine, the limiting precursor of GSH. Interestingly, xCT has been implicated in cocaine relapse (122). Sut/sut mice (xCT loss-of-function mutants) could also represent a valuable model. Interestingly, these mice develop brain atrophy by early adulthood, exhibiting ventricular enlargement, thinning of the cortex, and shrinkage of the striatum (123), as observed in schizophrenia. 3.3. Models of GSH Deficit to Investigate Comorbidity with Somatic Disorders

Alterations of the GSH system in schizophrenia have been found in not only brains but also blood and skin fibroblasts (15, 25, 27, 30, 96, 124). Therefore, a genetically compromised GSH system can affect all tissues and could represent a vulnerability factor for not only psychiatric diseases but also several somatic disorders. Genetic polymorphisms of GCL genes have been associated with various pathologies, including cardiovascular diseases (125–127) which are more prevalent in schizophrenia (128, 129). Compared to the general population, patients with schizophrenia also suffer more often from type 2 diabetes mellitus and have higher frequency of impaired glucose tolerance (130). Thus, patients with schizophrenia and relatives have higher levels of baseline plasma levels of insulin compared to control subjects. But 2 h after administration of oral glucose, levels of both glucose and insulin are higher in patients and relatives (130), suggesting a blunted response to insulin. These observations suggest either shared environmental or genetic predisposition in schizophrenia and in the impaired glucose tolerance. Interestingly, a dysregulation of the GSH system could be one candidate as a common vulnerability factor. Indeed, elevated oxidative stress and low GSH levels were reported in patients suffering from diabetes (131, 132). Hyperglycemia and insulin deficiency can lead to decreased GSH levels via reduced expression of GCLC (see (7)). Alteration in the mutual interactions between the GSH system and the glucose metabolism might be central to the observed comorbidity between schizophrenia, and more generally to the metabolic syndrome in schizophrenia (133–136). Further investigations on the role of the GSH system in the comorbidity of schizophrenia with various somatic disorders are needed. In this context, GCLM (−/−) mice and other genetic models of GSH deficit could be used to address these aspects.

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4. Summary Data collected (Table 7.1) using various animal models of GSH deficiency have revealed the following: • A GSH deficit affects the brain in a region-specific manner. The PFC (in particular the anterior cingulate cortex) and the ventral hippocampus are especially vulnerable, displaying elevated oxidative stress and impairment of fast-spiking PV-IR interneurons. • A GSH deficit renders the brain more vulnerable to high levels of catecholamine release, in particular dopamine. The most vulnerable brain regions to a GSH deficit appear to be regions that are densely innervated by catecholaminergic neurons. • A GSH deficit causes complex changes in dopaminergic transmission as evidenced by enhanced D2R-dependent signaling and enhanced amphetamine-induced dopamine release in the nucleus accumbens. • A GSH deficit causes hypofunction of NMDA receptors. • A GSH deficit during development disrupts cortical circuit in the anterior cingulate cortex, leading to permanent morphological anomalies (i.e., reduced number of PV-IR interneurons and altered myelination) and long-term behavioral impairment in object recognition and multi-sensory processing, but not in spatial memory and learning in water maze. • A chronic GSH deficit also causes a loss of PV-IR interneurons in the adult ventral hippocampus as oxidative stress increases selectively in this region after weaning age. Such impairment of PV-IR interneurons in the ventral hippocampus is accompanied by reduced β/γ-oscillations. • A chronic GSH deficit leads to impaired object recognition, inadequate response to emotion and stress-related situations with a lack of behavioral inhibition, and reduced responses to fear. Under acute GSH deficiency, adult animals display altered responses to psychostimulants and impairment in object recognition. Finally, a transitory GSH deficiency during postnatal development is sufficient to cause long-term behavioral effects, affecting mostly object recognition and multi-sensory processing. Overall, all these types of GSH deficit lead to impaired object recognition but tend to leave spatial abilities intact when tested in a water maze. Altogether, a redox dysregulation due to a compromised GSH synthesis leads to morphological, physiological, and

Non-mutant rats

BSO + GBR (PN days 5–24)

BSO and BSO + GBR

BSO + GBR

Morphology (in ACC)

Morphology (in ACC, not SSC)

Abnormal dendritic spines on pyramidal neurons of layer III at PN day 24

Reduced number of small PV-IR profiles at PN day 16 Reduced number of PV-IR interneurons at adult No significant change of calbindin and calretinin-IR at PN day 16

No significant change of PV-IR at PN day 16 Reduced number of PV-IR interneurons at adult

BSO

(65)

Cataract (partially reversible) (more severe in ODS rats)

Gheoghita et al., submitted

(70)

(70); Cabungcal, unpublished

(51, 65)

(51)

References

Normal neurological status at PN day 50

ODS rats

General health

BSO and BSO + GBR

Description of effects

In ODS rats only: loss of body weight at PN day 16 (reversible)

ODS and non-mutant rats

During postnatal development (PN days 5–16)

Types of effects

Method

ODS rats and non-mutant rats

Animal

GSH deficit

Table 7.1 Summary of the main effects of different induced GSH deficits on general health parameters, brain morphology, physiology and neurotransmission, and behavior in rodents. ACC, anterior cingulate cortex; SSC, somatosensory cortex; HP, hippocampus

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Acute at PN days 15–17

GSH deficit

Table 7.1 (continued)

Non-mutant rats

ODS and non-mutant rats

BSO

BSO

Physiology, neurotransmission (in HP)

NMDAR hypofunction Impaired LTP in CA1 Increased excitability of pyramidal neurons in CA1 Decreased paired-pulse facilitation

Impairment in homing board and radial maze when multiple sensory cues available

In ODS rats only: impaired object recognition (males and females) at ∼PN day 90

In ODS rats only: impaired object recognition (only in males) at ∼PN day 90

BSO and BSO + GBR

Behavior

Description of effects

In ODS rats only: transient impairment of performance in water maze in young rats (PN days 21–30) but not in adults

BSO

ODS rats and non-mutant rats

Types of effects

BSO

Method

Animal

(66)

(74)

(51, 65)

(74)

(51, 65)

(continued)

References

Glutathione Deficit and Redox Dysregulation in Animal Models of Schizophrenia 171

BSO

General health

Loss of body weight Normal neurological status and locomotor coordination Slight and short tremors/convulsions

Non-mutant rats

Semi-chronic in adults (12 days)

Physiology, neurotransmission

Enhanced amphetamine-induced dopamine release in nucleus accumbens (not in PFC) No effect on PCP-induced dopamine release in nucleus accumbens and PFC

BSO

Impaired object recognition and performance in a spontaneous alternation task

WT mice

Behavior

Description of effects

(2 days)

BSO

Types of effects

Enhanced PCP-induced locomotor activity; altered pattern of amphetamine-induced locomotor activity

WT mice

Acute in adults (1 day)

Method

(2 days)

Animal

GSH deficit

Table 7.1 (continued)

(84)

(52)

(64)

References

172 Steullet et al.

Chronic

GSH deficit

Table 7.1 (continued)

GCLM (−/−)

GCLM (−/−)

GCLM (−/−) + GBR (PN days 30–40) GCLM (−/−)

Mice

Mice

Mice

BSO

Non-mutant rats

Mice

Method

Animal

Morphology (in ventral, not dorsal HP)

Morphology (in ACC, not SSC)

Behavior

Types of effects

Reduced number of PV-IR interneurons (defect emerges after weaning) Signs of oxidative stress (8-oxo-Dg) emerging after weaning

(105)

Sign of oxidative stress (8-oxo-Dg) at PN day 20 Reduced PV-IR interneurons and signs of oxidative stress at PN day 40

103

103

(continued)

Cabungcal, unpublished

(105); Cabungcal, unpublished

(105)

(86)

References

Altered myelination at PN day 20

Delayed expression of PV at PN days 10–20 (aggravated by GBR)

Normal spatial learning and memory in water maze (impairment when combined with dopamine brain injection)

Normal locomotor activity (impairment when combined with dopamine brain injection)

Description of effects

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GSH deficit

Table 7.1 (continued)

Method GCLM (−/−)

GCLM (−/−)

Animal

Mice

Mice

Behavior

Physiology, neurotransmission (in ventral, not dorsal HP)

Types of effects

Impaired object recognition

Novelty-induced hyperactivity

Reduced expression of contextual and auditory-cued fear

Reduced freezing during delay fear conditioning

More time in light chamber of a light/dark box

More time in open arm of elevated plus maze

Normal performance in rewarded alternation task

103

103

Reduced γ-oscillations in adults

Normal spatial learning and memory in water maze

References

Description of effects

174 Steullet et al.

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behavioral changes in rodents that are for many analogous to pathologies observed in schizophrenia and possibly other psychiatric diseases (Table 7.2). Thus, a genetic GSH synthesis impairment represents a risk factor in schizophrenia. Redox dysregulation may constitute a “hub” where genetic and environmental vulnerability factors converge and their timing during neurodevelopment plays a decisive role on schizophrenia phenotypes. The described preclinical models that are based on pathophysiology and recent genetic evidence will allow to reveal underlying mechanisms and to test new developed drugs.

5. Technical Notes 5.1. Glutathione Measurements

There are different methods to measure GSH in tissue/cell extracts, other biological samples (i.e., blood), and even in vivo in individual cells or within a certain volume of tissue in live animals/humans. Description of non-invasive GSH measurements in brain tissue using proton magnetic resonance spectroscopy methodologies is however beyond the scope of this review. GSH is found in high concentrations in the cytoplasm and organelles, such as mitochondria and nuclei. Free glutathione is found in either reduced (GSH) or oxidized (GSSG) state (Fig. 7.1); GSH represents in normal conditions >95% of the total amount of free glutathione. Quantification of both GSH and GSSG can be important in some studies as the thiol redox state of a cell depends on the relative concentration of the GSH and GSSG. In addition, GSH can bind to NO to form nitrosoglutathione (GSNO) and to proteins to form protein mixed disulfides (for reviews, see (2, 7); see Fig. 7.1). HPLC methods can be used to separate and quantify these various thiols. In this review however, we will only present colorimetric and fluorometric assays that are often based on enzymatic reactions and have the advantages compared to HPLC techniques to be simpler, require less instrumentation, and be less time consuming. The choice of the method will depend on whether one is interested in quantifying: the total thiols, or total free glutathione (GSH + GSSG), or both GSH and GSSG, or GSH bound to proteins. Thus, a careful reading of the technical spreadsheets of the various commercially available glutathione assay kits is necessary before making a choice. Below is a brief description of some of the most commonly used assays that are commercially available. Most assays require preliminary deproteination of the samples using various acids (sulfosalicylic acid, perchloric acid, or metaphosphoric acid), which prevents oxidation of GSH and its degradation by glutamyl

Physiology

Myelination

Dendritic spines

Morphology PV-IR

Consistent with the NMDA model of schizophrenia which assumes hypofunction of NMDA receptors ? (no data available) Reduced γ-oscillations in cortex (110;111)

Hypofunction of NMDA receptors

Impaired LTP in CA1 (HP)

Reduced kainate-induced γ-oscillations in slices of ventral HP

In PFC, HP: Deficit in myelin-associated mRNA and proteins; however MBP expression deficit not seen in all studies (see (110))

In dorsolateral PFC: Reduced number of dendritic spines in deep layer III (72)

In HP: Reduced number of PV-IR cell bodies (145) Consistent with the emergence of symptoms in adulthood

In ventral HP : Reduced number of PV-IR cell bodies in adult GCLM (−/−) mice Anomalies of PV-IR emerge after weaning age

In ACC : Abnormal dendritic spines on pyramidal neurons of layer III at PN day 24 In ACC : Reduced number of and size of MBP-IR profiles in adult GCLM (−/−) mice

In PFC: Reduced expression of PV in fast-spiking interneurons (69) Consistent with the neurodevelopment hypothesis of schizophrenia

Schizophrenia

In ACC: Reduced number of PV-IR cell bodies in adult GCLM (−/−) mice when challenged with a GBR treatment After a transient GSH deficit during development, reduced number of PV-IR cell bodies in adult rats that are unable to synthesize ascorbic acid

Models of GSH deficit

Table 7.2 Comparisons between the major effects of GSH deficit observed in rodent models and pathologies described in schizophrenic patients. The effects of GSH deficit on NMDAR function, dopamine signaling, parvalbumin-immunoreactive (PV-IR) fast-spiking interneurons, and myelination mimic morphological and functional anomalies that might be central to the disconnectivity in schizophrenia. On the other hand, the lack of behavioral inhibition in GCLM (−/−) mice may be more related to behavioral disinhibition observed in other disorders. Note that all morphological data on schizophrenia are from post-mortem brains

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Behavior (in adults)

Impaired object recognition (78) Impairment in a hippocampal-dependent virtual Morris water task (147) PCP exacerbates symptoms in stabilized chronic patients (148) Amphetamine induces heterogeneous changes in psychotic behavior, which may be state dependent (149) Behavioral inhibition in patients (150)

Impaired object recognition

Intact spatial memory in water maze in adult rats and mice

Enhanced PCP-induced locomotion during acute GSH deficit in adult mice

Altered pattern of amphetamine-induced locomotion during acute GSH deficit in adult mice

Lack of behavioral inhibition in GCLM (−/−) mice

Enhanced amphetamine-induced dopamine release in striatum (146)

Enhanced amphetamine-induced dopamine release in nucleus accumbens

Consistent with the neurodevelopment hypothesis of schizophrenia and the emergence of symptoms in adulthood Impaired multi-sensory processing (82)

D2R-type antagonists are antipsychotics

Altered dopamine modulation of calcium responses (can be normalized by D2R-type antagonists)

A GSH deficit during postnatal development leads to behavioral impairments in adulthood GSH deficit during postnatal development causes impaired performance in homing board and radial maze when multiple sensory cues are available

Schizophrenia

Models of GSH deficit

Table 7.2 (continued)

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transpeptidase. It is usually recommended however to perform measurements as soon as possible, but some assay kits mention that samples following appropriate preparation can be stored for 1 month at –80◦ C before measurements. 5.1.1. Colorimetric Glutathione Assay Kits

The most commonly used assay is a colorimetric method originally described by Tietze (137, 138). Assay kits using this method are available from numerous commercial providers (i.e., Luminos, Ann Arbor, MI, USA; Cayman Chemical, Ann Arbor, MI, USA; Oxis International, Inc., Beverly Hills, CA, USA; and Biovision Incorporated, Mountain View, CA, USA). This method measures the total free glutathione, but can be also adapted to measure GSH and GSSG. In this method, GSH reacts with DTNB (5,5 dithio-bi-2-nitrobenzoic acid or Ellman’s reagent) to produce a yellow compound, 2-nitro-5-thiobenzoic acid (TNB) and GSSG. In the presence of GR and non-limiting amount of NADPH, the generated GSSG is reduced back to GSH, which can react again with DTNB to produce more TNB. The rate of TNB production, measured by absorbance at 405 or 412 nm, is directly proportional to the concentration of total free glutathione. To measure GSSG and therefore to deduct GSH, one has to first preincubate an aliquot of the sample with 2-vinylpyridine (2-VP; 2–3 μl/100 μl sample), which will slowly derivatize and scavenge free GSH (reaction time, about 60 min). After subsequent adjunction of DTNB, GR, and NADPH, the rate of TNB production will then be proportional to GSSG present in the sample. In some assay kits, another thiol-scavenging reagent (1-methyl2-vinylpyridinium trifluoromethanesulfonate) is provided to scavenge more rapidly and efficiently GSH than does 2-VP (i.e., kit from Oxis International, Inc.). Another colorimetric method commercially available (Calbiochem Merck, Darmstadt, Germany; Oxis International inc.) allows measurement within the same well of total thiols and GSH. This assay does not depend on enzymatic reactions. A patented reagent (4-chloro-1-methyl-7-trifluuromethylquinolinium methylsulfate) is first added to the sample where it reacts with all thiols to produce thiolethers that can be quantified at an absorbance of 356 nm. The second step consists of adding 30% NaOH to convert the thiolether obtained specifically with GSH (not with other thiols) into a chromophoric thione that has a maximal absorbance at 400 nm.

5.1.2. Fluorometric Glutathione Assay Kits

Several fluorometric assays can also be used. One of them (from Biovision Incorporated) allows detection of GSH, GSSG, and total glutathione individually using three aliquots of the same sample. In this assay, o-phthalaldehyde (OPA) reacts with GSH (not GSSG), generating fluorescence (Ex/Em: 340/420) that is proportional to GSH concentration. Adding a reducing agent,

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which converts GSSG to GSH prior to OPA incubation, will allow determination of total free glutathione (GSH + GSSG). To measure GSSG specifically, a GSH quencher is first added to remove GSH and then a reducing agent is added to destroy excess of quencher and to convert GSSG to GSH. Subsequent adjunction of OPA will allow quantification of GSH formed from GSSG. Another fluorometric assay (from Luminos) uses a nonfluorescent molecule, ThioStarTM , that covalently binds to the GSH to yield a highly fluorescent product (Ex/Em: 390/510). In the same aliquot, total free glutathione (GSH + GSSG) can also be subsequently quantified following addition of a reaction mixture that converts all GSSG into GSH. Finally, it is worth mentioning a last assay (i.e., from SigmaAldrich, St Louis, MO, USA; Chemicon International Millipore, Billerica, MA, USA) that utilizes a thiol probe (monochlorobimane, MCB), which becomes fluorescent (Ex/Em: 380/460) when bound to thiols. The formation of these adducts is very slow. However, in the presence of GST, the specific formation of fluorescent adduct between MCB and GSH is much faster, resulting in a fluorescence intensity which is closely correlated with the amount of GSH in the sample. This last method can also be adapted to determine GSH levels in living cells. Cells are permeable to MCB and the incorporated MCB rapidly forms adducts with free GSH via the constitutive GST of the cells. The fluorescence measured within the cells with epifluorescent or confocal microscopes is therefore correlated with the amount of GS–MCB adduct. However, the rate of formation of this adduct also depends on the enzymatic activity of GST within the cell. In our hands, maximal fluorescence is reached after 3–5 min in astrocytes but can take more than 60 min in neurons. Furthermore the time course of GS–MCB formation can change with the age of cells (139). GS–MCB adduct can be expelled from the cell via multidrug-resistance proteins (MRPs); this efflux is particularly prominent in astrocytes (140). Therefore, this method should be considered as semiquantitative and should be adapted for each type of cells and experiments (duration of MCB incubation and MCB concentration, use of inhibitor of MRPs). Thus, we would recommend to first characterize the time course of the fluorescence increase. The method is also inadequate to measure GSH levels when GST activity change is expected. For more information on this technique, see (139–142). Using a similar approach, Molecular Probes (Invitrogen, Carlsbad, CA, USA) proposes a new free thiol detection reagent (ThiolTrackerTM Violet) in order to measure intracellular GSH levels in alive or fixed cells.

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5.2. Use of Brain Slices for Physiological Measurements

The use of brain slices is a valuable in vitro approach to study neurotransmission, neuromodulation, and synaptic plasticity. However, when interested in the effect of a GSH deficiency, one should be cautious regarding the effect of dissecting and slicing procedures on tissue GSH levels. Since strong activation of neuronal tissue produces GSH release (143, 144), the preparation of brain slices could lead to subsequent depletion of GSH. In our hands, the following protocol of hippocampal slice preparation using 2- to 3-week-old rats led to only 10% decrease of total GSH after slicing, but GSH levels remained then stable in an interface recording chamber for at least 6 h (66). Brain was quickly removed and placed in an ice-cold modified artificial cerebrospinal fluid (ACF for slicing) that was aerated with 5% CO2 /O2 . The ACF for slicing contained the following: 118 mM NaCl, 2 mM KCl, 4 mM MgCl2 , 0.5 mM CaCl2 , 1.2 mM NaH2 PO4 , 25 mM NaHCO3 , and 10 mM glucose. Brain was divided into two hemispheres. One hemisphere was kept at −80◦ C for subsequent determination of GSH content (control GSH values before slice preparation) and the other was mounted onto a vibroslicer (Campden Instruments, Loughborough, UK) to make 350-μm-thick slices in aerated ice-cold ACF for slicing. Slices were transferred into an interface chamber superfused with aerated ACF for slicing (∼2 ml/min) at RT. After 15 min, temperature was slowly increased to 30±2◦ C and ACF for slicing replaced by normal aerated ACF (composition: 118 mM NaCl, 4 mM KCl, 2 mM MgCl2 , 2 mM CaCl2 , 1.2 mM NaH2 PO4 , 25 mM NaHCO3 , and 10 mM glucose). Electrophysiological experiments were undertaken after slices had been in normal ACF for at least 60 min. This protocol was adequate for 2- to 3-week-old rats. However, for some unknown reasons, GSH depletion induced by slicing tended to increase with animal age (Steullet, unpublished). In 4–6-month-old mice, slicing could lead to >30% GSH depletion. However, after the initial GSH depletion following slicing, levels of GSH remained stable over many hours. Up to date, all our attempts to prevent or limit such GSH depletion in slices of adult mice have failed. Improved slicing protocol is still needed to investigate the effects of GSH depletion on most aspects of neurotransmission using slices of adult rodents. Therefore, in the absence of a better protocol, which would spare GSH in brain slices of adult rodents, many effects of GSH might be underestimated or even not observed.

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Acknowledgments We would like to acknowledge all collaborators who contributed significantly to the development and study of some of the models described in this review. This includes in particular Beatrix Benz, Vincent Castagné, Ying Chen, Adeline Cottier, Timothy P. Dalton, Fulvia Gheorghita, Gilbert Grima, Jean-Pierre Hornung, Rudolf Kraftsik, Suzie Lavoie, Delphine Preissmann. We are also grateful to Pierre Magistretti for his constant encouragement and Paul Herrling for his support in the initial phase of the project. Work supported by the Swiss Research Foundation (grants nos. 31-55924.98 and 31-116689 to K.Q. Do; grant no. 3100A0–105765 to F. Schenk) and the foundation “Loterie Romande.” References 1. Valko, M., Leibfritz, D., Moncol, J., Cronin, M. T., Mazur, M., and Telser, J. (2007) Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 39, 44–84. 2. Jones, D. P. (2008) Radical-free biology of oxidative stress. Am J Physiol Cell Physiol 295, C849–C868. 3. Kwon, Y. W., Masutani, H., Nakamura, H., Ishii, Y., and Yodoi, J. (2003) Redox regulation of cell growth and cell death. Biol Chem 384, 991–996. 4. Menon, S. G. and Goswami, P. C. (2007) A redox cycle within the cell cycle: ring in the old with the new. Oncogene 26, 1101–1109. 5. Shi, Z. Z., Osei-Frimpong, J., Kala, G., Kala, S. V., Barrios, R. J., Habib, G. M., Lukin, D. J., Danney, C. M., Matzuk, M. M., and Lieberman, M. W. (2000) Glutathione synthesis is essential for mouse development but not for cell growth in culture. Proc Natl Acad Sci USA 97, 5101–5106. 6. Lipton, S. A., Choi, Y. B., Takahashi, H., Zhang, D., Li, W., Godzik, A., and Bankston, L. A. (2002) Cysteine regulation of protein function – as exemplified by NMDA-receptor modulation. Trends Neurosci 25, 474–480. 7. Lu, S. C. (2009) Regulation of glutathione synthesis. Mol Aspects Med 30, 42–59. 8. Forman, H. J., Zhang, H., and Rinna, A. (2009) Glutathione: overview of its protective roles, measurement, and biosynthesis. Mol Aspects Med 30, 1–12. 9. Dringen, R. (2000) Metabolism and functions of glutathione in brain. Prog Neurobiol 62, 649–671.

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Chapter 8 Psychiatric Genetics and the Generation of Mutant Animal Models P. Alexander Arguello and Joseph A. Gogos Abstract Accumulating evidence indicates that the genetic architecture of psychiatric disorders does not strictly conform to the common disease/common allele hypothesis. The contribution of common genetic variants, while likely, may be fundamentally different from those of rare genetic variants. It is possible that common alleles do not increase disease risk per se but are disease modifiers sculpting the psychopathological landscape produced by rare alleles. Unlike common alleles, the statistical association of rare alleles is usually more robust and their functional effects more translatable into etiologically valid animal models. Although rare alleles may not be shared across individuals with the same diagnosis, the comparison of multiple animal models of rare risk alleles can identify common pathogenetic mechanisms. Thus, paradoxically, the cumulative evidence gathered from these animal models is currently poised to offer more insight into common psychiatric disorders than are models of common alleles. Key words: Risk alleles, rare genetic variants, neuregulin, DISC1, 22q11 microdeletions.

1. Introduction Genetic variation influences almost all phenotypes from the simple to the complex, including the risk for and manifestation of mental illnesses. In light of recent successes and failures, psychiatric genetics is currently at a crossroads concerning how to efficiently identify causal genetic factors and how to translate these findings into meaningful insights for disease prevention and treatment (1). Clearly, teasing apart the contribution of genetic variants to neural development and function, and ultimately disease risk, necessitates the use of model systems. Mutant animal models are thus becoming increasingly instrumental in basic psychiatric P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_8, © Springer Science+Business Media, LLC 2011

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research (2). In this chapter we summarize the current and future roles of animal models in this process with a particular focus on schizophrenia. Genetic manipulation in animal models is commonly used to investigate either a hypothesized disease state or a disease process, yet we discuss only the latter. A disease state is a more proximate cause of overt symptoms and, in the case of psychiatric disorders, reflects pathophysiological brain function, whereas a disease process is more distal in the causal chain and results from an initial etiological factor setting off a pathogenetic cascade [for a recent review of pathophysiological models, see Desbonnet et al. (3)]. Unlike models of disease states, models based on psychiatric genetics are not limited by incomplete knowledge of pathophysiology, are more closely linked with the initial pathogenic event, and hold more promise for identifying targets for early intervention. Before describing specific animal models, however, we first discuss recent findings from psychiatric genetics. This is critical because mutant animal models are only as reliable as the clinical data upon which they are based and choosing wisely among genetic findings is the first step in creating valid models. We argue that for both theoretical and empirical reasons, the current methods for identifying genetic risk variants determine the types of etiologically valid mutant models possible. Finally, we illustrate these points by comparing currently available mutant animal models and offer suggestions for future research.

2. Genetic Architecture of Psychiatric Disorders

Psychiatric disorders, like other common diseases, are multifactorial in nature with complex genetic etiologies. The genetic architecture underlying disease susceptibility is characterized by both the frequency and the penetrance of risk alleles (Fig. 8.1). The common disease-common allele (CDCA) hypothesis emphasizes the importance of relatively common alleles, each of small effect, acting together to increase disease risk. The common disease-rare allele (CDRA) hypothesis instead emphasizes the impact of individually rare yet highly penetrant alleles. It is likely that both common and rare alleles contribute to the risk of psychiatric disorders, although the relative impact of each remains unknown. In this section, we discuss the emerging evidence supporting both of these hypotheses in relation to schizophrenia and then address how these data can best guide the creation of mutant animal models.

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Fig. 8.1. Genetic architecture of common psychiatric disorders. Based on current findings, genetic variants that are relatively common in the population (>5%) have minimal impact on disease risk with most of these variants having low odds ratios (OR). In contrast, the rare genetic variants (<1%) so far identified have a more substantial impact on disease risk with most having high OR.

2.1. The Impact of Common Genetic Variants

Modern association studies exploit ancestral genetic variants that are now common (>5% minor allele frequency) in the population. These studies can focus on candidate genes based on a priori evidence or identify candidates in an unbiased manner using genome-wide association (GWA) studies (4). So far, based on the CDCA hypothesis, more than 1400 studies have investigated nearly 800 candidate genes for association with schizophrenia (www.szgene.org). Many of these are considered strong susceptibility genes, but none have unequivocal statistical support (5, 6). Combined, well-powered, GWA studies of psychiatric disorders have identified two candidate loci for bipolar disorder (10q21.2 and 12p13.3) (7), one candidate locus shared between bipolar disorder and schizophrenia (10q26.13) (8), and one locus for autism (15p14.1) (9). It is interesting to note that these loci do not include any top candidate genes. While there is some debate about the relative merits of candidate gene versus GWA approaches, both suffer from similar limitations. They both use a restricted set of polymorphic markers that may or may not be linked with other unseen variants. Given that the markers genotyped represent a small fraction of all common genetic variants, significantly associated markers in GWA studies are a priori unlikely to be the true causative variants (10). Moreover, these variants often occur in regulatory regions whose effects are currently unknown or lie distal to genic regions leaving open the question of which genes are affected. Thus, even if statistically unequivocal common risk alleles are found in the near future, these are likely far removed from the actual causative allele. Follow-up studies, including replication and fine mapping

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studies, may help in identifying the causal alleles, but even then the functional impact of these may remain uncertain (11). 2.2. The Impact of Rare Genetic Variants

The rarity of families afflicted with psychiatric disorders in a strictly Mendelian fashion limited, until recently, acceptance that rare mutations play a significant etiological role. Taking into account variable penetrance and de novo occurrences, however, rare alleles may contribute substantially to both familial and sporadic cases (12–14). It is not surprising that the alleles with strongest statistical support for association with schizophrenia are rare, highly penetrant structural mutations. These include copy number mutations of 22q11.2 (14–16) and possibly 1q21.1, 5q13.3 (14, 16) as well as a balanced chromosomal translocation t(1;11)(q42.1;q14.3) (17). Moreover, recent surveys indicate that rare copy number mutations as a whole are over-represented in cases of schizophrenia and autism (13, 14, 18). These data suggest that rare alleles contribute substantially to the risk of psychiatric disorders. Unlike common variants, a rare allele may be population, family, or even individual specific. In the latter case, it is not possible to prove unequivocally that the mutation is pathogenic since individuals carry many private mutations. It is possible, however, to determine whether cumulative mutational load for a given locus or biological pathway is greater for affected versus unaffected individuals collectively (4). While current estimates indicate that only a minority of cases (≤1%) are attributable to any given rare, recurrent allele (14, 16), this is likely to be a lower limit. Improvements in the resolution and efficiency of genotyping platforms will increase detection of rare alleles and their association with disease. Moreover, common variants account for only a small proportion of familial risk for common diseases, leaving the possibility that rare alleles contribute significantly to the remaining risk (19). Nevertheless, it is possible that the penetrance or the expressivity of rare mutations is determined by common genetic variation (11).

2.3. Implications for Mutant Animal Models

In order for mutant animal models to uncover a genuine pathogenetic link between a candidate gene and disease risk, the association of the genetic variant must be unequivocal, its functional effect known, and the risk allele modeled accurately. None of these conditions are met for current candidate genes or variants identified through GWA studies and thus the prospect of creating valid animal models based on common alleles is strikingly low (2). Unlike association studies, assessments of rare variants identify specific, and very likely causative, disease-associated alleles. These rare alleles are far more likely to have deleterious functional consequences than are common alleles due to clear effects on gene products. As a result they have higher potential for revealing

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insights into disease etiology than do less penetrant common alleles (12). In fact, the few candidate genes with common alleles of clear functional effect (e.g., COMT rs4680 and BDNF rs6265 ) show the most inconsistent clinical associations (20, 21). Therefore, because the evidence of disease association for rare alleles is often more statistically sound and the effects of the mutation more transparent, it is possible to engineer genetically faithful, and thus etiologically valid, animal models.

3. Animal Models of DiseaseAssociated Loci

3.1. Neuregulin 1 (NRG1) Susceptibility Locus

Although the selection of candidate genes is controversial and their validation problematic, it is possible to dissect the functional roles of candidate genes and investigate the biological pathways in which they participate. This allows the generation of hypotheses regarding how these pathways may dysfunction and give rise to psychiatric disorders. When interpreting results from animal models of candidate genes, it is important to realize that the selection and the verification of candidate genes are not always independent of each other, making gene validation a potentially circular and self-bootstrapping process. Moreover, the fact that behavioral tests used to characterize models lack specificity raises the issue of whether a suite of behavioral changes can validate a mutant model in the absence of strong corroboration from human genetics. In this section we describe mutant animal models from four candidate genes, including three genes found in genomic loci showing genetic linkage with schizophrenia (NRG1, DTNBP1, and PPP3C). While linkage signals may reflect rare as well as common alleles, current technology allows only the efficient genotyping of common makers. Thus, association studies that follow-up linkage results assume a significant contribution of common variants within these loci. A significant linkage peak at 8p22 implicated Neuregulin 1 (NRG1) as a schizophrenia susceptibility locus. Despite a lack of robust statistical support for specific alleles, NRG1 and its receptor, ERBB4, are among the leading candidate susceptibility genes for schizophrenia (22, 23). NRG1 is a trophic factor that, through binding of receptor tyrosine kinases, is involved in cellular proliferation, migration, neurite outgrowth, synaptic transmission, and intracellular signaling (24). This high level of pleiotropy suggests that genetic variants affecting NRG1 signaling could influence disease risk through any number of biological pathways.

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Several groups have created mutant animal models affecting various domains of the Nrg1 gene to assess their impact on behavior. The first group to describe an Nrg1 mutant mouse deleted the EGF-like domain of the encoded protein and showed that these mice displayed increased locomotor activity in multiple contexts but intact short-term memory (25). Stefansson et al. (22) analyzed mutant mice lacking the transmembrane domain of Nrg1 and found that they had increased locomotor activity in an open field and impaired sensorimotor gating as measured with PPI, but normal anxiety-related behaviors. These mice were further analyzed to show abnormal social interactions but normal short-term spatial memory (26, 27). Rimer et al. (28) targeted a subset of Nrg1 isoforms by deleting the Ig domain and found that these mice showed impaired latent inhibition, but normal locomotor activity and short-term memory. Other mutant animal models have focused on Nrg1 receptors. Similar to Nrg1 hypomorphs, homozygous ErbB4 knockout mice, but not ErbB2 or ErbB3 knockouts, showed increased locomotor activity and impaired PPI. Roy et al. (29) used a transgenic approach to express a dominant-negative ErbB4 receptor specifically in oligodendrocytes. In addition to decreased complexity of oligodendrocytes and reduced myelin thickness, these mice showed decreased locomotor activity, increased anxiety-related behaviors, abnormal social interactions, and hyperactive midbrain dopamine function. Barros et al. (30) crossed floxed ErbB2 and ErbB4 mice with CRE-expressing mice to knock out ErbB2/4mediated Nrg1 signaling within the CNS and showed that these mice had increased locomotor activity, increased aggressivity, and decreased PPI. Surprisingly, cortical development of these mice was normal. They did, however, show decreased spine maturation of both neocortical and hippocampal neurons. Savonenko et al. (31) investigated Nrg1 function by assessing knockout mice of one of its proteolytic processors, Bace1, which were previously used as an animal model of Alzheimer’s disease prevention (32). These mice showed increased locomotor activity in several contexts, impaired PPI, abnormal social interactions, impaired fear-associative memory, and impaired spatial short-term memory, but normal long-term spatial reference memory. Mutant mice also showed changes in dendritic complexity and spine density of hippocampal neurons. It is unclear, however, if these effects were due to Nrg1 or one of the other downstream substrates of Bace1. Overall, these studies show that disruptions in Nrg1 signaling affect locomotor activity and sensorimotor gating but have less reliable impact on cognitive function. Given that there is, as of yet, no clear disease-associated functional allele within the NRG1 locus, it is uncertain which, if any, of the current mutant animal models are relevant to schizophrenia.

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DTNBP1 was first identified as a candidate gene by fine mapping of a significant linkage signal at 6p22. Several subsequent association studies indicated that common variants within this locus may influence disease risk. As in the case of NRG1, however, the statistical support is equivocal (5, 6). DTNBP1 is widely expressed across tissues and organs, including the CNS, and is involved in the trafficking of lysosome-related organelles. Although disputed as a strong susceptibility locus, DTNBP1 remains a popular candidate gene and the availability of a naturally occurring null allele in a mouse strain makes it an attractive candidate to model. The sdy strain of mice carries a spontaneous Dtnbp1 null allele resulting from a ∼38-kb deletion that abolishes production of the dysbindin-1 protein (33). These mice show systemic abnormalities in vesicle trafficking to lysosomes, resulting in albinism and pulmonary fibrosis. Takao et al. (34) carried out a behavioral screen of homozygous sdy mice and found that mutant mice had decreased locomotor activity, impaired long-term spatial reference memory, and impaired acquisition of a short-term memory task, but normal anxiety-related behaviors and intact neurological functions. Bhardwaj et al. (35) also characterized sdy mice and showed that homozygous mutant mice had decreased locomotor habituation and increased fear-associative memory. While both heterozygous and homozygous mutants showed impaired shortterm recognition memory and a blunted locomotor response to acute amphetamine, only homozygous mice were hypersensitized to amphetamine after chronic treatment. Feng et al. (36) examined homozygous sdy mice and found they had normal locomotor activity and normal anxiety-related behaviors but impaired social interactions and long-term recognition memory. Cox et al. (37) backcrossed sdy mice into a C57BL/6 background and used wild-type littermate controls. Homozygous mutant mice showed increased locomotor activity, decreased locomotor habituation, impaired spatial navigation, and impaired long-term spatial reference memory but less anxiety-related behaviors and improved motor learning. Given its role in vesicle trafficking, it is not surprising that dysbindin mutant mice show deficits in neurotransmitter release (38). These deficits as well as dopamine dysfunction in these animals (39) may be relevant to the putative role of DTBNP1 in schizophrenia. Although methodological differences, such as the use of littermate controls, may account for contradictory findings from sdy mice, further studies are needed to confirm dysbindin’s role in the CNS and define its neurobiological functions. Like NRG1, however, the lack of clear disease-associated, functional alleles within the DTBNP1 locus makes dysbindin’s role in disease risk unclear. Thus, even though there is evidence of reduced dysbindin expression in schizophrenic brain tissue (40), this makes the relevance of sdy mice to schizophrenia pathogenesis uncertain.

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3.3. Other Susceptibility Loci

4. Animal Models of DiseaseAssociated Alleles

Near the 8p22 linkage peak that implicated NRG1 as a candidate gene resides PPP3CC, which encodes for a calcineurin catalytic subunit. There is some support that common variants within this locus are associated with schizophrenia (41). Mice with a conditional knockout of calcineurin’s regulatory subunit provide additional support because they display behavioral deficits similar to animal models of other schizophrenia candidate genes. These deficits include increased locomotor activity, decreased PPI, impaired short-term spatial memory, and impaired reversal learning (42, 43). Variants within the regulatory subunit gene itself, however, have not been associated with schizophrenia. Nevertheless, taken together, genetic and animal model findings point to a potentially important role of this pathway in disease susceptibility, possibly via integrating glutamatergic and dopaminergic signaling as well as modulating synaptic vesicle recycling (44, 45). AKT1, a functional candidate gene, provides another relevant example. While AKT1 was not identified from a linkage peak, AKT1 protein levels and phosphorylation of its substrate GSK3β were reduced in brains and lymphocytes of individuals with schizophrenia (46). AKT1 it is an attractive candidate primarily because of its functional role in dopaminergic signaling (47, 48). Similar to other candidate genes, the support for common variants within this locus increasing disease risk is mixed (www.szgene.org). Nevertheless, mice deficient in Akt1 show a greater sensitivity to the disrupting effects of amphetamine on PPI (46) and have altered dendritic complexity in medial prefrontal cortex (mPFC) and impaired working memory performance under various pharmacological challenges. Whether any other changes produced by Akt1 deficiency (48) are also relevant to disease risk remains to be determined.

The increasing ability to detect rare genetic lesions strongly associated with psychiatric disorders provides a unique opportunity for generating etiologically valid mutant animal models. In this section we discuss animal models of the strongest inherited and de novo genetic risk factors for psychiatric disorders known: a familial truncation of the DISC1 gene and sporadic copy number mutations of 22q11.2, respectively. Although these lesions are individually rare, the ability to model them accurately allows identification of the exact cellular pathways and neural circuits likely involved in disease pathogenesis.

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DISC1 was identified through a balance chromosomal translocation t(1;11)(q42.1;q14.3) segregating with schizophrenia and mood disorders in a large Scottish pedigree. Like NRG1, DISC1 is a highly pleiotropic gene with effects on cellular proliferation, migration, dendritogenesis, synaptogenesis, and intracellular signaling (49). Association studies suggest that common variants within the DISC1 locus may influence the risk of psychotic and mood disorders in general (50). Like other association studies, however, these studies of DISC1 have not produced any statistically unequivocal risk alleles. This means the well-defined mutation in the Scottish family is currently the only disease-associated allele within the DISC1 locus capable of being modeled validly in animals. While the biology of DISC1 is being interrogated with many analytical tools, such as shRNA (51–53), these are not meant to recapitulate the integrated effects of a genetic lesion. Thus, the relevance of these findings to schizophrenia pathogenesis remains uncertain and, in fact, has produced opposing results from animal models carrying germline mutations (54). We therefore mainly discuss DISC1 mutant models that have attempted to recapitulate the Scottish mutation. We summarize the main findings from these models with a focus on the different methodological approaches used by investigators. Koike et al. (55) were the first to describe a DISC1 animal model. They used a knock-in approach to introduce a truncating lesion in the endogenous murine Disc1 orthologue to resemble the putative effects of the translocation (Fig. 8.2, Disc1tm1Kara ). This abolishes expression of the major Disc1 isoforms and, despite an artificial polyadenylation signal, causes low expression of the truncated protein, indicating its instability under physiological conditions (54). These mutant mice showed no changes in locomotor activity or PPI (55). They did, however, show a unique profile of cognitive impairments. A battery of tests revealed no changes in fear-associative, object recognition, long-term spatial reference, or short-term spatial memories (54). Rather, both heterozygous and homozygous mutant mice exhibited consistent impairments in working memory tests (i.e., short-term memory tests with high demands on cognitive control) (54, 55). Interestingly, the cognitive deficits in mutant mice were in the context of minimal histopathological changes in mPFC, with more evident changes in the hippocampus including changes in the distribution and organization of young and mature dentate gyrus granule cells. While the cognitive profile of translocation carriers is unknown, the cognitive impairments in these mice may relate to the cognitive control deficits prominent in psychotic and mood disorders.

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Fig. 8.2. Animal models of the DISC1 locus. (Top) Chromosomal location and genetic structure of the human DISC1 locus. The t(1;11) translocation break point occurs between exons 8 and 9 (arrow) with exons 9–13 (black) relocated to chromosome 11. Below it are the allele symbols and transgenic constructs used to model the functional effects of the translocation. These models interfere with endogenous mouse Disc1 function in a dominant-negative [Tg(Camk2a-DISC1)10Asaw and Tg(tetO/CMVDISC1∗ 1001Plet + Tg(CAmk2a-tTA)1Mmay] or transdominant-negative [Tg(camk2aESR1/DISC1∗ )2698.1Sva] manner. (Bottom) Syntenic chromosomal location and genetic structure of the mouse Disc1 locus and the corresponding break point location (arrow). Below it are the various mouse Disc1 alleles so far described. Disc1del arose from a spontaneous 25-bp deletion within exon 6 that introduces a premature stop codon in

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Other mouse models were designed under the assumption that the (1;11) translocation produces a truncated protein and thus overexpresses a truncated form of human DISC1. Hikida et al. (56) were the first to produce such a model and used a αCAMKII promoter to drive truncated DISC1 expression in forebrain principal neurons (Fig. 8.2, Tg(Camk2aDISC1)10Asaw). These transgenic animals showed increased lateral ventricular volume in adulthood, reduced numbers of parvalbumin-positive cells in the mPFC, abnormal locomotor activity, subtle deficits in PPI, impaired foraging, and increased immobility in the forced swim test (FST). There were no changes in measures of anxiety-related behaviors, long-term spatial reference memory, or short-term spatial memory as assessed in the Y maze. Pletnikov et al. (57) used the Tet-off double transgenic system to drive expression of truncated human DISC1 under the CMV promoter with tetracycline under the αCAMKII promoter (Fig. 8.2, Tg(tetO/CMV-DISC1∗ )1001Plet). These double transgenic mice showed increased lateral ventricular volume, abnormal locomotor activity, and increased aggression in males but no changes in measures of anxiety-related behaviors, long-term spatial reference memory, olfaction, or PPI. Females did, however, show deficits in long-term spatial reference memory. Unlike the previous studies that expressed an N-terminal fragment of DISC1, Li et al. (58) expressed a C-terminal fragment of DISC1 under the αCAMKII promoter using a single transgenic inducible and reversible system (Fig. 8.2, Tg(Camk2aESR1/DISC1∗ )2698.1Sva). This approach was designed to transiently disrupt Disc1 interactions occurring at the C terminus. Mice briefly exposed to this truncated DISC1 peptide at postnatal day 7 showed abnormal responses to the FST, spatial working memory deficits, and decreased sociability but no changes in locomotor activity or measures of anxiety-related behaviors. Interestingly, no deficits were apparent when the mutant DISC1 fragment was expressed during adulthood.



Fig. 8.2. (continued) exon seven (∗ ) of 129 and related strains of mice. Disc1tm1Kara was engineered to carry a premature stop codon at the end of exon 8 (∗ ) followed by a polyadenylation signal in an attempt to recapitulate the translocation, but since this was created in a 129S6/SveV background, the allele also carries the endogenous 25-bp deletion. Disc1Rgsc1390 and Disc1Rgsc1393 have missense mutations in exon 2 identified through a mutagenesis screen and mice carrying these alleles show divergent behavioral phenotypes. Although not meant to model the translocation and thus not discussed in the main text, these mutations indicate that allelic heterogeneity of the DISC1 locus may account for diverse psychopathologies. Tg(Disc1∗ -eGFP)unannotated is a transgenic allele constructed from an artificial bacterial chromosome consisting of exons 1–8 of the mouse Disc1 fused to enhanced green fluorescent protein (eGFP). Mice carrying two copies of this allele also have two wild-type, endogenous Disc1 copies.

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It is important to note that the above transgenic models do not necessarily recapitulate the effect of the diseaseassociated allele found in the Scottish family. The ectopic expression of mutant DISC1 does not reflect Disc1 expression in terms of absolute levels or spatial and temporal patterns. In an attempt to circumvent these limitations, Shen et al. (59) engineered transgenic mice to express two copies of truncated mouse Disc1 using a BAC system, thus, presumably, preserving the temporal and spatial profiles of Disc1’s endogenous expression (Fig. 8.2, Tg(Disc1∗ -EGFP)unannotated). These mice showed increased lateral ventricular volume, decreased neuronal proliferation, thinner cortices, agenesis of the corpus callosum, reduced parvalbumin-positive cells in the PFC and the hippocampus, increased immobility in the FST, and abnormal latent inhibition. While presumably the ratio of mutant to normal Disc1 copies is similar in this model to that in translocation carriers, the absolute dosage is increased by twofold and altered function of N-terminal interacting partners is possible. Although this model has advantages over other transgenic approaches, further clinical characterization of translocation carriers (e.g., MRIs) is needed to determine how accurately this model reflects the human condition. 4.2. The 22q11 Susceptibility Locus

Recent studies of human genetic variation indicate that largescale genomic structural rearrangements are common in the population. One of the most common abnormalities is de novo microdeletion of the 22q11.2 locus. Carriers of this microdeletion are at inordinately high risk of developing schizophrenia with odds ratios exceeding ∼25-fold. Currently, 22q11.2 microdeletions represent the only confirmed recurrent mutation responsible for introducing sporadic cases of schizophrenia into the population. The most common deletion spans 3 Mb, but a nested 1.5 Mb deletion seems to be the schizophrenia critical region (15). Premorbid children with the microdeletion present with variable symptoms including impairments in attention, memory, and cognitive control, suggesting that these cognitive deficits reflect genetic liability to schizophrenia. Fortunately, the human 22q11.2 locus is conserved within the syntenic region of mouse chromosome 16 and harbors nearly all orthologues of the human genes (Fig. 8.3). This provides a unique opportunity to create mutant animal models with a high degree of etiological validity. Kimber et al. (60) were the first to behaviorally characterize a model of the 22q11.2 microdeletion. They engineered mice deficient in seven genes spanning ∼150 kb of the 1.5-Mb deletion syntenic region (Fig. 8.3, Del(16Zpf520Slc25a1)1Awb). Hemizygous mice showed increased PPI but normal locomotor activity, anxiety-related behaviors, fear-associative memory, and neurological function. Paylor et al. (61) characterize a 22q11.2 model spanning ∼1 Mb containing 18

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Fig. 8.3. Animal models of the 22q11.2 locus. (Top) Chromosomal location and genetic organization of the 22q11.2 locus. Each open circle represents one gene. This 1.5-Mb critical region is flanked by low copy repeat sequences (black boxes), making it prone to non-homologous recombination. PRODH-P and DGCR6-like are pseudogenes. (Bottom) Syntenic region of mouse chromosome 16 and the genetic organization of the corresponding orthologues. Single gene deletion models that have been analyzed with PPI are indicated in solid black circles. Below are the various multigene deletion models that have been characterized behaviorally labeled by their allele symbols. Note that only two models contain all the genes within the 1.5 Mb deletion associated with schizophrenia.

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orthologues of the human genes within the 1.5 Mb deletion (Fig. 8.3, Del(16Es2el-Ufd1l)217Bld). They found decreased PPI and impaired fear-associative memory but no changes in measures of anxiety-related behaviors or neurological function. Long et al. (62) used mice with a hemizygous deletion spanning ∼1.3 Mb and containing all of the orthologous genes within the 1.5 Mb deletion (Fig. 8.3, Del(Dgcr2-Hira)1Rak) and found decreased PPI and neurological dysfunction but normal fear-associative memory. Finally, Stark et al. (63) also created mice with a hemizygous deletion syntenic to the human 1.5 Mb deletion (Fig. 8.3, Del(Dgcr2-Hira)2Aam). This deletion resulted in increased locomotor activity, decreased PPI, decreased fear-associative memory, and impaired acquisition of a working memory task. These behavioral changes were in the context of decreased dendritic complexity, reduced spine density and fewer excitatory inputs to CA1 hippocampal neurons. To identify which of the many genes within the 22q11.2 microdeletion may be responsible for the increased risk of schizophrenia, several groups have created single gene deletion models. Gogos et al. (64) described a knockdown model of Prodh, which encodes a mitochondrial enzyme that metabolizes L -proline. These mice showed decreased PPI, increased locomotor activity, hypersensitivity to amphetamine and impaired fearassociative memory but normal working memory (64, 65). Interestingly, transcriptional profiling revealed an increased expression of Comt, a gene within the 22q11.2 microdeletion that catabolizes dopamine. This seemed to be a compensatory response to underlying dopamine dysfunction as pharmacological inhibition of Comt exacerbated the sensitivity to amphetamine and produced working memory deficits in Prodh-deficient mice (65). This animal model suggests that the risk for schizophrenia in 22q11.2 deletion carriers is mediated, at least in part, by an inability to deploy a genetic feedback loop residing within the deletion. Two subsequent clinical studies of human carriers showed an interaction between proline levels and low COMT activity thus providing additional support for this hypothesis (66, 67). Several other orthologues of genes within the microdeletion seem to contribute to the phenotypic profile of the 22q11.2 deletion model. Mukai et al. (68) and Stark et al. (63) showed that mice deficient in Zdhhc8, a palmitoyltransferase, and Dgcr8, a microRNA processor, each account for distinct aspects of the reduced dendritic complexity and spine density of hippocampal neurons from complete deletion mice. Furthermore, Dgcr8 deficiency accounts for the reduced microRNA biogenesis observed in these mice and on its own causes working memory deficits (63). Other groups have shown that several genes within the 1.5-Mb region differentially affect PPI. While heterozygous deficiency of Gscl, Dgcr8, and Tbx1 decreases PPI (62, 63, 69), Rtn4r,

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Zdhhc8, Comt, and Sept5 deficiencies have no effect (69–72). It is interesting that deletion mice described by Kimber et al. (60) have increased PPI, even though Gscl deficiency itself decreases PPI, thereby suggesting that other genes within this region have opposing influences. Although PPI is not diagnostically specific or predictive of clinical outcome, it is a relatively robust assay and thus underscores the difficulty in teasing apart genotype– phenotype relationships from models of large structural mutations. Most importantly, and despite disparate results due to the various genes deleted within the 22q11.2 locus, mouse models of the 22q11.2 microdeletion have identified specific pathways affecting hippocampal neuronal connectivity and neuromodulation that, in combination, may be critical for the dramatic increase in schizophrenia rates seen in 22q11.2 deletion carriers. Moreover, studies from these models will serve as guidelines for dissecting out the contribution of specific genes within other contiguous gene deletion syndromes that may be associated with psychiatric disorders in the future.

5. Integrating Human Genetics and Mutant Models

5.1. Identifying Disease Pathways

Unlike most common diseases, psychiatric disorders do not have clear etiologies or diagnostic tests. Instead they are syndromes, constellations of commonly co-occurring symptoms that are classified by outward behavioral disturbances. This presents a unique challenge to psychiatric research not evident for other common diseases. In this last section we describe the difficulties in integrating human genetics with animal models in order to validate genetic findings and identify pathogenetic pathways. One major advantage of GWA studies is their unbiased approach to identifying candidate genes. They are not limited by hypotheses about etiology or pathophysiology and can identify previously unsuspected biological pathways. Indeed, this has happened for many common diseases (4). Identifying biological pathways, however, is not equivalent to deciphering disease mechanisms. This is most evident in the case of pleiotropic genes involved in multiple biological pathways across development and adulthood. It is essential to determine how and when specific alterations in these pathways underlie disease pathogenesis. This is a major obstacle for mutant models of genes without clear functional risk alleles because psychiatric disorders lack biological markers, or even behavioral tests, that can validate the model (73).

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In order to circumvent these limitations, findings from multiple, etiologically valid animal models are needed. It is likely that no single mutant animal model will provide clear etiological insight. For any individual genetic variant, whether common or rare, only a subset of its downstream functional alterations may be relevant to disease etiology (74). Taken collectively, however, various mutant animal models may identify a final common pathway, leading to psychopathology. The exact level of this convergence, whether at the molecular, cellular, or systems level, remains to be determined. Fortunately, as genotyping technologies advance, so will the ability to create valid animal models and the ability to integrate their phenotypes into a clearer picture of disease pathogenesis. 5.2. Challenges for Moving Beyond Single Genes

The challenge of unraveling biological pathways into disease mechanisms is compounded when interactions between different risk alleles and environmental factors are taken into account. For example, a candidate gene affecting one biological pathway may point to other genes within the same pathway as potential candidates. Without, however, corroborating human genetic findings implicating specific functional risk alleles in those genes, this added layer of complexity will likely create false leads and dead ends. The same applies to modeling gene–gene and gene– environment interactions and will be especially problematic when using animal models of genetic loci instead of specific risk alleles. It remains an open empirical question if risk factors, whether environmental or genetic, will interact with only disease-relevant pathways or also have non-specific effects. Moreover, simply looking for changes that are exaggerated in an animal model of multiple risk factors may be misleading and miss relevant mechanisms. This is because interacting factors may either worsen a pathogenetic process or mitigate a compensatory response (2). In both cases, this would lead to a more severe pathophysiology. Thus, caution is warranted when adding layers of higher order interactions atop shaky foundations of unreliable mutant models as this may increase the difficulty of unearthing relevant disease mechanisms.

Acknowledgments Work in the authors’ laboratory is supported by grants from NIMH, NARSAD, and the Simons Foundation. We thank M. Karayiorgou, B. Levy, and L. Drew for critical readings of the manuscript.

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Chapter 9 DISC1 Mouse Models Yavuz Ayhan, Hanna Jaaro-Peled, Akira Sawa, and Mikhail V. Pletnikov Abstract Disrupted-in-Schizophrenia 1 (DISC1) is a strong candidate gene for schizophrenia and major mental disorders. After its discovery in the Scottish chromosomal translocation, DISC1 has gained considerable attention in neuropsychiatric research. Recent studies have implicated DISC1 in fundamental processes of neurodevelopment and adulthood neuroplasticity. To get more insights into the functions of DISC1 in vivo, several mouse DISC1 models have been generated based on different approaches, including constitutive and inducible over-expression of different fragments of DISC1, targeted mutagenesis, and viral vector knockdown. Each model has provided important information regarding DISC1 functions and helped in elucidating the molecular pathways underlying behavioral disorders. The existing models also serve as valuable tools to address complex issues of the pathogenesis of schizophrenia, including gene– gene and gene–environment interactions. Here, we critically overview current DISC1 mouse models. Future directions in DISC1 mouse models and alternative approaches are discussed. Key words: DISC1, schizophrenia, depression, mouse models, neurodevelopment.

1. Introduction Animal models have been instrumental in advancing psychiatric research (1–4). Various pharmacological or lesion-based animal models of schizophrenia have been proposed (5). The progress in human psychiatric genetics and identification of several candidate genes for schizophrenia has stimulated generation of a number of genetic mouse models of the disease (6–8). In this chapter, we will evaluate Disrupted-in-Schizophrenia 1 (DISC1) genetic mouse models. We will first overview the discovery of the DISC1

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gene and its significance for psychiatry and neurodevelopment, and we will critically discuss existing DISC1 mouse models. The DISC1 gene was first identified in a t(1;11) translocation in a Scottish pedigree with high load of major mental disorders (9). The LOD ratio for this translocation is 7.1 for all mental disorders and 3.6 for schizophrenia (10). Fine mapping and cloning resulted in identification of a disrupted gene at chromosome 1, and hence the name disrupted-in-schizophrenia 1. The breakpoint was in the middle of an open reading frame region for the gene, suggesting the possibility of expression of a truncated product, although its existence remains to be demonstrated (9, 11). The existence of a clear, identifiable mutation with high LOD scores has put DISC1 in a unique position in schizophrenia research. In the neuropsychiatry literature, there are examples of how such mutations have been instrumental in enlightening the biology of diseases, including familial forms of Parkinson disease and Alzheimer disease (12–15). Thus, the familial DISC1 mutation stimulated studies of the gene’s functions and effects of the mutation on neurodevelopment (16–18). Human genetic research has associated different haplotypes or SNPs of DISC1 with various mental disorders such as schizophrenia, bipolar disorder, depression, and autism; also with neurocognitive features; and with structural brain changes in human studies (19–22). Early in vitro studies have demonstrated a role of DISC1 in neuronal proliferation, migration, and synaptogenesis (23, 24). In order to provide better understanding of the function of DISC1 in the developing and adult brain in vivo, animal models were generated to evaluate both the outcomes of the translocation mutation and the effects of different genetic manipulations. In the following text, we will introduce DISC1 models published to date and discuss their strengths and weaknesses. Models that over-express the proposed mutant DISC1 protein product are presented as a separate group.

2. Methods 2.1. Genetic Manipulation In Utero 2.1.1. Background

2.1.2. Approach

This model was reported when the DISC1 neurobiology research was mostly limited to cell culture techniques. At the time, the structure of the gene and its main interactors were identified (25, 26). DISC1 was shown to play a role in neurite outgrowth via interaction with NDEL (17) and participate in axonal elongation by binding to FEZ1 (27). In order to characterize the effects of DISC1 on neuronal migration and dendrite arborization in vivo, in utero electroporation approach was used to knock down expression of endogenous

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mouse DISC1 or to over-express mutant human DISC1 (18). Small interference (si) RNAs were injected directly into the ventricular zones of mouse embryos at embryonic day 14.5. The brain morphology was assessed at postnatal day (P)2 and P14. 2.1.3. Results

In utero electroporation of Disc1 siRNA or human mutant DISC1 resulted in deficits of neuronal maturation and differentiation of cortical neurons. Knockdown of Disc1 expression produced delayed migration of neurons as evidenced by fewer cells that reached the superficial cortical layers at P2. When evaluated at P14, mis-positioned neurons with shorter arbors were found at cortical layers II/III. Similar effects were also observed after electroporation of mutant DISC1.

2.1.4. Conclusion

This model is the first in vivo model to demonstrate the role of DISC1 in neurodevelopment of the cortex. On-going investigations are evaluating the long-term effects of knockdown of Disc1 in developing cortex, including resultant behavioral alterations. In addition, future research should attempt to determine what Disc1 isoforms may be responsible for the observed effects.

2.2. DISC1 Knockdown In Vivo 2.2.1. Adult Neurogenesis 2.2.1.1. Background

The effects of knockdown of Disc1 on cortical development had suggested a possible role for this gene in adult neurogenesis in the hippocampus, a phenomenon thought to be involved in some mental disorders, e.g., mood disorders (28, 29).

2.2.1.2. Approach

Duan et al. used RNAi approach to identify the effects of knockdown of Disc1 on neuronal progenitor cells in the adult hippocampus when postnatal neurogenesis takes place (30). Oncoretroviral constructs engineered to co-express green fluorescent protein (GFP) and Disc1 shRNA were injected in the hilar region of the hippocampus of 2-month-old female C57BL/6 mice.

2.2.1.3. Results

The authors found no difference in the number of immature neurons. However, there were significant morphological differences in newborn cells. Neurons with Disc1 knockdown exhibited multiple primary dendrites, a significant amount of neurons maintained their basal dendrites, and variable degrees of the ectopic dendrite phenotype were observed. The migration of newly born neurons was augmented without affecting differentiation. Disc1 knockdown also altered structure of dendrites. By reconstruction of dendrite arborization, dendrite complexity was found to be increased after treatment with Disc1 shRNA. The functional maturation and integration of these newborn neurons into existing networks was assessed by

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electrophysiological analysis of membrane resistance and the ability to fire repetitively, as functional markers of maturation. Knockdown of Disc1 produced significantly lower cell membrane resistance and increased cells’ ability to fire repetitively, indicating accelerated functional maturation. Also, dendritic spines were formed earlier and faster. Furthermore, the electrophysiological data indicated accelerated formation of functional GABAergic and glutamatergic synaptic inputs to new neurons. 2.2.1.4. Conclusions

The effects on adult neurogenesis reported by this chapter have suggested multiple and varying functions of DISC1 across different stages of prenatal and postnatal neurodevelopment. In addition, given the growing literature on the role of adult neurogenesis in mood disorders and schizophrenia, this study has suggested a new mechanism whereby DISC1 can be involved in the pathogenesis of major psychiatric diseases.

2.2.2. Progenitor Proliferation and a New DISC1 Pathway

Further evidence of the role of DISC1 in neurogenesis has been provided by the observation of high expression of DISC1 in neural progenitor cells in the ventricular (VZ) and subventricular zones (SVZ), with expression being reduced when cells exit the cell cycle (28).

2.2.2.1. Background 2.2.2.2. Approach

Disc1 shRNAs were electroporated at E13 into the embryonic brains to evaluate the effects of DISC1 on proliferation of neuronal progenitor cells and their fate. The brain samples were processed at postnatal day 7. Another approach included injections of Disc1 shRNAs into the hippocampus to assess the effects on adult neurogenesis.

2.2.2.3. Results

Knockdown of Disc1 at E13 decreased proliferation of neuronal progenitors as indicated by reduced numbers of cells at VZ/SVZ, reduced BrdU labeling, and decreased mitotic index. Disc1 overexpression had the opposite effects. Knockdown also caused premature neuronal differentiation and increased differentiation. These effects of DISC1 were shown to be related to a direct interaction of DISC1 with GSK3β, leading to decreased activity of the enzyme and reduced phosphorylation of β-catenin and its rescue from ubiquitinization. Increased levels of β-catenin in neural progenitors elevated levels of targets of this transcriptional factor and rescued the effects of Disc1 knockdown on cell cycle. In vitro experiments demonstrated that DISC1 regulated β-catenin abundance and stability. Consistent with the prior report, knockdown of Disc1 in granule cells of the adult dentate gyrus decreased neurogenesis and, additionally, was associated with novelty-induced hyperlocomotion and increased floating time in forced swim test. Remarkably, all these brain and behavioral effects of knockdown of Disc1 were rescued by administration of a selective GSK3β inhibitor.

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2.2.2.4. Conclusions

The study has demonstrated a role of DISC1 in proliferation of embryonic and adult neural progenitor cells and identified a new molecular pathway whereby DISC1 is involved in regulation of proliferation of neuronal progenitor cells, lending additional support for the proposed role of neurogenesis in human mental disorders (29, 30). Given some opposite effects of DISC1 on adult and embryonic neurogenesis (18, 28, 31, 32), it would be interesting to further compare morphological, neurochemical, electrophysiological, and behavioral effects of embryonic vs. adult knockdown of DISC1 in mice at different time points of prenatal and postnatal development.

2.3. DISC1 Polymorphism Model

An unexpected discovery of 25 bp deletion mutation in exon 6 and a stop codon in exon 7 in Disc1 in 129S6/EvSv substrain during a gene targeting process led to an attempt to generate a mouse model of the translocation. In this mouse model, expression of a full-length Disc1 isoform was absent when assayed with Western blotting using a custom-made polyclonal antibody against the N-terminal portion. Modified by introducing a polyadenylation site in intron 7 and a stop codon in exon 8, the mutation was transferred to C57Bl/6 J background. This modification aimed to generate a fragment similar to the Scottish translocation in which the breakpoint is between exons 8 and 9 (33, 34).

2.3.1. Background

2.3.2. Results

The first report with this model (35) characterized the morphological and behavioral abnormalities in mice. The authors used a delayed non-match to place task (T-maze) to evaluate the function of the prefrontal cortex such as visuospatial working memory known to be impaired in schizophrenia (36, 37). No differences between mutant and control mice were seen in the learning phase of the test. In contrast, during the test phase, significant impairment in working memory was found at the 15- and 30-s choice delays for both heterozygous and homozygous animals. In addition, a deficit was seen at the 5-s delay in homozygous mutants. No differences between groups were detected in novelty-induced activity or PPI. The low magnification evaluation of the brain morphology showed no gross abnormalities. A follow-up study (38) evaluated the pathology of the hippocampus and medial prefrontal cortex (mPFC) in more detail. A 14% decrease in volume of mPFC and no changes in volume of the hippocampus were observed. In order to get a better resolution of the cortical morphology, DISC1 mice were mated with GFP-M and Thy-1-GFP transgenic mice that express GFP in neurons (39, 40). The authors found increased quantities of immature neurons in the dentate gyrus of the hippocampus in mutant mice and increased rates of neuronal migration indicated

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by the presence of a larger number of cells localized in the outer layers of the granule cell layer. These cells were misoriented and tended to have decreased apical dendritic branch points and dendritic length. Morphology of mature granule cells was analyzed in DISC1–Thy-1 mutant mice that express GFP in post-mitotic neurons. The similar misorientation phenotype was also seen in mature granule cells in mutant mice. In addition, a subset of mature granule cells showed decreased total dendritic length and numbers of dendritic pines. The structural abnormalities were associated with altered short-term potentiation detected in CA1– CA3 synapses. The authors also performed a comprehensive behavioral evaluation of mutant mice, including contextual fear conditioning, novel object recognition, Morris water maze and a win-shift version of the eight-arm radial maze, and two-choice delayed nonmatch-to-position (DNMP) task. Mutant mice showed a significant deficit in DNMP test only. DNMP is a measure of working memory and consists of training and testing phases. Both heterozygous and homozygous mutants displayed a deficit in the learning phase and compared to control mice, the performance of mutant mice was significantly worse at the 70-s delay during the testing phase. 2.3.3. Conclusions

This model with a loss of one full-length Disc1 isoform provides important clues on the function of DISC1. The main strength of this model is a modification of the endogenous gene whose expression is regulated by the natural promoter. However, the possible existence of other full-length isoforms of the protein in these mice as suggested by a different study may complicate mechanistic interpretations of neurobehavioral alterations. This also highlights a difficulty in generating complete knockout models for genes with multiple spliced isoforms of RNA (41, 42).

2.4. ENU-Induced Mutagenesis Mouse Model

In the Scottish family, schizophrenia has not been diagnosed in all translocation carriers. Some members suffer from recurrent major depression, bipolar disorder, or alcohol and substance abuse. Recent postmortem and genetic epidemiology studies have also implicated DISC1 in mood disorders and autism (43–47). Thus, different mutations in the gene may lead to variable clinical and pathological manifestations.

2.4.1. Background

2.4.2. Approach

Mutagenesis with N-nitroso-N-ethylurea (ENU) was used to generate several mouse lines, carrying different mutations of the gene (48). Since all isoforms of the gene have exon 2, and exon 2 has the binding site for PDE4B (49, 50), mutants in exon 2 were screened. Two novel, independent missense mutations were identified, with one resulting in thymine instead of adenine at 127th position and the other resulting in cytosine instead of Thymine

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in 334th position. These single nucleotide mutations produced Q31L and L100P amino acid exchanges, respectively. Mutant mice were backcrossed to C57BL/6 J background and homozygous (31L/31L; 100P/100P), heterozygous (31L/+; 100P/+), and compound heterozygous (31L/100P) mutants were tested. 2.4.3. Results

The 31L mutant mice showed increased immobility in forced swim test (FST), decreased sociability and social novelty, and decreased sucrose consumption, resembling that of depression in humans. The 100P mutant mice predominantly exhibited increase in horizontal activity, decreased prepulse inhibition (PPI), decreased latent inhibition (LI), and decreased performance in T-maze, consistent with abnormalities in schizophrenia. Interestingly, bupropion, an antidepressant with properties of a dopamine and norepinephrine reuptake inhibitor, decreased immobility in FST and normalized PPI deficit in 31L homozygous animals. Rolipram, a phosphodiesterase-4 inhibitor, ameliorated PPI deficit in “schizophrenic-like” 100P mice, so did clozapine and haloperidol. Of note, clozapine but not other drugs significantly increased PPI in control mice. The biochemical assay found reduced binding of PDE4B to both mutant forms of Disc1, with 100P type being affected to a greater extent. The activity of PDE4B was found decreased in brains of 31L mice without altering levels of the protein. The decreased activity of the enzyme has been suggested to explain lower responsiveness to rolipram in these mice as the drug inhibits phosphodiesterase.

2.4.4. Conclusion

The study has demonstrated associations of two different mutations in Disc1 with distinct neurobehavioral and pharmacological phenotypes, resembling schizophrenia and mood disorders. In addition, the new result of the study is an association of “schizophrenia-like” phenotype of 100P mice with decreased levels of PDE4. A caveat of the model is that the mutated region is not conserved in humans, somewhat decreasing relevance of the induced mutations to human disorders.

2.5. Transgenic Models

Although the Scottish translocation mutation is a rare genetic variant, the potential outcome of the mutant, i.e., a production of mutant protein, is an intriguing possibility for mechanistic research. Indeed, in vitro studies demonstrated that mutant DISC1 might produce its effects via dominant-negative mechanisms (51). Thus, several transgenic mouse models of mutant human DISC1 have been generated.

2.5.1. Constitutive Expression 2.5.1.1. Background 2.5.1.2. Approach

Hikida et al. generated a transgenic mouse model with constitutive expression of mutant DISC1 under the CaMKII promoter (72). Expression of mRNA for the mutant protein product was

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demonstrated with Northern blotting and in situ hybridization in the olfactory bulb, frontal cortex, hippocampus, and basal ganglia at different developmental time points. 2.5.1.3. Results

Compared to control littermates, mutant DISC1 mice were more active in open field test and displayed a deficit in PPI when the intensity of prepulse was set at 74 dB. Mutant mice were more immobile in FST than control animals, suggesting depression-like phenotype. Also, it took longer for mutant mice to find the hidden food, indicating impaired olfaction or decreased motivation for food. When immunoreactivity of parvalbumin, calretinin, and calbindin was evaluated in the medial prefrontal cortex, decreased numbers of parvalbumin-positive cells were found, with no significant changes in numbers of calbindin- or calretinin-positive neurons. In vivo and ex vivo MRI analysis demonstrated enlargement of the lateral ventricles in two different lines of 6-week-old mice, although this pathology was no longer present in 3-month-old DISC1 mice, suggesting a compensatory process. The lateral ventricle enlargement was accompanied with a decreased left/right ratio for the hippocampus but not the cortex or basal ganglia.

2.5.1.4. Conclusions

Expression of mutant DISC1 under the CaMKII promoter resulted in the schizophrenia-like phenotypic features associated with schizophrenia and provides a useful experimental tool for future translational studies, including testing novel therapeutics.

2.5.2. Inducible Expression of the C-Terminal Fragment

As expression of DISC1 is high during neurodevelopment, an early disruption of DISC1 functions may result in long-lasting abnormalities present even in adulthood. A model of inducible expression of the truncated human DISC1 has been generated to test a hypothesis that a transient perturbation in the function of endogenous DISC1 by dominant-negative effects could have permanent neurobehavioral effects.

2.5.2.1. Background

2.5.2.2. Approach

The group of investigators led by T. Cannon and A. Silva described an interesting mouse model of inducible expression of the fragment of DISC1 (DISC1-cc) that is deleted in the affected members of the Scottish family. DISC1-cc expression is regulated by the CaMKII promoter, which is active in forebrain neurons. DISC1-cc spans residues 671–852, which is the portion of DISC1 crucial for binding to NUDEL and Lis1 (16–18). The DISC1-cc protein was fused to a HA-tagged mutant (G521R) estrogen receptor ligand-binding domain (LBD). In this inducible system, the transgenic protein is degraded after sequestration by heat-shock chaperone proteins. When tamoxifen (tam) is administered to transgenic mice, it binds the LBD, the fusion protein complex, which includes DISC1-cc,

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undergoes a conformational switch such that the transgenic protein dissociates from the chaperone proteins and becomes functional. After a quick metabolism of tam, the transgenic protein acquires non-functional state again (56). 2.5.2.3. Results

DISC1-cc was found to be bound to Nudel1 and Lis1 for 2 days, transiently decreasing expression of DISC1 interacting partners in transgenic mice. The neurobehavioral effects of DISC1-cc were evaluated in adult mice after tam administration at postnatal day (PND) 7 or in adulthood. Expression of DISC1-cc at PND 7 but not in adulthood impaired working memory assayed by DNMPT test with a 10-s delay. No effects were found when 1-, 5-, 10-, or and 20-s delay intervals were used. In similar vein, sociability of mice was significantly attenuated by early postnatal but not adult expression of the fragment. Mice treated with tam at PND 7 spent significantly less time with a live mouse than an inanimate object, while all control mice displayed an expected pattern of interaction, spending more time with live partners. In addition, early postnatal expression of DISC-cc led to a shorter latency to immobilization in forced swim test compared to other groups. The behavioral alterations induced by early postnatal expression of DISC1-cc were found to be associated with reduced dendritic complexity and decreased hippocampal synaptic transmission, without affecting paired-pulse facilitation, a marker of presynaptic activity and long-term potentiation.

2.5.2.4. Conclusions

This model has provided the first evidence that an early postnatal transient perturbation in the DISC1 function can have lasting permanent effects on brain morphology and behavior in adult mice. Although the observed effects convincingly support the hypothesis put forward by the authors, there is a caveat in the study related to expression of the fragment that is not found in humans. Additional control experiments would be necessary to directly compare the effects of early postnatal expression period (Tg/Tam/P7) and adulthood expression only (Tg/Tam/A) after a longer period to time (not 6 hours) as was done for the postnatal group, as well as to evaluate the effects of adult expression of this mutation.

2.5.3. Inducible Expression of Mutant DISC1

Given potential multiple functions of DISC1 across different stages of neurodevelopment to contribute to the pathogenesis of psychiatric disorders (52–54), studying selective contributions of prenatal vs. postnatal expressions of mutant human DISC1 is of great interest.

2.5.3.1. Background 2.5.3.2. Approach

Inducible DISC1 mouse model is a standard bi-transgenic Tetoff system. In order to turn off transgene expression, DOX is added to mouse food or drinking water. As transcription of tTA

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is regulated by the α-calmodulin kinase II (CaMKII) promoter, expression of mutant DISC1 is present in neurons of the olfactory bulbs, cortex, hippocampus, striatum but not cerebellum. It was found that expression of mutant DISC1 starts prenatally as early as embryonic day (E) 15 as detected by Western blot and E9 when assayed by RT-PCR (unpublished data). Thus, this model provides the opportunity to regulate both prenatal and postnatal expression of mutant DISC1. 2.5.3.3. Results

The initial study with the model (55) evaluated the neurobehavioral effects of constitutive expression of mutant DISC1. Compared to littermate control mice that did not express mutant DISC1, transgenic male mice exhibited increased spontaneous locomotor activity, decreased social interaction, and increased aggressive behaviors, while transgenic female mice demonstrated poorer spatial recognition memory in Morris water maze. These behavioral alterations were accompanied by enlargement of the lateral ventricles when assayed in 9-month-old mice. Evaluation of neurite outgrowth in primary cortical neurons showed a reduction in dendritic complexity measured with Scholl method.

2.5.3.4. Conclusions

The advantages of this model lie in the ability to turn on and off gene expression via doxycycline administration. Expression of the gene can be regulated in utero and across postnatal development for varying durations. Regulation of expression in developing and adult animals may be used in drug screen aimed at targeting stable and reversible aspects of the behavioral phenotype. Potential caveats of the model include unwanted effects of doxycycline on the brain, particularly during development, and multiple control groups for assessing non-specific effects of transgenesis and tTA. In addition, the feasibility to generate large cohorts might be an issue if several experimental groups are needed.

2.5.4. BAC Transgenic DISC1 Model

The transgenic DISC1 models described above utilize the CaMKII promoter to drive expression of the mutant gene in forebrain neurons. A different mouse system has been generated with regulation of expression by the endogenous Disc1 promoter (56).

2.5.4.1. Background 2.5.4.2. Approach

The construct was prepared from mouse bacterial artificial chromosome (BAC) RP23–236F19 containing Disc1 exons 1–9 with its entire upstream sequences fused with an EGFP cDNA to the end of exon 8 as an identifying tag, followed by a SV40 polyA signal.

2.5.4.3. Results

Transcripts were detected at E17.5. The authors observed comparable levels of expression of full-length and mutant DISC1 by RT-PCR. At E17.5, expression of mutant DISC1 was predominantly observed in the hippocampus and cortex, while cerebellar

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expression was also noted in adult mice. Nissl staining revealed enlarged lateral ventricles and reduced volumes of the cortex in male mice. In addition, thinning of the cortical layers II and III and a partial agenesis of the corpus callosum were noted. Immunohistochemical analysis revealed reduced numbers of parvalbumin-positive cells in the hippocampus and medial prefrontal cortex. Neurogenesis was reduced in the cortex of newborn mutant mice as shown by BrdU staining. Primary cortical neurons derived from P1 pups had fewer and shorter neurites. Compared to control animals, mutant mice exhibited latent inhibition and increased immobility in FST and TST. Significantly fewer stress calls by mutant mice were detected during a maternal separation test. 2.5.4.4. Conclusions

The expression of mutant DISC1 under the endogenous promoter resulted in the neurobehavioral abnormalities reminiscent of those observed in schizophrenia and mood disorders. The phenotype reported for these mice is similar to that found in other DISC1 mouse models. In addition, the study was the first one to demonstrate decreased cortical neurogenesis in a mutant DISC1 mouse model.

3. Summary In this chapter, current mouse models of DISC1 were reviewed (Table 9.1). These models need further characterization and confirmation to be accepted as reliable DISC1 mouse models. This particularly relates to the mutant DISC1 mouse models as, although not unexpectedly, both consistent and discordant phenotypic changes have been reported for mice of different models. It seems that a head-to-head comparison would be interesting. As interplay between different susceptibility genes and/or genetic × environmental factors seems to contribute to the pathogenesis of major mental illnesses, DISC1 mouse models may serve as valuable experimental tools to evaluate the mechanisms whereby those complex interactions take place (57). The DISC1 mutation may act as an additive or synergistic factor in interactions with environmental pathogens which by themselves would cause small if any abnormalities in mice without the mutant gene. The current models are based on expression of the Scottish mutation or a natural polymorphism of the endogenous mouse gene. A knockout model, which would have a complete deletion of the gene and main Disc1 isoforms, would be very valuable for a better understanding of the gene function. New models of DISC1 will advance our knowledge of the gene. One approach can include generating models with cell

In utero E14.5 KD function of endogenous Disc1

Oncoretrovirus KD endogenous injected into Disc1 the adult dentate gyrus

KD endogenous Disc1

KD endogenous Disc1

In utero E13, BrdU 2 h before sacrificing at E16

Lentivirus injected into the adult dentate gyrus, BrdU 5 weeks later

Kamiya

Duan

Mao

Function

Approach

Model

Table 9.1 DISC1 mouse models

Disc1 shRNA

Disc1 shRNA

Disc1 RNAi

Disc1 RNAi or express C-terminally truncated DISC1

Construct n/d

Hippocampus n/d

Hippocampus n/d

Reduced BrdU positive cells, rescued by GSK3-β inhibitor

Reduced BrdU positive cells, rescued by degradation of resistant β-catenin or GSK3-β inhibitor

Soma hypertrophy, multiple primary dendrites, overextended migration, and accelerated synaptogenesis

P2: Inhibition of neuronal migration. P14: Shorter, misoriented dendrites

Gross Cellular anatomy abnormalities

Hippocampus n/d

Pyramidal cortical neurons

Expression pattern

Locomotor hyperactivity increased immobility in forced swim test

n/d

n/d

n/d

Behavior

DISC1 regulates neuronal progenitor proliferation by inhibiting GSK3-β

DISC1 is involved in embryonic and adult neuronal migration

KD of Disc1 either with RNAi or with mDISC1 leads to abnormal development of the cortex

Significance

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Approach

Function

Construct

Spontaneous mutation in 129S6/SvEv, transferred to C57BL/6

Transgenic

Koike Kvajo

Hikida

Express C-terminally truncated human DISC1, dominant negative

Some Disc1 isoforms are missing

Endogenous

Expression pattern

Reduced prefrontal cortex volume

Reduced brain volume

Gross anatomy

Constitutive Olfactory Enlarged under bulbs, lateral CaMKII frontal ventripromoter cortex, hipcles pocampus, basal ganglia postnatally

25 bp Endogenous deletion in exon 6, results in premature stop codon in exon 7

Clapcote ENU Missense point n/a mutagenesis, mutations: screening of Q31L, L100P DISC1 exon 2

Model

Table 9.1 (continued) Behavior

Significance

Decreased parvalbumin+ immunoreactivity in the medial prefrontal cortex

Altered organization of the dentate gyrus, No difference in parvalbumin+ or calbindin+ staining in medial prefrontal cortex

Increased immobility in forced swim test, mild hyperactive in the open field, PPI deficit

Impaired working memory

First DISC1 model based on a human mutation

Loss of some isoforms altered hippocampal morphology and memory performance. Note that all 129 substrains have the mentioned mutation which may affect the phenotypes of the models generated in them.

Reduced binding Q31L: greater A point mutation in to PDE4B immobility in the Disc1 produced forced swim test, various reduced abnormalities. sociability, The two point reduced sucrose mutants had consumption. different behavioral L100P: phenotypes (Q31L novelty-induced depression-like, activity, deficits in L100P PPI, latent schizophrenia-like) inhibition, and working memory

Cellular abnormalities

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BAC (bacterial artificial chromosome) transgenic

Shen

Express Fused GFP C-terminally in-frame to the truncated end of exon 8 mouse DISC1

Express the Transgenic under C-terminus of CaMKII DISC1, which promoter, competes on inducible by binding to tamoxifen for NDEL1 and <2 days LIS1

Transgenic

Bi-transgenic regulatory plasmid expresses tTA under CAMKII promoter

Construct

Li

Function

Express C-terminally truncated human DISC1, dominant negative

Approach

Pletnikov Transgenic

Model

Table 9.1 (continued) Gross anatomy

Endogenous

Cortex, hippocampus, striatum, cerebellum. Activated at P7

Reduced hippocampal dendritic complexity

Decreased neurite outgrowth in primary neurons

Cellular abnormalities

Smaller Thinning of cortex, layers II/III enlarged due to lateral reduced ventrineuronal cles, proliferation corpus callosum agenesis

n/d

Hippocampus, Enlarged cortex, lateral striatum and ventriolfactory bulb. cles Expression is highest at embryonic life, and decreases with age

Expression pattern

Impaired latent inhibition, greater immobility in the forced swim test

Impaired working memory, greater immobility in the forced swim test, reduced sociability

Altered spatial memory in females; decreased social interaction and increased aggression in males

Behavior

Mouse transgene regulated by endogenous promoter

Short induction at P7 is sufficient to elicit significant abnormalities in adulthood

First inducible DISC1 model

Significance

224 Ayhan et al.

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type-specific expression. For example, as DISC1 is proposed to be expressed in glial cells (58), mouse models with changes in expression of DISC1 in astrocytes or oligodendrocytes would provide new information on the function of DISC1 in those glial cells. Glia–neuron interaction is necessary for effective synaptic transmission and altered interaction might be associated with mental disorders (53, 59, 60). Abnormalities in glial expression of DISC1 might cause dysfunction in glial cellular operations and eventually disrupt glia–neuron interaction. DISC1 is not the only susceptibility gene in schizophrenia in particular and for other major mental disorders. Distinct roles of NRG1, NRGIII, RGS4, DAO, AKT1, and PICK1 have been also demonstrated (61–65) and corresponding animal models have been generated (66–69). These mouse models may provide in vivo working models of how these genes interact (70–73), how the common pathways are affected by a mutation in any of these genes, and what outcomes those interactions have at cellular, circuitry, systems, and behavioral levels. Evaluation of interactions between susceptibility genes may provide identification of pathways that may be targeted for preventive measures or treatment approaches.

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57. Ayhan, Y., Sawa, A., Ross, C. A., and Pletnikov, M. V. (2009) Animal models of geneenvironment interactions in schizophrenia, Behav Brain Res 204, 274–281. 58. Austin, C. P., Ma, L., Ky, B., Morris, J. A., and Shughrue, P. J. (2003) DISC1 (Disrupted in Schizophrenia-1) is expressed in limbic regions of the primate brain, Neuroreport 14, 951–954. 59. Sawa, A., Pletnikov, M. V., and Kamiya, A. (2004) Neuron-glia interactions clarify genetic-environmental links in mental illness, Trends Neurosci 27, 294–297. 60. Fujii, K., Maeda, K., Hikida, T., Mustafa, A. K., Balkissoon, R., Xia, J., Yamada, T., Ozeki, Y., Kawahara, R., Okawa, M., Huganir, R. L., Ujike, H., Snyder, S. H., and Sawa, A. (2006) Serine racemase binds to PICK1: potential relevance to schizophrenia, Mol Psychiatry 11, 150–157. 61. Thiselton, D. L., Vladimirov, V. I., Kuo, P. H., McClay, J., Wormley, B., Fanous, A., O’Neill, F. A., Walsh, D., Van den Oord, E. J., Kendler, K. S., and Riley, B. P. (2008) AKT1 is associated with schizophrenia across multiple symptom dimensions in the Irish study of high density schizophrenia families, Biol Psychiatry 63, 449–457. 62. Corfas, G., Roy, K., and Buxbaum, J. D. (2004) Neuregulin 1-erbB signaling and the molecular/cellular basis of schizophrenia, Nat Neurosci 7, 575–580. 63. Chen, P. L., Avramopoulos, D., Lasseter, V. K., McGrath, J. A., Fallin, M. D., Liang, K. Y., Nestadt, G., Feng, N., Steel, G., Cutting, A. S., Wolyniec, P., Pulver, A. E., and Valle, D. (2009) Fine mapping on chromosome 10q22-q23 implicates Neuregulin 3 in schizophrenia, Am J Hum Genet 84, 21–34. 64. Yamada, K., Ohnishi, T., Hashimoto, K., Ohba, H., Iwayama-Shigeno, Y., Toyoshima, M., Okuno, A., Takao, H., Toyota, T., Minabe, Y., Nakamura, K., Shimizu, E., Itokawa, M., Mori, N., Iyo, M., and Yoshikawa, T. (2005) Identification of multiple serine racemase (SRR) mRNA isoforms and genetic analyses of SRR and DAO in schizophrenia and D-serine levels, Biol Psychiatry 57, 1493–1503. 65. Mirnics, K., Middleton, F. A., Stanwood, G. D., Lewis, D. A., and Levitt, P. (2001) Disease-specific changes in regulator of Gprotein signaling 4 (RGS4) expression in schizophrenia, Mol Psychiatry 6, 293–301. 66. Hashimoto, A., Yoshikawa, M., Niwa, A., and Konno, R. (2005) Mice lacking D-amino acid oxidase activity display marked attenuation of stereotypy and ataxia induced by MK801, Brain Res 1033, 210–215.

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Chapter 10 Genetically Engineered Mice for Schizophrenia Research Juan E. Belforte and Kazu Nakazawa Abstract NMDA receptor (NMDAR) hypofunction theory of schizophrenia has been assessed in rodents with pharmacological intervention and global knockout strategy of NMDAR blockade. However, these manipulations of NMDAR function have been relatively coarse, affecting all NMDA receptors throughout the brain. Here we tested the effects of eliminating NMDA receptors in about half the interneurons located in the cortex and the hippocampus in early postnatal development by engineering “conditional knockout” mice. Remarkably, the mutant mice produced a variety of schizophrenia-related phenotypes. Key words: NMDA receptor, GABAergic neurons, conditional knockout, schizophrenia, animal model, ketamine, MK-801, NMDAR hypomorph, Cre/loxP.

1. Introduction The NMDA receptor (NMDAR) hypofunction theory of schizophrenia has gained support over the last two decades from not only human studies but also the studies of animal disease models. Based on this theory, different manipulations, from pharmacological to genetic, have been used to alter the NMDAR signaling in order to replicate various features of the disorder in animal models. An animal model should ideally mimic the human condition of interest with respect to its etiology, symptomatology, and the treatment in order to attain construct, face, and predictive validity, respectively. The model phenotypes should also be reproducible (reliability). Schizophrenia, as well as other psychiatric disorders, has been a difficult case because its etiology is unknown, preventing the attainment of true construct validity. Its symptoms are also diverse and complex, involving multiple brain areas and requiring extensive characterization of the animal model P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_10, © Springer Science+Business Media, LLC 2011

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from the cellular level to the systems level in order to obtain face validity. Even the predictive validity remains problematic, since no single drug or class of drugs is effective for all patients (1, 2). In this chapter, we compare the advantages and problems of different methodological approaches used in the animal modeling of schizophrenia based on the NMDAR hypofunction theory.

2. Traditional Approach 2.1. Pharmacological Approach

The discovery that phencyclidine (PCP), a strong psychomimetic drug acting as a non-competitive antagonist of the NMDA subtype of glutamate receptors, established the glutamatergic theory of schizophrenia as a major hypothesis (3–7). Other noncompetitive NMDA receptor antagonists, such as ketamine and MK-801, were also found to induce a range of schizophrenia symptoms in normal volunteers at low doses, including the characteristic cognitive deficits, such as working memory deficits (6, 8). These observations help to consolidate the NMDAR hypofunction theory, and the development of animal models of schizophrenia has begun. Animal models with relevance to schizophrenia have historically been driven by studies combining pharmacologically induced conditions by the treatment of NMDAR antagonists with relevant measures. A comprehensive list of behavioral phenotypes related to schizophrenia has been published in rats, mice, and monkeys, including locomotor hyperactivity, deficits in working memory and social interaction, impaired prepulse inhibition (PPI), and latent inhibition. Non-behavioral aspects of the disease, including reduced levels of GAD67 and parvalbumin and impaired cortical gamma band oscillations, have also been reported in animals after chronic treatment with NMDAR antagonists. Most of this literature is based on rodent study and is reviewed extensively elsewhere (9–13). In general, the advantages of the pharmacological approach appear to be the simplicity of procedure, the induced manipulation at a given time point, and the low cost of development. On top of these advantages, PCP has attracted the strong attention as a reagent for an animal model for schizophrenia because of its ability to produce, in humans, a range of symptoms remarkably similar to those of schizophrenia patients. However, the ability of PCP to model the symptoms of schizophrenia varies greatly depending on the treatment regimen. Only chronic, rather than acute, exposure to PCP seems to produce in animals behavioral and neurochemical changes observed in schizophrenia (14). Moreover, dose-dependent effects of PCP may be extremely difficult to assess in vivo because it has limited receptor selectivity with

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binding affinities for dopamine D2 receptors and 5-HT2 receptors just one order of magnitude lower than those for NMDAR (15). Furthermore, other studies show a more complex pharmacological profile of PCP than originally thought (reviewed in (16)). The ketamine model of schizophrenia has also been used due to its specific psychotomimetic properties and excellent safety profile in humans (6). In particular, induction of negative symptoms such as blunted affect and social withdrawal is the main argument in favor of the ketamine model. However, the literature using ketamine in rodents shows conflicting results in terms of face and predictive validities, depending on different administration schemes, including acute, subchronic, and chronic manipulation with animal ages from early postnatal to adulthood. For example, intraperitoneal administration of ketamine for two consecutive days reduces GAD67 levels but a single dose does not (17). Indeed, a low-dose infusion of ketamine has rather been suggested to be worth a trial in patients who suffer from treatment-resistant depression (18). Moreover, different from another NMDA receptor antagonist, MK-801, ketamine exerts a strong hypnotic effect, perhaps due to its direct inhibition of hyperpolarization-activated, cyclic nucleotide-modulated (HCN1) cation channels (19). MK-801 (dizocilpine) is a more potent and selective NMDAR antagonist than is PCP (20). However, while there is strong evidence that PCP and ketamine can produce effects in humans that parallel the positive, negative, and cognitive deficits of schizophrenia, the evidence for MK-801 is limited. For instance, withdrawal from subchronic PCP has been shown to cause a delay-dependent impairment of working memory, reduced social interaction, and enhanced D-amphetamineinduced motor activity; these results were not replicated in animals sub-chronically treated with MK-801 (21). Overall, MK-801 models are highly valuable since they attain a certain level of face validity. However, it appears that as the drug becomes more specific to antagonizing NMDARs, pharmacological antagonism at NMDAR is somehow not sufficient to explain the full spectrum of PCP psychomimetic properties. 2.2. Global NMDAR Genetic Manipulation in Mice

The genetic manipulation of the NMDARs appeared as a refined transition toward a more reliable animal model of schizophrenia based on its glutamatergic theory. The first genetically engineered transgenic mouse strain, NMDAR hypomorph mutant, was published in 1999, in which the overall level of NMDA receptor subunit NR1 (GluN1) protein is reduced to ~10% (22). Contrary to the NR1 null knockout mice which die perinatally due to breathing impairments, the NMDAR hypomorph mutants were viable and displayed no gross anatomical abnormalities. However, they

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exhibited phenotypes similar to those observed in the PCP- or MK-801-treated animals. For instance, locomotor activity and stereotypy were augmented and social interaction and sexual behaviors were impaired, and many of them were ameliorated by clozapine treatment (22). Prepulse inhibition of acoustic startle was also impaired but mutants also showed increased startle reactivity (23). Several other null knockout or “knock-in” mouse strains carrying mutations in the genes encoding NMDAR subunit proteins or their downstream pathway proteins also displayed phenotypes compatible with schizophrenia symptoms. For instance, NR2A subunit (GluN2A) null knockout mice showed an increased locomotor response to novelty and metabolism of both dopamine and serotonin was increased in the frontal cortex and the striatum (24). As in the NR1 hypomorph mutants, antipsychotics haloperidol and risperidone can attenuate the hyperlocomotion. However, face validity is compromised by the finding that these mice show normal PPI (25). Interestingly, the administration of the specific NR2B antagonist Ro 63-1908 to NR2A knockout mice induced a marked disruption of the PPI, raising a possibility that both NR2A- and NR2B-containing receptors must be affected for the PPI impairment (26). Two point mutations in NR1 subunit (GluN1) were also described targeting the glycine-binding site of the receptor (27). These mice, GluN1(D481N) and GluN1(K483Q), show a 5- and 86-fold reduction in glycine binding, respectively. Grin1(K483Q) homozygously mutated mice usually die within a few days. However, Grin1(D481N) homozygous mutants are viable and fertile, and showed a non-habituating hyperactivity, as well as increased stereotyped behavior, social interaction, and decreased sociability (27, 28). Consistently, heterozygously combined Grin1(D481N/K483Q) mutation displayed stronger phenotypes including a nest construction impairment. However, these mutants did not exhibit an abnormal prepulse inhibition of the startle reflex (29). While global NMDAR genetic manipulation in mice clearly supports the notion of NMDAR hypofunction for schizophrenia pathogenesis, certain precautions seem advisable in interpreting the behavioral data. Due to its critical importance for the NMDAR function of many brain areas, the global manipulation of NMDAR subunits often disturbed primary sensory or motor functions. For instance, the Grin1(D481N/K483Q) mutants were impaired in the cued version of the Morris water maze, suggesting a visual impairment. On the other hand, some phenotypes following NMDAR deletion may be potentially masked due to their spatial and/or temporal compensation, as the genetic manipulation is not temporally or spatially restricted. Obviously, it is also inappropriate to address the issue of cell-type, brain region specificity or whether there is a critical period for NMDAR hypofunction. These pitfalls may preclude correct interpretation

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of the underlying neural mechanisms following NMDAR hypofunction. To overcome these limitations, a second generation of gene-targeting technology, i.e., conditional transgenic approach, is required.

3. New Approach: Cell Type-Specific/ Brain Region-Restricted Genetic Manipulation

The results obtained after the global genetic manipulations support the notion of an NMDAR malfunction involved in the pathophysiology of schizophrenia. However, the intrinsic nature of the global manipulation precludes a direct understanding of the underlying mechanisms involved in the abnormal behaviors. In which brain areas could an NDMAR deficit lead to behavioral symptoms? Do all cell types equally contribute to the development of the disorder? Are developmental changes involved? These are all fundamental questions in understanding the role of NMDAR in the schizophrenia pathophysiology that cannot be addressed in conventional global knockouts (Table 10.1).

Table 10.1 Comparison among various animal models of schizophrenia Systemic NMDAR antagonist administration

Global NMDAR genetic manipulation

Cell type-specific/brain region-restricted genetic manipulation of NMDAR

Molecular specificity

Variable (low PCP, high MK-801)

High

High

Cellular/regional specificity

No

No

Yes

Pros

Simple, fast, cheap, can be compared with equivalent studies in humans

Very reliable, gene specific

Very reliable, gene specific, area specific, cell type specific. Potentially inducible

Cons

Large methodological variations, multiple administration schemes. Potential off-target effects

Potential temporal and/or spatial compensatory mechanisms. Not inducible

Expensive and hard to develop. Time consuming for initial characterization.

Access

Widely available through commercial suppliers

Collaboration New initiative

Collaboration New initiative

Hypothesis-driven approach with a specific gene of interest

Hypothesis-driven approach with a specific gene and cell type/brain area

(22, 24, 27)

(45)

Best use

References

(9–13)

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Among many brain areas/cell types that may potentially serve as a target of NMDAR hypofunction, we have focused on the cortical and hippocampal GABAergic interneurons. Basically, three lines of evidence suggest the idea that NMDAR hypofunction occurs preferentially in cortical GABAergic neurons in schizophrenia; first, acute systemic administration of NMDAR antagonists results in hyperactivity of cortical pyramidal neurons (30), and spillover of cortical acetylcholine (31) and glutamate (32). These paradoxical cellular changes concur with brain imaging data, showing net cortical excitation after NMDAR antagonist treatment in human subjects (33–35). Second, GABAergic interneurons are disproportionally more sensitive to NMDAR antagonists than are pyramidal neurons (36, 37), although the precise mechanism for this difference is unresolved. NMDAR antagonist-induced cortical excitation may be due to a preferential reduction in the firing of fast-spiking interneurons and resultant disinhibition of cortical excitatory neurons. Third, repeated administration of NMDAR antagonists reduces the levels of GAD67 and parvalbumin expression in cortical GABAergic neurons (38–42), directly linking NMDAR hypofunction to dysfunction of GABAergic neurons. To directly test the role of NMDARs in GABAergic neurons in the development of schizophrenia-like phenotypes, we created a conditional NMDAR knockout mouse strain in which early postnatal ablation of NR1 was targeted to corticolimbic GABAergic neurons using the Cre/loxP system. At first glance, the approach may appear inappropriate because genetic predispositions of human schizophrenia are not manifested solely in corticolimbic GABAergic neurons. Nonetheless, we expected that by bypassing the temporal and spatial compensations inherent to the global knockout approach, the conditional transgenic approach may evoke the selective and characteristic impairments, thereby providing insights into the mechanisms of cellular and behavioral manifestation of schizophrenia-related phenotypes.

4. Methodological Challenge in Targeting Transgene Expression to Certain Cell Types of the Brains

The ability to target genetic manipulations to specific brain cell types is essential for analysis of genes, cells, and circuits that play key roles in behavior and disease. In particular, Cre recombinase driver transgenic mouse lines, which target the Cre

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expression to particular brain areas/cell types of interest, would be extremely useful by crossing them to a loxP-flanked transgenic line (“floxed”) of the targeted gene. Unfortunately, only a few Cre lines are available in which Cre expression is confined to particular cell types of brain areas and certain brain areas (e.g., 43, 44). As such, no Cre driver lines had been previously reported where the recombination is restricted to cortical and hippocampal GABAergic neurons. This is because no endogenous gene promoters have been discovered to drive the transgene expression to cortical and hippocampal GABAergic neurons throughout the development. Therefore, lack of appropriate promoter prevented our attempt from generating Cre lines using ES cellderived homologous recombination approach (knock-in strategy). Alternatively, it is possible to use conventional transgenic approach of Cre recombinase over-expression by oocyte injection. This approach is to aim at screening out a particular line of desired Cre expression pattern, which may be determined by the combinatory effects of short transcriptional regulatory elements injected and positional effect at the transgene integration site in the genome. In particular, bacterial artificial chromosome (BAC) transgenic approach appears to be more feasible because the transcriptional enhancer/promoter elements contained in the BAC fragment would direct the transgene expression faithfully mimicking to the endogenous pattern. Note that this method would also allow us to pick the mouse line with slightly different expression pattern, due to the positional effect at the transgene integration site. Thus, it seems ideal to utilize this BAC transgenic approach if endogenous expression pattern is close to what you wish to obtain but not completely the same. Here we adopted the BAC transgenic approach to target the Cre expression to corticolimbic interneurons. We utilized a BAC fragment carrying Ppp1r2 gene [protein phosphatase 1, regulatory (inhibitor) subunit 2] (45). Endogenous Ppp1r2 is expressed in GABAergic neurons in the striatum, the cortex, and the hippocampus and, to a lesser degree, pyramidal neurons in hippocampal CA1 throughout the development. Using this promoter for BAC-transgenic approach, we tried to obtain the Cre line, in which Cre is not expressed in the striatum. To the end, we inserted the Cre cDNA prior to the initial ATG of the promoter region of the gene by homologous recombination method in BAC (46) and injected the BAC fragment carrying Cre cDNA to mouse-fertilized oocytes to generate several transgenic lines. After screening by intercrossing with loxP-flanked Rosa26LacZ reporter line, we chose one line showing the desired pattern of Cre expression.

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5. A Postnatal NMDA Receptor Ablation in Corticolimbic Interneurons Confers SchizophreniaLike Phenotypes

Using the new generated Cre line, Ppp1r2-Cre, we successfully deleted NR1 subunit (GluN1) in 40–50% of cortical and hippocampal interneurons, predominantly parvalbumin-positive ones, in early postnatal development. This was confirmed by double in situ hybridization histochemistry and patch clamp recordings of NMDA-mediated currents. Consistent with the NMDAR hypofunction theory of schizophrenia, distinct schizophrenia-like symptoms emerged after adolescence in mutants. These included novelty-induced hyperlocomotion, which may reflect psychomotor agitation, a reduced preference for sweet solution (anhedonia), deficits in nesting and mating that suggest social withdrawal, and deficits in spatial working and short-term social memory that echo cognitive impairments; taken together, these deficits are reminiscent of the positive, negative, and cognitive symptoms of human schizophrenia. Impaired sensorimotor gating, as assessed by the PPI of the startle reflex, was also observed in these mutants. In addition, the mutants exhibited reduced GAD67 and parvalbumin levels in the NR1-deleted cortical GABAergic neurons, which concur with postmortem studies in the cortex of schizophrenia individuals (47, 48). Notably, deficits in social nest building, anxiety-like behaviors, and anhedonia-like behaviors were exacerbated by social isolation stress resembling stress precipitation of psychotic states in schizophrenia. Furthermore, the phenotypes of disrupted nest building and mating, as well as the anhedonic and anxiety-like behaviors, were most prominent after 12 weeks of age, suggesting the existence of a latency period before their emergence. This latency period is reminiscent of the premorbid stage before the emergence of symptoms characteristic of major psychiatric disorders (49). Spatial working memory and PPI deficits were ameliorated by the antipsychotic risperidone, conferring some degree of predictive validity to the model. Strikingly, post-adolescent deletion of NR1 subunit in the same interneuron population did not result in such abnormalities, demonstrating a fundamental role of NMDAR during early postnatal stages in the development of schizophrenia-like phenotypes later in adulthood. In summary, our model not only reproduces positive, negative, and cognitive aspects of schizophrenia but also mirrors three additional features of human schizophrenia: stress-dependent precipitation of symptoms, a latency period before the development of symptoms, and a critical period for disease acquisition. It also exhibits non-behavioral features compatible with schizophrenia, like GAD67/parvalbumin decrease increasing the face validity of the model. All these features give our animal model unique

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properties, making it suitable for the study of schizophrenia pathophysiology and the testing of new approaches for treating human psychiatric illnesses and schizophrenia in particular.

6. Future Directions While a number of schizophrenia susceptibility genes have been implicated in the modulation of NMDAR activity (50), genetic linkage studies have not directly associated polymorphisms in the NR1 subunit with schizophrenia (51). It is also unlikely that genetic predispositions of human schizophrenia are manifested solely in corticolimbic GABAergic neurons. Nevertheless, we expect that our strategy to spatially and temporally restrict NMDAR hypofunction will provide insights into the mechanisms of cellular and behavioral manifestation of schizophrenia-related phenotypes. Indeed, restricting the genetic deletion of NR1 to corticolimbic GABAergic neurons during early postnatal development was sufficient to trigger the development of schizophreniarelated phenotypes after adolescence in mice. Future studies will address what could cause reduced NMDAR function in people with schizophrenia; how disrupted regulation of this receptor causes symptoms and how stress exacerbates them; and how currently used medications work to alleviate symptoms. Ultimately, understanding these processes will provide the basis for developing new targeted medications.

Acknowledgments We would like to thank Veronika Zsiros, Elyse R. Sklar, Zhihong Jiang, Gu Yu, Yuqing Li, and Elizabeth M. Quinlan for their significant contributions as co-authors to our work cited in Ref. 45. We also acknowledge the colleagues for suggesting improvements to aspects of this review. References 1. Swartz, M. S., Perkins, D. O., Stroup, T. S., Davis, S. M., Capuano, G., Rosenheck, R. A., Reimherr, F., McGee, M. F., Keefe, R. S., McEvoy, J. P., Hsiao, J. K., and Lieberman, J. A. (2007) Effects of antipsychotic medications on psychosocial functioning in patients

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Chapter 11 Epigenetic Animal Models of GABAergic Deficit in Mental Disorders Patricia Tueting, Erminio Costa, and Alessandro Guidotti Abstract There is substantial evidence that psychosis is characterized by GABAergic gene promoters that are in a closed chromatin state, leading to reduced transcription of proteins essential for GABAergic and synaptic function in the forebrain. Two critical genes that are downregulated in cortical and hippocampal interneurons by 30–50% in psychotic postmortem brain are GAD1, the gene for GAD67, and RELN, the gene for reelin. The promoter region of these genes is vulnerable to cytosine methylation by DNA methyltransferases, enzymes that in psychosis are elevated in GABAergic interneurons. An alteration in histone deacetylase or methyltransferase expression provides further evidence for an underlying epigenetic etiology. Animal models aimed at studying the epigenetic basis for key phenotypic components of psychiatric illness have involved the following: (1) administering drugs affecting epigenetic processes, e.g., repeated corticosterone or L-methionine administration to induce a disease state or histone deacetylase inhibitors or nicotinic agonists to reverse the abnormality, or (2) manipulating the natural environment, e.g., by social isolation/enrichment, social dominance/defeat, or chronic exposure to stress early in development prenatally or by maternal care. Key words: GAD67, reelin, DNA methyltransferase, L-methionine, histone deacetylase inhibitors, DNA demethylase, histone deacetylase, imidazenil, maternal care.

1. Introduction Several genes that interact in complex networks are implicated in vulnerability for major mental illness, and there is increasing evidence that epigenetic contributions are substantial (1). In schizophrenia (SZ) and bipolar (BP) disorder, a concordance rate of 40–60% in identical twins, fluctuating disease course, sex differences, onset associated with major hormonal changes, and parentof-origin effects provide epidemiological clues to significant P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_11, © Springer Science+Business Media, LLC 2011

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epigenetic influences (2). However, it is now obvious that simple gene × environment models make it nearly impossible to determine a cause–effect relationship between specific environmental risk factors and mental disorders. It is hoped that the newer epigenetic models will provide better estimates of environmental to mental disorders and lead to new drug interventions (3). In developing epigenetic models that provide a framework for the study of mental disorders, it is essential to regard that cause is distinct from consequence, compensation, or confound (4). For this review, epigenetics will refer to the regulation of gene expression that is mediated by DNA methylation/demethylation and changes in chromatin structure involving covalent modifications of histones. Micro mRNAs are undoubtedly involved in the epigenetics of mental disorders but will not be covered in this review. Gene promoters that contain cytosine-[phosphodiester] guanine (CpG) islands susceptible to methylation exist in different methylation states depending on the status of chromatin. The structure of chromatin – the DNA–nucleosome packages involved in gene transcription – is regulated by methylation of the DNA cytosine base and by histone acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and other chemical processes (5). The result of these DNA/histone remodeling processes is chromatin that can be either open, (euchromatin) allowing for transcription, or closed, (heterochromatin) limiting transcription. Transcriptional activation is associated with an open chromatin state that is characterized by low levels of DNA methylation and by histone–lysine acetylation or by other histone modifications, e.g., trimethylation of H3-lysine4 (5, 6). On the other hand, transcription is repressed by DNA hypermethylation at CpG sites in gene promoter regions. Methylation is accomplished by DNA methyltransferase enzymes (DNMTs) that require the methyl donor S-adenosyl methionine (SAM). Methylated cytosines facilitate the assembly of transcription repressor complexes, methyl-CpG-binding domain proteins (MBDs), and histone deacetylases (HDACs). The repressive processes of DNA methylation, histone deacetylation, and histone methylation, e.g., H3-lysine9 or H3-lysine27 trimethylation, cooperate in reducing transcription and gene expression (5, 6). The chemical processes underlying transcriptional regulation are all interrelated. The direct recruitment of HDACs and histone methyltransferases (HMTs) by DNMTs (7) and also the reverse, direct recruitment of DNMTs by HMTs (8) have been reported. Indirect interactions whereby methyl-CpG-binding domain proteins (MBD), such as methyl-CpG-binding protein (MeCP2), lead to increased histone methylation and histone deacetylation have also been documented (7, 9). In addition, while the existence of a DNA demethylase that can reverse promoter

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methylation and increase transcription is often assumed based on the epigenetic modifications observed (10, 11), the exact biochemical characterization of a DNMT demethylase is controversial and currently under intense investigation (12). Histone demethylases are also operative (13).

2. In Psychosis, Gene Expression Is Downregulated in Specific Populations of Telencephalic GABAergic Neurons

GABAergic interneurons, impinging on dendrites and cell bodies of the large glutamatergic pyramidal cells in the microcircuitry of the cortex and the hippocampus, orchestrate and control the pyramidal cell firing that underlies cognition and generates the gamma frequency of the EEG (14). Thus, the consequence of a GABAergic deficit in the frontal cortex and the hippocampus of psychotic patients is likely to be a fundamental disarray in the synaptic circuits underlying cognitive information processing including attention and memory. It has been proposed that this disarray is reflected in the decreased power of the gamma frequency in psychosis (15). Converging evidence from several lines of research suggests that psychosis is characterized by genes with promoters that are in a closed chromatin state, leading to reduced transcription of proteins essential for GABAergic and synaptic function (1, 16, 17). Two critical GABAergic genes whose expression is downregulated in psychotic postmortem brain are GAD67 (GAD1) and reelin (RELN). An approximate 50% expression downregulation of these genes in major mental disorder has been repeatedly demonstrated (5, 18–24). Glutamic acid decarboxylase (GAD) is the enzyme that synthesizes GABA from glutamate. There are two isoforms of this enzyme. Only GAD67 (GAD1) is downregulated in the postmortem brain of SZ and BP patients and it is the isoform that is most prevalent in human brain and is the isoform associated with neuronal activity, GABA synthesis, and non-vesicular GABA release. GAD65 (GAD2) is not decreased in the frontal cortex in psychotic postmortem brain, which is consistent with the failure to find an obvious loss of GABAergic neurons in telencephalic areas despite decreased GABA function. Another significant GABA protein that is substantially decreased in psychotic postmortem brain is reelin, which in the cortex and the hippocampus is critical for normal dendritic spine and glutamatergic synaptic function (25–29), for neural stem cell migration (30), and for normal neuronal guidance during embryonic development (31). Reelin is constitutively released into the extracellular matrix by specific types of GABAergic neurons located in the upper cortical layers (32). Dendritic spines are a major target

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of this large protein. In the cortex and the hippocampus, these spines are preferentially located on the dendritic trees of glutamatergic pyramidal cells (33). This location is consistent with the hypothesis that reelin is operative in the pathways and processes involved in the synaptic and microcircuit remodeling that occurs during adaptation to changing environments, i.e., learning and memory. The promoters of both GAD1 and RELN are rich in CpG islands, making them especially susceptible to methylation, and methylation of the RELN gene has been reported in psychotic postmortem brain (34, 35). However, there is conflicting evidence that DNA methylation is primarily responsible for the downregulation of these GABAergic genes in SZ or BP disorder and other epigenetic chromatin-remodeling processes may also be involved (1, 17, 36). The techniques for measuring methylation and the areas of the brain measured were different in the above studies and these differences may be important considerations. In addition, epigenetic processes are cell-type specific and may require laser microdissection of GABA neurons for accurate measurement (32). A methylation microarray technology that accounts for cell-type specificity still needs to be developed. In addition to GAD1 and RELN, it is likely that other genes involved in GABA function in the cortex and the hippocampus are also downregulated in psychosis. These genes include among others the N-methyl-D -aspartate (NMDA) receptor subunit NR2A (GRIN2a), the GABA transporter GAT 1 (SLC6A1), somatostatin, Trk-b, parvalbumin, and the nicotinic acetylcholine receptor (nAChR) subunits α4β2 and α7 (1, 23, 24, 37–42). Recent development of microarrays for the simultaneous measurement of gene expression and gene methylation of hundreds of genes has resulted in a surge of knowledge about gene expression and how genes interact with each other and with the environment. In a recent postmortem brain study using CpG-island microarrays, evidence was found for associated DNA methylation differences in the frontal cortex of SZ, BP, and control subjects in several loci involved in GABAergic and glutamatergic neurotransmission, brain development, and other processes functionally linked to mental illness, importantly the stress response (1). A further intriguing finding of the Mill et al. (1) methylation microarray study is the discovery that DNA promoter methylation may be coordinated into networks or clusters. The presence of networks would be consistent with the finding that GAD1 and RELN, but not GAD2, have been shown to be coordinately regulated in neuronal progenitor cells (43) and may account for the finding that in telencephalic areas, GAD67 but not GAD65 levels are decreased in the heterozygous reeler mouse (44) and in SZ (19).

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2.1. DNA Methyltransferases Are Elevated in Telencephalic GABAergic Neurons of Psychiatric Patients

DNA methyltransferase 1 (DNMT1) is highly expressed in the brain and preferentially in GABA neurons of the prefrontal cortex and the striatum (45, 46). Ruzicka et al. (32), using laser microdissection of GABA neurons, reported that DNMT1 was increased threefold in layer 1 prefrontal cortex of schizophrenia patients in the same tissue samples in which GAD67 and reelin mRNAs were decreased twofold. No differences in DNMT or GABAergic gene expression were found in layer 5. An increase in de novo DNA methyltransferase 3a (DNMT3a) in GABAergic interneurons of the frontal cortex was also found (46). In addition to an increase in DNMTs, DNA promoter hypermethylation is suggested by evidence of an increase in S-adenosyl methionine (SAM) in the prefrontal cortex of postmortem brain of psychotic patients (47); SAM is a universal methyl donor for the methylation of the 5-carbon of deoxycytosines in CpG islands of gene promoters and for the methylation of histones. Importantly, increases in DNMT and SAM were found to be significantly correlated with decreased expression of GAD67 and reelin in the prefrontal cortex of BP disorder patients (45, 47–49). Further evidence for epigenetic changes in GABAergic function in mental illness is provided by studies of the postmortem brain of depressed patients who committed suicide. DNMT1 and DNMT3b mRNA and protein levels were increased in the prefrontal cortex of these patients (50) and the increased expression of DNMT3b was correlated with increased DNA methylation of the promoter of the GABAA receptor α1 subunit gene.

2.2. Histone Deacetylase (HDAC) Expression Is Elevated in the Telencephalon of SZ Postmortem Brain

An increase in histone deacetylase 1 (HDAC1) expression in the frontal cortex and the hippocampus of schizophrenia postmortem brain provides additional evidence for an epigenetic downregulation of GABAergic function (51, 52). Presumably, HDACs recruiting DNMT1, MeCP2, and other repressor proteins form a chromatin repressor complex that may lead to a downregulation of the expression of reelin or GAD67. However, an alternative epigenetic explanation for GAD67 downregulation is based on data indicating that GAD67 gene expression downregulation is associated with a decrease in histone H3-lysine 4 (tri) methylation in the area of the GAD67 promoter (5, 17, 53). These results highlight how closely DNA methylation/demethylation and histone chromatin modifications are intertwined in determining whether transcription is activated or repressed. Postmortem brain findings are supported by the results of lymphocyte studies. These findings include increased DNMT1 and 3a expression in SZ (46), decreased levels of acetylated H3-lysine9, lysine14, decreased GAD67, and increased levels of dimethylated H3-lysine 9 (52). While postmortem brain samples

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and lymphocyte tissues of SZ patients can provide valuable information, there are many questions that cannot be answered with these approaches, especially with respect to brain region selectivity, circuitry, and drug responsivity. These questions can be better addressed using animal models.

3. Heterozygous Reeler and GAD67 Mice

Mice heterozygous for reelin or GAD67 were an obvious first choice to model the approximate 50% downregulation of GAD67 and reelin mRNA and protein levels in psychotic postmortem brain (54). Several abnormalities in neurochemistry, neuroanatomy, and behavior reminiscent of psychosis have been reported in heterozygous reeler mice (29, 55, 56). An early finding was that GAD67 was decreased in the frontal cortex of haploinsufficient reeler mice. In mice, low levels of reelin are associated with decreased GABA turnover, decreased cortical neuropil, deficits in learning and memory in behavioral tasks (57), and deficits in long-term potentiation (LTP) and other aspects of glutamatergic receptor function that underlie memory (25, 29, 58). The density of dendritic spines of cortical and hippocampal pyramidal neurons is considered to be a measure of synaptic plasticity underlying learning and memory. Thus, it is especially interesting that a decreased dendritic spine density of layer III pyramidal neurons in the frontal cortex is a characteristic of both SZ postmortem brain (59) and the heterozygous reeler mouse (44). Although GAD67 expression is decreased in heterozygous reeler mice, the reverse is not found; reelin is not decreased in GAD67 heterozygous mice and neither is spine density (44). The postsynaptic density of the spine contains glutamate receptors (including NMDA and AMPA receptors). Reelin, released into the extracellular matrix by GABA neurons in layers I/II by a constitutive process, decorates the spines on the dendrites of pyramidal neurons (33) and there is evidence that reelin may be operative in the regulation of proteins involved in spine morphology, such as activity-regulated cytoskeletal protein (Arc) (60). Studies with heterozygous mice have also demonstrated that reelin is required for long-term potentiation (LTP), is involved in synaptic changes underlying memory (29), and influences the relative expression of NR2A and NR2B subunits of the NMDA receptor (25). There are several recent reports indicating that polymorphisms involving the RELN gene (61–64) or the GAD1 gene (65) are associated with increased genetic vulnerability for major mental disorders. However, with the realization that the downregulation of GABAergic function in psychotic postmortem brain

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might be largely epigenetic, the focus is shifting from heterozygous mouse models to epigenetic mouse models. Nevertheless, the heterozygous reeler mouse may be valuable for studying epigenetic compensation for reelin and GABAergic downregulation and for understanding the interaction between genetic and epigenetic influences on reelin and GABA function. For example, in a recent study (56), we subjected wild-type mice (with high levels of prepulse inhibition of startle, or PPI) and heterozygous reeler mice (with low levels of PPI) to social isolation. Wild-type mice responded to social isolation in a typical manner with a decrease in PPI, while the heterozygous reeler mice did not. Another group, comparing the effects of maternal separation on wild-type versus heterozygous reeler mice, found that the reeler heterozygous genotype exhibited less sensitivity to environmental insult along with greater sensitivity to the atypical antipsychotic olanzapine (66, 67). The similarity of the findings on the effects of social isolation and maternal separation could be interpreted as supportive of the theory that the heterozygous reeler mouse (and perhaps SZ) is characterized by chromatin that is less responsive to adaptive change (68).

4. Environmental and Pharmacological Animal Models of Epigenetic Change in GABA Function

Every stimulus-driven neuronal activity (sensory, motor, drug, or food related) leaves its mark in the nervous system so that similar information in the future can be more quickly and successfully processed to insure survival. Animal studies with an epigenetic perspective have been conducted at different levels. Useful information about fundamental processes can be obtained using cultures of excised embryonic or adult tissues from specific brain regions of animals. However, the main focus has been on exposing live animals to different experiences or drug treatments and subsequently measuring behavior and/or neurochemical and neuroanatomical changes suggestive of chromatin remodeling. Animal models, instead of being specific for a diagnostic category, are usually aimed at key phenotypic components of mental disorders. Epigenetic animal models can be arbitrarily separated into two main methodological categories: (1) models that induce or reverse a specific phenotypic aspect of the disease, such as learning/memory deficit or fear/anxiety by manipulating the natural environment, and (2) models that induce or reverse a phenotypic aspect of the disease state by drug administration. Social isolation/enrichment, social dominance/defeat, chronic exposure to stress, and variations in maternal care are examples of the former. Repeated administration of corticosterone and repeated

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administration of L-methionine (MET) are examples of models using drug administration to induce a disease state. The objective of these models is to use the induced disease state to test ways in which the disease phenotype can be corrected by changing the environment or by drugs. We will briefly review the different types of models with respect to GABAergic function and particularly with respect to reelin and dendritic structure and spine density. 4.1. Natural or Environmental Epigenetic Models Implicate Reelin and Dendritic Spine Density Changes in Adaptive Response to New Experience

In the cortex and the hippocampus, adaptation to the environment and memory is encoded by changes occurring at the synapse. The synapse is also the focus in developing drugs that target the negative symptoms of SZ, which involve cognitive dysfunction and failure to adapt successfully to new environments. Changes in many neurotransmitter and neurochemical pathways are also involved, but findings point to changes in GABAergic function as playing a central role. Reelin, a protein constitutively released in cortex and hippocampus by GABAergic neurons, is central in the synaptic function underlying memory. Extracellular matrix reelin binds to integrin and the lipoprotein receptors VLDLR/APOER2 located on the spine head, resulting in signal cascades that induce changes in the NMDA receptor (25, 28) and lead to translation of various other resident proteins required for changes in receptor function and spine morphology (60). Significantly, studies using environmental epigenetic models often implicate epigenetic changes in GABA function, although this was not the original intent. Formation of a memory trace for a previously presented shock during contextual fear is accompanied by demethylation of the RELN gene (69) and is associated with a transient increase in dendritic spines on CA1 pyramidal neurons and sometime later, with an increase in spines on pyramidal neurons of the anterior cingulate area of the frontal cortex (70). We suspect that such dynamic changes in reelin levels and in dendritic spines that take place during learning may be under GABAergic epigenetic control. Similarly, the effect of early maternal care in shaping the stress response mediated by hypothalamic–pituitary–adrenal (HPA) axis function is the focus of a recent review (71). Maternal licking and grooming and other maternal behaviors have a profound influence on the stress response in offspring. Offspring of low licking and grooming dams exhibit anxiety and lower levels of reelin than do offspring of high licking and grooming rats (72). It is possible that differences in dendritic structure and spines that characterize offspring of low versus high maternal care dams (73, 74) are related to differences in the epigenetic control of reelin expression in GABAergic neurons. The effect of stress on reelin expression in rats has also been studied by chronically injecting corticosterone (75).

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Studies using drugs to induce epigenetic changes in GABA function may provide more experimental control compared to natural methods. An animal model developed by our group specifically for studying epigenetic downregulation of GABAergic function in psychosis entails repeated administration of Lmethionine (MET). MET is a dietary amino acid that leads to the coordinated methylation of promoters of several genes critical for GABAergic function including RELN and GAD1 (72, 76). In the 1960s and 1970s, several clinical experiments were conducted in the hope that administration of MET would improve psychotic symptoms. Unfortunately, MET exacerbated psychosis in ∼60% of patients, which remained a mystery for decades until an epigenetic explanation was proposed (39). Like the heterozygous reeler mouse, MET-treated mice have lower levels of reelin and GAD67 expression in the cortex and the hippocampus. MET-treated mice also exhibit a significant increase in steady-state levels of SAM and hypermethylation of GABAergic gene promoters, notably RELN and GAD1, which leads to a downregulation of the expression of reelin and GAD67 (77, 78). Significantly, the increase in methylation of the RELN and GAD1 promoters was not observed for GAD2. MET-treated mice also exhibit behavioral abnormalities (PPI and social interaction deficits) reminiscent of behavioral abnormalities exhibited by SZ patients and heterozygous reeler mice (55, 77, 78). The HDAC inhibitor valproic acid (VPA) reverses the MET-induced downregulation of reelin and GAD67, increases acetylation of histone H3 in mouse brain nearly fourfold, and corrects the SZ-like epigenetic behavioral modifications induced by MET (77, 78). The decreased expression of reelin and GAD67 induced by MET is accompanied by an increase in the association of the methyl CpG-binding protein MeCP2 with mouse reelin and GAD67 promoters (76). VPA co-administered with MET reduces the MeCP2 binding. In a subsequent study, Dong et al. (10) measured levels of RELN and GAD67 promoter hypermethylation following termination of repeated MET administration. Promoter methylation declined by 50% after 6 days of MET withdrawal, and the HDAC inhibitors VPA and MS-275, administered after MET treatment termination, greatly accelerated RELN and GAD67 promoter demethylation within 48–72 h and increased the binding of acetyl histone-3 to RELN and GAD67 promoters. These findings suggest that H3 covalent modifications are capable of modulating DNA demethylation in neurons, supporting the view that HDAC inhibitors may facilitate DNA demethylation.

Tueting, Costa, and Guidotti APICAL

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Fig. 11.1. L-Methionine (MET) decreases spine density on both the apical and the basilar dendrites. Mean spine density ±SE shown here for a distance of 50–100 μm from the pyramidal cell body located in frontal cortex layer III. Mice were treated for 7 days with VEH (saline twice daily s.c.) or MET (5.2 mmol/kg twice daily s.c.). VEH, 13 mice; MET, 12 mice. The difference in mean spine density was assessed by unpaired t-test, ∗∗ p < 0.01.

We have found that protracted MET treatment decreases spine density in the frontal cortex of mice, similar to the decrease in spine density of the frontal cortex layer III pyramidal neurons in the postmortem brain of patients with SZ and in heterozygous reeler mice (Fig. 11.1). The decrease in spine density induced by MET can be prevented by co-administration of VPA, supporting our previous finding that RELN promoter hypermethylation induced by MET can be prevented by co-administration of VPA (79). We also found that VPA administered alone increased spine density, which is consistent with our earlier finding that VPA increases acetylated histone 3 content in GABAergic interneurons (10, 76). Weaver et al. (72) administered MET for 7 days to offspring of high licking and grooming rat mothers. A microarray analysis of gene expression indicated that only a small fraction of genes analyzed in the hippocampus were affected by repeated administration of MET, and RELN was one of the few genes affected. The upregulation of reelin in the hippocampus of offspring of high licking and grooming dams was reversed by MET. This finding and the reversal of the downregulation of reelin in offspring of low licking and grooming dams by the HDAC inhibitor trichostatin A suggest that both DNA methylation and histone modifications are involved in the RELN chromatin remodeling attributable to maternal care. In addition to MET’s ability to downregulate reelin and GAD67 mRNAs and proteins in mice (77), MET added to primary neuronal cell cultures in vitro also downregulates reelin and GAD67 (80). This effect of MET is attenuated by co-transfection of DNMT1 antisense oligonucleotides (80), providing a link between the overexpression of DNMT1 and the reduced expression of reelin and GAD67 in GABAergic neurons found in psychosis and MET-treated mice (32, 42, 45, 49).

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The objective of developing an epigenetic animal model of GABAergic dysfunction is that once a phenotypic disease model has been induced, the model can be used to explore ways to pharmacologically correct the deficits using drugs that (a) enhance the expression of GABAergic genes reversing chromatin-remodeling abnormalities or (b) act as positive allosteric modulators of GABAA receptors directly to enhance defective GABAergic neurotransmission. (i) A possible approach would be to diminish DNA methyltransferase overactivity with inhibitors of DNMTs. However, the most potent DNMT inhibitors (5-azacytidine and zebularine) fail to readily cross the blood–brain barrier when administered systemically and are active only in the S phase of the cell cycle. Hence, there is a search for drugs that could have DNMT inhibitory activity in nondividing differentiated neurons. (ii) Nicotinic receptor agonists may have the ability to inhibit DNMT expression in cortical and hippocampal areas. The rate of tobacco smoking in psychiatric patients is very high, especially in SZ patients. Attempts to decrease this behavior have been unsuccessful because withdrawal from nicotine causes an exacerbation of SZ symptoms. Thus, smoking is often viewed as an attempt by patients to self-medicate. Satta et al. (42) administered nicotine to mice and found that nicotine downregulated DNMT1 expression in the frontal cortex and hippocampal areas that express cholinergic nicotinic receptors. Further, the same doses of nicotine that decreased DNMT1 expression also diminished the level of cytosine-5-methylation in the GAD1 promoter and prevented the MET-induced hypermethylation of this promoter (Fig. 11.2). The results suggest that nicotine or other nicotinic agonists, by activating nicotinic cholinergic receptors located on cortical or hippocampal GABAergic interneurons, can upregulate GAD67 expression via modification of an epigenetic mechanism. (iii) Another approach to correct abnormal GABAergic promoter methylation is to test drugs that activate DNA demethylation processes. Using the MET mouse model of SZ, we have shown that hypermethylated reelin and GAD1 promoters can be demethylated by the administration of clinically relevant doses of the HDAC inhibitors VPA or MS-275 (6, 81). Because the HDAC inhibitor VPA is frequently used clinically in combination with antipsychotics as an

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Fig. 11.2. Nicotine reduces GAD67 promoter methylation in the frontal cortex. VEH, received saline four times a day for 5 days; NIC, received nicotine 3.5 mg/kg s.c. four times a day for 4 days followed by saline on the fifth day; VEH+MET, received saline for 2 days followed by L-methionine (5 mmol/kg, twice a day) for 3 days; NIC+MET, received nicotine (3.5 mg/kg) for 4 days. On the third and fourth days of nicotine treatment, mice also received L-methionine. On the fifth day, nicotine was suspended and mice received only L-methionine. Each value is the mean and ±SE for three mice. Oneway ANOVA: Newman–Keuls multiple comparison. ∗∗ p < 0.01 VEH + MET vs VEH and VEH + MET vs NIC + MET ; ∗ p < 0.05 VEH vs NIC and VEH vs NIC + MET. (Modified from Satta et al., (42).)

augmentation strategy to treat multiple symptoms, especially cognitive symptoms in patients suffering from BP or schizoaffective disorders, Dong et al. (82) measured the ratio of 5-methylcytosine to unmethylated cytosine in RELN and GAD1 promoters in the mouse frontal cortex and striatum. Normal mice were compared to mice pretreated with MET to hypermethylate these promoters. The results were that clinically relevant doses of clozapine and sulpiride, but not haloperidol or olanzapine, induced doserelated increases in the cortical and striatal demethylation of hypermethylated RELN and GAD67 promoters. The effects of clozapine and sulpiride were dramatically potentiated by a clinically relevant dose of VPA. These findings in mice are consistent with the clinical efficacy in some BP patients of combining VPA with antipsychotic treatment. These data suggest that the induction of DNA demethylation by specific subtypes of antipsychotics or other agents that act at the level of chromatin structure should be further investigated. This may lead to the development of drug treatments effective not only on positive but also on negative symptoms of major psychosis. (iv) A further strategy to correct epigenetic GABAergic downregulation in psychosis is to enhance the defective GABAergic

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transmission using drugs acting as selective positive allosteric modulators of GABA action at pertinent GABAA receptor subtypes (35, 83). Benzodiazepines that are devoid of intrinsic activity at GABAA receptors that express α1 subunits but that act as full positive allosteric modulators of GABA action at GABAA receptors containing α2, α3, or α5 subunits should counteract GABAergic signal transduction deficits without eliciting sedation, amnesia, tolerance, or dependence liability (26). A benzodiazepine with the above-mentioned properties is imidazenil (26). Hence, we suggest that drugs like imidazenil, given alone or in combination with antipsychotics, should be tested in the treatment of GABAergic dysfunction operative in SZ and BP disorder. Support for the use of imidazenil in psychoses is provided by experiments with reeler heterozygous mice. In one such experiment (37), imidazenil corrected the deficiency in PPI in heterozygous reeler mice, while alprazolam failed to do so. In addition, administration of imidazenil reduced the PPI and social interaction deficits in mice receiving protracted MET treatment (78). PPI is often viewed as an endophenotype for psychosis vulnerability (84) and it has been shown to reflect α3 and α5 subunit activity (85, 86), which are the subunits of the GABAA receptor affected by imidazenil.

7. Conclusions This review has focused on epigenetic animal models of SZ or BP disorders characterized by hypermethylation and epigenetic downregulation of forebrain GABAergic genes, even though in the MET-treated mouse model, non-GABAergic promoters can also be hypermethylated. However, interrogating the genome using the ChIP on chip technique indicates that only a relatively small number of genes (<5%) are hypermethylated in the frontal cortex or the hippocampus of mice receiving protracted treatment with MET (82). This selectivity for an epigenetic regulation of a small number of genes has also been reported in rats as a function of variations in maternal care. Offspring show that the vast majority of genes did not change expression (72). Further, normalization by the HDAC inhibitor trichostatin A or methylation by MET involved changes in the expression of a relatively few select gene clusters (72). Hence, these observations would argue against a hypothesis proposing an overall general reprogramming of gene expression in the changes induced by the environment or protracted MET treatment in mice. A shortcoming of the MET model is that we have not yet found evidence for an upregulation of DNMTs by MET

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comparable to the elevation of DNMT levels in psychotic postmortem brain, although SAM, which is increased in SZ along with DNMTs, is increased by MET and contributes to the hypermethylation of selective gene promoters (77). In spite of shortcomings, the mouse methionine model continues to be useful, especially in regard to study of the combined use of HDAC inhibitors and antipsychotics. Using this model, we have recently demonstrated that specific antipsychotic subtypes (i.e., clozapine and sulpiride) can cause persistent epigenetic changes by affecting chromatin structure and by inducing GABAergic promoter demethylation (6). The goal of our studies is to develop a classification of antipsychotic drugs based on their epigenetic action. To achieve this goal, we propose using the MET-treated mouse model to study other typical and atypical antipsychotics to investigate whether antipsychotics that decrease GABAergic promoter methylation act by decreasing DNMTs or by activating a DNA demethylase. We have preliminary evidence suggesting that the molecular mechanisms underpinning GABAergic promoter demethylation caused by clozapine or sulpiride administration may include an activation of a putative DNA demethylation process (82). Because the biochemical identity and function of a putative DNA demethylase in neurons still requires clarification, in the future, its characterization will be crucial for the identification of a new line of pharmacological agents to treat Sz and related major psychiatric disorders.

Acknowledgments This research was partially supported by NIH grants MH071667 to E. Costa and by MH070855 to A. Guidotti. References 1. Mill, J., Tang, T., Kaminsky, Z., Khare, T., Yazdanpanah, S., Bouchard, L., Jia, P., Assadzadeh, A., Flanagan, J., Schumacher, A., Wang, S.-C., Petronis, A. (2008) Epigenomic profiling reveals DNA-methylation changes associated with major psychosis. Am. J. Hum. Genet. 82, 696–711. 2. Oh, G., Petronis, A. (2008) Environmental studies of schizophrenia through the prism of epigenetics. Schizophr. Bull. 34, 1122–1129. 3. Ptak, C., Petronis, A. (2008) Epigenetics and complex disease: from etiology to new therapeutics. Ann. Rev. Parmacol. Toxicol. 48, 257–276.

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Chapter 12 Modeling Schizophrenia in Neuregulin 1 and ErbB4 Mutant Mice Yisheng Lu, Dong-Min Yin, Wen-Cheng Xiong, and Lin Mei Abstract Schizophrenia, a most prevalent brain disorder, remains to be one of the least understood. Unlike Alzheimer’s disease or Parkinson’s disease, schizophrenia lacks clear pathological lesions, which has made it difficult to model in animals. However, genetic studies have recently identified many susceptibility genes of this disorder including neuregulin 1 (NRG1) and its receptor ErbB4. Arguably, relevant mutant mice have provided a unique opportunity to model “schizophrenia” in animals where expression or function of susceptibility genes is altered. This review summarizes recent studies of NRG1 and ErbB4 mutant mice and their implication in schizophrenic pathology. Key words: Neuregulin 1, ErbB4, schizophrenia, mouse model, rodents, psychiatric disorders, synaptic plasticity, neurotransmission, working memory, hyperactivity, prepulse inhibition.

1. Introduction Schizophrenia is a devastating disorder with a worldwide prevalence of 0.5–1.2% (1–2). Schizophrenic patients could have three categories of symptoms: positive symptoms (delusions, auditory hallucinations, and thought disorders), negative symptoms (blunted affect or emotion, unpleasure, or lack of motivation), and cognitive symptoms (deficits in executive function, working memory, and attention). Being a most prevalent of central nervous system (CNS) disorders, schizophrenia imposes a huge burden on individuals and society. Nevertheless, it continues to be one of the least understood. Unlike Alzheimer’s disease or Parkinson’s disease, schizophrenia lacks clear pathological lesions, Y. Lu and D.M. Yin have contributed equally P. O’Donnell (ed.), Animal Models of Schizophrenia and Related Disorders, Neuromethods 59, DOI 10.1007/978-1-61779-157-4_12, © Springer Science+Business Media, LLC 2011

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which has made it difficult to model in animals. On the other hand, the etiology of schizophrenia is complex (3–5). The heritability estimates of schizophrenia are between 60 and 80% (6), which highlights the importance of genetic factors in the pathogenesis of schizophrenia. Recent years have witnessed significant progress in understanding the pathology of schizophrenia, as a continuously increasing number of susceptibility genes, including neuregulin 1 (NRG1) and its receptor ErbB4, have been identified. Some of them encode proteins that have been implicated in neural development, in agreement with well-accepted hypothesis that schizophrenia may result from problems in neural development (7–8). Others have their function yet to be identified. Nevertheless, the identification of these genes, together with genetic tools available, provides a unique opportunity to model “schizophrenia” in animals where expression or function of the susceptibility genes is altered. At present, it is difficult to determine whether animals “see” or “hear” things that do not exist, but positive symptoms are thought to associate with increased locomotor activity (9), a characteristic rodent phenotype that might correspond to the psychomotor agitation of schizophrenic patients. On the other hand, negative symptoms could be modeled in mice by home–cage social interaction, nesting behavior, interaction with a juvenile conspecific, social dominance, and aggression behavior on resident intruder. Cognitive deficits in schizophrenic patients could be displayed as impaired attention and working memory (10). Among them, working memory deficits are thought to be central to poor cognitive performance in schizophrenia and could result from GABAergic dysfunction (11). Reduced PPI ability is thought to contribute to schizophrenic conditions including inattention, distractibility and cognitive deficits. This chapter focuses on NRG1 and ErbB4. We will briefly review advances in studies of their functions and discuss mouse models where NRG1 and ErbB4 are mutated in relevance to schizophrenia.

2. Imbalanced Activity in Schizophrenia

The brain activity is determined by an intricate balance between function of pathways of the excitatory neurotransmitter glutamate and inhibitory neurotransmitters such as GABA. Altered activity of glutamatergic and GABAergic pathways has been implicated in schizophrenia (12–13). Phencyclidine (PCP), an NMDAR uncompetitive antagonist (14), produces psychotic symptoms, thought disorder, blunted affect and cognitive impairments that resemble those in schizophrenic patients (15–16).

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In healthy individuals, ketamine, a PCP derivative, produces transient schizophrenia-like (positive and negative) symptoms and impairs prefrontal cortex (PFC)-dependent cognitive function (17–18). In schizophrenic patients, on the other hand, ketamine increases signs and symptoms of the disorder (18–19). Finally, in rodents, pharmacological or genetic manipulations of NMDA receptors cause behaviors relevant to schizophrenia (20– 22). These observations support a mechanism of glutamatergic hypofunction in schizophrenia. GABAergic interneurons are crucial for synchronization of pyramidal neuron activity. For example, different segments of a dorso lateral PFC (DLPFC) pyramidal neuron, including somata, dendritic branches and spines, and axonal initial segment, receive dense GABAergic synaptic innervation (23–28). These PFC interneurons, distinct in morphology and function, are critical for working memory (29–34). The alteration in perisomatic inhibition of pyramidal neurons contributes to a diminished capacity for the gamma-frequency-synchronized neuronal activity that is required for working memory function (35). In addition, cognitive abnormalities in schizophrenia may result from pathological alternations in cortical inhibitory circuits (36–37). Accordingly, expression of GAD65 and GAD67, enzymes that synthesize GABA, is reduced in DLPFC layers 3–5 of individuals with schizophrenia (35, 37–41). Both the synthesis and the reuptake of GABA seem to be greatly reduced in a subset of inhibitory neurons in the DLPFC of schizophrenia. In addition, parvalbumin (PV) expression was shown to be significantly decreased in schizophrenic cortex (35). These observations suggest that disruption of inhibitory GABAergic transmission in the PFC contributes to schizophrenia. In adult brains, both NRG1 and its receptors are distributed throughout different brain regions (42–45), suggesting a role of NRG1 in regulating neurotransmission and synaptic plasticity. In support of this hypothesis were observations that ErbB4 is present at the postsynaptic density of excitatory synapses presumably via interaction with PSD95 (46–48). Exogenous NRG1 suppresses the induction of LTP at Schaffer collateral–CA1 synapses in the hippocampus (47–49) or reverses it (50–51).

3. NRG1 Signaling in Neural Development and Synaptic Plasticity 3.1. NRG1 and ErbB Kinases

The NRG1 are a family of growth factors containing the epidermal growth factor (EGF)-like domain (52). There are at least six types of NRG1, each of which has a distinct aminoterminal region and varies in levels and patterns of expression. They act by activating type I transmembrane ErbB receptor tyrosine kinases (ErbB2/HER2, ErbB3/HER3, and ErbB4/

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HER4). NRG1 binds directly to both ErbB3 and ErbB4, but not ErbB2, induces homo- and heterodimerization of ErbB receptors, and thus stimulates the kinases (53–57). Unlike ErbB2 and ErbB4, ErbB3 is impaired in kinase activity. Therefore, ErbB4 homodimers are sufficient to initiate NRG1 signaling, whereas ErbB2 and ErbB3 need to form heterodimers between them or with ErbB4 or the EGF receptor. Downstream of ErbB kinases are the Ras-Raf-Erk and PI3 kinase/Akt pathways. NRG1 has been implicated in many aspects of neural development including neuron migration, axon projection, myelination, synapse formation, or upregulation of neurotransmitter receptor expression (52). Recently, when ErbB2 and ErbB4 are mutated in the CNS, mice appeared normal in layered structures of the cerebral cortex, the hippocampus, and the cerebellum or expression of NMDA receptor subtypes (58–59), suggesting that NRG1 signaling may not be as critical in these events as previously thought. 3.2. NRG1 in Synaptic Plasticity

ErbB4 mRNA is enriched in regions where interneurons are clustered (42) and GAD-positive neurons of the hippocampus express high levels of ErbB4 (47). In agreement, ErbB4 is shown to be selectively expressed in parvalbumin (PV)-positive interneurons (60–61), which supports the hypothesis that the physiological function of NRG1–ErbB4 signaling may be through PV-positive interneurons. A couple of years ago, we found that NRG1 facilitates activity-dependent release of GABA in the PFC in a manner dependent on ErbB4 (62). Recently, we found that the NRG1– ErbB4 pathway in PV-positive neurons is critical in regulating GABAergic transmission and the activity of pyramidal neurons (63). We demonstrated that exogenous NRG1 was able to suppress spontaneous spikes and evoked firing activity of pyramidal neurons in PFC slices, and this effect was abolished in brain slices isolated from PV-Cre;ErbB4−/− (PV–ErbB4−/− ) mutant mice where ErbB4 was specifically ablated in PV-positive interneurons. To investigate whether endogenous NRG1 was critical for pyramidal neuron activity, brain slices were treated with ectoErbB4, a neutralizing peptide, to clean up endogenous NRG1. Remarkably, it increased the activity of pyramidal neurons, suggesting a role of endogenous NRG1. Moreover, the firing rates of pyramidal neurons were increased in PV–ErbB4−/− brain slices. These observations indicate that NRG1 controls pyramidal neuron firing by promoting GABA release from PV-positive interneurons. Considering that PV is not expressed until postnatal day 10 (64–65), a time when the cortical lamination is nearly achieved (65–66), these results suggest that the schizophrenialike phenotypes in PV–ErbB4−/− mice may be not due to deficit in neuronal migration. Since NRG1–ErbB4 signaling is also involved in synapse formation (67–68) and interneuron survival (60) which can persist after postnatal day 10, it remains

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to be clarified whether the schizophrenia-relevant behavior in PV–ErbB4−/− mice is due to abnormal neural development or synaptic plasticity.

4. NRG1 and ErbB4 as Schizophrenia Susceptibility Genes

Fine mapping of the 8p, a susceptibility locus, and haplotype association analysis of schizophrenic patients in Iceland led to the identification of NRG1 as a candidate gene for schizophrenia (69). An independent study of Chinese Han schizophrenia family trios showed similar results (70). Subsequently, the association between NRG1 and schizophrenia has been replicated in various populations (71–74). Genome-wide association studies (GWAS) of African-American people indicate that both ErbB4 and NRG1 are two of the most related genes (75–76), and in some rare cases, ErbB4 is truncated and has lost the intracellular kinase domain (77). It is not known how the variation of NRG1 and ErbB4 gene predisposes to schizophrenia. Most at-risk genetic variations or SNPs in the NRG1 and ErbB4 genes are intronic, synonymous exonic substitutions, or in 5 - or 3 -end non-coding regions; it is therefore believed that they could alter NRG1 or ErbB4 expression in schizophrenic patients. In support of this hypothesis were observations that mRNA levels of type I and type IV NRG1 and JMa/CYT1 ErbB4 were upregulated in schizophrenic dorsolateral PFC (DLPFC) or hippocampus (78–81), two brain regions strongly implicated in schizophrenia. In some schizophrenic patients, however, the expression of NRG1 and ErbB4 is not altered, while the NRG1-induced activation of ErbB4 signaling is dramatically increased due to the enhanced interaction between ErbB4 and PSD95 (82). These observations suggest the necessity to use gain-of-function models to study the roles of NRG1–ErbB4 signaling in the pathogenesis of schizophrenia. There was no genetic evidence that ErbB2 is a susceptibility gene for schizophrenia. In terms of ErbB3, there appears to be contradictory results in the literature. Two groups reported no genetic association between ErbB3 and schizophrenia in Japanese population (83–84), whereas a recent study indicated the association between ErbB3 and schizophrenia in Caucasian population (85). One recent linkage study has implicated neuregulin 3 (NRG3) as a schizophrenia susceptibility gene in Ashkenazi Jewish (AJ) and Han Chinese populations in Taiwan (86). NRG3 is a paralog of NRG1 with an expression pattern more limited to the CNS and, like NRG1, encodes a single-pass membrane protein with an extracellular, N-terminal EGF domain

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(87). The full-length human NRG3 protein, 720 amino acids in length, has an extracellular EGF-like domain (31% identity with that of NRG1) that has been shown to be functional (87–88). Biochemical studies suggest that ErbB4 is the only receptor for NRG3, although ErbB4 also interacts with NRG1 (89–90). However, little is known about the physiological function of NRG3 in the brain.

5. SchizophreniaRelevant Behavioral Deficits in NRG1 and ErbB4 Mutant Mice

Null mutant mice of NRG1 and its receptors ErbB2, ErbB3, and ErbB4 die prematurely (91) (Table 12.1), but their hypomorphs survive into adulthood without observable difference in life span. Behavioral studies of NRG1 and ErbB4 hypomorphs support the notion that their mutations could be risk factors for schizophrenia. NRG1 has various domains, some of which such as the epidermal growth factor (EGF) domains and transmembrane (TM) domain are conserved in most NRG1 isoforms (52). The EGF domain is critical for its biological activity. The immunoglobulin (Ig) domain is conserved among all isoforms except type III that contains the cysteine-rich domain (CRD). Accordingly, deletion of the EGF, TM, or IG domains appears to cause more behavioral deficits than in mice without the CRD. Maturation of most, if not all, proNRG1 isoforms requires cleavage by BACE1 or a related protease (52). Not surprisingly, BACE1+/− mutant mice showed PPI deficits, hyperactivity, and cognitive impairments (92).

5.1. NRG1–EGF+/− Hypomorphs

NRG1–EGF+/− mice are hyperactive in the open field test (91). The mutant mice would complete the T-maze test faster. They also performed better in rotarod test, a test of forced motor activity that is associated with cerebellum and motor cortex. Ehrlichman (93) studied another line of NRG1–EGF+/− mice (94) where EGF domain was partially deleted. The mice display reduced mismatch negativity, contextual fear conditioning, and social interactions, although their prepulse inhibition and locomotion appear normal.

5.2. NRG1–TM+/− Hypomorphs

NRG1–TM+/− mice are hyperactive in the open-field test (69, 95–97). The aggressive behavior in NRG1–TM+/− mice is increased while the response to social novelty is reduced. The NRG1–TM+/− mice appear normal in spatial learning and working memory (98). Intriguingly, clozapine was able to reverse the hyperactive phenotype (69). Whether NRG1–TM+/− mice are impaired in prepulse inhibition is controversial (96–97).

↓ (69), = (97, 110) n.d.

= (93, 109)

↓ (93)

Fear conditioning

= (96, 112),↑ (97)

= (109)

Elevated plus maze

= (97), ↑ (112) n.d.

↓ (93) n.d.

Social interaction Clozapine

n.d. Decreased locomotor activity, rescued LI (99)

n.d.

n.d.

n.d.

n.d.

↓ (99), = (111)

= (99, 111)

= (99)

= (99), ↑ (111)

NRG1–Ig+/−

n.d. n.d.

↓ (100)

n.d.

n.d.

n.d.

↓ (100)

n.d.

n.d.

n.d.

= (100)

n.d. n.d.

n.d.

= (102)

= (102)

n.d.

↓ (102)

n.d.

↓ (102)

n.d.

= (102)

NRG1–CRD+/− NRG1type1-tg

↑, significantly increased; =, no significant difference; ↓, significantly decreased. n.d., not done. PPI, prepulse inhibition; LI, latent inhibition.

n.d.

n.d.

T-maze

Working memory

= (96, 112), ↑ (97)

= (109)

Light–dark test

Anxiety

n.d.

n.d.

PPI

n.d.

LI

Executive function

Rotarod

↑ (91)

n.d.

↑ (91)

Performance

↑ (69, 95–97, 110)

↑ (91, 109), =(93)

T-maze

NRG1–TM+/−

Open-field test

Locomotor activity

NRG1–EGF+/−

Table 12.1 NRG1 mutant mice phenotypes

↓ (103) n.d.

n.d.

n.d.

n.d.

n.d.

↓ (103)

↓ (103)

n.d.

n.d.

= (103)

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NRG1–TM+/− mice showed anxiety-like phenotype, but normal social interaction. 5.3. NRG1–Ig+/− Hypomorphs

NRG1–Ig+/− mice were normal in open-field and wheel rotation tests. They were impaired in latent inhibition and showed suppression of open-field and running wheel activity by clozapine (99).

5.4. NRG1–CRD+/− Hypomorphs

NRG1–CRD+/− mice appeared normal in open-field and Y-maze tests but were impaired in prepulse inhibition, latent inhibition and working memory (100).

5.5. NRG1type1-tg Hypermorphs

Type 1 neuregulin 1 transgenic mice showed overexpression of type I NRG1 in hippocampus and cerebral cortex neurons, under the control of thy-1 promoter (101). The mice appeared normal in open field test but impaired in the accelerating rotarod test and PPI (102).

5.6. Neonatal Injection of NRG1 Ectodomain

The extracellular domain of NRG1 could go through the blood– brain barrier after subcutaneous injection in neonatal mice. These mice showed impaired prepulse inhibition, latent inhibition and social interaction at adult stage (103). After being treated with methamphetamine, the mice showed hyperactive in open field test (103).

5.7. ErbB2+/− and ErbB3+/− Hypomorphs

Because null mutations of ErbB2 and ErbB3 are embryonically lethal, only heterozygous mutant mice have been analyzed before NRG1 was identified as a schizophrenia susceptibility gene (91). In a limited set of behavioral paradigms, they appeared to be normal in open-field exploratory activity, spontaneous alternation, and rotarod performance. It was unknown whether they are impaired in prepulse inhibition or social interaction.

5.8. ErbB4 Mutant Mice

Expression of an ErbB4 transgene in the heart (i.e., in ErbB4−/− ;ht+ mice) prevents premature death and allows for behavioral analysis. ErbB4−/− ;ht+ mice are hyperactive but show no deficits in prepulse inhibition (69) (Table 12.2). When ErbB4 is specifically mutated in neurons by crossing the floxed ErbB4 mice with Nestin Cre mice, nestin–ErbB4−/− mice appeared to be impaired in long-term spatial memory. They were hyperactive during postnatal days 19–20 but became hypoactivity at adult stage (104). Transgenic mice were generated to express dominant negative-ErbB4 (DN-ErbB4), a truncated ErbB4 without the entire intracellular C-terminal region, specifically in oligodendrocytes. DN-ErbB4 was shown to block NRG1 signaling by competing with endogenous ErbB proteins. DN-ErbB4 transgenic mice exhibit hypoactivity, with increased anxiety-like behavior

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Table 12.2 ErbB4 mutant mice phenotypes Nestin– ErbB4−/− ;ht+ ErbB4−/−

DN–ErbB4

hGFAP– ErbB2/4−/−

PV–ErbB4−/−

↑ (69)

↑ (104)

↓ (105)

= (59)

↑ (63)

= (69)

n.d.

n.d.

↓ (59)

↓ (63)

n.d.

n.d.

n.d.

n.d.

↓ (63)

Water maze test

n.d.

↓ (104)

n.d.

n.d.

n.d.

Clozapine

n.d.

n.d.

n.d.

Rescued PPI (59)

n.d.

Locomotor activity Open-field test Executive function PPI Short term memory Working memory Long-term memory

↑, significantly increased; =, no significant difference; ↓, significantly decreased. n.d., not done. PPI, prepulse inhibition.

(105). When ErbB2 and ErbB4 are specifically mutated in CNS by crossing the floxed ErbB2, ErbB4 mice with the hGFAP Cre mice, hGFAP–ErbB2−/− /ErbB4−/− mice showed normal in open field test but reduced prepulse inhibition which could be rescued by clozapine (59). 5.9. PV–ErbB4−/− Mutant Mice

To study the role of ErbB4 in PV-positive interneurons, we recently ablated ErbB4 specifically in PV-positive interneurons by crossing the floxed ErbB4 mice with the PV Cre mice (named thereafter PV–ErbB4−/− mice) (63). As described above, this mutation prevented NRG1 from promoting activity-dependent GABA release and from suppressing the activity of pyramidal neurons. PV–ErbB4−/− mutant mice do not exhibit differences in weight, whisker number, and rectal temperature in comparison with control littermates, and there were no significant differences in motor coordination. The mutant mice are able to adapt to a novel environment, but they exhibit increased locomotor activity and stereotypical activity. To examine whether the loss of ErbB4 in PV-positive interneurons resulted in cognitive deficits, PV–ErbB4−/− mice were trained to retrieve food pellets from the end of each arm in an eight-arm radial maze (63). They were scored for the total wrong entries (repeated entries into a previously visited arm or omission of an arm) and the total time to retrieve all pellets. Both mutant and control mice are able to learn to retrieve food pellets by reducing wrong entries and exploration time. However, PV–ErbB4−/− mice showed an increased number of total errors, suggesting possible deficits in working memory. Because the

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mutant mice are hyperactive, the increase in total errors could be due to random hyperactivity. To exclude this possibility, we monitored correct entries during the first four entries, eliminating effects of total travel distance and time (106). The percentage of correct entries within the first four entries is significantly lower in mutant mice in comparison with controls. Together these observations provide strong evidence for impaired working memory in PV–ErbB4−/− mice. Finally, although mutant mice produced similar startle responses as the control littermates, prepulse inhibition was significantly lower in PV–ErbB4−/− mice. Remarkably, acute treatment with low-dose diazepam, a GABA enhancer, was able to ameliorate disrupted PPI in PV–ErbB4−/− mice, providing evidence for impaired GABAergic transmission. These observations could be interpreted as evidence that PV-positive interneurons are a major cellular target of abnormal NRG1/ErbB4 signaling in schizophrenia. They are in line with the idea that disrupted NRG1 signaling may cause imbalance in neuronal activity in the brain, providing insight into possible pathogenic mechanisms of schizophrenia. Due to late onset of the PV promoter activity, levels of ErbB4 and function of NRG1 in PV-positive interneurons are not expected to alter until almost 2 weeks after birth. These behavioral deficits are in PV–ErbB4−/− mice likely to result from abnormal neurotransmission or synaptic plasticity. In light of higher expression of NRG1 and ErbB4 in schizophrenic PFC, we believe that these results support a working hypothesis that increased NRG1 activity promotes GABA release and thus suppresses the activity of pyramidal neurons, providing an underpinning mechanism of hypofunction of glutamergic pathways in schizophrenia. Finally, unlike NRG1 and ErbB4 null mutant mice that often die prematurely, PV–ErbB4−/− mice survive into adulthood and thus could serve as a valuable model to study schizophrenia and relevant brain disorders.

6. Conclusions While recent advances in understanding NRG1 function are exciting, many critical questions remain to be addressed. Due to limited availability of specific antibodies against NRG1 isoforms and different ErbB kinases, little is known when and where they are expressed. NRG1 and ErbB4 are found increased expression in schizophrenic patients. Yet, studies in the past have been carried out mainly in mutant mice where the expression of NRG1 isoforms or ErbB4 was reduced. Moreover, the schizophrenia-like

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phenotypes in NRG1 and ErbB4 mutant or transgenic mice could result from the defects in neural development as well as in neurotransmission or synaptic plasticity. Therefore, mouse models whose overexpression or mutant of NRG1 and ErbB4 are controlled in space and time could be extremely valuable in understanding how the alternation of NRG1 ErbB4 signaling contributes to schizophrenia pathology. Such models should also be invaluable for the development of therapeutic intervention of this devastating disorder. 6.1. What Is a Good Model for Schizophrenia?

An animal model of schizophrenia needs face validity, constructive validity, and predictive validity. Face validity means that the animal model should resemble the symptoms from schizophrenic patient. Because the delusions and hallucinations could not be measured in animal models, the sensory-motor gating, attention, learning and memory, psychomotor excitement, and social interactions can be evaluated in animal models. Psychomotor excitement and sensory-motor gating are the most wildly used criteria for evaluating NRG1 and ErbB4 mutant mouse models, because it is not usual for a mouse model to match all the phenotypes. What is more, general health should also be observed, including their water and food consumption, body weight, and circadian behavior. It will be more convincing for a mouse model to have behavioral defect only after late adolescence and early adulthood, when the schizophrenic symptoms usually appears in patients. Constructive validity requests that the animal model should have similar pathophysiological mechanism as the schizophrenic patients. Since many genetic and environmental factors might be involved in the etiology of schizophrenia, it is impossible for one schizophrenic model to mimic all the factors, but the mouse model should mimic the underlying mechanism at different levels, including molecular, cellular and brain circuitry levels. Several hypothesizes for the pathogenesis of schizophrenia were proposed, including abnormal development, hyperdopaminergic, hypoglutamatergic and hypo-GABAergic hypothesis (107). Further studies need to investigate how the NRG1/ErbB4 is involved in the balance of the neurotransmissions among GABA, glutamate and dopamine (108). Predictive validity means that the animal model should have similar efficacy for the treatment drugs for schizophrenia and can be used to explore new drugs for patients. Atypical antipsychotics such as clozapine are the most commonly used drugs for evaluating the mouse models. Since the downstream pathways of NRG1/ErbB4 signaling are not fully understood, whether any drugs that target these pathways can rescue the schizophrenialike phenotypes in the NRG1 or ErbB4 mutant mice needs to be further tested.

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SUBJECT INDEX Note: The letters ‘f’ and ‘t’ following the locators refer to figures and tables respectively. A Acoustic Startle response. . . . . . . . . . . . . . . . 41, 42f, 82, 95–96, 100, 234 aCSF (artificial cerebrospinal fluid) . . . . . . . . . . . 3–6, 8, 11, 13, 21, 59, 180 ActiMot software . . . . . . . . . . . . . . . . . . . . . . . . . . . 97, 100 Addiction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 AIN93G rodent diet . . . . . . . . . . . . . . . . . . . . . . 115, 123 AKT1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196, 225 AlexaFluor488 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62, 141 ALZ (Alzheimer’s disease). . . . . . .154, 194, 212, 261 Amphetamine . . . . . . . . . . . . . . . 29, 29f, 57–58, 84–85, 96, 163, 172t, 177t, 195–196, 202, 233 Anatomical anomalies in E17 MAM-exposed rats hippocampus and subcortical regions . . . . . . . . . . . . . . . . . . . . . . . . 64–67, 65f prefrontal cortex and other cortices . . . . . . . 64, 65f Anesthesia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23 chemical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 deep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 hypothermic . . . . . . . . . . . . . . . . . . . . . . . 8, 11–13, 22 Anhedonia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 Animal preparation . . . . . . . . . 102, 117–120, 135–142 Animal selection/litter management, NVHL . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10–11 gender differences in genitalia in PD-3–7 pups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10f gender-typing pups . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 NVHL vs. SHAM ratios . . . . . . . . . . . . . . . . . . . . . . 10 ANOVA (analysis of variance) . . . . . . . . . . . . . 106, 254f Anterior cingulate cortex . . . . . . . . . 63, 102, 159–160, 165–166, 169, 170t Antioxidant defense systems . . . . . . . . . . . . . . . 151, 153 Antipsychotics . . . . . . . . . 85, 108, 164, 177t, 253–256 clozapine . . . . . . . . . . . . . . . . . . . . . . . . . . 95, 107, 271 haloperidol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115, 234 olanzapine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 risperidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234, 238 Anti-rabbit AlexaFluor 568 . . . . . . . . . . . . . . . . . 62, 141 Anxiety . . . . . . . . . . . . . 36, 55, 82, 194–195, 199–200, 202, 238, 249–250, 267t, 268–269 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85–86, 115 Arabidopsis thaliana . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Astrocyte dissection procedure/preparation . . . . . . . 135–138 growth media . . . . . . . . . . . . . . . . . . . . . 134, 138–139

monolayer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138, 143 plating media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Attention impaired . . . . . . . . . . . . . . . . . . . . . . . . . . . 26, 200, 262 and information processing . . . . . . . . . . . . . . . 40–49 attentional set shifting . . . . . . . . . . . . . . . . . 44–46 5-CSRT task . . . . . . . . . . . . . . . . . . . . . . . . . . 46–49 LI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42–43 PPI of Startle reflex. . . . . . . . . . . . . . . . . . . . 40–42 Atypical antipsychotics . . . . . . . . . . . . . . . 249, 256, 271 Auditory hallucinations . . . . . . . . . . . . . . . . . . . . . . . . . 261 Autism . . . . . . . . . . . . . . . . . . . . . . . . . 191–192, 212, 216 Autoclaving . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116, 122 Automatic P/ACETM MDQ system . . . . . . . . . . . . . . 59 Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . 101–102 B BAC (bacterial artificial chromosome) . . . . . 200, 220, 224t, 237 BACE1−/+ mutant mice . . . . . . . . . . . . . . . . . . . . . . . 266 BALB/c mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Basal ganglia . . . . . . . . . . . . . . . . . . . . . . . . . 64, 218, 223t Basket and chandelier cells . . . . . . . . . . . . . . . . . . . . . . 128 Behavior(s) abnormal. . . . . . . . . . . . . . . . . . . . . . . .33, 51, 60, 235 aggression . . . . . . . . . . . . . . . . . . . 220, 262, 266–270 anhedonia-like . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 anxiety-like . . . . . . . . . . . . . . . . . . . . 36, 55, 194–195, 199–200, 202, 238, 268–269 anxiogenic-like . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 BSO-induced GSH deficit effects on . . . . 162–163 compulsive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 exploratory . . . . . . . . . . . . . . . . . . . . . . . . . 36, 82, 160 impulsive-like . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 long-term effects on. . . . . . . . . . . . . . . . . . . .160–161 maternal . . . . . . . . . . . . . . . . . . . . . . . 98–99, 107, 250 persevering . . . . . . . . . . . . . . . . . . . . . . . 27–28, 36, 86 rodent. . . . . . . . . . . . . . .156, 170t–174t, 222t–224t social . . . . . . . . . . . . . . . . . . . . . . . . . 2, 31, 33, 82, 100 Behavioral testing attentional set shifting . . . . . . . . . . . . . . . . . . . . 44–46 delayed non-match to position . . . . . . . . . . . . 37–39 five-choice serial reaction time . . . . . . . . . . . . . 46–49 latent inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . 42–43 locomotor activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Morris water maze . . . . . . . . . . . . . . . . . . . . . . . . 39–40

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NIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS 280 A Subject Index

Behavioral testing (continued) object recognition task . . . . . . . . . . . . . . . . . . . . 51–52 prepulse inhibition of startle . . . . . . . . . . . . . . 40–42 radial arm maze . . . . . . . . . . . . . . . . . . . . . . . . . . 34–35 reversal learning . . . . . . . . . . . . . . . . . . . . . . . . . . 49–50 social interaction test . . . . . . . . . . . . . . . . . 28, 31, 55 spatial recognition . . . . . . . . . . . . . . . . . . . . . . . . 53–55 spontaneous alternation . . . . . . . . . . . . . . . . . . . 35–36 Bi-transgenic Tet-off System . . . . . . . . . . . . . . . . . . . . 219 Brain cutting/histology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 fetal . . . . . . . . . . . . . . . . . . . . . . . . 3, 81, 83, 85–87, 89 GSH levels, effects. . . . . . . . . . . . . . .151, 155f, 156, 158–159, 162, 167 HDAC expression. . . . . . . . . . . . . . . . . . . . . .247–248 investigations . . . . . . . . . . . . . . . . . . . . . . . . . . 101–106 of 31L mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 post-mortem . . . . . . . . . . . . . . . . . . . . . 151, 160, 167, 176t, 245–248, 252, 256 of preweaned mice . . . . . . . . . . . . . . . . . . . . . . . . . . 158 region-restricted genetic manipulation . . 235–236 removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17, 21 slices, use of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 transgene expression to cell types of . . . . . 236–237 BrdU staining . . . . . . . . . . . . . . . . . . . . . . . 214, 221, 222t BSO (DL-buthionine-(SR)-sulfoximine) . . . . . . . . 156 -induced acute GSH deficit in adulthood . . . . . . . . . . . . . . . . . . . . . . . 161–164 behavioral effects . . . . . . . . . . . . . . . . . . . 162–163 methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161–162 neurotransmission, effects . . . . . . . . . . . 163–164 oxidative stress/physical parameters, effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 -induced transient postnatal GSH deficit . . . . . . . . . . . . . . . . . . . . . . . . . . . 157–161 animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 behavior/cortical circuits, long-term effects . . . . . . . . . . . . . . . . . . . . . . . . . . . 160–161 BSO treatment . . . . . . . . . . . . . . . . . . . . . 157–158 cortical circuits, developmental effects . . . . . . . . . . . . . . . . . . . . . . . . . . . 159–160 GBR12909 treatment . . . . . . . . . . . . . . . . . . . . 158 oxidative stress/physical parameters, effects . . . . . . . . . . . . . . . . . . . . . . . . . . . 158–159 C Calcineurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163, 196 CaMKII (α-calmodulin kinase II) promoter . . . . . . . 217–218, 220, 223t–224t Cardiovascular and kidney abnormalities . . . 121, 168 Cataracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158–159, 170t CAT (catalase) . . . . . . . . . . . . . . . . . . . . . . . 150, 152, 158 Cavum septum pellucidum . . . . . . . . . . . . . . . . . . . . . . . 88 CB (calbindin) . . . . . . . . . 61–63, 159, 170t, 218, 223t C57Bl/6 J and C57BL/6 mice . . . . . . . . 82, 165, 213 CDCA (common disease-common allele) hypothesis . . . . . . . . . . . . . . . . . . . . . . . 190–191 CDRA (common disease-rare allele) hypothesis . . 190 ChIP on chip technique . . . . . . . . . . . . . . . . . . . . . . . . 255

Cholecalciferol . . . . . . . . . . . . . . . . . 115–116, 117f, 122 Chromosomes . . . . . . . . . . . . . . . . . 198f, 200, 201f, 212 Clozapine . . . . . . . . . . . . . . . . . . . . . . . 95–101, 107, 217, 234, 254, 256, 267t, 268–269, 269t, 271 CNS (central nervous system) disorders . . . . . . . . . . . 3, 26, 128, 194–195, 261, 264–265, 269 Cocaine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .168 Cognitive impairments . . . . . . . . . . . . . . . . . . 26, 33, 45, 113, 160, 197, 238, 262, 266 Cognitive symptoms of schizophrenia. . . . . . . . .33–57 attention and information processing . . . . . . 40–49 behavioral flexibility . . . . . . . . . . . . . . . . . . . . . . 49–51 episodic memory . . . . . . . . . . . . . . . . . . . . . . . . . 51–55 spatial reference memory. . . . . . . . . . . . . . . . . . 55–57 working memory . . . . . . . . . . . . . . . . . . . . . . . . . 34–40 Colorimetric Glutathione Assay Kits. . . . . . . . . . . . .178 Colorimetric method . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 Confocal microscopy . . . . 60, 62, 135, 138, 141, 179 Copy number mutations of 22q11.2 . . . . . . . 192, 196 Cortex cerebral. . . . . . . . . . . . .53, 102, 167–168, 264, 268 entorhinal . . . . . . . . . . . . . . . . . . . . . . . . . . . 20, 64, 66f frontal . . . . . . . . . . . . 82, 218, 223t, 234, 245–248, 250, 252–254, 252f, 254f mPFC. . .22, 34, 58–60, 63, 65f, 67, 84–85, 102, 151, 196–197, 199, 215, 218, 221, 223t perirhinal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65f prefrontal . . . . . . . . . . . . . . . . . . . . 20, 26, 34, 38–39, 44–46, 48–49, 52, 57–64, 65f, 67, 94f, 95, 131, 159, 215, 223t, 247, 263 CpG (cytosine-[phosphodiester] guanine) islands . . . . . . . . . . . . . . . . 244, 246–247, 251 CR (calretinin) . . . . . . . . . . . . . . 60–63, 159, 170t, 218 CRD (cysteine-rich domain) . . . . . . . . . 266, 267t, 268 Cre/loxP system . . . . . . . . . . . . . . . . . . . . . . 236, 268–269 Cross-fostering . . . . . . . . . . . . . . . . . . . 99, 106–107, 122 CSF (cerebrospinal fluid) . . . . . . . . . . . . . . . . . . 151, 153 Cultures bacterial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 of MAM rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60–61 neuronal, see Primary neuronal cultures organotypic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Cytokines . . . . . . . . . . . . . . . . . . . 80–81, 83, 85–88, 132

D D-amphetamine-induced hyperlocomotion . . . 57–58 Delusions . . . . . . . . . . . . . . . 25–27, 113, 127, 261, 271 Depression. . . . . . . . . . .153, 212, 216–218, 223t, 233 Development abnormal brain . . . . . . . . . . . . . . . . . . . . . . . . . . . 57, 95 of CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 of cortical circuits . . . . . . . . . . . . . . . . . . . . . . 159–160 fetal brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3, 85 neural . . . . . . . . . 10, 131, 139, 144, 189, 263–265 postnatal brain . . . . . . . . . . . . . . . 94, 128, 130–131, 135, 154, 155f, 156–161, 163, 165, 167, 169, 170t, 177t, 215, 220, 238–239 of post-pubertal hippocampus . . . . . . . . . . . . . . . . 60 pre- and perinatal . . . . . . . . . . . . . . . . . . . . . . 154, 215

ANIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS Subject Index 281 Diaminobenzidine immunoperoxidase method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60, 62 Diet AIN93G rodent . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 deplete . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115–116 vitamin D-deficient . . . . . . . . . . . . . . . . . . . . . . . . . 122 weanling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116, 122 DISC1 (disruptedin-schizophrenia 1) BAC transgenic model . . . . . . . . . . . . . . . . . 220–221 knockdown in vivo . . . . . . . . . . . . . . . . . . . . . 213–215 adult neurogenesis . . . . . . . . . . . . . . . . . . 213–214 progenitor proliferation and new pathway . . . . . . . . . . . . . . . . . . . . . . . . . 214–215 mouse models, see Mouse models, DISC1 polymorphism model. . . . . . . . . . . . . . . . . . .215–216 susceptibility locus . . . . . . . . . . . . . . . 197–200, 198f BAC system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 effects of (1;11) translocation . . . . . . . . . . . . 197, 198f, 199 shRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 single transgenic inducible and reversible system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Tet-off double transgenic system . . . . . . . . . 199 Disconnection hypothesis . . . . . . . . . . . . . . . . . . . . . . . . 26 DLPFC (dorsal lateral PFC) . . . . . . . . . . . . . . . 263, 265 DNA demethylase . . . . . . . . . . . . . . . . . . . . . . 244–245, 256 histone remodeling processes . . . . . . . . . . . . . . . . 244 methylation/demethylation . . . . . . . . . . . . . . . . . 244, 246–247, 252 methyltransferases DNMT1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 DNMT3b mRNA . . . . . . . . . . . . . . . . . . . . . . . 247 SAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 DN-ErbB4 transgenic mice . . . . . . . . . . . . . . . . 268–269 DNMT (DNA methyltransferase) . . . . 244–245, 247, 253, 255–256 DNMT1 (DNA methyltransferase 1) . . . . . . . . . . . 247, 252–253 DNMTP (delayed non-match to position) . . . . 37–39 Dopamine extracellular . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159–160 levels of . . . . . . . . . . . . . . . . . . 58–59, 155f, 157–158 receptor agonist-induced hyperactivity . . . . . . . . 29 light-dark cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 rats (Wistar and Fisher 344) . . . . . . . . . . . . . . . 29 release . . . . . 57–58, 84, 157, 163, 169, 172t, 177t signaling . . . . . . . . . . . . . . . . . . . . . . . . . 164, 176t, 196 type 1 and type 2 receptors . . . . . . . . . . . . . . . . . . . 84 DTNBP1 (dystrobrevin-binding protein 1) susceptibility locus . . . . . . . . . . . . . . . . . . . . 195 DVD (developmental vitamin D) model . . . . 113–124 materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115–116 control diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 deplete diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 weanling diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 methods dams, preparation of . . . . . . . . . . . . . . . . 116–117

DVD-deficient/control offspring, preparation of . . . . . . . . . . . . . . . . . . . 117–120 female Sprague–Dawley rat of 25OHD3 . . . . . . . . . . . . . . . . . . . . . . . . . 117f of maternal vitamin D deficiency. . . . .120–121 measures of pup health . . . . . . . . . . . . . . 120–121 vitamin D-deficient dam . . . . . . . . . . . . . . . . . 118f

E EDS (extradimensional shift) . . . . . . . . . . . . . 44–46, 49 E17 (embryonic day 17) MAM-exposed rats anatomical anomalies . . . . . . . . . . . . . . . . . . . . . 64–67 behavioral changes observed in cognitive symptoms of schizophrenia . . . 33–57 negative symptoms of schizophrenia . . . . 31–33 positive symptoms of schizophrenia . . . . 27–31 functional anomalies . . . . . . . . . . . . . . . . . . . . . . . . . . 67 neurochemical changes observed in abnormal GABAergic markers. . . . . . . . . .61–63 D-amphetamine-induced hyperlocomotion . . . . . . . . . . . . . . . . . . . 57–58 MK-801-induced hyperlocomotion . . . . 58–60 Reelin level in organotypic hippocampal cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60–61 EGF (epidermal growth factor) . . . . . . . . . . . 136, 194f, 200, 220, 263–266, 267t Electrophysiology . . . . . . . . . . . . . . . 138, 180, 214–215 Electroporation approach . . . . . . . . . . . . . . . . . . 212–213 ELISA kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 ENU (N-nitroso-N-ethylurea) . . . . . . . 216–217, 223t Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87–88 Epigenetic animal models of GABAergic deficit in mental disorders . . . . . . . . . . . . . . . . . 243–256 environmental and pharmacological animal models chromatin remodeling . . . . . . . . . . . . . . . . . . . 249 drug administration . . . . . . . . . . . . . . . . . 249–250 epigenetic animal models . . . . . . . . . . . . . . . . . 249 implicate Reelin/dendritic spine density changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 GAD67 promoter methylation . . . . . . . . . . . . . . 254f heterozygous Reeler and GAD67 mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248–249 density of dendritic spines . . . . . . . . . . . . . . . . 248 RELN gene or GAD1 gene . . . . . . . . . . . . . . 248 wild-type mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 pharmacological correction . . . . . . . . . . . . . 253–255 benzodiazepines . . . . . . . . . . . . . . . . . . . . . . . . . 255 DNMT inhibitory activity . . . . . . . . . . . . . . . . 253 imidazenil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 MET mouse model of SZ . . . . . . . . . . . 253–254 nicotinic receptor agonists. . . . . . . . . . . . . . . .253 in psychosis, gene expression in telencephalic GABAergic neurons DNA methyltransferases. . . . . . . . . . . . . . . . . .247 GAD67 (GAD1) and reelin (RELN) . . . . . 245 gamma frequency of EEG . . . . . . . . . . . . . . . . 245 HDAC expression . . . . . . . . . . . . . . . . . . 247–248 and repeated L-MET administration . . . 251–252, 252f

NIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS 282 A Subject Index

Episodic memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51–55 object recognition task . . . . . . . . . . . . . . . . . . . . 51–52 spatial recognition memory in Y Maze . . . . . 53–55 EpiTect Kit H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 ErbB4, see NRG1 (neuregulin 1) and ErbB4 mutant mice Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 ESMEH software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 F FDR (false discovery rate) method . . . . . . . . . . . . . . 106 Feeder layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137, 144 Fetal hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Fetal programming hypotheses . . . . . . . . . . . . . . . . . . . 87 5-CSRT (five-choice serial reaction time) task behavioral challenges . . . . . . . . . . . . . . . . . . . . . . . . . 48 nine-hole box operant chamber . . . . . . . . . . . . . . 47f procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46–48 task acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . 46–48 Fluorometric assay . . . . . . . . . . . . . . . . . . . 175, 178–179 Fluorometric Glutathione Assay Kits . . . . . . . 178–179 FS (fast-spiking) cells . . . . . . . . . . . . . . . . 127–145, 159, 166, 169, 176t, 236 FST (forced swim test). . . . . . . . . . . . . . .199–200, 214, 217–219, 221, 224t Functional anomalies in E17 MAM-exposed rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 G GABA GABAA receptor . . . . . . . . . . 3, 163, 247, 253, 255 GABAergic system . . . . . . . . . . . . . . . . . . . . . . . . . . 165 markers in MAM rats . . . . . . . . . . . . . . . . . . . . . 61–63 neurons/interneurons . . . . . . . . . . . . . . . . . . . . . . . 57, 61–63, 67, 84, 131, 236–239, 245–247, 250, 252–253, 263–264 phenotype of PV interneurons . . . . . . . . . . 131–132 See also Epigenetic animal models of GABAergic deficit in mental disorders GAD67 (GAD1) and reelin (RELN) . . . . . . . . . . . . . . . . . 245 gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 heterozygous Reeler and . . . . . . . . . . . . . . . 248–249 density of dendritic spines . . . . . . . . . . . . . . . . 248 RELN gene or GAD1 gene . . . . . . . . . . . . . . 248 wild-type mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 promoter methylation . . . . . . . . . . . . . . . . . . . . . . 254f in PV interneurons . . . . . . . . . . . . . . . 141–142, 142f image analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 GAD-1 (glutamic acid decarboxylase-1) . . . . . . . . . . 61 GAG-TNR (GAG trinucleotide repeat). . . . . . . . . .153 GBR12909 . . . . . . . . . . . . . . . . . . . . . . . . . 155f, 157–159 GCLC (catalytic subunit of GCL) . . . 151f, 152, 156, 164, 167–168 GCL (glutamate cysteine ligase) . . . . 128, 152f–153f, 154–156, 164, 167–168 GCLM knockout (GCLM−/−) mice . . . . . . 164–167 behavioral phenotype. . . . . . . . . . . . . . . . . . .166–167 impaired object recognition memory . . . . . 167

lack of behavioral inhibition . . . . . . . . . . . . . . 167 normal spatial learning and memory capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 novelty-induced hyperactivity. . . . . . . . . . . . .167 reduced acquisition of delay fear conditioning . . . . . . . . . . . . . . . . . . . . . . . . . 167 GABAergic system . . . . . . . . . . . . . . . . . . . . . 165–166 methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 morphological anomalies . . . . . . . . . . . . . . . 165–166 myelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 reduction of γ-oscillations . . . . . . . . . . . . . . . . . . . 166 GD (gestational day) . . . . . . . . . . . . . . . . . . 3, 26, 82, 86 Gene expression . . . . . . 87–88, 95–97, 106–107, 220, 244–248, 252, 255 Genetically engineered mice for research . . . 231–239 animal models of schizophrenia, comparison . . . . . . . . . . . . . . . . . . . . . . . . . . 235t cell types of brains, transgene expression . . . . . . . . . . . . . . . . . . . . . . . 236–237 BAC transgenic approach . . . . . . . . . . . . . . . . 237 Cre recombinase driver transgenic mouse lines. . . . . . . . . . . . . . . . . . . . . . . . . . . . .236–237 Ppp1r2 gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 cell type-specific/brain region-restricted genetic manipulation . . . . . . . . . . . . . . . . . . . . 235–236 cortical and hippocampal GABAergic interneurons . . . . . . . . . . . . . . . . . . . . . . . . . 236 Cre/loxP system . . . . . . . . . . . . . . . . . . . . . . . . . 236 global NMDAR genetic manipulation in mice . . . . . . . . . . . . . . . . . . . . . . . . . . 233–235 GluN1(D481N/K483Q) mice . . . . . . . . . . . 234 Grin1(D481N/K483Q) homozygous mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 NMDAR hypomorph mutant . . . . . . . 233–234 NR2A subunit (GluN2A) null knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234 pharmacological approach . . . . . . . . . . . . . . 232–233 advantages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .232 behavioral/non-behavioral phenotypes . . . 232 ketamine model of schizophrenia . . . . . . . . . 233 MK-801 (dizocilpine) . . . . . . . . . . . . . . . . . . . . 233 phencyclidine (PCP) . . . . . . . . . . . . . . . . 232–233 postnatal NMDA receptor ablation . . . . . 238–239 impaired sensorimotor gating . . . . . . . . . . . . 238 NMDAR hypofunction theory . . . . . . . . . . . 238 psychotic states . . . . . . . . . . . . . . . . . . . . . . . . . . 238 spatial working memory and PPI deficits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 Genetic models of GSH deficit excitatory amino acid carrier-1 (EAAC1). . . . . . . . . . . . . . . . . . . . . . . .167–168 GCLM knockout (GCLM−/−) mice . . . 164–167 to investigate comorbidity with somatic disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Na+ -independent glutamate–cysteine exchanger (xCT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 Genetics and mutant models, integrating human challenges for moving beyond single genes . . . 204 identifying disease pathways . . . . . . . . . . . . 203–204

ANIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS Subject Index 283 Germline mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Gestational MAM administration anatomical/functional anomalies in E17 MAM-exposed rats anatomical anomalies . . . . . . . . . . . . . . . . . . 64–67 functional anomalies . . . . . . . . . . . . . . . . . . . . . . 67 behavioral changes observed in E17 MAM-exposed rats cognitive symptoms of schizophrenia . . . 33–57 negative symptoms of schizophrenia . . . . 31–33 positive symptoms of schizophrenia . . . . 27–31 neurochemical changes observed in E17 MAM-exposed rats abnormal GABAergic markers. . . . . . . . . .61–63 D-amphetamine-induced hyperlocomotion . . . . . . . . . . . . . . . . . . . 57–58 MK-801-induced hyperlocomotion . . . . 58–60 reelin level in organotypic hippocampal cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60–61 Gestational timing of infection . . . . . . . . . . . . . . . 86, 88 GFAP (glial fibrillary acid protein) . . . . . . . . . . . . . . . 85 GFP (green fluorescent protein) . . . . . 199f, 200, 213, 215–216, 224t Glutamate chips, preparation of DNA. . . . . . . . . . . . . .104–105 neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . 28, 130f, 246 NMDA antagonist-induced hyperactivity MK-801 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 PCP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29–30 receptors . . . . . . . . . . . . . . . . . . . . 131–132, 232, 248 Glutathione deficit and redox dysregulation . . . . . . . . . . . . . . . . . . . . 149–181 genetic models of GSH deficit GCLM knockout (GCLM−/−) mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164–167 to investigate comorbidity with somatic disorders . . . . . . . . . . . . . . . . . . . . . . . . 168–169 other models . . . . . . . . . . . . . . . . . . . . . . . 167–168 GSH measurements . . . . . . . . . . . . . . . . . . . . 175–180 Colorimetric Glutathione Assay Kits . . . . . . 178 Fluorometric Glutathione Assay Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178–179 pharmacological models of GSH deficit BSO-induced acute GSH deficit in adulthood . . . . . . . . . . . . . . . . . . . . 161–164 BSO-induced transient postnatal GSH deficit . . . . . . . . . . . . . . . . . . . . . . . . . . . 157–161 electrophile compounds . . . . . . . . . . . . . . . . . . 155 specific inhibitors . . . . . . . . . . . . . . . . . . . . . . . . 155 synthesis, GSH . . . . . . . . . . . . . . . . . . . . . . . 152f–153f types of induced GSH deficits . . . . . . . . . . . . . . . 155f effects of . . . . . . . . . . . . . . . . . . . . . . . . . . 170t–174t in rodent models . . . . . . . . . . . . . . . . . . 176t–177t use of brain slices for physiological measurements . . . . . . . . . . . . . . . . . . . . . . . . 180 GNU-R statistics software . . . . . . . . . . . . . . . . . . . . . . 106 γ-oscillations . . . 128–129, 166, 169, 174t, 176t, 232 GPX (glutathione peroxidases) . . . . . . . . . . . 150, 152f, 153, 158

GSH (γ-glutamylcysteine–glycine) . . . 150–151, 152f animal models of . . . . . . . . . . . . . . . . . . . . . . . 169–174 See also Glutathione deficit and redox dysregulation GSSG (glutathione disulphide) . . . . . . . . . . . 150, 152f, 153, 175, 178–179 GST (glutathione transferases) . . . . . . . 150, 152f, 179 GWAS (genome-wide association studies) . . . . . . . . 88, 191–192, 203, 265 H Hallucinations . . . . . . . . . . . . . . 27, 113, 127, 261, 271 Haloperidol . . . . . . . . . . . . . . . . . 82, 115, 217, 234, 254 HDAC (histone deacetylase) expression in telencephalon of SZ postmortem brain GAD67 gene expression . . . . . . . . . . . . . . . . . 247 HDAC1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 inhibitors . . . . . . . . . . . . . . . . . . . . 251–253, 255–256 HDAC1 (histone deacetylase 1) expression . . . . . . 247 Head stabilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 HF (hippocampal formation) . . . . . . . . . 26, 60–61, 64 Hippocampectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Hippocampus . . . . . . . . . . . . . . . . . . . . . . . . . . . 222t–224t dorsal . . . . . . . . . . . . . . . . . 18f, 19–20, 65f, 165–167 of GCLM (−/−) mice . . . . . . . . . . . . . . . . . 165–166 of MAM rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62–63 and subcortical regions . . . . . . . . . . . . . . . . . . . 64–67 ventral . . . . . . . . . . . . . . 2, 19, 65f, 67, 85, 166–167 Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9, 17–18, 21 HMT (histone methyltransferases) . . . . . . . . . . . . . . 244 House-keeping gene (Beta-actin) . . . . . . . . . . . . . . . 106 HPA (hypothalamic–pituitary–adrenal) axis function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 HPLC methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58, 175 HSV-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80–81 Huntington’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 25OHD3 (25-hydroxyvitamin D3) . . . . . . . . . . . . . 116, 118f, 120, 122 Hyperactivity . . . . . . . . . . . . . 27–31, 33, 60, 167, 174t, 222t, 232, 234, 236, 266, 270 Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 Hypothermia . . . . . . . . . . . . . . . . . . . . 12, 16, 21, 23, 60 Hypovitaminosis D . . . . . . . . . . . . . . . . . . . . . . . . 114, 122 Hypoxia cerebral . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 chronic neonatal. . . . . . . . . . . . . . . .95–98, 101, 107 postnatal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 pre- or perinatal . . . . . . . . . . . . . . . . . . . . . . . . . 95, 114 rat model, see Obstetric complications, hypoxic rat model for short and long-term . . . . . . . . . . . . . . . . . . . . . . . . . . 96 I IBO (ibotenic) acid blast zone intact . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Eppendorf tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . 4, 5f for neonatal lesioning . . . . . . . . . . . . . . . . . . . . . . . . . . 4 preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4–5, 21

NIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS 284 A Subject Index

IDS (intradimensional shift). . . . . . . . . . . . . . . . . . . . . .44 Image analysis system . . . . . . . . . . . . . . . . . . 62, 102, 142 Imidazenil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Immune activation advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 maternal models LPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 poly I:C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80–81 and in utero infection. . . . . . . . . . . . . . . . . .85–86 in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Immune models, see Prenatal infection/immune models of schizophrenia Immunohistochemistry . . . . . . . . . . . . . 57, 60, 62, 159 Infection gestational timing of. . . . . . . . . . . . . . . . . . . . . . . . . .86 maternal . . . . . . . . . . . . . . . . . . . 80–81, 94, 114, 153 prenatal, see Prenatal infection/immune models of schizophrenia in utero, see In utero infection Influenza animal models of schizophrenia . . . . . . . . . . . . . . . 85 findings from Poly I:C models . . . . . . . . . 84–85 gestational timing of infection . . . . . . . . . . . . . 86 influenza models . . . . . . . . . . . . . . . . . . . . . . . . . . 85 LPS models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 maternal immune activation and in utero infection . . . . . . . . . . . . . . . . . . . . . . . . . . . 85–86 MIA and in utero infection . . . . . . . . . . . . . . . . 87 poly I:C models . . . . . . . . . . . . . . . . . . . . . . . 84–85 experimental protocols . . . . . . . . . . . . . . . . . . . . 82–83 cell counting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 influenza A/NWS/33 (H1N1) virus . . . . . . . 82 pregnant female C57BL/6 mice . . . . . . . . . . . 82 inoculation with infectious microbe . . . . . . . 83–84 advantages and disadvantages . . . . . . . . . . 83–84 models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85 behavioral abnormalities . . . . . . . . . . . . . . . . . . . 85 in mid-gestation . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Influenza A/NWS/33 (H1N1) virus . . . . . . . . . . . . . 82 Information processing . . . . . . . . . . . . 33, 40, 166, 245 In situ hybridization . . . . . . . 101–103, 105f, 218, 238 Interleukin IL-6 . . . . . . . . . . . . . . . . . . . . . . . 81, 85–87, 132–133 IL-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80–81 IL-10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81, 86 IL-1β . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81, 132 Interneurons corticolimbic . . . . . . . . . . . . . . . . . . . . . . . . . . 237–239 fast spiking, see PV (parvalbumin)-positive fast-spiking interneurons GABAergic . . . . . . . . . . . . . . . . . . . . . 61, 63, 67, 236, 245, 247, 252–253, 263 PFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 In utero infection animal models of advantages of poly I:C/LPS models. . . . . . . . 81 direct infectious insult . . . . . . . . . . . . . . . . . . . . . 80 maternal immune activation models . . . . 80–81

non-infectious maternal immune activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 reverse translational studies . . . . . . . . . . . . . . . . 80 electroporation approach . . . . . . . . . . . . . . . . . . . . 212 genetic manipulation . . . . . . . . . . . . . . . . . . . 212–213 and maternal immune activation. . . . . . . . . . . 85–86 and MIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 fetal programming hypotheses . . . . . . . . . . . . . 87 long-term effects on expression of cytokines. . . . . . . . . . . . . . . . . . . . . . . . . . . .87 in vivo microdialysis techniques . . . . . . . . . . . . . . . . . . 57 Irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116, 122 K Ketamine . . . . . . . . . . . . . 23, 129, 132, 141, 143f, 161, 232–233, 263 Kolmogorov–Smirnov test . . . . . . . . . . . . . . . . . 105–106 L LBD (ligand-binding domain) . . . . . . . . . . . . . . . . . . 218 LC/MS/MS technology . . . . . . . . . . . . . . . . . . . . . . . 116 Lesion confirmation, NVHL . . . . . . . . . . . . . . . . . 17–21 dorsal blade of hippocampus in coronal section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18f grading lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . 19–21 bi-laterality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 largest vs. smallest lesion . . . . . . . . . . . . . . . . . . . 21 lesioned and SHAM rats. . . . . . . . . . . . . . . . . . . 19 location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17–18 dehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 fixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 rehydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 sectioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17–18 lesion damage and SHAM vs. NVHL . . . . . . . . . 20f lesioning using dye . . . . . . . . . . . . . . . . . . . . . . . . . . 22f primary target areas (cross hairs) . . . . . . . . . . . . . 19f typical zone of tissue loss. . . . . . . . . . . . . . . . . . . . .19f Lesion verification/histology materials . . . . . . . . . . . . . 9 animal sacrifice/brain extraction. . . . . . . . . . . . . . . .9 brain cutting/histology . . . . . . . . . . . . . . . . . . . . . . . . 9 Light/dark cycle . . . . . . . . . . . . . . . . . . . . 29, 33, 98, 116 LI (latent inhibition) . . . . . . . . . . . . . 2, 40, 42–43, 43f, 84, 86, 194, 200, 217, 221, 223t–224t, 232, 267t, 268 31L mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217, 223t Locomotor activity . . . . . . . . . . . . . 28, 30f, 31–32, 32f, 53–54, 54f, 57, 59, 82, 96, 107, 162–163, 172t–173t, 194–197, 199–200, 202, 220, 234, 262, 267t, 269, 269t LOESS method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Long term memory . . . . . . . . . . . . . . . 38, 55, 268, 269t LPS (lipopolysaccharide) animal models of in utero infection. . . . . . . . . . . .81 experimental protocols . . . . . . . . . . . . . . . . . . . . . . . 82

ANIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS Subject Index 285 findings from animal model study . . . . . . . . . . . . . 85 maternal immune activation models . . . . . . . 80–81 models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85 astrogliosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 dopaminergic abnormalities . . . . . . . . . . . . . . . 85 PPI deficits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 LTP (long-term potentiation) . . . . . . . . . . . . 163, 171t, 176t, 219, 248, 263

M MAM (methylazoxymethanol acetate), see Gestational MAM administration Maternal care . . . . . . . . . . . . . . . . . . . 249–250, 252, 255 Maternal vitamin D depletion . . . 119–121, 120t, 123 MBD (methyl-CpG-binding domain proteins) . . . 244 MeCP2 (methyl-CpG-binding protein) . . . . 244, 247, 251 Memory episodic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51–55 spatial reference . . . . . . . 51, 55–57, 156, 162, 167, 194–195, 197, 199 working, see Working memory Mental disorders . . . . . . . . . . . . . 2, 212–213, 215, 225 See also Epigenetic animal models of GABAergic deficit in mental disorders Methods of NVHL rat model animal selection and litter management . . . . 10–11 gender-typing pups. . . . . . . . . . . . . . . . . . . . . . . .10 NVHL vs. SHAM ratios . . . . . . . . . . . . . . . . . . . 10 lesion confirmation . . . . . . . . . . . . . . . . . . . . . . . 17–21 grading lesions . . . . . . . . . . . . . . . . . . . . . . . . 19–21 histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17–18 NVHL and SHAM control surgeries . . . . . . 11–17 inducing hypothermic anesthesia . . . . . . . 12–13 lesioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13–17 surgical setup. . . . . . . . . . . . . . . . . . . . . . . . . .11–12 MET (L-methionine) . . . . . . . . . . 250–256, 252f, 254f behavioral abnormalities . . . . . . . . . . . . . . . . . . . . . 251 DNMT1 antisense oligonucleotides . . . . . . . . . . 252 RELN and GAD67 . . . . . . . . . . . . . . . . . . . . . . . . . 251 -treated mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 treatment, 252MHC . . . . . . . . . . . . . . . . . . . . . . . . . 88 MIA (maternal immune activation) models LPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 poly I:C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80–81 and in utero infection . . . . . . . . . . . . . . . . . . . . . 85–87 cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85–86 fetal brain development . . . . . . . . . . . . . . . . . . . 85 IL-6 gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85–86 maternal LPS administration . . . . . . . . . . . . . . . 85 Microdeletions, 22q11.2 . . . . . . . . . . . . . 200, 202–203 MicroGrid II Arrayer . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Micro mRNAs. . . . .61, 94, 102, 104, 118, 132, 164, 176t, 217, 244, 247–248, 252, 264–265 Micro-punching tool . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 MK-801 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 administration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84 evoked locomotor activity . . . . . . . . . . . . . . . . . . . . 32f -induced hyperlocomotion . . . . . . . . . . . . . . . . 58–60

NMDA receptor antagonists . . . . . . . . . . . . 232–233 Mouse models, DISC1. . . . . . . . .211–225, 222t–224t ENU-induced mutagenesis . . . . . . . . . . . . . 216–217 genetic manipulation in utero. . . . . . . . . . .212–213 knockdown in vivo . . . . . . . . . . . . . . . . . . . . . 213–215 adult neurogenesis . . . . . . . . . . . . . . . . . . 213–214 progenitor proliferation and new pathway . . . . . . . . . . . . . . . . . . . . . . . . . 214–215 polymorphism model. . . . . . . . . . . . . . . . . . .215–216 transgenic models . . . . . . . . . . . . . . . . . . . . . . 217–221 BAC transgenic DISC1 model. . . . . . . 220–221 constitutive expression . . . . . . . . . . . . . . 217–218 C-terminal fragment, inducible expression of . . . . . . . . . . . . . . . . . . . . 218–219 mutant DISC1, inducible expression of . . . . . . . . . . . . . . . . . . . . 219–220 mPFC (medial prefrontal cortex) . . . . . . . . . . . . . . . . 22, 34, 58–60, 63, 65f, 67, 84–85, 102, 151, 196–197, 199, 215, 218, 221, 223t Multi Probe II pipetting robot . . . . . . . . . . . . . . . . . . 106 Mutagenesis . . . . . . . . . . . . . . . . . . . 199f, 216–217, 223t MWM (Morris Water Maze) reference memory . . . . . . . . . . . . . . . . . . . . 56–57, 56f reversal learning . . . . . . . . . . . . . . . . . . . . . . . . . . 50–51 spatial recognition memory . . . . . . . . . . . . . . . . . . 220 working memory . . . . . . . . . . . . . . . . . . . . . . . . . 39–40

N NanoDrop ND-1000 spectrophotometer. . . . . . . .103 Negative symptoms of schizophrenia. . . . . . . . . . . . .26, 31–33, 233, 250, 261, 263 Neonatal hypoxia . . . . . . . . . . . . . . . . . . 95–98, 101, 107 Neurochemical changes observed in E17 MAM-exposed rats abnormal GABAergic markers . . . . . . . . . . . . . 61–63 D-amphetamine-induced hyperlocomotion . . . . . . . . . . . . . . . . . . . 57–58 MK-801-induced hyperlocomotion. . . . . . . .58–60 Reelin level in organotypic hippocampal cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60–61 Neurodevelopment . . . . . . . 106, 175, 177t, 212–214, 218–219 methods . . . . . . . . . . . . . . . 2, 26, 57, 59, 64, 94–96, 114, 128, 154 Neurogenesis. . . . . . . . . . . .63, 85, 115, 213–215, 221 Neuromorphometric anomalies . . . . . . . . . . . . . . . . . . 88 Neuronal cultures, primary, see PV (parvalbumin)positive fast-spiking interneurons Neuropsychiatric syndrome . . . . . . . . . . . . . . . . . . . . . . . 1 Neurotransmission . . . . . . . . . . . . . . . . . . . . . . . 170t–174t effects on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163–164 dopamine signaling . . . . . . . . . . . . . . . . . . . . . . 164 NMDA receptors . . . . . . . . . . . . . . . . . . . . . . . . 163 glutamatergic or dopaminergic . . . . . . . . . . . 28, 57, 97, 104, 115, 246, 253 Nissl staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9, 221 NMDA (N-methyl-D-aspartate) . . . . 58–59, 115, 120 antagonist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29–31 receptor ablation, postnatal . . . . . . . . . . . . . 238–239

NIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS 286 A Subject Index

NMDA (N-methyl-D-aspartate) (continued) impaired sensorimotor gating . . . . . . . . . . . . 238 NMDAR hypofunction theory of schizophrenia . . . . . . . . . . . . . . . . . . . . . . . . 238 psychotic states . . . . . . . . . . . . . . . . . . . . . . . . . . 238 spatial working memory and PPI deficits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 NMDAR (NMDA receptor) . . . . . . . . . . . . . . . . 95, 231 antagonists PCP/ketamine/MK801 . . . . . . . . . . . . . . . . . 129 blockade, in vivo . . . . . . . . . . . . . . . . . . . . . . . 131–132 dysfunction of PV interneurons in vitro and in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 hypomorph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 NMTP (non-match-to-position) . . . . . . . . . 37–38, 38f Normoxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98–101 NORT (novel object recognition task) . . . . . . . . . . . 52 Nox2 (NADPH oxidase-2) . . . . . . . . . . . . . . . . 132–133 NPE (non-pre-exposed) rats. . . . . . . . . . . . . . . . . .42–43 NRG1 (neuregulin 1) and ErbB4 mutant mice. . . . . . . . . . . . . . . . .261–271, 267t, 269t imbalanced activity in schizophrenia . . . . 262–263 DLPFC pyramidal neuron . . . . . . . . . . . . . . . . 263 PCP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262–263 NRG1 signaling in neural development and ErbB kinases . . . . . . . . . . . . . . . . . . . 263–264 in synaptic plasticity . . . . . . . . . . . . . . . . . 264–265 schizophrenia-relevant behavioral deficits ErbB2−/+ and ErbB3−/+ hypomorphs . . 268 ErbB4 mutant mice . . . . . . . . . . . . . . . . . 268–269 neonatal injection of NRG1 ectodomain . . . . . . . . . . . . . . . . . . . . . . . . . . 268 NRG1–CRD−/+ hypomorphs . . . . . . . . . . . 268 NRG1–EGF−/+ hypomorphs . . . . . . . . . . . 266 NRG1–Ig−/+ hypomorphs . . . . . . . . . . . . . . 268 NRG1–TM−/+ hypomorphs . . . . . . . 266–268 NRG1type1-tg hypermorphs . . . . . . . . . . . . . . . 268 PV–ErbB4 mutant mice . . . . . . . . . . . . . 269–270 schizophrenia susceptibility genes . . . . . . . 265–266 ErbB3 and schizophrenia. . . . . . . . . . . . . . . . .265 GWAS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .265 NRG3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 NRG1 and ErbB4 gene . . . . . . . . . . . . . . . . . . 265 NVHL (neonatal ventral hippocampal lesion) rat model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1–23 materials ibotenic acid and artificial CSF. . . . . . . . . . . .3–5 lesion verification/histology materials . . . . . . . 9 rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 surgical equipment . . . . . . . . . . . . . . . . . . . . . . . 5–8 methods animal selection and litter management . . . . . . . . . . . . . . . . . . . . . . . 10–11 lesion confirmation . . . . . . . . . . . . . . . . . . . . 17–21 NVHL and SHAM control surgeries . . . 11–17 problems and pitfalls alternative approaches, cautions . . . . . . . . 22–23 anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 head stabilization . . . . . . . . . . . . . . . . . . . . . . . . . 23

wound closure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 ibotenic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 lesioning, practice . . . . . . . . . . . . . . . . . . . . . 21–22 SHAM controls . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 O Object recognition task . . . . . . . . . . . . . . . . . . 51–52, 53f Obstetric complications, hypoxic rat model for . . . . . . . . . . . . . . . . . . . . 93–108, 94f animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98–99 cross-fostering . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 neonatal hypoxia . . . . . . . . . . . . . . . . . . . . . . . . . . 98 oxymeter/separate oxygen/nitrogen inflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99f brain investigations. . . . . . . . . . . . . . . . . . . . .101–106 data processing . . . . . . . . . . . . . . . . . . . . . 105–106 deep-frozen brains . . . . . . . . . . . . . . . . . . . . . . . 101 gene transcription and translation . . . . . . . . 102f hybridization and scanning of slides . . . . . . 105 liquid nitrogen-cooled isopentane frozen brains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 preparation of DNA (glutamate) chips . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104–105 qRT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 receptor autoradiography . . . . . . . . . . . . . . . . 102 RNA isolation and reverse transcription . . . . . . . . . . . . . . . . . . . . . 103–104 in situ hybridizations . . . . . . . . . . 102–103, 104f 35 S-stained rat brain sections . . . . . . . . . . . . 105f tritium-labeled sections . . . . . . . . . . . . . . . . . . 102 in vitro transcription and labeling . . . . . . . . . 104 materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97–98 methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98–106 testing procedure. . . . . . . . . . . . . . . . . . . . . . . .99–101 on baseline Startle magnitude and PPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100–101 on motor activity . . . . . . . . . . . . . . . . . . . . . . . . 100 on social interaction/recognition . . . . . . . . . 100 ODS (osteogenic-disorder Shionogi) rats . . . . . . 155f, 157–161, 170t–171t Olanzapine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249, 254 Orofacial dyskinesia . . . . . . . . . . . . . . . . . . . . . 29–30, 31f Oxidative stress . . . . . . 132, 151, 153, 157–158, 162, 164–166, 168–169, 173t P Packard ScanArray 5000 scanner . . . . . . . . . . . . 98, 105 Paranoia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25–26 Parkinson’s disease . . . . . . . . . . . . . . . . . . . 154, 212, 261 PCP (phencyclidine) . . . . . . . . . . . . . . . . . . . . . . . . . 29–31 -evoked ataxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30f -evoked locomotor activity . . . . . . . . . . . . . . . . . . . 30f -evoked orofacial dyskinesias . . . . . . . . . . . . . . . . . 31f NMDA-R antagonists . . . . . . . . . . . . . . . . . . 129, 262 PD (postnatal day) . . . . . . . . . . . . . . . . . . . . . . . 3, 10, 17, 21, 60, 94–96, 98–99, 101–102, 106–107, 118, 130, 142, 154, 157, 199, 213–214, 219, 264–265, 268 PE (pre-exposed) rats . . . . . . . . . . 7, 7f, 29, 42–43, 43f

ANIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS Subject Index 287 PFC (prefrontal cortex) . . . . . . . . . . 20, 26, 34, 38–39, 44–46, 48–49, 52, 57–64, 65f, 67, 94f, 95, 131, 159, 215, 223t, 247, 263 Piezoelectric accelerometers . . . . . . . . . . . . . . . . . . . . 100 PI3 kinase/Akt pathways . . . . . . . . . . . . . . . . . . . . . . . 264 Poly I:C (polyinosinic:polycytidylic acid) models . . . . . . . . . . . . . . . . . . . . . . . . . 80, 84–85 administration of MK-801 . . . . . . . . . . . . . . . . . . . . 84 animal models of in utero infection. . . . . . . . . . . .81 experimental protocols . . . . . . . . . . . . . . . . . . . . . . . 82 exposed offspring and abnormalities . . . . . . . . . . . 84 maternal immune activation models . . . . . . . 80–81 increases cytokines . . . . . . . . . . . . . . . . . . . . . . . . 81 neuropathologic abnormalities . . . . . . . . . . . . . . . . 85 study findings from animal models . . . . . . . . 84–85 Positive symptoms of schizophrenia. . . . . . . . . . .27–31 dopaminergic receptor agonist-induced hyperactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 glutamatergic NMDA antagonist-induced hyperactivity . . . . . . . . . . . . . . . . . . . . . . . 29–31 spontaneous locomotor activity . . . . . . . . . . . . . . . 28 stereotyped behaviors . . . . . . . . . . . . . . . . . . . . . 29–31 Post mortem studies . . . 2, 26, 61–64, 128, 151, 160, 167, 176t, 216, 238, 245–248, 252, 256 PPI (prepulse inhibition). . . . . . . . . . . . . .41f–42f, 267t and baseline Startle magnitude. . . . . . . . . .100–101 deficits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85, 238 rat brain development . . . . . . . . . . . . . . . . . . . . 95–96 of Startle reflex in Fischer 344 rats . . . . . . . . . . . . . . . . . . . . . . . . 41 in Sprague Dawley rats . . . . . . . . . . . . . . . . . . . . 41 Prefrontal cortical dysfunction . . . . . . . . . . . . . . . . . . . . . 2 Premorbid children. . . . . . . . . . . . . . . . . . . . . . . . . . . . .200 Prenatal E17 MAM- vs. saline-exposed rats attentional set-shifting task in . . . . . . . . . . . . . . . . 45f LI performances in . . . . . . . . . . . . . . . . . . . . . . . . . . 43f object recognition in. . . . . . . . . . . . . . . . . . . . . . . . .53f phencyclidine-evoked ataxia in . . . . . . . . . . . . . . . 30f reference memory in Morris’ water maze in . . . 56f social interactions in . . . . . . . . . . . . . . . . . . . . . . . . . 33f spatial recognition memory (Y-maze paradigm) in . . . . . . . . . . . . . . . . . . . . . . . . . . 54f spontaneous locomotor activity in . . . . . . . . . . . . 28f working memory performance in delay-interposed/eight-arm radial maze task . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36f in delay NMTP task . . . . . . . . . . . . . . . . . . . . . . 38f in reversal learning paradigm of Morris’ water maze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40f Prenatal infection/immune models of schizophrenia . . . . . . . . . . . . . . . . . . . 79–89 animal models of in utero infection advantages of poly I:C/LPS models. . . . . . . . 81 maternal immune activation models, poly I:C/LPS . . . . . . . . . . . . . . . . . . . . . . . . . . . 80–81 back translation to epidemiology . . . . . . . . . . 87–88 experimental protocols influenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82–84 LPS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

poly I:C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 study findings from animal models gestational timing of infection . . . . . . . . . . . . . 86 influenza models . . . . . . . . . . . . . . . . . . . . . . . . . . 85 LPS models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 maternal immune activation and in utero infection . . . . . . . . . . . . . . . . . . . . . . . . . . . 85–86 MIA and in utero infection . . . . . . . . . . . . . . . . 87 poly I:C models . . . . . . . . . . . . . . . . . . . . . . . 84–85 Pre- or perinatal hypoxia. . . . . . . . . . . . . . . . . . . . . . . .114 Presynaptic genes. . . . . . . . . . . . . . . . . . . . . . . . . . .97, 107 Primary cortical neurons, dissection/preparation of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137–142 dissociated neuronal cultures . . . . . . . . . . . 138–139 one-step cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 parvalbumin/GAD67 in PV interneurons . . . . . . . . . . . . . . . 141–142, 142f image analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 parvalbumin interneurons in culture fixation and blocking. . . . . . . . . . . . . . . . . . . . . 139 immunocytochemistry . . . . . . . . . . . . . . 140–141 Primary neuronal cultures . . . . . . . . . . . . . . . . . 127–145 cultured cortical neurons . . . . . . . . . . . . . . . . . . . 130f materials coverslips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133–135 methods dissection/preparation of astrocytes . . . . . . . . . . . . . . . . . . . . . 135–137 dissection/preparation of primary cortical neurons . . . . . . . . . . . . . . . . . . . . . . . . . 137–142 schizophrenia pathophysiology dysfunction of PV interneurons in vitro and in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 loss of function of PV interneurons. . . . . . . . . . . . . . . . . . . . .132–133 NMDA receptor blockade, in vivo . . . 131–132 Prostaglandins or growth factors . . . . . . . . . . . . . . . . . 88 Psychiatric disorders, genetic architecture of . . . . . . . . . . . . . . . . . . . 190–191 CDCA/CDRA hypothesis. . . . . . . . . . . . . . . . . . .190 common genetic variants . . . . . . . . . . . . . . . 191–192 bipolar disorder . . . . . . . . . . . . . . . . . . . . . . . . . . 191 GWA studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 mutant animal models . . . . . . . . . . . . . . . . . . 192–193 rare genetic variants . . . . . . . . . . . . . . . . . . . . . . . . . 192 Psychiatric genetics and generation of mutant animal models . . . . . . . . . . . . . . . . . . . . . . . . . . 189–204 animal models of disease-associated alleles DISC1 susceptibility locus . . . . . . . . . . 197–200 22q11 susceptibility locus . . . . . . . . . . . 200–203 animal models of disease-associated loci AKT1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196 DTNBP1 susceptibility locus . . . . . . . . . . . . . 195 NRG1 susceptibility locus . . . . . . . . . . . 193–194 genetic architecture of psychiatric disorders . . . . . . . . . . . . . . . . . . 190–191, 191f impact of common genetic variants . . 191–192

NIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS 288 A Subject Index

Psychiatric genetics and generation (continued) impact of rare genetic variants . . . . . . . . . . . . 192 implications for mutant animal models . . . . . . . . . . . . . . . . . . . . . . . . . . 192–193 integrating human genetics and mutant models challenges, beyond single genes . . . . . . . . . . 204 identifying disease pathways . . . . . . . . . 203–204 Psychosis onset anatomical/functional anomalies in E17 MAM-exposed rats . . . . . . . . . . . . . . . . . 64–67 behavioral changes in E17 MAM-exposed rats cognitive symptoms of schizophrenia . . . 33–57 negative symptoms of schizophrenia . . . . 31–33 positive symptoms of schizophrenia . . . . 27–31 neurochemical changes in E17 MAM-exposed rats abnormal GABAergic markers. . . . . . . . . .61–63 D-amphetamine-induced hyperlocomotion . . . . . . . . . . . . . . . . . . . 57–58 MK-801-induced hyperlocomotion . . . . 58–60 reelin level in organotypic hippocampal cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60–61 Psychostimulant or hallucinogenic drugs . . . . . . . . . . . 1 PV–ErbB4−/− mice . . . . . . . . . . . . 264–265, 269–270 PV (parvalbumin)-positive fast-spiking interneurons. . . . . . . . . . . . . . . . . . . . .127–145 cultured cortical neurons . . . . . . . . . . . . . . . . . . . 130f materials coverslips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133–135 methods dissection/preparation of astrocytes . . . . . . . . . . . . . . . . . . . . . 135–137 dissection/preparation of primary cortical neurons . . . . . . . . . . . . . . . . . . . . . . . . . 137–142 schizophrenia pathophysiology dysfunction of PV interneurons in vitro and in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 loss of function of PV interneurons . . 132–133 NMDA receptor blockade, in vivo . . . 131–132 Q 22q11 locus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .200–203 animal models . . . . . . . . . . . . . . . . . . . . . . . . 199f, 201f de novo microdeletion . . . . . . . . . . . . . . . . . . . . . . 200 expression of Comt gene . . . . . . . . . . . . . . . . . . . . 202 hemizygous mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 heterozygous deficiency . . . . . . . . . . . . . . . . 202–203 knockdown model of Prodh . . . . . . . . . . . . . . . . . 202 1.5-Mb deletion . . . . . . . . . . . . . . . . . . . . . . . 200–202 22q11.2 microdeletion . . . . . . . . . . . . . . . . . 202–203 Zdhhc8 and Dgcr8 deficiency. . . . . . . . . . . . . . . . .202 Quantitative real time PCR (qRT-PCR) . . . . 102, 106 R Radial Arm Maze . . . . . . . . . . . . . . . . . . . . 34–35, 35f, 39 Radio-immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 Rare genetic variants . . . . . . . . . . . . . . . . . . . . . . 191f, 192 Rat 7-day-old rat pups (PD-7) . . . . . . . . . . . . . . . . . . . . . 3

lesioned and SHAM . . . . . . . . . . . . . . . . . . . . . . . . . . 19 MAM, see Gestational MAM administration NVHL, see NVHL (neonatal ventral hippocampal lesion) rat model ODS . . . . . . . . . . . . . . . . . 155f, 157–161, 170t–171t pups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2–3, 5, 21, 96 Sprague-Dawley . . . . . 3, 116, 117f, 118, 121, 123 Wistar . . . . . . . . . . . . . . . . . . 28, 33, 56, 58, 160–161 Rat brain development model of sensorimotor gating . . . . . . . . . . . . . . . . . 96 neuropeptide Y and complexins . . . . . . . . . . . . . . . 97 NMDA receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . .95 hypofunction in schizophrenia . . . . . . . . . . . . . 95 NR2 subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 obstetric complications . . . . . . . . . . . . . . . . . . . . . . . 95 postnatal day (PD) . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 postnatal period . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 prepulse inhibition (PPI) . . . . . . . . . . . . . . . . . . 95–96 SNARE complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Receptor autoradiography . . . . . . . . . . . . . . . . . 101–102 Redox dysregulation, see Glutathione deficit and redox dysregulation Reelin (RELN) and dendritic spine density changes . . . . . . . . . . 250 and GAD67 (GAD1) . . . . . . . . 245–248, 251–252 level in organotypic hippocampal cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60–61 double fluorescence immunohistochemistry . . . . . . . . . . . . . . . . . 60 and methylation levels . . . . . . . . . . . . . . . . . 60–61 neuronal marker NeuN . . . . . . . . . . . . . . . . . . . . 61 polymerase chain reaction (PCR) . . . . . . . . . . 61 post-pubertal hippocampus, development of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Stoppini method . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Reversal learning in Morris Water Maze . . . . . . . . . . . . . . . . 40f, 50–51 working memory performance . . . . . . . . . . 40f, 129 in Y Maze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49–50 Reverse transcription . . . . . . . . . . . . . . . . . . . . . . 103–104 Risk alleles . . . . . . . . . . . . . . . . . 190–192, 197, 203–204 Risk factors . . . 26, 67–68, 80, 83, 94, 94f, 106–108, 114–115, 164, 175, 200, 202, 266 Risperidone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234, 238 RNA double-stranded. . . . . . . . . . . . . . . . . . . . . . . . .80, 114 extraction kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 influenza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 isolation and reverse transcription . . . . . . . 103–104 messenger. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103f RNAMaxx High Yield Transcription Kit . . . . . . . . . 104 RNS (reactive nitrogen species) . . . . . . . . . . . . 150, 154 Rodents . . . . . . . . . . . . . . . . . . . . . 27, 31, 33–35, 39–40, 44, 52–53, 55, 57–58, 83, 114–115, 123, 129–130, 154, 156–157, 161–162, 166–167, 170t, 175, 176t, 180, 232–233, 262–263 ROS (reactive oxygen species) . . 132, 150, 152–154, 157, 163, 165

ANIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS Subject Index 289 S SAM (S-adenosyl methionine) . . . 244, 247, 251, 256 Schizophrenia cognitive symptoms. . . . . . . . . . . . . . . . . . . . . . .33–57 attention and information processing . . . 40–49 behavioral flexibility . . . . . . . . . . . . . . . . . . . 49–51 episodic memory . . . . . . . . . . . . . . . . . . . . . . 51–55 spatial reference memory . . . . . . . . . . . . . . 55–57 working memory . . . . . . . . . . . . . . . . . . . . . . 34–40 negative symptoms . . . . . . . . . . . . . . . . . . . . . . . 31–33 positive symptoms . . . . . . . . . . . . . . . . . . . . . . . . 27–31 dopaminergic receptor agonist-induced hyperactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 glutamatergic NMDA antagonist-induced hyperactivity . . . . . . . . . . . . . . . . . . . . . . . 29–31 spontaneous locomotor activity . . . . . . . . . . . . 28 stereotyped behaviors . . . . . . . . . . . . . . . . . . 29–31 psychosis, see Psychosis onset Schizophrenic-like 100P mice. . . . . . . . . . . . . . . . . . .217 Scholl method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 Sdy strain of mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .195 Sham inducing hypothermic anesthesia . . . . . . . . . . 12–13 lesioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 mounting the rat . . . . . . . . . . . . . . . . . . . . . . 13–14 post-operative recovery . . . . . . . . . . . . . . . . 16–17 puncture and infusion . . . . . . . . . . . . . . . . . 14–16 and NVHL control surgeries . . . . . . . . . . . . . . 11–17 rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19, 54, 56 surgical setup cannula preparation . . . . . . . . . . . . . . . . . . . 11–12 hypothermic anesthesia bucket . . . . . . . . . . . . . 11 post-op chamber . . . . . . . . . . . . . . . . . . . . . . . . . . 12 pre-op ready chamber . . . . . . . . . . . . . . . . . . . . . 12 Shapiro–Wilk test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Short term memory . . . . . . . . . . . . . . . . 35–36, 39, 156, 163, 194–197, 199, 216, 238, 269t Single-neuron transcriptional analysis . . . . . . . . . . . 138 si (small interference) RNAs . . . . . . . . . . . . . . . . . . . . 213 SNARE complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Social interaction test . . . . . . . . . . . . . . . . . . . . 28, 31, 55 SOD (superoxide dismutase) . . . . . . . . . 132, 151, 158 Spatial recognition memory in Y Maze . . . 53–55, 54f Spatial reference memory . . . . . . 51, 55–57, 156, 162, 194–195, 199 Spontaneous alternation . . . . . . . . . . . 35–36, 37f, 160, 163, 172t, 268 Sprague–Dawley rat. . . . .3, 116, 117f, 118, 121, 123 Startle magnitude/PPI, test on baseline . . . . 100–101 Stereology software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Stereotypies . . . . . . . . . . . . . . . 27–31, 31f, 96, 234, 269 Stoppini method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Strokes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2, 103 Sulpiride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164, 254, 256 Superscript II reverse transcriptase . . . . . . . . . . . . . . 102 Surgery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5–8 anesthesia. . . . . . . . . . . . . . . . . . . .8–9, 11–13, 17, 22 cannula lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5–8

as backup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 injection needle . . . . . . . . . . . . . . . . . . . . . . . . . 7, 7f 10-μl syringe with thin metal plunger . . . . . . . 6 for NVHL lesioning . . . . . . . . . . . . . . . . . . . . . . . . 8 for practice lesioning . . . . . . . . . . . . . . . . . . . . . . . 8 for SHAM lesioning . . . . . . . . . . . . . . . . . . . . . . . . 8 tubing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 equipment . . . . . . . . . . . . . . . . . . . . . . . . . . 5, 8–9, 133 Harvard Apparatus microinfusion pumps . . . . . . . 8 infusion pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 miscellaneous material . . . . . . . . . . . . . . . . . . . . . . . . . 8 stereotaxic instrument . . . . . . . . . . . . . . . . . . . 5–6, 13 custom designing guidelines . . . . . . . . . . . . . . . . 6 VD stereotactic measuring/calculation . . . . . . . . . 7 SVZ (subventricular zones) . . . . . . . . . . . . . . . . . . . . . 214 Synaptic plasticity . . . . . . 95, 180, 248, 263–265, 270 T tam (tamoxifen) . . . . . . . . . . . . . . . . . . . . . 218–219, 224t Thalamus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20, 52 mediodorsal nucleus . . . . . . . . . . . . . . . . . . . . . . 64–65 Thy-1-GFP transgenic mice. . . . . . . . . . . . . . . . . . . . .215 TM (transmembrane) domain . . . . . . . . 194, 263, 266 Toxoplasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80–81 Transgenic models . . . . . . . . . . . . . . . . . . . 200, 217–221 BAC transgenic DISC1 model . . . . . . . . . . 220–221 constitutive expression . . . . . . . . . . . . . . . . . 217–218 C-terminal fragment, inducible expression of . . . . . . . . . . . . . . . . . . . . 218–219 mutant DISC1, inducible expression of . . . . . . . . . . . . . . . . . . . . 219–220 Trizol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103 TTX (tetrodotoxin) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 V VD (ventral–dorsal) stereotactic measuring and calculation . . . . . . . . . . . . . . . . . . . . . . . 7, 7f, 15 Ventral hippocampus . . . . . . . . . 2, 19, 22, 65f, 67, 85, 166–167 Video tracking system . . . . . . . . 32, 39, 51–52, 54, 56 Vitamin D deficiency . . . . . . . . . . . . . . . . . . . 114, 119–122, 119t -deficient dam . . . . . . . . . 118–119, 118f, 119t, 121 depletion, maternal . . . . 119, 120t, 121, 123–124 neonatal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 prenatal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 See also DVD (developmental vitamin D) model VZ (ventricular zones). . . . . . . . . . . . . . . . . . . . .213–214 W Wisconsin Card Sorting Task . . . . . . . . . . . . . . . . . 44, 49 Working memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34–40 in delayed NMTP task. . . . . . . . . . . . . . . .37–38, 38f impairments . . . . . . . . . . . . . . . . 196–197, 215, 219, 233, 268, 270 in MAM rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37, 39 Morris Water Maze . . . . . . . . . . . . . . 36f, 39–40, 40f

NIMAL MODELS OF SCHIZOPHRENIA AND RELATED DISORDERS 290 A Subject Index

Working memory (continued) Radial Arm Maze . . . . . . . . . . . . . . . . . . . . . . . . . 34–35 in reversal learning paradigm . . . . . . . . . . . . . . . . . 40f spatial . . . . . . . . . . . . . . . 2, 34–35, 38f, 39, 56, 129, 199, 215, 238 spontaneous alternation . . . . . . . . . . . . . . . . . . . 35–36 Wound closure . . . . . . . . . . . . . . . . . . . . . . . . . . . 8, 16–17, 23

Y Y Maze for MAM rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35–36 reversal learning in . . . . . . . . . . . . . . . . . . . 49–50, 50f short-term spatial memory . . . . . . . . . . . . . 156, 199 spatial recognition memory . . . . . . . . . . . . . . 54f, 55 Z ZETALIF detector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59