Handbook of the Autonomic Nervous System in Health and Disease (Neurological Disease and Therapy)

Handbook of the Autonomic Nervous System in Health and Disease edited by

C. Liana Bolis University of Milan Milan, Italy, and AIREN WHO Collaborating Centre for Research and Training in Neuroscience Geneva, Switzerland

Julio Licinio David Geffen School of Medicine at UCLA Los Angeles, California, U.S.A.

Stefano Govoni University of Pavia Pavia, Italy

Marcel Dekker, Inc. TM

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

New York • Basel

Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. ISBN: 0-8247-0842-3 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-260-6300; fax: 41-61-260-6333 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright © 2003 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

NEUROLOGICAL DISEASE AND THERAPY Advisory Board Louis R. Caplan, M.D.

Jerome Murphy, M.D.

Professor of Neurology Harvard University School of Medicine Beth Israel Deaconess Medical Center Boston, Massachusetts

Professor of Neurology University of Missouri School of Medicine Chief of Neurology Children’s Mercy Hospital Kansas City, Missouri

William Koller, M.D. National Parkinson Foundation Miami, Florida

John C. Morris, M.D. Friedman Professor of Neurology Co-Director, Alzheimer’s Disease Research Center Washington University School of Medicine St. Louis, Missouri

Kapil Sethi, M.D. Professor of Neurology Director, Movement Disorders Program Medical College of Georgia Augusta, Georgia

Mark Tuszynski, M.D., Ph.D. Associate Professor of Neurosciences Director, Center for Neural Repair University of California–San Diego La Jolla, California

1. Handbook of Parkinson's Disease, edited by William C. Koller 2. Medical Therapy of Acute Stroke, edited by Mark Fisher 3. Familial Alzheimer's Disease: Molecular Genetics and Clinical Perspectives, edited by Gary D. Miner, Ralph W. Richter, John P. Blass, Jimmie L. Valentine, and Linda A. Winters-Miner 4. Alzheimer's Disease: Treatment and Long-Term Management, edited by Jeffrey L. Cummings and Bruce L. Miller 5. Therapy of Parkinson's Disease, edited by William C. Koller and George Paulson 6. Handbook of Sleep Disorders, edited by Michael J. Thorpy 7. Epilepsy and Sudden Death, edited by Claire M. Lathers and Paul L. Schraeder 8. Handbook of Multiple Sclerosis, edited by Stuart D. Cook 9. Memory Disorders: Research and Clinical Practice, edited by Takehiko Yanagihara and Ronald C. Petersen 10. The Medical Treatment of Epilepsy, edited by Stanley R. Resor, Jr., and Henn Kutt 11. Cognitive Disorders: Pathophysiology and Treatment, edited by Leon J. Thal, Walter H. Moos, and Elkan R. Gamzu 12. Handbook of Amyotrophic Lateral Sclerosis, edited by Richard Alan Smith 13. Handbook of Parkinson's Disease: Second Edition, Revised and Expanded, edited by William C. Koller 14. Handbook of Pediatric Epilepsy, edited by Jerome V. Murphy and Fereydoun Dehkharghani 15. Handbook of Tourette's Syndrome and Related Tic and Behavioral Disorders, edited by Roger Kurlan 16. Handbook of Cerebellar Diseases, edited by Richard Lechtenberg 17. Handbook of Cerebrovascular Diseases, edited by Harold P. Adams, Jr. 18. Parkinsonian Syndromes, edited by Matthew B. Stern and William C. Koller 19. Handbook of Head and Spine Trauma, edited by Jonathan Greenberg 20. Brain Tumors: A Comprehensive Text, edited by Robert A. Morantz and John W. Walsh Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

21. Monoamine Oxidase Inhibitors in Neurological Diseases, edited by Abraham Lieberman, C. Warren Olanow, Moussa B. H. Youdim, and Keith Tipton 22. Handbook of Dementing Illnesses, edited by John C. Morris 23. Handbook of Myasthenia Gravis and Myasthenic Syndromes, edited by Robert P. Lisak 24. Handbook of Neurorehabilitation, edited by David C. Good and James R. Couch, Jr. 25. Therapy with Botulinum Toxin, edited by Joseph Jankovic and Mark Hallett 26. Principles of Neurotoxicology, edited by Louis W. Chang 27. Handbook of Neurovirology, edited by Robert R. McKendall and William G. Stroop 28. Handbook of Neuro-Urology, edited by David N. Rushton 29. Handbook of Neuroepidemiology, edited by Philip B. Gorelick and Milton Alter 30. Handbook of Tremor Disorders, edited by Leslie J. Findley and William C. Koller 31. Neuro-Ophthalmological Disorders: Diagnostic Work-Up and Management, edited by Ronald J. Tusa and Steven A. Newman 32. Handbook of Olfaction and Gustation, edited by Richard L. Doty 33. Handbook of Neurological Speech and Language Disorders, edited by Howard S. Kirshner 34. Therapy of Parkinson's Disease: Second Edition, Revised and Expanded, edited by William C. Koller and George Paulson 35. Evaluation and Management of Gait Disorders, edited by Barney S. Spivack 36. Handbook of Neurotoxicology, edited by Louis W. Chang and Robert S. Dyer 37. Neurological Complications of Cancer, edited by Ronald G. Wiley 38. Handbook of Autonomic Nervous System Dysfunction, edited by Amos D. Korczyn 39. Handbook of Dystonia, edited by Joseph King Ching Tsui and Donald B. Calne 40. Etiology of Parkinson's Disease, edited by Jonas H. Ellenberg, William C. Koller, and J. William Langston 41. Practical Neurology of the Elderly, edited by Jacob I. Sage and Margery H. Mark 42. Handbook of Muscle Disease, edited by Russell J. M. Lane 43. Handbook of Multiple Sclerosis: Second Edition, Revised and Expanded, edited by Stuart D. Cook 44. Central Nervous System Infectious Diseases and Therapy, edited by Karen L. Roos 45. Subarachnoid Hemorrhage: Clinical Management, edited by Takehiko Yanagihara, David G. Piepgras, and John L. D. Atkinson 46. Neurology Practice Guidelines, edited by Richard Lechtenberg and Henry S. Schutta 47. Spinal Cord Diseases: Diagnosis and Treatment, edited by Gordon L. Engler, Jonathan Cole, and W. Louis Merton 48. Management of Acute Stroke, edited by Ashfaq Shuaib and Larry B. Goldstein 49. Sleep Disorders and Neurological Disease, edited by Antonio Culebras 50. Handbook of Ataxia Disorders, edited by Thomas Klockgether 51. The Autonomic Nervous System in Health and Disease, David S. Goldstein 52. Axonal Regeneration in the Central Nervous System, edited by Nicholas A. Ingoglia and Marion Murray 53. Handbook of Multiple Sclerosis: Third Edition, edited by Stuart D. Cook 54. Long-Term Effects of Stroke, edited by Julien Bogousslavsky 55. Handbook of the Autonomic Nervous System in Health and Disease, edited by C. Liana Bolis, Julio Licinio, and Stefano Govoni 56. Dopamine Receptors and Transporters: Function, Imaging, and Clinical Implication, Second Edition, edited by Anita Sidhu, Marc Laruelle, and Philippe Vernier Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

57. Handbook of Olfaction and Gustation: Second Edition, Revised and Expanded, edited by Richard L. Doty 58. Handbook of Stereotactic and Functional Neurosurgery, edited by Michael Schulder 59. Handbook of Parkinson’s Disease: Third Edition, edited by Rajesh Pahwa, Kelly E. Lyons, and William C. Koller

Additional Volumes in Preparation Clinical Neurovirology, edited by Avi Nath and Joseph R. Berger

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Preface

The autonomic nervous system (ANS) consists of an integrated neuronal network that reaches all organs and tissues and is essential for the maintenance of homeostasis. The ANS ensures rapid adjustment of biological functions that are crucial for survival, adaptation, and evolution. It integrates afferent information that originates from both the environment and the internal milieu and, through its efferent pathways, regulates the functions of various organs and systems according to the needs of the organism. Because of its role in the regulation of biological functions that are essential to life, the ANS was the subject of some of the earliest research work in neurology and neuroscience. The earliest recorded reference to the visceral nervous system was made by Galen in the second century. The considerable amount of neuroanatomical and neurophysiological work performed over the last 18 centuries has helped elucidate the structure and key functions of the ANS. However, much remains to be done. Recent progress in molecular medicine and imaging has led to new approaches in neuroscience research and has provided elegant descriptions of the ANS and its functions. Increasingly sophisticated approaches using the tools of contemporary biology have allowed the molecular dissection of the mechanisms through which the ANS ensures rapid and effective integration between the central nervous system and visceral inputs, thereby establishing and maintaining homeostasis. Dysregulation of such a key system is associated with disease either linked to a primary ANS dysfunction or as an element in the pathophysiology of complex disorders such as cardiovascular diseases, asthma, and depression. The role of the ANS in disease goes beyond primary and secondary dysfunction. Pharmacological approaches to important diseases are based on drugs that are agonists and an-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

tagonists of various components of ANS function. The treatment of conditions that are highly prevalent and represent public health problems worldwide involves the use of drugs that modulate ANS function. Such diseases include various types of cardiovascular disorders, such as hypertension, heart failure, arrhythmias, ischemia, and myocardial infarction, as well as conditions such as asthma, diabetes, urinary retention, and shock. Thus, a better understanding of ANS biology and further work in this field will lead to improved knowledge of biology, pathophysiology, and pathogenesis in general. It will also lead to more efficacious treatment for human diseases of major public health significance. The volume includes views from leading scientists in the field of ANS research dealing with both classical neuroanatomical and neurochemical issues. It also discusses some less traditional aspects of the molecular organization of the ANS and its participation in functions such as sleep, thermoregulation, and neuroendocrine and neuroimmune control in health and in disease, including novel data on the role of the ANS in depression. Of particular interest is the discussion by various contributors of the ANS and cardiovascular disease, including modern clinical techniques used to study and analyze ANS function in the affected patient. This book offers a comprehensive view of the status of current research on the autonomic nervous system, directions for future investigation, and implications for clinical medicine and public health. It is a progress report on ANS research and its implications for health and disease. C. Liana Bolis Julio Licinio Stefano Govoni

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Contents

Preface Contributors 1.

Structural and Chemical Organization of the Autonomic Neuroeffector System Geoffrey Burnstock

2.

Immune Function and Regulation of the Autonomic Nervous System: A Molecular Point of View Serge Rivest

3.

Organ Specificity of Autonomic Nervous System Responses Shaun F. Morrison

4.

Plasticity in the Autonomic Nervous System: Responses of Adult Sympathetic Neurons to Injury Richard E. Zigmond

5.

The Adrenal Medulla: Physiology and Pathophysiology Graeme Eisenhofer, Monika Ehrhart-Bornstein, and Stefan R. Bornstein

6.

Autonomic Nervous System–Leptin Interactions: Impact on Metabolic Rate and Body Weight Regulation Bulent Yildiz, Metin Ozata, Ma-Li Wong, and Julio Licinio

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

7.

The Autonomic Nervous System and Thermoregulation Quentin J. Pittman

8.

Phylogenesis of the Autonomic Nervous System from Fish to Mammals C. Liana Bolis, Andrea Zenone Dalla Valle, and Margherita Piccolella

9.

Clinical Studies of the Sympathetic Nervous System Murray Esler and Magdalena S. Rumantir

10.

Autonomic Regulation and Dysregulation of the Heart: Insights from Spectral Analysis of Cardiovascular Oscillations Massimo Pagani and Daniela Lucini

11.

Cardiovascular Autonomic Dysregulation Vilho V. Myllylä, Juha T. Korpelainen, Tarja H. Haapaniemi, Uolevi Tolonen, Timo H. Mäkikallio, Mikko P. Tulppo, Kyösti A. Sotaniemi, and Heikki V. Huikuri

12.

Uremic Cardiac Autonomic Neuropathy: Clinical Evaluation with Heart Rate Variability and Metaiodobenzylguanidine Chinori Kurata

13.

The Function of the Autonomic Nervous System in Hypertension Guido Grassi and Giuseppe Mancia

14.

Autonomic Control of the Airways Peter J. Barnes

15.

The Parasympathetic Nervous System in the Pathophysiology of the Gastrointestinal Tract Yvette Taché

16.

The Autonomic Nervous System in the Normal Control and Pathophysiology of the Exocrine–Endocrine Pancreas Osvaldo M. Tiscornia

17.

Diabetes and the Autonomic Nervous System Maria Grazia Natali-Sora and Guido Pozza

18.

Sympathetic Innervation of the Kidney in Health and Disease Lars Christian Rump, Kerstin Amann, and Eberhard Ritz

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

19.

Nerve Control of Bladder Function Giorgio Gabella

20.

Sleep Regulation and the Autonomic Nervous System Harvey Moldofsky and Wah-Ping Luk

21.

Major Depression and the Autonomic Nervous System Gina Rinetti and Ma-Li Wong

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Contributors

Kerstin Amann, M.D. Professor, Department of Pathology, University of Erlangen, Erlangen, Germany Peter J. Barnes, D.M., D.Sc., F.R.C.P. Professor and Head, Department of Thoracic Medicine, National Heart & Lung Institute, Imperial College, London, United Kingdom C. Liana Bolis, M.D., Ph.D. Professor, Laboratory of Comparative Biology, Department of Pharmacological Sciences, University of Milan, Milan, Italy, and AIREN, Geneva, Switzerland Stefan R. Bornstein, M.D., Ph.D. Professor of Medicine and Associate Director, Department of Endocrinology, University of Düsseldorf, Düsseldorf, Germany Geoffrey Burnstock, Ph.D, D.Sc., F.A.A., F.R.C.S.(Hon.), F.R.C.S.(Hon.), F.Med.Sci., F.R.S. Autonomic Neuroscience Institute, Royal Free & University College Medical School, London, United Kingdom Andrea Zenone Dalla Valle, Ph.D. Research Fellow, Laboratory of Comparative Biology, Department of Pharmacological Sciences, University of Milan, Milan, Italy Monika Ehrhart-Bornstein, Ph.D. German Diabetes Institute, University of Düsseldorf, Düsseldorf, Germany

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Graeme Eisenhofer, Ph.D. Staff Scientist, Clinical Neurocardiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, U.S.A. Murray Esler, M.B.B.S., Ph.D., F.R.A.C.P. Associate Director, Baker Medical Research Institute, and Professor of Medicine, Monash University, Melbourne, Victoria, Australia Giorgio Gabella, M.D., D.Sc. Professor of Histology and Cytology, Department of Anatomy and Developmental Biology, University College London, London, United Kingdom Stefano Govoni, Ph.D. Professor of Pharmacology, Head, Department of Experimental and Applied Pharmacology, School of Pharmacy, University of Pavia, Pavia, Italy. Guido Grassi, M.D. Associate Professor, Department of Clinical Medicine, University of Milan-Bicocca, and San Gerardo Hospital, Monza (Milan), Italy Tarja H. Haapaniemi, M.D., Ph.D. Department of Neurology, University of Oulu, Oulu, Finland Heikki V. Huikuri, M.D.,Ph.D. Professor, Division of Cardiology, Department of Medicine, University of Oulu, Oulu, Finland Juha T. Korpelainen, M.D., Ph.D. Assistant Professor, Department of Neurology, University of Oulu, Oulu, Finland Chinori Kurata, M.D. General Manager, Yamaha Health Care Center, Hamamatsu, Japan Julio Licinio, M.D. Director, Interdepartmental Clinical Pharmacology Center, and Professor of Psychiatry and Medicine, Neuropsychiatric Institute, David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Daniela Lucini Centro di Ricerca sulla Terapia Neurovegetativa, University of Milan and L. Sacco Hospital, Milan, Italy Wah-Ping Luk, B.Sc. Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada Timo H. Mäkikallio, M.D., Ph.D. Division of Cardiology, Department of Internal Medicine, University of Oulu, Oulu, Finland

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Giuseppe Mancia, M.D. Professor of Medicine and Head, Department of Clinical Medicine, University of Milan-Bicocca, and San Gerardo Hospital, Monza (Milan), Italy Harvey Moldofsky, M.D., F.R.C.P.C. Director, Sleep Disorders Clinics, Centre for Sleep and Chronobiology, and Professor Emeritus, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada Shaun F. Morrison, Ph.D Senior Scientist, Neurological Sciences Institute, Oregon Health and Sciences University, Beaverton, Oregon, U.S.A. Vilho V. Myllylä, M.D., Ph.D. Professor of Neurology and Vice Rector, Department of Neurology, University of Oulu, Oulu, Finland Maria Grazia Natali-Sora, M.D. Department of Neurology, Vita-Salute San Raffaele University, Milan, Italy Metin Ozata, M.D. Associate Professor, Division of Endocrinology and Metabolism, Department of Medicine, Gulhane School of Medicine, Ankara, Turkey Massimo Pagani, M.D., F.A.C.C. Professor, Department of Medicine, University of Milan and L. Sacco Hospital, Milan, Italy Margherita Piccolella, Ph.D. Research Fellow, Laboratory of Comparative Biology, Department of Pharmacological Sciences, University of Milan, Milan, Italy Quentin J. Pittman, Ph.D. Professor of Physiology, Neuroscience Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada Guido Pozza, M.D., Ph.D. Professor of Clinical Medicine, Vita-Salute San Raffaele University, Milan, Italy Gina Rinetti, M.D. Cousins Center for Psychoneuroimmunology, Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Eberhard Ritz, M.D., F.A.H.A, F.R.C.P., F.A.C.P. Professor of Medicine and Chief, Division of Nephrology, Department of Internal Medicine, Ruperto Carola University of Heidelberg, Heidelberg, Germany

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Serge Rivest, Ph.D. Professor and MRC Scientist, Laboratory of Molecular Endocrinology, CHUL Research Center, and Professor, Department of Anatomy and Physiology, Laval University, Quebec, Quebec, Canada Magdalena S. Rumantir, M.D., Ph.D. Research Associate, Human Neurotransmitter Research Laboratory, Baker Medical Research Institute and Monash University, Melbourne, Victoria, Australia Lars Christian Rump, M.D. Professor of Medicine and Chief, Division of Nephrology, Department of Internal Medicine, Ruhr-University Bochum, Bochum, Germany Kyösti A. Sotaniemi, M.D., Ph.D. Associate Professor and Head, Department of Neurology, University of Oulu, Oulu, Finland Yvette Taché, Ph.D. Associate Director, CURE: Digestive Diseases Research Center, VA Greater Los Angeles Healthcare System, and Professor, Division of Digestive Diseases, Department of Medicine, and Brain Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Osvaldo M. Tiscornia, M.D., F.A.C.G. Consultant Professor, Department of Gastroenterology, University of Buenos Aires, and Chairman, Pancreatic Studies Program, José de San Martin Clinical Hospital, Capital Federal Buenos Aires, Argentina Uolevi Tolonen, M.D., Ph.D. Associate Professor, Department of Clinical Neurophysiology, University of Oulu, Oulu, Finland Mikko P. Tulppo, Ph.D. Head, Exercise Laboratory, Merikoski Rehabilitation and Research Centre, Oulu, Finland Ma-Li Wong, M.D. Professor, Department of Psychiatry and Biobehavioral Sciences, and Director, Laboratory for Pharmacogenomics, Neuropsychiatric Institute, David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Bulent Yildiz, M.D. Postdoctoral Fellow, Departments of Psychiatry and Medicine and Laboratory for Pharmacogenomics, Neuropsychiatric Institute, David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Richard E. Zigmond, Ph.D. Professor, Department of Neurosciences, Case Western Reserve University School of Medicine, Cleveland, Ohio, U.S.A.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

About the Editor

C. Liana Bolis is Professor of Comparative Endocrinology, Department of Pharmacological Science, University of Milan, Italy; Director of AIREN, Geneva, Switzerland; and Codirector of the Alert Bay Marine Laboratory, Cormoran Island, Canada. Dr. Bolis is the author, coauthor, editor, or coeditor of numerous journal articles, book chapters, and books. Dr. Bolis received the M.D. degree cum laude from the University of Milan, Italy, and several M.D. Honours Cause from the University of Marseille and Aix-en-Provence, France; the University of Dakar Medical School, Senegal; and the Moron University Medical School, Buenos Aires, Argentina. Dr. Bolis also received the Ph.D. Honours Cause in zoology from the University of Goteborg, Sweden, and the academic qualification of Honour Professor in neuroscience from the Beijing Medical University and from the Shanghai Medical University, P.R.C. Since 1996 she has been a member of the Pontifical Academia for life, Vatican City. Julio Licinio is Professor of Psychiatry and Biobehavioral Sciences and Medicine/Endocrinology, Director of the Interdepartmental Clinical Pharmacology Center and the Graduate Training Program in Translational Investigation, Associate Program Director of the General Clinical Research Center, and Codirector of the Laboratory of Pharmacogenomics, David Geffen School of Medicine at UCLA, Los Angeles, California. The author, coauthor, editor, or coeditor of numerous journal articles, book chapters, and books, he is the editor of The Pharmacogenomics Journal and Molecular Psychiatry. Dr. Licinio received the M.D. degree (1982) from the University of Bahia, Brazil. Stefano Govoni is Professor of Pharmacology and Chairman of the Department of Experimental and Applied Pharmacology, School of Pharmacy, University of Pavia, Italy. Dr. Govoni is the author, coauthor, or coeditor of numerous journal articles, book chapters, and books. Since 1974, he has worked in the field of neurobiology and neuropharmacology of brain aging. Dr. Govoni received the Ph.D. degree (1974) in pharmacology from the University of Milan, Italy.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

1 Structural and Chemical Organization of the Autonomic Neuroeffector System Geoffrey Burnstock Autonomic Neuroscience Institute, Royal Free & University College Medical School, London, United Kingdom

Within the last 30 years, new discoveries have changed our understanding of the organization of the autonomic nervous system. The structure of the autonomic neuroeffector junction and the multiplicity of neurochemicals that take part in the process of autonomic neuroeffector transmission are described, as well as cotransmission, neuromodulation, receptor expression, and trophic factors. Taking into consideration the distribution and colocalization of neurotransmitters and neuromodulators in autonomic nerves, it is clear that the terms adrenergic, cholinergic, purinergic, nitrergic, and peptidergic nerves should no longer be used, although reference to adrenergic, cholinergic, purinergic, nitrergic, or peptidergic neurotransmission is appropriate when describing a particular component of neural signaling. An outstanding feature of autonomic neurotransmission is the inherent plasticity afforded by its structural and neurochemical organization and the interaction between expression of neural mediators and environmental factors. In this way autonomic neurotransmission is matched to ongoing changes in demands and can sometimes be compensatory in pathophysiological situations. Selective neurochemical changes have been demonstrated in disorders of the autonomic nervous system. An understanding of the mechanisms governing the expression of neurotransmitters and their receptors will allow selective manipulation to promote beneficial changes for the therapeutic treatment of diseases that feature autonomic dysfunction.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

I. A.

FUNCTIONAL ANATOMY Autonomic Neuroeffector Junctions

The autonomic neuroeffector junction between autonomic nerve fibers and smooth muscle cells differs in several ways from the neuromuscular junction in skeletal muscle and from the synapses in the central and peripheral nervous systems (1–3). A major difference is that the autonomic effector is a muscle bundle rather than a single cell. Only a certain percentage of smooth muscle cells are directly innervated, and low-resistance pathways between individual muscle cells allow electrotonic spread of activity within the effector muscle bundle. Morphologically, the sites of electrotonic coupling are represented by gap junctions or nexuses (Fig. 1) (4). These gap junctions vary in size from punctate junctions to junctional areas of more than 1
m in diameter. Gap junctions are not static, but undergo a continual process of formation and removal.

Figure 1 Gap junction between two cultured smooth muscle cells (M1 and M2) from embryonic chick gizzard. A gap of up to about 30 Å can be seen between the outer leaflets of the unit membrane; there are a few short areas of fusion. The inner leaflets of the membranes are lined by an accumulation of electro-opaque material. Inset: High magnification (135,000) of a freeze-fracture preparation of a gap junction showing 80–90 Å particles on the A face and 30–40 Å pits on the B face. The hexagonal arrangement of the pits on the B face also shows some dislocations of the lattice. (From Ref. 4, with permission of Rockefeller University Press.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 A scanning electron micrograph of a single terminal varicose nerve fiber lying over smooth muscle of the small intestine of a rat. The intestine was pretreated to remove connective tissue components by digestion with trypsin and hydrolysis with hydrochloric acid. Scale bar  3
m. (From Ref. 5, with permission of Marcel Dekker, Inc.)

Another characteristic of the autonomic neuromuscular junction is that a synapse does not have a well-defined structure with pre- and postjunctional specializations like the skeletal muscle motor endplate. Unmyelinated, highly branched, postganglionic autonomic nerve fibers reaching the effector smooth muscle become beaded or varicose (Fig. 2). These varicosities are not static in their relationships to smooth muscle, consistent with the lack of postjunctional specialization. They are 0.5–2
m in diameter and about 1
m in length and are packed with vesicles and mitochondria. Neurotransmitters and neuromodulators from autonomic nerve fibers are released from these varicosities that occur at intervals of 5–10
m along axons. The minimum distance of the cleft between the varicosity and smooth muscle varies considerably depending on the tissue, from 20 nm in densely innervated structures such as the vas deferens to 1–2
m in large Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

elastic arteries. Thus pre- and postjunctional sites are particularly accessible to neuromodulatory influences, where local agents may reduce or increase the release of neurotransmitter or may alter the extent or time course of neurotransmitter action. During conduction of an impulse along an autonomic axon, neurotransmitter is released en passage from varicosities at variable distances from effector cells. A given impulse evokes release from only some of the varicosities that it encounters (6). Release of neurotransmitter causes a transient change in membrane potential of the postjunctional cell. If the result of a single pulse is a depolarization, the response is called an excitatory junction potential (EJP). EJPs summate and facilitate with repetitive stimulation, and upon reaching sufficient amplitude, the threshold for the generation of an action potential is reached, which results in mechanical contraction. If the result of a single pulse of neurotransmitter release is a hyperpolarization, the response is called an inhibitory junction potential (IJP). IJPs prevent action potential discharge in spontaneously active smooth muscle and thus cause relaxation. B.

Neurotransmitters

A neurotransmitter is a chemical substance released from nerves upon electrical stimulation which acts on specific receptors on adjacent effector cells to bring about a response, thus acting as a chemical messenger of neural activation. In early studies (7,8), acceptance of a substance as a neurotransmitter required satisfaction of the following criteria: 1) the presynaptic neuron synthesizes and stores the transmitter; 2) the transmitter is released in a calcium-dependent manner; 3) there should be a mechanism for terminating the activity of the transmitter, either by enzymatic degradation or by cellular uptake; 4) local exogenous application of the substance should mimic its effects following release due to electrical nerve stimulation; and 5) agents that block or potentiate the endogenous activity of the transmitter should also affect local exogenous application in the same way. Since the late 1960s, studies on autonomic neurotransmission have revealed a multiplicity of neurotransmitters in the autonomic nervous system (ANS). Neurally released substances, including monoamines, amino acids, neuropeptides, adenosine 5-triphosphate (ATP), and nitric oxide (NO), have been identified (see Table 1). Since NO does not conform to the constraints of the criteria outlined above, although it certainly acts as a rapid chemical messenger in the ANS, a reappraisal of the criteria for defining a neurotransmitter has been proposed (9), taking into account evidence for nonvesicular, Ca2+-independent release of some classical neurotransmitters (10) and the intracellular site of action of NO. The classical view of autonomic nervous control as antagonistic actions of noradrenaline (NA) and acetylcholine (ACh) causing either contraction or relaxation, depending on the tissue, was changed in the early 1960s when clear evidence of nonadrenergic, noncholinergic (NANC) neurotransmission was presented (see Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Table 1 Putative Neurotransmitters/Neuromodulators in the Autonomic Nervous System Noradrenaline (NA) Acetylcholine (ACh) Adenosine 5-triphosphate (ATP) and other nucleotides Nitric oxide (NO) Carbon monoxide (CO) 5-Hydroxytryptamine (5-HT) Dopamine (DA) -Aminobutyric acid (GABA) Glutamate (GLU) Neuropeptides Neuropeptide Y (NPY)/pancreatic polypeptide (PP) Enkephalin (ENK)/endorphin (END)/dynorphin (DYN) Vasoactive intestinal polypeptide (VIP) and related peptides PHI and PHM Pituitary adenylate cyclase-activating peptide (PACAP) Substance P (SP)/neurokinin A (NKA)/neurokinin B (NKB) Calcitonin gene-related peptide (CGRP) Somatostatin (SOM) Galanin (GAL) Gastrin-releasing peptide (GRP)/bombesin (BOM) Neurotensin (NT) Cholecystokinin (CCK)/gastrin (GAS) Angiotensin II (AII) Adrenocorticotrophic hormone (ACTH) Secretoneurin Endothelin (ET)

Refs. 2, 11). IJPs blocked by tetrodotoxin were recorded in intestinal smooth muscle during stimulation of guinea pig enteric nerves in the presence of adrenergic and cholinergic blocking agents (12). At about the same time, other researchers showed that relaxation of the cat stomach following vagal stimulation was resistant to adrenergic and cholinergic blockade (13). Similar observations were subsequently made in a wide variety of tissues, including urinary bladder, lung, esophagus, seminal vesicles, trachea, and blood vessels. In 1970, the purine nucleotide ATP was proposed as a principal transmitter in NANC neurotransmission (14,15). The recent identification of the molecular structure of purine receptor subtypes has reinforced the concept of purinergic neurotransmission (see Refs. 16, 17). Ultrastructural studies of the enteric nervous system offered the first suggestion that there were several different neurotransmitters in autonomic nerves: at least nine distinguishable types of axon profile were described in the guinea pig myenteric plexus (18). Subsequently, using the then newly available immunohistochemical techniques, several biologically active peptides were localized in Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

neural elements of the gut (19). In addition to the many polypeptides, 5-hydroxytryptamine (5-HT), dopamine, and -amino butyric acid (GABA) were proposed as autonomic neurotransmitters (see Refs. 20, 21). More recently, NO has been added to the list of neurotransmitters in the autonomic nervous system (see Refs. 22–24). The rapid expansion of the number of proposed autonomic neurotransmitters in recent years, including endothelin (25,26), secretoneurin (27,28), pituitary adenylate cyclase-activating peptide (PACAP), which is similar in structure to vasoactive intestinal polypeptide (VIP) (29), glutamate (30), and carbon monoxide (31,32), makes it likely that the list is still incomplete. 1.

Noradrenaline

The synthesis of NA is catalyzed by three enzymes, tyrosine hydroxylase (TH), L-dopa decarboxylase, and dopamine--hydroxylase (DBH). The rate-limiting enzyme, TH, requires tyrosine, oxygen, and the cofactor tetrahydrobiopterin and is subject to multiple regulatory mechanisms mediated by phosphorylation by protein kinases in addition to regulation at the level of gene transcription (33,34). NA exists in the neuronal cytosol but is stored in small and large dense core vesicles together with other cotransmitters, chromogranins, and DBH. Thus, in sympathetic neurones, the vesicles are involved in not only storage and release of NA, but also with the last stage of its synthesis. Following electrical stimulation, the vesicular contents are released by exocytosis, in a Ca2+-dependent manner, into the extracellular space (35). After interaction with specific receptors, the action of NA is rapidly terminated by reuptake into the nerve varicosity or into nonneuronal cells, where it is metabolized by the intracellular enzymes, monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) (see Ref. 36). NA produces a variety of effects by interaction with a number of different adrenoceptor subtypes. Several subtypes of 1-, 2-, and -adrenoceptors have now been characterized and cloned (37). These receptors have different effector mechanisms via heterotrimeric guanosine-5-triphosphate (GTP)–binding proteins (G proteins). Generally, 1-adrenoceptors are coupled with Go and stimulation of phospholipase C and inositol trisphosphate (IP3), 2-adrenoceptors are coupled with Gi and inhibition of adenyl cyclase and reduced cyclic AMP levels, and 1-, 2-, and 3-adrenoceptors are coupled with Gs and activation of adenyl cyclase and increased cyclic AMP levels (38). There are many regulatory systems inherent to the adrenergic machinery, including autoinhibition of NA release via presynaptic 2 receptors, regulation of NA synthesis, and adrenoceptor desensitization and supersensitivity dependent on agonist exposure (for review see Ref. 36). A schematic of noradrenergic transmission is presented in Figure 3A. 2.

Acetylcholine

The synthesis of ACh from choline and acetyl coenzyme A is catalyzed by choline acetyltransferase (ChAT) and takes place in the neuronal cytoplasm. ACh is then Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

pumped into small agranular vesicles, which have a specific ACh transporter in their membranes, and stored until Ca2+-dependent exocytotic release upon electrical stimulation. The vesicular ACh transporter has been characterized at the molecular and functional level (39). The observation that the transporter gene is contained entirely within the first intron of the gene that encodes ChAT opens up interesting possibilities of coordinated regulation of these two proteins (40). ACh released during neurotransmission is inactivated by hydrolysis due to the action of acetylcholinesterase (AChE), which is localized on both pre- and postsynaptic membranes. The choline that results from this breakdown is recycled by transport back into the nerve varicosities by a metabolically driven high-affinity choline uptake mechanism for resynthesis and vesicular storage of ACh. Choline uptake into the presynaptic terminal is the rate-limiting factor for ACh synthesis (41). A schematic of cholinergic autonomic neurotransmission is presented in Figure 3B. ACh acts on two different classes of receptors. Nicotinic receptors are ionotropic receptors consisting of subunits that constitute multimeric ligand-gated Na channels, which mediate fast responses. There are four different polypeptide chains arranged as a pentamer (2 ): these were the first neurotransmitter receptors to be cloned (42). In the autonomic nervous system, nicotinic receptors (subtype N2) are mainly found within ganglia. In contrast, muscarinic receptors are metabotropic receptors coupled with G proteins, with slower responses, and are widespread throughout autonomic effector tissues and smooth muscle. Five distinct subtypes of muscarinic receptor have now been cloned, and three (M1, M2, and M3) have been pharmacologically characterized (43). Generally, M1 receptors facilitate ganglionic transmission after nicotinic stimulation, M2 receptors exert a prejunctional negative feedback on transmitter release on parasympathetic and sympathetic neurones, and M3 receptors cause contraction of visceral smooth muscle, secretion from exocrine glands, and release of NO from the endothelium. They have different signal transduction mechanisms: M1 and M3 occupation leads to stimulation of phospholipase C and increased IP3 levels and M2 occupation leads to inhibition of adenyl cyclase, reduced cyclic AMP levels, and changes in K and Ca2+ conductance (44). 3.

ATP

The purine nucleotide ATP was the first substance that was found to best satisfy the criteria for a neurotransmitter in NANC nerves (14,15). There is now substantial evidence to support the original hypothesis for purinergic neurotransmission and mechanisms of storage, release, and inactivation of ATP (see Refs. 16, 45, 46) as proposed by Burnstock in 1972 (15) (see Fig. 3C). ATP synthesized in nerve terminals is stored in vesicles, often colocalized with other neurotransmitters. After its release and activation of purine receptors, ATP is rapidly broken down to adenosine by Mg2+-activated ATPase (a ubiquitous membrane-bound ectoATPase) and ecto-5-nucleotidase located at sites of ATP release. Ecto-5-nuCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

A

B

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

C Figure 3 Simplified schematic representation of synthesis, storage, release, receptor activation, and neurotransmitter inactivation at neuromuscular junctions: (A) noradrenergic neurotransmission; (B) cholinergic neurotransmission; (C) purinergic neurotransmission. (Modified from Ref. 15, with permission of Lippincott, Williams and Wilkins.)

cleotidase is strongly inhibited by ADP. Adenosine is transported into neurons and nonneuronal cells via a nucleoside carrier high-affinity uptake system and either phosphorylated to ATP and reincorporated into physiological stores or broken down by adenosine deaminase to inosine, which is inactive and leaks into the circulation. In addition to ATP, there is now evidence that small amounts of other nucleotides such as ADP, AMP, GTP, UTP, and diadenosine polyphosphates are stored in synaptic vesicles and may play neuromodulatory roles in signaling in the nervous system (see Ref. 47). Based on the relative potencies of purine nucleosides and nucleotides and second messenger systems on a variety of tissues, two major types of purine receptor were distinguished (48). P1 receptors are most sensitive to adenosine and are competitively blocked by methylxanthines. P2 receptors are most sensitive to ATP, and their occupation may lead to prostaglandin synthesis. Pharmacological, biochemical, receptor binding, and, more recently, cloning studies have enabled subdivision of these two types of receptor. There are four subtypes of P1 receptors, namely A1, A2A, A2B, or A3 subtypes. A1 receptors are preferentially actiCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

vated by N6-substituted adenosine analogs, and their occupation leads to decreased cyclic AMP levels, whereas A2 receptors show preference for 5-substituted compounds and cyclic AMP levels are increased; occupation of A3 receptors does not lead to changes in adenylate cyclase. Selective agonists and antagonists for these P1 receptor subtypes have been identified (49). Following expression cloning, transduction mechanism studies and the use of newly synthesized agonists and antagonists, in keeping with other neurotransmitters, P2 receptors have been divided into two major families, a P2X receptor family, which are ligand-gated ion channel receptors mediating fast transmission, and a P2Y receptor family, which are G protein–coupled receptors mediating slower responses (50,51). Currently, seven P2X (P2X1–7) subclasses and six P2Y (P2Y1,2,4,6,8,11) subclasses have been recognized (see Ref. 17). These incorporate the receptors that respond to the pyrimidine derivative UTP as well as to ATP and also receptors that respond to adenine dinucleotide polyphosphates (16,52). 4.

Neuropeptides

Peptides involved in neurotransmission in the autonomic nervous system are a large and diverse group (Table 1). Like the classical neurotransmitters, they are stored in vesicles and are released on depolarization to act on specific receptors to produce an effector response. However, by virtue of their structure, there are important differences from classical neurotransmission in their mode and site of synthesis and in their inactivation after release: namely, they are synthesized and packaged into vesicles in the nerve cell body rather than in nerve varicosities, and there are no mechanisms for reuptake and recycling of neuropeptides after receptor activation (53). Neuropeptides are stored in large electron-dense cored vesicles and released by exocytosis. The regulation of neuropeptide neurotransmission is quite different from the classical neurotransmitters as replacement of neuropeptides after release is dependent on new synthesis in the nerve cell body and axonal transport, which are relatively slow processes compared to local synthesis in nerve terminals by enzymatic activity and replacement by efficient reuptake mechanisms. Neuropeptide release is more easily exhausted by repeated or prolonged stimulation. There is no known reuptake mechanism for removal of neuropeptides from the site of action; their action is terminated by internalization and degradation of the receptor-bound peptide but mainly by metabolism by proteolytic enzymes (54). A few key ectoenzymes, including endopeptidase 24.11 and angiotensinconverting enzyme, are thought to account for the degradation of most neuropeptides. The regulation of expression of these ectopeptidases is another level of modulation of peptidergic neurotransmission. Neuropeptide receptors are G protein–coupled receptors, which activate either adenyl cyclase or phospholipase C as signal tranducers. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

5.

Nitric Oxide

NO has only recently been added to the list of putative neurotransmitters in the autonomic nervous system. The nature of these molecules and their actions means that it has been necessary to amend the criteria for defining a neurotransmitter. NO is not stored within neurons since it can travel freely through membranes. Furthermore, NO does not act on extracellular receptors on the postjunctional membrane of the target, but rather at intracellular sites (see Fig. 4) (55).

Figure 4 Current model for the nitrergic neurotransmission process. The arrival of an action potential at the terminal region opens voltage-operated Ca2+ channels, allowing calcium to enter the neuronal cytoplasm and activate NOS. The enzyme converts L-arginine to L-citrulline, with the concomitant production of NO. The NO rapidly diffuses out of the nerve cell, across the gap, and into the postjunctional cell (usually smooth muscle), where it binds to the heme group of soluble guanylate cyclase and consequently activates the conversion of GTP to cyclicGMP. Nitrergic transmission may be inhibited by -conotoxin ( CgTx; inhibits calcium channel), L-NG-monomethyl-arginine and L-NG nitro-arginine (LNMMA and L-NOARG; inhibit NOS), hemoglobin (traps NO in the junctional gap), or 1 H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; inhibits soluble guanylate cyclase). Exogenous NO mimics the relaxant effect of nitrergic stimulation and is similarly inhibited by the presence of hemoglobin. Several substances (superoxide anions; hydroquinone; carboxy-PTIO) strongly inhibit the relaxation to exogenous NO but have little effect on nitrergic responses, raising doubts about whether NO is released from the nerve as a free radical or in some other form. (From Ref. 55, with permission from Elsevier Science BV.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

NO is synthesized in a reaction in which L-arginine is converted to L-citrulline by nitric oxide synthase (NOS). The reaction is dependent on NADPH, which is a cosubstrate with molecular oxygen. NOS can exist in three main isoforms (Types I–III). Type I is constitutively expressed and was first identified in neurons (see Refs. 24, 56). Immunohistochemical studies have shown that Type I NOS is present in a variety of autonomic neurons (see Refs. 22, 24, 56). Type I NOS only synthesizes small amounts of NO during conditions of raised intracellular Ca2+ such as those that occur during an action potential. This is of fundamental importance to the function of NO as a neurotransmitter. It is the synthesis of NO that is stimulated during transmission by a Ca2+-dependent mechanism, not its release from intracellular stores (24,56–58). Analogs of Larginine compete with L-arginine for binding of NOS and have been used as inhibitors of NOS in pharmacological studies. Such analogs have been instrumental in demonstrating NO-dependent responses in autonomic transmission (56,59). Once NO has been synthesized, it can diffuse freely through membranes to the postjunctional target. Being a free radical, NO is unstable. Thus, for ending NO-dependent responses, there is no need for the mechanisms such as degradative enzymes or reuptake that are required for other neurotransmitters. NO binds readily to the heme group of hemoglobin, which can thus inhibit NO-dependent responses (60). Similarly, free radical generators such as hydroquinone and pyrogallol are frequently used to inhibit nitrergic transmission by the ability of free radicals to react with and inactivate NO. Conversely, superoxide dismutase, which removes superoxide anions, can enhance NO-dependent responses (22,56). Due to its instability, NO release on autonomic nerve stimulation is difficult to establish directly. However, using a superfusion bioassay or chemiluminescence, NO release has been demonstrated during autonomic neuromuscular transmission in the gastrointestinal and urogenital tracts (see Ref. 58). A variety of substances, including sodium nitroprusside, S-nitroso-N-penicillamine, and sydnonimins such as SIN-1 can release NO (61). Such agents have been used as NO donors together with free NO to demonstrate that exogenous application of NO can mimic a nerve-mediated response. It has been suggested that during transmission NO may be released as a nitrosothiol rather than in its free form (62); however, this is still controversial (see Ref. 58). In autonomic transmission, NO produces its effects in the target predominantly by its interaction with intracellular guanylate cyclase (63,64). 6.

Other Neurotransmitters

5-HT is an indolamine synthesized from tryptophan via 5-hydroxytryptophan by two enzymes, tryptophan hydroxylase and L-aromatic amino acid decarboxylase. Neuronal synthesis of 5-HT has been demonstrated in myenteric neurons (20), but it is often regarded as false neurotransmitter as it is taken up and employed as a neurotransmitter by sympathetic nerves (65–67). 5-HT is taken up into small, Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

clear and large granular vesicles and is released in a Ca2+-dependent manner. After release it is catabolized by MAO-A to 5-hydroxyindoleacetaldehyde and subsequently to 5-hydroxyindoleacetic acid. There are multiple 5-HT receptors (5-HT3 ionotropic; 5-HT1,2,4,5,6,7 metabotropic) in sympathetic, parasympathetic, and sensory ganglia (see Ref. 21). GABA, glutamate, and dopamine, classical neurotransmitters in the central nervous system, are also autonomic neurotransmitters (21,30,68). GABA plays a role in enteric neurotransmission via excitatory GABAA and prejunctional inhibitory GABAB receptors. The GABA synthesizing and catabolizing enzymes (glutamate decarboxylase and 4-aminobutyrate-2-oxoglutarate transaminase, respectively), GABA itself, and high-affinity GABA uptake sites have all been localized in gastrointestinal tissue (21,69,70). After convincing evidence that NO can act as a neurotransmitter was presented, it was soon proposed that carbon monoxide (CO) could behave in an analogous way to NO as a neuronal messenger (see Ref. 71, 72). While there are some striking similarities between the synthesis of NO and CO and their effects, full and unequivocal evidence for a neurotransmitter for CO is still largely lacking (see Refs. 32, 73, 74). There is recent evidence for endothelin in perivascular nerves in cerebral blood vessels (26). C.

Cotransmission/Neuromodulation/Chemical Coding

Since the early 1970s, new concepts in neurotransmission including cotransmission and neuromodulation have been introduced (2,57,75–82). 1.

Cotransmission

The concept of cotransmission was first formulated by Burnstock in 1976, incorporating hints in the earlier literature for both vertebrate and invertebrate systems. Immunohistochemical evidence of coexistence of more than one neurotransmitter should not necessarily be interpreted as evidence of cotransmission, since in order for substances to be termed cotransmitters it is essential to show that postjunctional actions to each substance occur via their own specific receptors (77). For example, many neuropeptides have slow trophic actions on surrounding tissues, and this may be their primary role (see Ref. 83), or they may act as neuromodulators (84). The relative contribution of each transmitter to neurogenic responses is dependent on the parameters of stimulation. For example, short bursts (1 s) of electrical stimulation of sympathetic nerves at low frequency (2–5 Hz) favor ATP release, whereas longer periods of nerve stimulation (30 s or more) favor NA release (85). Peptides, purine nucleotides, and NO (identified by localization of NOS) are often found together with the classic neurotransmitters NA and ACh. In fact, the majority, if not all, of nerve fibers in the autonomic nervous system contain a mixture of different neurotransmitter substances that vary in proportion in different tissues Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

and species and during development and disease. The widespread use of double and triple immunohistochemical labeling techniques has been critical to the demonstration of colocalization of potential cotransmitters within the same nerve fiber and has been invaluable when combined with electron microscopy. Different neurotransmitters within the same varicosity may be localized in the same or separate vesicular populations using postembedding colloidal gold techniques. In the gastrointestinal tract, many neurons contain multiple neuropeptides (86). ATP is a cotransmitter with calcitonin gene–related peptide (CGRP) and substance P (SP) in many sensorimotor nerves (87) and ATP, NO, and vasoactive intestinal peptide (VIP) in enteric NANC-inhibitory nerves (88). Transmitters with seemingly diverse and opposing effector action are sometimes colocalized in the same neurone, but generally they act in the same way and usually synergistically. 2.

Neuromodulation

Some substances stored and released from nerves do not have direct actions on effector muscle cells but alter the release and/or the actions of other transmitters; these substances are termed neuromodulators. Many other substances (e.g., circulating neurohormones, locally released agents such as prostanoids, bradykinin, histamine, and endothelin, and neurotransmitters from nearby nerves) are also neuromodulators in that they modify the process of neurotransmission. Many substances that are cotransmitters are also neuromodulators. The wide and variable cleft characteristic of autonomic neuroeffector junctions makes them particularly amenable to the mechanisms of neural control mentioned above. The many different ways in which cotransmitters and neuromodulators interact to effect neurotransmission have been described previously (89) and are outlined below and in Figure 5. 1.

Autoinhibition: A transmitter, in addition to its postjunctional effects, reduces its own release via prejunctional receptors; release of cotransmitters may also be inhibited (Fig. 5A). 2. Cross-talk: A neuromodulator may act on closely juxtaposed terminals (Fig. 5B). 3. Synergism: Each of two transmitters, from either different nerve terminals or cotransmitters, have the same postjunctional effect so that there is a reinforcement of their individual effects (Fig. 5C,D). 4. Opposite actions: Occasionally, a transmitter may have opposite actions in different postjunctional effector cells; sensitivity of the responses of cotransmitters depends on the tone of the effector cell (Fig. 5E,F). 5. Prolongation of effect: A neuromodulator may act on degradative enzymes (e.g., peptidases responsible for removal of neuropeptides from the junctional cleft) to prolong the time course of their effect. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 5 Schematic representation of different types of interactions between two neurotransmitter substances, X and Y. Examples of: (A) autoinhibition, where, in addition to the neurotransmitter (X) acting postjunctionally to either contract () or relax () the muscle, it acts on prejunctional receptors (usually of a different subclass) to form a negative feedback system that inhibits release of the transmitter; (B) cross-talk, where transmitters X and Y in separate varicosities not only act on receptors in the muscle but also on prejunctional receptors on each other’s terminals to modulate transmitter release; (C) synergism, where transmitters X and Y in separate varicosities have the same contractile () action on the muscle cell and potentiate each other’s action by the process of postjunctional neuromodulation; (D) synergism, where transmitters X and Y, released as cotransmitters from a single varicosity, potentiate each other’s action on the postjunctional effector; (E) opposite actions, where cotransmitters X and Y have opposite actions on different effector sites; and (F) opposite actions depending on the tone of the effector tissue. (From Ref. 89, with permission of Lippincott, Williams and Wilkins.) Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

6.

Trophic effects: A neurotransmitter may effect the expression of another transmitter or receptor within a population of neurons (e.g., in ganglia) at the level of gene transcription.

All these mechanisms of control of neurotransmission reflect the versatility of the peripheral components of the autonomic nervous system. 3.

Chemical Coding

The precise combinations of neurotransmitters (and neuromodulators) contained in individual neurons and their projections and central connections, termed their “chemical coding,” has been defined in studies of the enteric nervous system (19,90) and peripheral autonomic and sensory ganglia (82,91). For example, in guinea pig sympathetic ganglia, which innervates a variety of tissues in the head, pilomotor, vasoconstrictor, vasodilator, and different classes of secretomotor neurons, can be distinguished from each other by their peptide content (92). The specificity of innervation even extends to different levels of the arterial tree; large arteries supplying the head contain NA and neuropeptide Y (NPY), small arteries and arterioles to the ear contain NA, NPY, -neo-endorphin, dynorphin (DYN) A1–8, and DYN B, whereas those to arteriovenous anastomoses are similar to arterioles but contain no NPY (92). There is considerable variation in chemical coding between species, but some aspects appear to be constant; e.g., NA, ATP, and NPY are found in sympathetic nerves and in the enteric nervous system, excitatory motor neurons always utilize ACh and tachykinins as transmitters (although other substances within these neurons vary), while ATP, NO, and VIP are found in most NANCinhibitory nerves. The chemical codings in the major component of the nervous system are summarized in Figure 6. a. Sympathetic Nerves. It is now recognized that the main neurotransmitters/neuromodulators in postganglionic sympathetic nerves are NA, ATP, and NPY (57,77,93–96). These substances are coreleased in varying proportions, depending on the tissue and species and also on the parameters of stimulation (97). Short bursts at low frequency particularly favor the purinergic component, whereas longer periods of nerve stimulation favor the adrenergic component, and NPY release is optimal with high-frequency intermittent bursts of stimulation (85,98). A considerable variability in the contribution of a purinergic component to sympathetic neurotransmission has been demonstrated in different blood vessels. For example, rabbit saphenous and mesenteric arteries have a substantial purinergic component, whereas in the rabbit ear artery the purinergic component is relatively small (77). In submucosal arteries the responses to sympathetic nerve stimulation are mediated solely by ATP, with NA acting as a prejunctional modulator via 2-adrenoceptors (99,100). The initial electrophysiological postjunctional response to sympathetic nerve stimulation is a rapid, transient EJP, which is mediated by ATP (see Refs. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 6 Schematic representation of the principal cotransmitters in autonomic and sensory nerve varicosities and in CNS and retinal nerve terminals. (From Ref. 79 with permission of Elsevier Science, Inc.)

16, 96). In some vessels the EJP is followed by a slow depolarization, which is mediated by NA. The mechanical constrictor response to sympathetic stimulation can be biphasic, as in the vas deferens, or monophasic, as in most blood vessels (96). Postjunctionally, the effects of ATP and NA released as sympathetic cotransmitters are generally cooperative, since both typically act as smooth muscle constrictors and usually act synergistically (101). NA and ATP also act cooperatively following sympathetic stimulation of coronary arteries, where the response is vasodilatation rather than vasoconstriction: here NA acts on -adrenoceptors and ATP acts on P2Y receptors located on the smooth muscle (102). NA and ATP can depress sympathetic neurotransmission by prejunctional modulation, NA via 2-adrenoceptors and ATP predominantly via P1 receptors following extracellular breakdown to adenosine, but also via P2 receptors in some vessels (103). Prejunctional P2 receptor–mediated increase in NA release has been reported in the rabbit ear artery. In most tissues, including the vas deferens and many blood vessels, NPY does not act as a genuine neurotransmitter, having little direct postjunctional action, but rather acts as a neuromodulator, often by prejunctional attenuation of NA and ATP release and/or postjunctional potentiation of responses to adrenergic and purinergic components of sympathetic nerve responses (57,104). It has been suggested that the mode of neuromodulation by NPY is related to the width of the junctional cleft. A narrow cleft (20 nm; e.g., vas deferens) favors prejunctional modulation of release of cotransmitters. A medium-sized cleft (100–500 nm; most blood vessels) favors postjunctional modulation when concentrations of NPY are low; subsequently, as NPY concentrations increase, prejunctional modulation can also occur. A wide cleft (1000–2000 nm; large elastic arteries) favours postjunctional modulation (77). In Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

tissues where NPY does have a direct vasoconstrictor effect, for example, in blood vessels of the spleen and kidney and coronary and cerebral arteries, the response is characteristically slow in onset and is long-lasting (105). Other substances localized within sympathetic nerves include 5-HT, which is largely taken up by sympathetic nerves and released as a false transmitter (66). Opioid peptides are also widely distributed in sympathetic neurons (92), where their functional role appears to be related to their prejunctional inhibitory effects on sympathetic neurotransmission. Sympathetic nerves can interact with both sensorimotor and parasympathetic nerves in close proximity. The activity of sensorimotor nerves is subject to prejunctional inhibitory neuromodulation by the sympathetic cotransmitters, NA and NPY, and by adenosine, following breakdown of released ATP (106). Opioids also elicit inhibitory effects on sensorimotor nerves. Prejunctional inhibitory effects of NPY on cholinergic neurotransmission may explain the prolonged attenuation of the action of the vagus in controlling heart rate following sympathetic stimulation. b. Parasympathetic Nerves. ACh, VIP, ATP, and NO are cotransmitters commonly synthesized in and released from parasympathetic nerves (22,57,107). As with sympathetic cotransmission, the relative importance of the cotransmitters to the functional responses to parasympathetic neurotransmission in different tissues and species is variable. For example, NO may be the main mediator of neurogenic vasodilatation in cerebral vessels, whereas VIP may be of more importance during neurogenic vasodilatation in the pancreas (108,109). The coordinated roles of VIP and ACh in parasympathetic neurotransmission were demonstrated in an elegant study of the cat exocrine salivary gland innervation by Lundberg (107). This showed that VIP and ACh were stored in separate vesicles in the same nerve terminal and were both released upon transmural nerve stimulation but with different stimulation parameters. ACh was released during low-frequency stimulation to increase salivary secretion from acinar cells and to elicit some minor dilatation of blood vessels in the gland. At high stimulation frequencies, VIP was released to produce marked dilatation of the blood vessels in the gland and to act as a neuromodulator postjunctionally on the acinar gland to enhance the actions of ACh and prejunctionally on the nerve varicosities to enhance the release of ACh. ACh was also found to have an inhibitory action on the release of VIP. VIP has since been shown to have a direct vasodilator action in the submandibular gland in humans. PACAP also seems to be present in VIP-containing parasympathetic nerves. Two classes of VIP receptor with different affinity and specificity have been described. PHI (peptide with N-terminal histidine and C-terminal isoleucine) and PACAP also interact with these receptors (see Ref. 57). NOS is often colocalized with ACh and VIP in parasympathetic nerves innervating blood vessels; NO and VIP have been proposed as the fast and slow components, respectively, of neurogenic vasodilatation (91). Evidence for NO enhancement of sympathetic Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

neurotransmission opens up the possibility of parasympathetic modulation of sympathetic activity. Parasympathetic cotransmission involving NO and VIP may be particularly important in cerebral vessels, where VIP- and NOS-containing nerve fibers originate from the cranial parasympathetic ganglia, in particular, from the sphenopalatine ganglion (110). The anterior vessels of the circle of Willis receive a more dense innervation of VIP-containing nerve fibers than those in the posterior circulation (111). Postganglionic nerves from pelvic ganglia containing VIP, ACh, and NOS project to the urethra, colon, and penis. The human bladder body receives a dense parasympathetic innervation comprised predominantly of ACh-containing nerves. In the rodent bladder, ATP is a cotransmitter in these nerves (112); however, pharmacological studies have demonstrated only a small purinergic component in response to electrical field stimulation in human bladder (113), except in pathological conditions (see later). There are several reports that some cranial, ciliary, and paracervical parasympathetic ganglia that supply VIP/ACh-containing nerves to cerebral arteries, iris, and uterine artery also contain NPY (111). Dopamine--hydroxylase, DYN, somatostatin, and CGRP have also been localized in guinea pig paracervical ganglia (114,115). c. Sensorimotor Nerves. The tachykinins SP and neurokinin A and CGRP are the principal cotransmitters in unmyelinated, primary afferent nerves. They often coexist in the same large granular vesicles in capsaicin-sensitive nerve terminals (105). The level of coexistence of SP and CGRP varies with species; for example, in the guinea pig most sensory neurons containing CGRP also contain SP, but in the rat about 50% of CGRP-containing neurons do not contain SP (105). In human beings, SP and other tachykinins have been shown to coexist with CGRP in sensory neurones. Capsaicin is uniquely selective for vanilloid receptors localized on the membrane of unmyelinated primary afferent nerves, causing dose-dependent depolarization, desensitization, and neurotoxicity (116); consequently, this drug has been used extensively to investigate sensory components of the ANS. ATP is now also established as a cotransmitter in small primary sensory nerves mediating mechanical and/or nociceptive signals (117). ATP is released from sensory nerves during antidromic stimulation of the great auricular nerve to bring about vasodilatation of the ear artery (118) and has more recently been shown to be released with CGRP during electrical field stimulation of capsaicinsensitive sensory nerves to bring about vasodilatation of small arteries of the mesenteric bed (119). The motor function of sensory nerves, whereby antidromic impulses result in local release of sensory neurotransmitters, is widespread in autonomic effector systems and forms an important physiological component of autonomic control (87,120). To distinguish these nerves from the other subpopulation of afferent fibers that have an entirely sensory role and have terminals containing few vesiCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

cles and a predominance of mitochondria, they have been termed sensorimotor nerves (1). Chronic capsaicin treatment leads to degeneration of small afferent nerves and a marked loss of SP- and CGRP-containing nerves from most tissues of the cardiovascular system, urinogenital system, and airways (105). In the vasculature, SP does not appear to act directly on receptors of the vascular smooth muscle, but rather acts via occupation of receptors on endothelial cells at the lumen to bring about NO release and consequent vasodilatation. This action of neurally released SP may be particularly important in the microvasculature, but access of neurally released SP to the endothelium in large vessels is questionable where it is largely released from endothelial cells (121,122). SP is also a mediator of neurogenic inflammation; its actions include plasma extravasation, histamine release from mast cells, and recruitment and/or stimulation of inflammatory cells. CGRP too has some pro-inflammatory actions (57). Other neuropeptides and transmitters have been localized in sensorimotor nerves. In the human urinary bladder, VIP, cholecystokinin (CCK), and DYNs are present together with SP and CGRP in the afferent projections to the lumbosacral spinal cord. These neuropeptides have also been localized in dorsal root and trigeminal ganglia neurons of several species. In the guinea pig, dorsal root ganglia neurons containing SP, CGRP, CCK, and DYN project to the epidermis and small dermal blood vessels, those containing SP, CGRP, CCK, but not DYN, project to small blood vessels in skeletal muscle, and those containing SP, CGRP, and DYN mostly supply the pelvic viscera: neurons with SP and CGRP only project mainly to the heart, large arteries, and veins (123). NOS has been localized in populations of primary sensory neurons of trigeminal and dorsal root ganglia (124). Endothelin, a potent vasoconstrictor peptide with mitogenic actions, is also localized in neurons of these sensory ganglia, often colocalized with SP (125,126). There are increasing examples in the literature of cross-talk between sensorimotor sympathetic, and parasympathetic nerves. In the heart, SP has excitatory effects on cardiac parasympathetic innervation, in contrast to CGRP, which is inhibitory (127). d. Intrinsic Nerves. Many intrinsic neurons localized within autonomic neuroeffector tissues are part of the postganglionic parasympathetic system, but there are also intrinsic neurons derived from neural crest tissue, which differs from the tissue that forms sympathetic and parasympathetic neurons. Intrinsic neurons are abundant in the ANS, where they have been demonstrated by persistence after extrinsic denervation, and by tissue culture techniques in the heart, bladder, airways, pancreas, kidney, and gastrointestinal tract. The most extensive system of intrinsic neurons is in the myenteric and submucous plexuses of the gastrointestinal tract. These enteric neurons contain numerous neuroactive substances, of which the majority are involved with neuromodulation at the ganglion level and/or have a trophic role; only a small percentage are involved in neuromuscular transmission (128). The chemical coding of enteric neurons has been examined in detail, particularly in the guinea pig (19,90). Some enCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

teric neurons project from the intestine to innervate the mesenteric arteries and arterioles of the colon; however, most form a complex network of interconnections between the two major plexuses and the mucosa. The motor neurons for the circular muscle are located entirely in the myenteric plexus, whereas most of those for the mucosa are located in the submucosal ganglia. Intrinsic submucosal neurons, by regulating neurogenic vasodilatation of submucosal arterioles, are involved in local physiological control of mucosal blood flow (129). Enteric motility reflexes stimulated by localized distension of the intestine or mechanical or chemical stimuli can be initiated through entirely intrinsic mechanisms. Definitive identification of intrinsic primary afferent neurons has recently been established by immunohistochemistry and by intracellular recordings in response to stimuli (130–132). ATP, NO, and VIP mediate NANC-inhibitory neurotransmission in the gut in varying proportions depending on the region: VIP is claimed to be the prominent inhibitory transmitter in the stomach, while ATP is the main transmitter in the intestine. As noted earlier, ATP is a fast neurotransmitter, producing fast IJPs, NO elicits slower IJPs, while VIP produces slow tonic relaxations. ACh and SP are cotransmitters in enteric excitatory neurones (see Ref. 128). There are many intrinsic neurons in the heart, particularly in the right atrium, where guanethidine sympathectomy leads to a depletion of only 46% of the tissue content of NPY in adult rats (133). NPY-containing intrinsic nerves do not innervate the coronary arteries but may innervate the small resistance vessels (134). The neurochemical makeup of the intrinsic cardiac ganglia is heterogeneous and includes a variety of neurochemical markers, often in addition to ChAT (135). Subpopulations of atrial intrinsic neurons from newborn guinea pigs immunostain for NPY, 5-HT, heme oxygenase-2, and NOS, and there are indications that these neurons also utilize ACh and ATP (136–138). Ganglion cells from adult guinea pig atrial preparations that immunostain for NPY do not immunostain for TH, unlike the majority of NPY-containing nerve fibers that envelope the intrinsic neurones (139). Intrinsic cardiac neurons also express a range of receptors for classical transmitters and neuropeptides, demonstrating their potential for complex interactions with extrinsic nerves supplying the heart (127,140). Intramural ganglia within the tracheobronchial tree show a high degree of electrophysiological specialization, which would support a role in integration or modulation of extrinsic neural input (141). NOS-containing nerve fibers originating from intrinsic neurons have been described in human trachea (142). Most airway intrinsic neurons contain ChAT, but NOS and VIP are also found in these neurons in human beings (143): their cholinergic characteristics indicate that these may be part of the parasympathetic system. Intrinsic ganglia in the human urinary bladder wall contain a number of neuroactive substances (VIP, NOS, NPY, galanin, and occasionally TH) but are devoid of other selected neuropeptides, enkephalin (ENK), SP, and CGRP (144); in the bladder neck, only very few intrinsic neurons contain ENK and SP (145). Intramural ganglia containing NPY and VIP have been identified in human urethra (146). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

While it is well established that intrinsic nerves in the gut can sustain local reflex activity, independent of the central nervous system (i.e., they are not simple mesenteric relay stations), it remains to be resolved whether similar local reflexes operate in other visceral organs such as the heart, lung, and bladder. D.

Plasticity of the ANS During Physiological Events

The particular combination and quantity of neurotransmitters/neuromodulators expressed by autonomic neurones is partly preprogrammed and partly determined by the influence of molecules of the neural environment, such as trophic factors and hormones, that modulate the expression or suppression of the appropriate genetic machinery. During a normal lifespan, there is a constant potential for these factors to change, e.g., during growth and during pregnancy. The plasticity of expression of neural substances coordinated to environmental cues allows rapid and precise matching of neurotransmission to altered demands. Several neurotransmitters/neuromodulators are themselves trophic molecules, with mitogenic or growth-promoting/-inhibiting properties (see Ref. 83). 1.

Development

Developmental changes in the density of NA -and NPY-containing nerve fibers supplying the basilar artery of the young rat do not proceed in synchrony, even though these substances are coexpressed: increased expression of NA occurs between 4 and 6 weeks, while increased expression of NPY occurs later, at 6–8 weeks (147). Vascular developmental innervation patterns vary considerably with the location and species. For example, in guinea pig mesenteric and carotid arteries there are large increases in innervation by neuropeptide-containing nerves, particularly CGRP, which occur before an increase in the density of NA-containing nerves, at 4 weeks after birth, while in renal and femoral arteries noradrenergic nerve density reaches a peak before peptide-containing nerve plexuses (148). A widely studied example of naturally occurring change in neuronal phenotype is the innervation of the eccrine sweat glands. During development, NA-containing sympathetic nerves innervating sweat glands acquire cholinergic and peptidergic function. When axons first contact the developing glands, they exhibit only catecholamine fluorescence and immunoreactivity to TH. With maturation, cholinergic markers (AChE and ChAT) and VIP are detected, followed by CGRP-immunoreactivity several days later, while adrenergic markers disappear (149). These changes are dependent on interactions with the appropriate target tissue (150). Transplantation studies have shown that sweat glands from mature animals retain the capacity to induce cholinergic characteristics in neonatal and adult sympathetic nerves, although a few catecholaminergic characteristics remain (150,151). Visceral targets also specify neurotransmitter expression in parasympathetic and sensory nerves during development. For example, somatostatin expression is stimuCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

lated in developing ciliary ganglion cells by cells of the choriod plexus and CGRP and SP expression in trigeminal afferents is regulated by the target (152). The innervation of the mammalian uterus shows considerable plasticity around puberty: following puberty there is a marked reduction in the density of NA-containing nerves supplying nonvascular smooth muscle of the uterine horn and parametrial tissue, while sensory nerve innervation does not change during the transition from prepubertal to the adult period (153). There is plasticity too in the responses to cotransmitters during development. For example, during the transition from neonate to adult, the relative contribution of ATP and ACh to contractile responses of the urinary bladder changes; in the neonate rabbit, responses to ATP are significantly greater than in the adult while those to ACh remain constant (154). 2. Aging. The innervation of autonomic target tissues varies considerably with increasing age (155–158). Age-related change in the pattern of innervation of blood vessels by sympathetic nerves varies from one vessel to another. For example, the adrenergic innervation of small arteries (mesenteric, femoral, and basilar) of aging rabbits changes only slightly, while the larger elastic arteries (renal and carotid) experience a significant reduction in nerve density in old age. Immunohistochemical studies have shown that there is a decrease in vasoconstrictor and increase in vasodilator neurotransmitters in cerebrovascular nerves in old age: in rat cerebral vessels there is a reduction in the density of nerve fibers containing NA with age, as in other vessels, and an increase in the density of CGRP and VIP-immunoreactive nerves. In cerebral vessels in human beings, there is a decrease in the levels of NPY, VIP, and SP between the ages of 1 and 46 years (159). In the mesenteric arterial bed, decreased sensory nerve vasodilator function occurs in old age (160) in the femoral artery, CGRP levels are reduced, while NPY levels are constant (161). Altered patterns of nerve markers of perivascular innervation with age may be influenced by age-related changes in selective sensitivity for target-derived nerve growth factor (NGF). The responsiveness of sensory CGRP-positive neurons to NGF does not vary with age, but sympathetic nerves require more NGF in old animals to increase nerve density (158). In the gastrointestinal tract, there is extensive loss of enteric neurons and extrinsic sympathetic innervation in old age. While the total number of myenteric neurons in the intestine declines with age (162), an increasing proportion that remain contain NOS (163,164). This age-induced profile of enteric innervation occurs in human beings (165). There are marked changes in the pattern of innervation of the gastrointestinal sphincters in the aged compared to mature rat, and this varies according to the particular sphincter (166). Age-related up- or downregulation of neurotransmitter receptor expression adds to overall changes in autonomic function. For example, increased A1 adenosine receptor density in the rabbit heart with aging may explain the increased sensitivity of senescent heart to the negative inotropic action of adenosine (167). DeCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

creased expression of this receptor in the testis of the aged rat may be related to deficiencies in spermatogenesis accompanying the aging process (168). 3.

Pregnancy

During pregnancy the uterine wall undergoes considerable hypertrophy and hyperplasia with profound changes in innervation, particularly in the fetus-bearing regions. There is a progressive loss of NA-containing nerves innervating the uterus leading to a disappearance of these sympathetic nerves in late pregnancy (169–171) in parallel with a decrease in NPY- and VIP-containing nerves (see Ref. 83). There is also a decrease in the density of sensorimotor nerves in the uterine myometrium associated with the large increase in uterine size, but there is an overall threefold increase in total SP content of the rat uterine horn at term (172). The increase is more marked in uterine horns with several fetuses and more hypertrophy suggestive of a correlation between afferent innervation of the uterus and fetal growth. Afferent rather than motor responses of sensorimotor nerves may predominate in late pregnancy as the relaxant responses to CGRP are diminished and absent at term: these changes are restricted to the uterine myometrium and do not occur in the systemic vasculature (173). In the term-pregnant rat the uterine denervation that occurs is seen in all layers of the body of the uterus with a few remaining nerves associated only with blood vessels (174). In the rat, the number and density of nerve bundles increases within 48 hours after parturition, but restoration of the normal pattern of innervation takes several months (170,171,174). In late pregnancy, there is also hypertrophy and hyperplasia of the uterine artery, but the density of innervation of this vessel remains high because of the growth of many new axons (175). There is, however, a marked decrease of NA-containing nerves and NA content, while at the same time there is an increase in NPY, resulting in the predominance of the latter. These changes are not mimicked by systemic progesterone treatment (176). 5-Hydroxydopamine is normally taken up by the high-affinity NA uptake mechanism in sympathetic nerves. There is an increase in the proportion of 5-hydroxydopamine–labeled varicosities in the uterine artery during pregnancy. Interestingly, in late pregnancy there is evidence for uptake of 5hydroxydopamine into nonsympathetic nerves (177). In late pregnancy there is a switch from adrenergic to predominantly cholinergic responses in this vessel (178). In pregnant human uterine arteries, sympathetic responses to electrical field stimulation and endogenous NA levels are reduced and neuronally mediated dilatation, mediated in part by NO, is increased (179). Pregnancy is associated with marked changes in hemodynamics and vascular remodeling of the systemic circulation. Generalized vasodilatation associated with increased circulating estrogen levels causes a fall in total peripheral resistance and systemic blood pressure despite a 50% rise in cardiac output. Decreased systemic resistance may be attributed to reduced sympathetic neurotransmission at the presynaptic level and blunted responses to vasoconstrictors rather than increased sensory motor or endothelium-dependent vasodilatation (180). NPY, CGRP, and SP Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

perivascular expression in the kidney cortex is diminished in late pregnancy. Dysfunction of these mechanisms during pregnancy may contribute to the development of preeclampsia. 4.

Acute and Chronic Altitude Hypoxia

Increasing altitude is accompanied by decreased oxygen concentration. Studies on altitude natives and on sojourners at altitude have revealed the physiological and biological adaptations to short-term and long-term residency at high altitude that ensure adequate oxygen supply to tissues. Notable changes include altered ventilation rates, increased red blood cell number, and increased capillary volume. In recent years, the role of the autonomic nervous system in adaptation to altitude hypoxia has become apparent (181). Acute altitude exposure is associated with sympathetic activation and skin vasoconstriction. There is a blunted hypoxic pulmonary vasoconstriction response during the initial days of altitude exposure that has been shown to be independent of the NO/cyclic GMP pathway (182). Chronic hypobaric hypoxia increases sympathetic activation and causes contraction of vascular smooth muscle leading to a rise in pressure in the pulmonary arterial circulation, and remodeling of the pulmonary vasculature and pulmonary hypertension (183,184). Persistence of increased sympathetic activity and raised NA levels may account for the downregulation of adrenergic receptors after prolonged high-altitude exposure (185,186). This may explain the decreased maximal heart rate in altitude acclimatized subjects. Elevated plasma NPY and adrenaline levels can be attributed to an effect of chronic hypoxia on the adrenal gland rather than an overactive sympathetic nervous system (187). During chronic hypoxia there is decreased myocardial NA uptake by the NA transporter, an event that precedes cardiac hypertrophy (188). Studies at altitude have relevance in clinical situations at sea level. Tissue hypoxia is a feature of many diseases, such as asthma, emphysema, coronary artery disease, and cancer. Knowledge of compensatory autonomic mechanisms to hypoxia may be harnessed to alleviate the symptoms of pathogenic hypoxia.

II.

PATHOPHYSIOLOGY

There are often remarkable changes in the organization and neurotransmitter expression in the autonomic nervous system as a result of pathophysiological situations such as trauma, surgery, or disease (156). Some of this plasticity is for compensatory advantage and some lead to altered neural control of effector tissues, which is not beneficial. Manipulation of the ANS to encourage beneficial compensatory changes in nerve growth and the expression of neurotransmitters/neuromodulators and their receptors is an attractive means of therapeutic advance for autonomic dysfunction. Diseases in which the autonomic nervous system is the primary target of the pathological process are relatively rare. It is more usual for autonomic neuropathy Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

to occur in a variety of different conditions as a secondary phenomenon. Such conditions include spinal cord injury, radical surgery, obstruction and inflammation, diabetes, and chronic alcohol abuse. A.

Trauma and Surgery

Guanethidine and capsaicin are two neurotoxins that have been used as tools to study the effects of either selective long-term sympathectomy or sensory (primary afferent) denervation, respectively (116,189,190). Chemical denervation and selected ganglionectomy studies have shown that loss of sympathetic or sensory innervation induces remarkable changes in the nerves that remain. Some of these studies are outlined below. 1.

Sympathectomy

Unilateral removal of the superior cervical ganglion (SCG) results in the reinnervation of the denervated cerebral vessels by sprouting nerves from the contralateral ganglion (191). Following chronic guanethidine sympathectomy there is complete depletion of sympathetic cotransmitters NA and NPY from the dura mater but an increase in the expression of NPY in nonsympathetic axons supplying cerebral vessels and the iris (111). The source of increased cerebrovascular NPY is thought to be preexisting parasympathetic cranial ganglia, which normally express both NPY and VIP (192). Upregulation of NPY expression in these neurons may be a compensatory mechanism, as NPY has direct constrictor actions in cerebral vessels (159). In certain situations there may be a switch in phenotype from cholinergic to adrenergic (193). For example, sympathectomy-induced increased DBH immunoreactivity in the sphenopalatine (parasympathetic) ganglion occurs at the same time as a loss in VIP immunoreactivity (194). In the cerebral artery and uterine artery, loss of sympathetic nerves also leads to increased DBH immunoreactivity in nonsympathetic nerves that lack TH and NA (195,196). In contrast to these seemingly compensatory changes, up to 4 months after neonatal guanethidine sympathectomy there is no reinnervation of the mesenteric vein, urinary bladder, vas deferens, atria ventricles, or large coronary arteries by NPY-containing nerves of intrinsic origin and no evidence for increased synthesis or de novo synthesis of NA or NPY in these nonsympathetic nerves (133,134). The loss of sympathetic neurons and nerve fibres is also accompanied by striking increases in sensory innervation. This has been attributed to increased availability to NGF (as there are no sympathetic nerves with which to compete for uptake), which promotes the growth of sensory nerves (197). SCG ganglionectomy leads to a rise in SP and CGRP in the iris (198,199). One month after neonatal guanethidine sympathectomy, there is notable hyperinnervation by CGRPcontaining nerves of several tissues, including the SCG, atrium, vas deferens, bladder, and mesenteric vessels of the rat: this persists for up to 5 months (200). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Chemical sympathectomy of the mature rat rather than the neonate also leads to sensory hyperinnervation, although there are a few differences (201). In the lung, sympathectomy induces a marked increase in CGRP-immunoreactive nerve density around the airways, blood vessels, and also in the vicinity of the neuroepithelial bodies of the pulmonary epithelium (202). Probably due to the predominance of intrinsic neurons, the enteric nervous system responds more slowly to long-term chemical sympathectomy. Five months after guanethidine sympathectomy of neonatal rats, levels of CGRP and SP, presumably representing extrinsic sensory nerves, increase in the myenteric plexus and surrounding smooth muscle of the ileum while NPY and VIP levels are unchanged (203). Transient expression of catecholaminergic phenotypic traits has been described in the ganglionated plexuses of the gut during embryonic development (204), but there are no intrinsic NA-containing neurons in the postnatal rat ileum. This suggests that chronic loss of extrinsic sympathetic innervation to the gut may rekindle features of this early phenotype in the adult enteric nervous system (203). Immunosympathectomy by neonatal administration of antiserum to NGF leads to an increase in VIP, galanin, and SP in nerves of the myenteric plexus of the ileum of the rat at 4 and 8 weeks of age but has no effect on NA-, CGRP-, and NPY-containing nerves (205). Thus, NGF availability may be an important regulatory agent in the postnatal expression of at least some enteric neuropeptides. 2.

Sensory Denervation

Neonatal sensory denervation facilitates reinnervation of smooth muscle by sympathetic nerves and results in sympathetic hyperinnervation of selected tissues such as the small blood vessels in the rat lung (202). These capsaicin-induced changes in sympathetic innervation are mirrored by increased perivascular sympathetic neurotransmission (206), although cardiac sympathetic neurotransmission is unaffected (207). Chronic sensory denervation has a differential effect on nonvascular and vascular components of the uterus: the sympathetic nerve supply to the uterine myometrium increases, but perivascular sympathetic nerves in the uterine parametrial tissue are unaffected (208). The innervation of the vas deferens by NPY-containing sympathetic nerves is also unaffected by neonatal capsaicin treatment (209). Guanethidine treatment of capsaicin treated rats reverses the loss of CGRP- but not SP- immunoreactive nerves (210). 3.

Total Extrinsic Denervation

Total denervation of extrinsic nerves to the gut provokes marked plasticity of enteric neurons. Submucosal arteries normally innervated by extrinsic sympathetic and sensory nerves are reinnervated by an abundance of SP- and VIP-containing nerves of intrinsic origin. Loss of extrinsic nerves to the gut is associated with an increase in NOS-containing neurons in the myenteric plexus and potentiation of

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

inhibitory neurotransmission to longitudinal smooth muscle. This has been shown to be due to loss of the sensory innervation (211). VIP levels in the small intestine are also increased following denervation, probably as a result of enhanced transcriptional regulation in the intrinsic ganglia (212). Following extrinsic denervation of the human respiratory tract by heart-lung transplantation, there are significant changes to the neurochemical makeup of the intrinsic neurons that remain: namely, there is the appearance of NPY- and THpositive neurons that are not found in nontransplanted respiratory tract. This may be another example of adaptive phenotypic plasticity of the ANS (213). Autonomic function tests indicate that parasympathetic reinnervation of transplanted hearts occurs more readily than sympathetic reinnervation (214). 4.

Spinal Cord Injury and Decentralization

Following transection of preganglionic autonomic nerves or in spinal cord injury, there are marked changes in the nerves that remain. Such changes can be manifested not only as nerve growth and changes in neurotransmitter expression but, remarkably, in reorganization of nerve pathways and their function. The most dramatic examples of such plasticity occur in the urogenital tract, and it is probable that the anatomical organization of nerve pathways in this region accounts for this (see Refs. 215–218) Not all preganglionic sympathetic nerves synapse with postganglionic neurons in the prevertebral or paravertebral ganglia. Thus, some of the sympathetic nerves traveling to the pelvic viscera in the hypogastric nerve are preganglionic and also synapse with local ganglia in the pelvic region and the pelvic plexus. Since sprouting is a common response of the nerves that remain following nerve injury, the close association of the different divisions of the autonomic nervous system in the pelvic region opens up the possibility for new connections to form new pathways. Spinal cord injury can unmask spinal reflexes that are normally inhibited by input from higher centers in the brain (219). Spinal cord injury can also lead to changes in the sensory nerves supplying the bladder (220–222). Complete decentralization of the rat bladder by simultaneous transection of the hypogastric and pelvic nerves results in increased levels of NGF in the bladder (223). Since NGF acts predominantly on unmyelinated sensory nerves and sympathetic nerves, it could well play a role in plasticity following decentralization or spinal cord injury. These changes in sensory nerves and reflexes on spinal cord injury do not restore normal function. In many cases, detrusor-sphincter dyssynergia can occur (215,216). Plasticity of bladder afferents under pathological conditions may well contribute to bladder hyperactivity (216). Intravesical administration of capsaicin has been investigated with respect to the treatment of bladder hyperactivity (224,225). Parasympathetic decentralization by either transection of the pelvic nerve or ventral roots in the cat has been shown to result in an increase of NA-containing nerves in the bladder and urethra (226–228). Similar changes have been found in Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

human bladders from patients with sacral spinal cord lesions (229). In addition, the response to hypogastric nerve stimulation switches from the normal response of bladder contraction followed by relaxation to one of sustained bladder contraction after preganglionic parasympathetic denervation in cats (226,228). It has been suggested that denervation results in a switch in receptor expression from predominantly -adrenoceptors to predominantly -adrenoceptors (226,227). VIP levels and VIP-containing nerve fibers also increase in the bladder after transection of the pelvic nerves (230). Increased expression of NA-containing nerves has also been observed in the striated muscle of the external urethral sphincter in humans following sacral spinal cord injury (231). Remarkably, experimental studies have shown that following somatic denervation there is sprouting of local autonomic nerves and reinnervation of motor endplates by autonomic cholinergic and/or adrenergic axons (232). Furthermore, removal of the somatic and parasympathetic input to the urethra results in a change of function such that sympathetic hypogastric nerve stimulation can result in activation of striated muscle fibres, a response that is never seen under normal conditions (233). Denervation may not be the only stimulus for autonomic plasticity in the bladder. Hypertrophy of the detrusor muscle and local inflammation can also induce changes in autonomic nerves under pathological conditions. In the rat, under normal conditions, hypogastric nerve stimulation has no effect on penile pressure, which is controlled by the pelvic nerve. Following removal of the parasympathetic input by transection of the pelvic nerve, the pattern changes such that hypogastric nerve stimulation induces erection (234). Reorganization of autonomic pathways does not only occur after removal of parasympathetic input. After sympathetic decentralization in rats, pelvic nerve stimulation can cause contraction of the vas deferens, a response that normally can only be elicited by the hypogastric nerve (235). It has been proposed that decentralized local sympathetic postganglionic neurons are reinnervated by preganglionic axons in the pelvic nerve (235). B.

Disorders of the Urogenital Tract

Disorders of the urogenital tract can occur for a wide variety of reasons (see Refs. 236, 237). Those involving the autonomic nervous system range from trauma and diseases such as multiple sclerosis that affect the preganglionic autonomic neurons in the spinal cord to iatrogenic causes such as radical surgery or x-irradiation, which can result in local nerve damage, and, finally, metabolic disorders such as diabetes, which affect autonomic neuromuscular transmission. In some cases the precise nature of the changes that occur in human beings has proved difficult to determine and, where possible, much information has been derived from animal models. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

1.

Bladder Obstruction

The most common cause of bladder dysfunction in males is obstruction due to benign prostatic hypertrophy. During the course of obstruction there is hypertrophy of the smooth muscle, and, in many cases, the bladder becomes hyperactive (238). In the rat, partial urethral ligation results in muscle hypertrophy (239), enhanced spinal micturition reflexes, and increased frequency of voiding (240). Following hypertrophy, there is an increase in the size of autonomic postganglionic neurons in the major pelvic ganglion of the rat (239,241,242). Neural input is not a prerequisite for neuronal growth since it still occurs in obstruction when the major pelvic ganglion has been decentralized or following destruction of unmyelinated sensory nerves by capsaicin. Receptor expression may also be affected following obstruction. Increased expression of -adrenoceptors has been observed in human bladder strips from patients with prostatic bladder obstruction and NA can cause contraction of the muscle, a response that does not usually occur in unobstructed bladder (243). Similarly, under normal conditions, the contractile response to nerve stimulation in human bladder does not contain a significant purinergic component indicated by atropine resistance. However, increased purinergic transmission can be demonstrated in patients with unstable obstructed bladders (244,245; see also Ref. 246). Upregulation of purinergic receptors appears to occur in hyperreflexia whether this is due to obstruction (244,245), interstitial cystitis (247), or neurogenic bladders (248). DRG neurons with projections to the bladder also increase in size in bladder hypertrophy of the rat. In addition, there is increased expression of a growth associated protein (GAP-43) in afferent projections to the spinal cord including regions surrounding the parasympathetic nucleus (240). NGF levels are significantly increased in obstruction-induced hypertrophy of the bladder in rats (238,242). Furthermore, autoimmunity to NGF can prevent the increase in size of both major pelvic ganglion neurons and dorsal root ganglion neurons induced by obstruction (240,242). Autoimmunity to NGF also abolishes increased GAP-43 expression and can eliminate enhancement of the micturition reflex in obstruction (240). 2.

Bladder Inflammation

Inflammatory conditions of the bladder such as chronic cystitis and interstitial cystitis are characterized by symptoms of urgency and increased frequency of voiding, both associated with pain (249,250). The association with pain has implicated the involvement of bladder afferents in the process, although both sensory and autonomic nerves may be involved in abnormal detrusor muscle responses in inflammation. The two conditions should be distinguished in that chronic cystitis results from chronic inflammation due to persistent infection, whereas in intersti-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

tial cystitis it has been proposed that neurogenic inflammation in the absence of infection may be a primary mechanism in its pathogenesis (249,251). Proliferation of nerve fibers in both the suburothelium and smooth muscle has been reported in the bladder from patients with interstitial cystitis but not chronic cystitis (252,253). The suburothelium contains predominantly sensory nerve fibers. Experimental inflammation in rats has been shown to increase the expression of Type I NOS in dorsal root ganglion neurons identified as projecting to the bladder (254) and to induce hyperreflexia (255). NGF has been implicated in altered nerve function in bladder inflammation. NGF levels are increased in human bladder in both chronic and interstitial cystitis (250). Furthermore, NGF sensitizes bladder afferents (256), and blockade of NGF prevents hyperreflexia associated with experimental inflammation (255). Purinergic neuromuscular transmission is enhanced in human bladder in interstitial cystitis (247). Furthermore, bradykinin and histamine (both released from mast cells) and substance P potentiate purinergic transmission in guinea pig bladder (257,258). Thus abnormal secretion from mast cells could contribute to abnormal autonomic neuromuscular transmission in the bladder during inflammation. P2X purinoceptors have also been localized in nociceptive C-fibers, and ATP also has the potential for contributing to visceral pain in inflammatory conditions (259,260) as well as the micturition reflex (261). 3.

Impotence

Penile erection is a vascular event, and it is important to recognize that local vasodilatation in the penis is under dual control by autonomic nerves and the endothelium (262). In the periphery major causes of impotence are vascular disease and diabetic autonomic neuropathy. Both neurogenic and endothelium-dependent relaxation of erectile smooth muscle can be impaired in diabetic men with impotence and in experimental diabetes (263,264). Within the corpus cavernosum, reduced levels of NA and decreased innervation by VIP-containing and AChE-positive nerve fibers have been reported in diabetic patients with impotence (265). NO has been proposed as an important neurotransmitter in erection. The production of NO by the corpus cavernosum following nerve stimulation is reduced in patients with vasculogenic impotence (266). In patients with neurogenic impotence a significant reduction in NOS-containing nerve fibers in the corpus cavernosum has been observed (267). Sildenafil, the latest drug (Viagra) used for the treatment of impotence, acts by inhibiting the breakdown of cGMP, the intracellular mediator of NO-dependent responses (268). Intraurethral infusion of capsaicin can induce penile erection in men (269), indicating that local sensory nerves may also perform a motor function in penile erection. Erectile tissue is densely innervated by CGRP-containing nerves and CGRP can induce erection (270).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

C.

Gut Motility Disorders

1.

Slow Transit Constipation

Patients with idiopathic constipation with normal bowel diameter have an increased whole-gut transit time. This may be related to an imbalance of enteric transmitter release. For example, VIP levels are reduced in the myenteric plexus and muscle layers of patients with this colonic motility disorder, while levels of SP and NPY are normal (271,272). Disturbances in the function of cholinergic innervation of the taenia coli of the colon have also been reported. 5-HT levels are elevated in circular smooth muscle and mucosa in this disorder but unchanged in preparations containing the plexuses (273). Reduced numbers of SP- and VIP-immunoreactive nerve fibers in colonic circular muscle have been reported in biopsy samples taken from children with severe intractable constipation (274). Reduced levels of SP have also been reported in the mucosa and in submucosa isolated from rectal biopsies from patients with slow transit constipation showing normal levels of VIP and somatostatin (275). It appears then that disturbances or imbalances of both excitatory and inhibitory elements of intestinal innervation may contribute to bowel motility disorders associated with chronic constipation (128). 2.

Megacolon

In constipation associated with idiopathic megacolon or megarectum, abnormalities in inhibitory systems may contribute to abnormal gut function and subsequent bowel dilatation. In the rectum of these patients the density of innervation by nerves containing VIP and NADPH-diaphorase (a marker for NOS) is increased in the muscularis mucosae and lamina propria but decreased in the longitudinal muscle layer (276). D.

Disorders of the Cardiovascular System

1.

Hypertension

Irregularities of the sympathetic nervous system, renin-angiotensin system, and endothelial factors have all been implicated in the development of hypertension. Studies on experimental models of hypertension have been particularly useful in defining early vascular changes that precede the onset of elevated blood pressure. One such model, the spontaneously hypertensive rat (SHR), is widely recognized as an excellent model of genetically prone hypertension. There are several lines of evidence to support an involvement of the sympathetic nervous system in the genesis and maintenance of hypertension in the SHR: there is an increase in the density of sympathetic innervation of cerebral arteries, which precedes the onset of hypertension and associated medial hypertrophy. Sympathetic hyperinnervation and increased sympathetic activity have been reported in several

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

vessels (147,277,278). Examination of sympathetic neurotransmission in the tail and mesenteric arteries of the SHR has revealed a greater cotransmitter role for ATP compared to NA, such that ATP is the dominant component of the sympathetic response (279,280). In line with the animal models, enhanced sympathetic activity has also been reported to be involved in the pathogenesis of hypertension in human beings (281). Plasma levels of NPY are elevated in patients with moderate and severe hypertension, and increased systemic NA levels have also been shown (281). While arterial responses are unaffected, postjunctional contractile responses to NPY and the ATP agonist, ,-methylene ATP, are attenuated in subcutaneous veins in essential hypertension (282). All three sympathetic cotransmitters, NA, NPY, and ATP, invoke a mitogenic response in human vascular smooth muscle cells (281). There are indications that ATP regulates SHR smooth muscle cell proliferation via P2Y4 receptors (283). Decreased sensorimotor nerve innervation may contribute to the development of hypertension. In the mesenteric arterial bed, the density of CGRP-containing nerve fibers and sensorimotor vasodilatation is decreased in the SHR and plasma CGRP levels are decreased (277). The potential role of endothelin in the genesis of hypertension has been extensively investigated as it is a potent vasoconstrictor, mitogenic, and antiapoptotic agent (284). Most studies have concentrated on an endothelial source for this peptide, but recent reports of a perivascular localization in nerve varicosities opens up the possibility of a neural source and altered neural expression as a factor in the development of hypertension (26). 2.

Atherosclerosis

Sympathetic overactivity in hypertension, independent of blood pressure, is a risk factor for the development of atherosclerosis. Endothelial damage is widely regarded as the critical initiating factor in atherogenesis (285). Impaired endothelium-mediated vasodilatation has been widely reported in human beings and in animal models of this hypercholesterolemic disorder, although enhanced endothelial-mediated responses occur in vessels with small areas of atheromatous lesion (286). In addition to these endothelial changes, there are also disturbances in the neural control of vascular tone. There are reduced nerve-mediated constrictor responses, even in vessels with little lesion development (286). Diminished sympathetic activity is indicated by an inhibition of NA release from sympathetic nerves innervating atherosclerosed aortas and pulmonary arteries (287). In the mesenteric artery, NPY- and CGRP-containing nerves appear less varicose and reduced sympathetic neurotransmission is accompanied by enhanced potentiating and inhibitory neuromodulatory actions of NPY and CGRP, respectively (288). Vascular responses in models of atherosclerosis are also dependent on gender and age.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

3.

Systemic Sclerosis

Raynaud’s phenomenon, in which there is inappropriate constriction of blood vessels to the digits on exposure to cold, is often an early event in the development of systemic sclerosis. Systemic sclerosis is characterized by vascular damage and dermal fibrosis. An early functional deficit of the vascular endothelium is present before extensive visceral and skin involvement (289). ANS dysfunction has also been linked with systemic sclerosis (290,291). Defective neurovascular sensory motor control as an early event is suggested by the reduction of CGRP-containing nerve fiber density around capillaries in dermal papillae in Raynaud’s phenomenon and systemic sclerosis (292). Reduced sensory innervation may affect the endothelial expression of vasoconstrictor and vasodilator substances and contribute to the pathology of this disorder (122,293). E.

Diabetic Neuropathy

Diabetes is the most common cause of autonomic neuropathy in humans (294,295). Diabetic autonomic neuropathy has been implicated in dysfunction of the cardiovascular, gastrointestinal, and urogenital systems (295,296). Animal models have been used extensively in research in the field of diabetes. Studies of rats made diabetic by administration of streptozotocin (STZ) have provided a wealth of evidence for diabetes-induced changes in autonomic nerves throughout the vasculature and visceral organs. In erectile tissue a similar loss of VIP and neurogenic relaxation has been demonstrated in human male diabetics with impotence and in experimental diabetes (264,297). In the rat proximal colon, an initial increase in NA and VIP is followed by a loss of NA and VIP at a later stage in STZ-induced diabetes (298). Similarly, in human skin from patients with diabetes of different duration, an early increase of VIP in autonomic nerve fibers precedes a later depletion (299). In the rat ileum 8 weeks after induction of diabetes, no VIP release can be detected on electrical field stimulation of myenteric nerves despite the fact that VIP levels in diabetic tissue are more than twice that of controls (300). Thus, an early feature of autonomic nerve damage in diabetes may be a failure in release mechanisms resulting in accumulation of a neurotransmitter within the nerve. At a later stage, there is overt degeneration of nerve terminals resulting in loss of neurotransmitter. Not all autonomic nerves are affected in the same way by diabetes. Thus in rat cerebral vessels, there is a reduction of VIP and 5-HT but not NPY or NA in perivascular nerves in STZ-induced diabetes (301). Similarly, in the proximal colon, intrinsic VIP- and extrinsic NA-containing nerves undergo degeneration in STZ-induced diabetes, while 5-HT–, SP-, and CGRP-containing nerves display altered levels of their neurotransmitters without undergoing degeneration. NPYcontaining nerves appear to be unaffected by diabetes in this region (298,302,303). In addition, the same types of nerve may be affected differently by Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

diabetes depending on the target they innervate. Thus, NA-containing nerves supplying the ileum degenerate, whereas those supplying the distal colon appear unaffected by diabetes (304). A number of theories have been proposed to account for the development of neuropathy in diabetes. These include activation of the polyol pathway, depletion of myoinositol, impaired fatty acid metabolism, a reduction in the blood supply to nerves, and inadequate trophic support by the target (see Refs. 305–308). There is some evidence that all of these factors contribute to diabetic neuropathy, and they are not mutually exclusive. However, they cannot adequately explain why some nerves degenerate in diabetes while others do not. One factor that may be of significance in this is oxidative stress, which is known to occur in diabetes (309,310). The potential importance of oxidative stress is indicated by the fact that clinical studies are already investigating the administration of antioxidants as treatment for diabetic autonomic neuropathy (311).

REFERENCES 1. 2. 3.

4. 5.

6.

7. 8. 9.

10. 11. 12.

Burnstock G. Autonomic neuromuscular junctions: current developments and future directions. J Anat 1986; 146:1–30. Burnstock G. The changing face of autonomic neurotransmission (The first von Euler lecture in physiology). Acta Physiol Scand 1986; 126:67–91. Gabella G. Fine structure of post-ganglionic nerve fibres and autonomic neuroeffector junctions. In: Burnstock G, Hoyle CHV, eds. Autonomic Neuroeffector Mechanisms. Chur, Switzerland: Harwood Academic, 1995:1–31. Campbell GR, Uehara Y, Mark G, Burnstock G. Fine structure of smooth muscle cells grown in tissue culture. J Cell Biol 1971; 49:21–34. Burnstock G. Autonomic neural control mechanisms, with special reference to the airways. In: Kaliner MA, Barnes PJ, eds. The Airways: Neural Control in Health and Disease New York: Marcel Dekker, 1988:1–22. Brock JA, Cunnane TC. Electrophysiology of neuroeffector transmission in smooth muscle. In: Burnstock G, Hoyle CHV, eds. Autonomic Neuroeffector Mechanisms. Chur, Switzerland: Harwood Academic, 1995:121–213. Paton WDM. Central and synaptic transmission in the nervous system (pharmacological aspects). Annu Rev Physiol 1958; 20:431–455. Eccles JC. The Physiology of the Synapses. New York: Academic Press, 1964. Hoyle CHV, Burnstock G. Criteria for defining enteric neurotransmitters. In: Gaginella TS, ed. Handbook of Methods in Pharmacology. London: CRC Press, 1996:123–140. Attwell D, Barbour B, Szatkowski M. Nonvesicular release of neurotransmitter. Neuron 1993; 11:401–407. Burnstock G. Evolution of the autonomic innervation of visceral and cardiovascular systems in vertebrates. Pharmacol Rev 1969; 21:247–324. Burnstock G, Campbell G, Bennett M, Holman ME. Inhibition of the smooth muscle of the taenia coli. Nature 1963; 200:581–582.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

13. 14.

15. 16. 17. 18. 19. 20.

21.

22.

23. 24. 25.

26. 27.

28.

29.

30.

Martinsen J, Muren A. Excitatory and inhibitory effects of vagal stimulation on gastric motility in the cat. Acta Physiol Scand 1963; 57:309–316. Burnstock G, Campbell G, Satchell D, Smythe A. Evidence that adenosine triphosphate or a related nucleotide is the transmitter substance released by non-adrenergic inhibitory nerves in the gut. Br J Pharmacol 1970; 40:668–688. Burnstock G. Purinergic nerves. Pharmacol Rev 1972; 24:509–581. Burnstock G. The past, present and future of purine nucleotides as signalling molecules. Neuropharmacology 1997; 36:1127–1139. Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 1998; 50:413–492. Cook RD, Burnstock G. The ultrastructure of Auerbach’s plexus in the guinea-pig. 1. Neuronal elements. J Neurocytol 1976; 5:171–194. Furness JB, Costa M. The Enteric Nervous System. Edinburgh: Churchill Livingstone, 1987. Gershon MD, Mawe GM, Branchek TA. 5-HT and enteric neurones. In: Fozard JR, ed. The Peripheral Actions of 5-HT. Oxford: Oxford University Press, 1989:247–273. Hills JM, Jessen KR. Transmission: -amino butyric acid (GABA), 5-hydroxytryptamine (5-HT) and dopamine. In: Burnstock G, Hoyle CHV, eds. Autonomic Neuroeffector Mechanisms. Chur, Switzerland: Harwood Academic, 1995:465–507. Rand MJ. Nitrergic transmission: nitric oxide as a mediator of non-adrenergic, noncholinergic neuro-effector transmission. Clin Exp Pharmacol Physiol 1992; 19:147–169. Sanders KM, Ward SM. Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission. Am J Physiol 1992; 262:G379–393. Lincoln J, Hoyle CHV, Burnstock G. Nitric Oxide in Health and Disease. Cambridge: Cambridge University Press, 1997. Inagaki H, Bishop AE, Escrig C, Wharton J, Allen-Mersh TG, Polak JM. Localization of endothelin-like immunoreactivity and endothelin binding sites in human colon. Gastroenterology 1991; 101:47–54. Loesch A, Milner P, Burnstock G. Endothelin in perivascular nerves. An electronimmunocytochemical study of rat basilar artery. NeuroReport 1998; 9:3903–3906. Schmid KW, Kunk B, Kirchmair R, Totsch M, Bocker W, Fischer-Colbrie R. Immunohistochemical detection of secretoneurin, a novel neuropeptide endoproteolytically processed from secretogranin II, in normal human endocrine and neuronal tissues. Histochem J 1995; 27:473–481. Dunn NJ, Dun SL, Lin HH, Hwang LL, Saria A, Fischer-Colbrie R. Secretoneurinlike immunoreactivity in rat sympathetic, enteric and sensory ganglia. Brian Res 1997; 760:8–16. Sundler F, Ekblad E, Absood A, Hakanson R, Koves K, Arimura A. Pituitary adenylate cyclase activating peptide: a novel vasoactive intestinal peptide-like neuropeptide in the gut. Neuroscience 1992; 46:439–454. Burns GA, Stephens KE. Expression of mRNA for the N-methyl-D-aspartate (NMDAR1) receptor and vasoactive intestinal polypeptide (VIP) co-exist in enteric neurons of the rat. J Auton Nerv Syst 1995; 55:207–210.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

31. 32.

33. 34.

35.

36.

37.

38. 39.

40. 41.

42.

43.

44. 45. 46.

47.

Rattan S, Chakder S. Inhibitory effect of CO on internal anal sphincter: heme oxygenase inhibitor inhibits NANC relaxation. Am J Physiol 1993; 265:G799–G804. Werkström V, Ny L, Persson K, Andersson K-E. Carbon monoxide-induced relaxation and distribution of haem oxygenase isoenzymes in the pig urethra and lower oesophagogastric junction. Br J Pharmacol 1997; 120:312–318. Thoenen H. Induction of tyrosine hydroxylase in peripheral and central adrenergic neurones by cold exposure of rats. Nature 1970; 228:861–862. Zigmond R, Mohney R, Schreiber R, Shadiack A, Sun Y, Vaccariello YS, Zhou Y. Changes in gene expression in adult sympathetic neurons after axonal injury. Adv Pharmacol 1998; 42:899–903. De Potter WP, De Schaepdryver AF, Moerman EJ, Smith AD. Evidence for the release of vesicle proteins together with noradrenaline upon stimulation of the splenic nerve. J Physiol 1969; 204:102–104P. Fillenz M. Transmission: noradrenaline. In: Burnstock G, Hoyle CHV, eds. Autonomic Neuroeffector Mechanisms. Chur, Switzerland: Harwood Academic, 1995:323–365. Bylund DB, Eikenberg DC, Hieble JB, Langer SZ, Lefkowitz RJ, Minneman KP, Molinoff PB, Ruffolo RR Jr, Trendelenburg U. International Union of Pharmacology nomenclature of adrenoceptors. Pharmacol Rev 1994; 46:121–136. Gilman AG. G proteins: transducers of receptor generated signals. Annu Rev Biochem 1987; 56:615–649. Erickson JD, Varoqui H, Schafer MK, Modi W, Diebler MF, Weihe E, Rand J, Eiden LE, Bonner TI, Usdin TB. Functional identification of a vesicular acetylcholine transporter and its expression from a “cholinergic” gene locus. J Biol Chem 1994; 269:21929–21932. Usdin TB, Eiden LE, Bonner TI, Erickson JD. Molecular biology of the vesicular ACh transporter. Trends Neurosci 1995; 18:218–224. Birks RI, Worsley KJ. Activation of acetylcholine synthesis in cat sympathetic ganglia: dependence on external choline and sodium-pump rate. J Physiol 1985; 367:401–417. Noda M, Takahashi H, Tanabe T, Toyosato M, Furutani Y, Hirosa T, Asai M, Inayama S, Miyata T, Numa S. Primary structure of the a-subunit precursor of the Torpedo california acetylcholine receptor deduced from cDNA sequence. Nature 1982; 299:793–797. Kubo T, Maeda A, Sugimoto K, Akiba I, Mikami A, Takahashi H, Haga T, Haga K, Ichiyama A, Kangawa K, Matsuo H, Hirose T, Numa S. Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence. FEBS Lett 1986; 209:367–372. Goyal RK. Muscarinic receptor subtypes. Physiology and clinical implications. N Engl J Med 1989; 321:1022–1029. Zimmermann H. Signalling via ATP in the nervous system. J Auton Nerv Syst 1994; 17:420–426. Hoyle CHV. Transmission: purines. In: Burnstock G., Hoyle CHV, eds. Autonomic Neuroeffector Mechanisms. Chur, Switzerland: Harwood Academic, 1995:367–407. Miras-Portugal MT, Gualix J, Pintor J. The neurotransmitter role of diadenosine polyphosphates. FEBS Lett 1998; 430:78–82.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

48.

49. 50. 51. 52.

53.

54. 55. 56.

57.

58. 59. 60. 61.

62.

63.

64.

Burnstock G. A basis for distinguishing two types of purinergic receptor. In: Straub RW, Bolis L, eds. Cell Membrane Receptors for Drugs and Hormones: A Multidisciplinary Approach. New York: Raven Press, 1978:107–118. Jacobson KA, Suzuki F. Recent developments in selective agonists and antagonists acting at purine and pyrimidine receptors. Drug Dev Res 1996; 39:289–300. Burnstock G, Kennedy C. Is there a basis for distinguishing two types of P2purinoceptor? Gen Pharmacol 1985; 16:433–440. Abbracchio MP, Burnstock G. Purinoceptors: Are there families of P2X and P2Y purinoceptors? Pharmacol Ther 1994; 64:445–475. Burnstock G. P2 purinoceptors: historical perspective and classification. In: Chadwick DJ, Goode JA, eds. P2 Purinoceptors: Localization, Function and Transduction Mechanisms. Ciba Foundation Symposium 198. Chichester: John Wiley and Sons, 1996:1–34. Dockray GJ. Transmission: peptides. In: Burnstock G, Hoyle CHV, eds. Autonomic Neuroeffector Mechanisms. Chur, Switzerland: Harwood Academic, 1995:409–464. Konkoy CS, Davis TP. Ectoenzymes as sites of peptide regulation. Trends Neurosci 1996; 17:288–294. Gibson A, Lilley E. Superoxide anions, free-radical scavengers, and nitrergic neurotransmission. Gen Pharmacol 1997; 28:489–493. Lincoln J, Hoyle CHV, Burnstock G. Transmission: nitric oxide. In: Burnstock G, Hoyle CHV, eds. Autonomic Neuroeffector Mechanisms. Chur, Switzerland: Harwood Academic, 1995:509–539. Lundberg JM. Pharmacology of cotransmission in the autonomic nervous system: integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acids and nitric oxide. Pharmacol Rev 1996; 48:113–178. Boeckxstaens GE, Pelckmans PA. Nitric oxide and the non-adrenergic non-cholinergic neurotransmission. Comp Biochem Physiol 1997; 118A:925–937. Sneddon P, Graham A. Role of nitric oxide in the autonomic innervation of smooth muscle. J Auton Pharmacol 1992; 12:445–456. Lowenstein CJ, Dinerman JL, Snyder SH. Nitric oxide: a physiologic messenger. Ann Intern Med 1994; 120:227–237. Feelisch M. The biochemical pathways of nitric oxide formation from nitrovasodilators: appropriate choice of exogenous NO donors and aspects of preparation and handling of aqueous NO solutions. J Cardiovasc Pharmacol 1991; 17(suppl 3):S25–S33. Rand MJ, Li CG. Differential effects of cysteine and glutathione on NO- and stimulation-induced relaxations of the rat anococcygeus muscle (abstr). Proc Aus Physiol Pharmacol Soc 1992; 23:175P. Schmidt HHHW, Lohmann SM, Walter U. The nitric oxide and cGMP signal transduction system: regulation and mechanism of action. Biochim Biophys Acta 1993; 1178:153–175. McDonald LJ, Murad F. Nitric oxide and cyclic GMP signaling. Proc Soc Exp Biol Med 1996; 211:1–6.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

65. Griffith SG, Burnstock G. Immunohistochemical demonstration of serotonin in nerves supplying human cerebral and mesenteric blood vessels: some speculations about their involvement in vascular disorders. Lancet 1983; i:561–562. 66. Jackowski A, Crockard A, Burnstock G. 5-Hydroxytryptamine demonstrated immunohistochemically in rat cerebrovascular nerves largely represents 5-hydroxytryptamine uptake into sympathetic nerve fibres. Neuroscience 1989; 29:453–462. 67. Lincoln J. Innervation of cerebral arteries by nerves containing 5-hydroxytryptamine and noradrenaline. Pharmacol Ther 1995; 68:473–501. 68. Jessen KR, Mirsky R, Dennison ME, Burnstock G. GABA may be a neurotransmitter in the vertebrate peripheral nervous system. Nature 1979; 281:71–74. 69. Davanger S, Hjelle OP, Babaie E, Larsson LI, Houjaard D, Storm-Mattheson J, Ottersen OP. Colocalization of -aminobutyrate and gastrin in rat antrum: an immunocytochemical and in situ hybridization study. Gastroenterology 1994; 107:137–148. 70. Krantis A, Nichols K, Staines W. Neurochemical characterization and distribution of enteric GABA-ergic neurons and nerve fibres in the human colon. J Auton Nerv Syst 1998; 68:33–42. 71. Maines MD. Carbon monoxide: an emerging regulator of cGMP in the brain. Mol Cell Neurosci 1993; 4:389–397. 72. Dawson TM, Snyder SH. Gases as biological messengers: nitric oxide and carbon monoxide in the brain. J Neurosci 1994; 14:5147–5159. 73. Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol 1997; 37:517–554. 74. Farrugia G, Miller SM, Rich A, Liu X, Maines MD, Rae JL, Szurszewski JH. Distribution of heme oxygenase and effects of exogenous carbon monoxide in canine jejunum. Am J Physiol 1998; 274:G350–G358. 75. Burnstock G. Do some nerves release more than one transmitter? Neuroscience 1976; 1:239–248. 76. Burnstock G. Review lecture. Neurotransmitters and trophic factors in the autonomic nervous system. J Physiol 1981; 313:1–35. 77. Burnstock G. Co-transmission. The Fifth Heymans Lecture—Ghent, February 17, 1990. Arch Int Pharmacodyn Ther 1990; 304:7–33. 78. Burnstock G (guest ed.). Purinergic Neurotransmission. Semin Neurosci 1996; 8:171–257 79. Burnstock G. Purinergic cotransmission. Brain Res Bull 1999; 50:355–357. 80. Hökfelt T, Holets VR, Staines W, Meister B, Melander T, Schalling M, Schultzber M, Freedman J, Björklund H, Olson L, Pernow B. Coexistence of neuronal messengers—an overview new principle in chemical transmission. Prog Brain Res 1986; 68:33–70. 81. Kupfermann I. Functional studies of cotransmission. Physiol Rev 1991; 71:683–732. 82. Morris JL, Gibbins IL. Co-transmission and neuromodulation. In: Burnstock G, Hoyle CHV, eds. Autonomic Neuroeffector Mechanisms. Chur, Switzerland: Harwood Academic, 1995:33–119.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

83.

84.

85.

86.

87.

88.

89. 90.

91.

92. 93.

94.

95. 96.

97.

Milner P, Burnstock G. Trophic factors in the control of smooth muscle development and innervation. In: Raeburn D, Giembycz MA, eds. Airways Smooth Muscle: Development and Regulation of Contractility. Basel: Birkhäuser, 1994:1–39. Burnstock G. Cotransmission with particular emphasis on the involvement of ATP. In: Fuxe K, Hökfelt T, Olson L, Ottoson D, Dahlström A, Björklund A, eds. Molecular Mechanisms of Neuronal Communication. A Tribute to Nils-Åke Hillarp. Oxford: Pergamon Press, 1996:67–87. Kennedy C, Saville VL, Burnstock G. The contributions of noradrenaline and ATP to the responses of the rabbit central ear artery to sympathetic nerve stimulation depend on the parameters of stimulation. Eur. J Pharmacol 1986; 122:291–300. Furness JB, Pompolo S, Murphy R, Giraud A. Projections of neurons with neuromedin U-like immunoreactivity in the small intestine of the guinea-pig. Cell Tissue Res 1989; 257:415–422. Rubino A, Burnstock G. Capsaicin-sensitive sensory-motor neurotransmission in the peripheral control of cardiovascular function. Cardiovasc Res 1996; 31:467–479. Burnstock G. Purinergic signalling in gut. In: Abbracchio MP, Williams M, eds. Handbook of Experimental Pharmacology, Vol. 151/II. Purinergic and Pyrimidinergic Signalling II. Berlin: Springer-Verlag, 2001:141–238. Burnstock G. Mechanisms in interaction of peptide and nonpeptide vascular neurotransmitter systems. J Cardiovasc Pharmacol 1987; 10(suppl 12):S74–S81. Costa M, Brookes SJH, Steele PA, Gibbins I, Burcher E, Kandiah CJ. Neurochemical classification of myenteric neurons in the guinea-pig ileum. Neuroscience 1996; 75:949–967. Morris JL, Grasby DJ, Anderson RL, Gibbins IL. Neurochemical distinction between skeletal muscle vasodilator neurons and pelvic vasodilator neurons in guineapigs. J Auton Nerv Syst 1998; 71:64–68. Morris JL, Gibbins IL. Co-localization and plasticity of transmitters in peripheral autonomic and sensory neurons. Int J Dev Neurosci 1989; 7:521–531. Sneddon P, Burnstock G. Inhibition of excitatory junction potentials in guinea-pig vas deferens by ,-methylene ATP: further evidence for ATP and noradrenaline as co-transmitters. Eur J Pharmacol 1984; 100:85–90. Stjärne L, Lundberg JM, Åstrand P. Neuropeptide Y- a cotransmitter with noradrenaline and adenosine 5-triphosphate in the sympathetic nerves of the mouse vas deferens? A biochemical, physiological and electropharmacological study. Neuroscience 1986; 18:151–166. Burnstock G. Noradrenaline and ATP as cotransmitters in sympathetic nerves. Neurochem Int 1990; 17:357–368. Kennedy C, McLaren GJ, Westfall TD, Sneddon P. ATP as a co-transmitter with noradrenaline in sympathetic nerves—function and fate. In: Chadwick DJ, Goode JA, eds. P2 Purinoceptors: Localization, Function and Transduction Mechanisms. Ciba Foundation Symposium 198. Chichester: John Wiley and Sons, 1996:223–238. Kasakov L, Ellis J, Kirkpatrick K, Milner P, Burnstock G. Direct evidence for concomitant release of noradrenaline, adenosine 5-triphosphate and neuropeptide Y from sympathetic nerve supplying the guinea-pig vas deferens. J Auton Nerv Syst 1988; 22:75–82.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

98.

99.

100. 101. 102.

103. 104. 105. 106.

107.

108.

109. 110.

111.

112. 113.

114.

Lundberg JM, Rudehill A, Sollevi A, Theodorsson-Norheim E, Hamberger E. Frequency- and reserpine-dependent chemical coding of sympathetic transmission: differential release of noradrenaline and NPY from pig spleen. Neurosci Lett 1986; 63:96–100. Ramme D, Regenold JT, Starke K, Busse R, Illes P. Identification of the neuroeffector transmitter in jejunal branches of the rabbit mesenteric artery. Naunyn Schmeidebergs Arch Pharmacol 1987; 336:267–273. Evans RJ, Surprenant A. ATP mediates fast synaptic transmission in mammalian neurons. Nature 1992; 357:503–505. Ralevic V, Burnstock G. Roles of P2-purinoceptors in the cardiovascular system. Circulation 1991; 84:1–14. Corr L, Burnstock G. Vasodilator response of coronary smooth muscle to the sympathetic co-transmitters noradrenaline and adenosine 5-triphosphate. Br J Pharmacol 1991; 104:337–342. von Kügelgen I, Stoffel D, Starke K. P2-purinoceptors-mediated inhibition of noradrenaline release in rat atria. Br J Pharmacol 1995; 115:247–254. Ellis JL, Burnstock G. Neuropeptide Y neuromodulation of sympathetic co-transmission in the guinea-pig vas deferens. Br J Pharmacol 1990; 100:457–462. Wharton J, Gulbenkian S. Peptides in the mammalian cardiovascular system. Experientia 1987; 43:821–872. Rubino A, Ralevic V, Burnstock G. Contribution of P1 (A2b subtype) and P2purinoceptors to the control of vascular tone in the rat isolated mesenteric arterial bed. Br J Pharmacol 1995; 115:648–652. Lundberg JM. Evidence for coexistence of vasoactive intestinal polypeptide (VIP) and acetylcholine in neurons of cat exocrine glands. Morphological, biochemical and functional studies. Acta Physiol Scand Suppl 1981; 496:1–57. Toda N, Okamura T. Modification by L-NG-monomethyl arginine (L-NMMA) of the response to nerve stimulation in isolated dog mesenteric and cerebral arteries. Jpn J Pharmacol 1990; 52:170–173. Holst JJ, Rasmussen TN, Schmidt P. Role of nitric oxide in neurally induced pancreatic exocrine secretion in pigs. Am J Physiol 1994; 266:G206–G213. Nozaki K, Moskowitz MA, Maynard KI, Koketsu N, Dawson TM, Bredt DS, Snyder SH. Possible origins and distribution of immunoreactive nitric oxide synthasecontaining nerve fibres in cerebral arteries. J Cereb Blood Flow Metab 1993; 13:70–79. Mione MC, Cavanagh JFR, Lincoln J, Milner P, Burnstock G. Long-term chemical sympathectomy leads to an increase of neuropeptide Y immunoreactvity in cerebrovascular nerves and iris of the developing rat. Neuroscience 1990; 34:369–378. Burnstock G, Cocks T, Crowe R, Kasakov L. Purinergic innervation of the guineapig urinary bladder. Br J Pharmacol 1978; 63:125–138. Hoyle CHV, C Chapple, Burnstock G. Isolated human bladder: evidence for an adenine dinucleotide acting on P2x-purinoceptors and for purinergic transmission. Eur J Pharmacol 1989; 174:115–118. Morris JL, Gibbins IL. Neuronal colocalization of neuropeptides, catecholamines and catecholamine synthesizing enzymes in the guinea-pig paracervical ganglia. J Neurosci 1987; 7:3117–3130.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

115. 116. 117. 118. 119.

120.

121.

122.

123.

124. 125.

126.

127.

128.

129.

130.

Anderson RL, Gibbins IL, Morris JL. Five inhibitory transmitters coexist in pelvic autonomic vasodilator neurons. NeuroReport 1997; 8:3023–3028. Holzer P. Capsaicin: cellular targets, mechanisms of action and selectivity for thin sensory neurons. Pharmacol Rev 1991; 43:143–201. Burnstock G, Wood JN. Purinergic receptors: their role in nociception and primary afferent neurotransmission. Curr Opin Neurobiol 1996; 6:526–532. Holton P. The liberation of adenosine triphosphate on antidromic stimulation of sensory nerves. J Physiol 1959; 145:494–504. Kakuyama M, Vallance P, Ahluwalia A. Endothelium-dependent sensory NANC vasodilatation: involvement of ATP, CGRP and a possible NO store. Br J Pharmacol 1998; 23:310–316. Maggi CA. The dual, sensory and ‘efferent’ function of the capsaicin-sensitive primary sensory neurons in the urinary bladder and urethra. In: Maggi CA, ed. Nervous Control of the Urogenital System. Chur, Switzerland: Harwood Academic, 1993:387–422. Milner P, Ralevic V, Hopwood AM, Feher E, Lincoln J, Kirkpatrick KA, Burnstock G. Ultrastructural localisation of substance P and choline acetyltransferase in endothelial cells of rat coronary artery and release of substance P and acetylcholine during hypoxia. Experientia 1989; 45:121–125. Milner P, Bodin P, Loesch A, Burnstock G. Interactions between sensory perivascular nerves and the endothelium of brain microvessels. Int J Microcirc 1995; 15:1–9. Gibbins IL, Furness JB, Costa M. Pathway-specific patterns of coexistence of substance P, calcitonin gene-related peptide, cholecystokinin and dynorphin in neurons of the dorsal root ganglia of the guinea-pig. Cell Tissue Res 1987; 248:417–437. Aimi Y,. Fujimura M, Vincent SP, Kimura H. Localization of NADPH-diaphorasecontaining neurons in sensory ganglia of the rat. J Comp Neurol 1991; 306:382–392. Franco-Cereceda A, Rydh M, Lou Y-P, Dalsgaard C-J, Lundberg JM. Endothelin as a putative sensory neuropeptide in the guinea-pig: different properties in comparison with calcitonin gene-related peptide. Regul Pept 1991; 32:253–265. Giaid A, Gibson SJ, Ibrahim NBN, Legon S, Bloom SR, Yanagasawa M, Masaki T, Varndell IM, Polak JM. Endothelin-1, an endothelium-derived peptide, is expressed in neurons of the human spinal cord and dorsal root ganglia. Proc Natl Acad Sci USA 1989; 86:7634–7638. Rubino A, Hassall CJS, Burnstock G. Autonomic control of the myocardium: nonadrenergic non-cholinergic (NANC) mechanisms. In: Shepherd JT, Vatner SF, eds. Nervous Control of the Heart. Chur, Switzerland: Harwood Academic, 1996:139–177. Hoyle CHV, Milner P, Burnstock G. Neuroeffector transmission in the intestine. In: Brookes SJ, Costa M, eds. Nervous Control of the Gastrointestinal Tract. Chur, Switzerland: Harwood Academic, 2002. Neild TO, Shen KZ, Surprenant A. Vasodilation of arterioles by acetylcholine released from single neurones in the guinea-pig submucosal plexus. J Physiol 1990; 420:247–265. Furness JB, Kunze WAA, Bertrand PP, Clerc N, Bornstein JC. Intrinsic primary afferent neurons of the intestine. Prog Neurobiol 1998; 54:1–18.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

131. 132.

133.

134.

135.

136.

137.

138. 139.

140.

141. 142. 143.

144.

145.

146.

Kunze WAA, Furness JB. The enteric nervous system and regulation of intestinal motility. Annu Rev Physiol 1999; 61:117–142. Kunze WAA, Clerc N, Furness JB, Gola M. The soma and neurites of primary afferent neurons in the guinea-pig intestine respond differentially to deformation. J Physiol 2000; 526:375–385. Milner P, Lincoln J, Corr LA, Aberdeen JA, Burnstock G. Neuropeptide Y in nonsympathetic nerves of the rat: changes during maturation but not after guanethidine sympathectomy. Neuroscience 1991; 43:661–669. Corr LA, Aberdeen JA, Milner P, Lincoln J, Burnstock G. Sympathetic and nonsympathetic neuropeptide Y-containing nerves in the rat myocardium and coronary arteries. Circ Res 1990; 66:1602–1609. Horackova M, Kroll RP, Hopkins DA, Losier AM, Armour JA. Morphological and immunohistochemical properties of primary long-term cultures of adult guinea-pig ventricular cardiomyocytes with peripheral cardiac neurons. Tissue Cell 1996; 28:411–425. Crowe R, Burnstock G. Fluorescent histochemical localisation of quinacrine-positive neurones in the guinea-pig and rabbit atrium. Cardiovasc Res 1982; 16:384–390. Hassall CJS, Saffrey JM, Belai A, Hoyle CHV, Moules EW, Moss J, Schmidt HHHW, Mural F, Förstermann U, Burnstock G. Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in a subpopulation of intrinsic neurones of the guinea-pig heart. Neurosci Lett 1992; 143:65–68. Hassall CJS, Hoyle CHV. Heme oxygenase-2 and nitric oxide synthase in guineapig intracardiac neurones. NeuroReport 1997; 8:1043–1046. Sosunov AA, Hassall CJS, Loesch A, Turmaine M, Feher E, Burnstock G. Neuropeptide Y-immunoreactive intracardiac neurones, granule containing cells and nerves associated with ganglia and blood vessels in rat and guinea-pig heart. Cell Tissue Res 1997; 289:445–454. Hassall CJS, Stanford SC, Burnstock G, Buckley NJ. Co-expression of four muscarinic receptor genes by the intrinsic neurons of the rat and guinea-pig heart. Neuroscience 1993; 56:1041–1048. Allen TGJ, Burnstock G. A voltage-clamp study of the electrophysiological characteristics of the intramural neurones of the rat trachea. J Physiol 1990; 423:593–614. Belvisi MG, Ward JK, Mitchell JA, Barnes PJ. Nitric oxide as a neurotransmitter in human airways. Arch Int Pharmacodyn Ther 1995; 329:97–110. Fischer A, Canning BJ, Kummer W. Correlation of vasoactive intestinal peptide and nitric oxide synthase with choline acetyltransferase in the airway innervation. Ann NY Acad Sci 1996; 805:717–722. Smet PJ, Edyvane KA, Jonavicius J, Marshall VR. Neuropeptides and neurotransmitter-synthesising enzymes in intrinsic neurons of the human urinary bladder. J Neurocytol 1996; 25:112–124. Dixon JS, Jen PY, Gosling JA. A double-label immunohistochemical study of intramural ganglia from the human male urinary bladder neck. J Anat 1997; 190:125–135. Crowe R, Burnstock G, Light JK. Intramural ganglia in the human urethra. J Urol 1988; 140:183–187.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

147.

148.

149.

150. 151.

152.

153.

154. 155.

156. 157. 158. 159.

160.

161. 162. 163.

Dhital KK, Gerli R, Lincoln J, Milner P, Tanganelli P, Weber G, Burnstock G. Increased density of perivascular nerves to the major cerebral vessels of the spontaneously hypertensive rat: differential changes in noradrenaline and neuropeptide Y during development. Brain Res 1988; 444:33–45. Dhall U, Cowen T, Haven AJ, Burnstock G. Perivascular noradrenergic and peptidecontaining nerves show different patterns of changes during development and ageing in the guinea-pig. J Auton Nerv Syst 1986; 16:109–126. Landis SC, Siegel RE, Schwab M. Evidence for neuroplasticity in vivo. II. Immunocytochemical studies of the rat sweat gland innervation during development. Dev Biol 1988; 126:129–140. Stevens LM, Landis SC. Target influences on transmitter choice by sympathetic neurons developing in the anterior chamber of the eye. Dev Biol 1990; 37:109–124. Cowen T, Thrasivoulou C, Shaw SA, Abdel-Rahman T. Transplanted sweat glands from mature and aged donors determine cholinergic phenotype and altered density of host sympathetic nerves. J Auton Nerv Syst 1996; 60:215–224. Horgan K, Van der Kooy D. Visceral targets specify calcitonin gene-related peptide and substance P enrichment in trigeminal afferent projections. J Neurosci 1992; 12:1135–1143. Brauer MM, Lincoln J, Sarner S, Blundell D, Milner P, Passaro M, Corbacho A, Burnstock G. Maturational changes in sympathetic and sensory innervation of the rat uterus: effects of neonatal capsaicin treatment. Int J Dev Neurosci 1994; 12:157–171. Sneddon P, McLees A. Purinergic and cholinergic contractions in adult and neonatal rabbit bladder. Eur J Pharmacol 1992; 214:7–12. Cowen T, Burnstock G. Development, aging and plasticity of perivascular autonomic nerves. In: Gootman PM, ed. Development Biology of the Autonomic Nervous System. Clifton, NJ: Humana Press, 1986:211–232. Burnstock G. Changes in expression of autonomic nerves in aging and disease. J Auton Nerv Syst 1990; 30:S25–S34. Mione MC, Ralevic V, Burnstock G. Peptides and vasomotor mechanisms. Pharmacol Ther 1990; 46:429–468. Cowen T, Gavazzi I. Plasticity in adult and ageing sympathetic neurons. Prog Neurobiol 1998; 54:249–288. Edvinsson L, Edman R, Otteson A, Uddman R. Distribution, concentration and effects of neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal polypeptide (VIP) in human cerebral blood vessels. J Cereb Blood Flow Metab 1985; 5(suppl):S545. Li Y, Duckles SP. Effect of age on vascular content of calcitonin gene-related peptide and mesenteric vasodilator nerve activity in the rat. Eur J Pharmacol 1993; 236:373–378. Glen TC, Duckles SP. Vascular responses to neuropeptide Y in the rat: effect of age. Aging 1994; 6:277–286. Gabella, G. On the plasticity of form and structure of enteric ganglia. J Auton Nerv Syst 1990; 30:S59–S66. Santer RM. Survival of the population of NADPH-diaphorase stained myenteric neurons in the small intestine of aged rats. J Auton Nerv Syst 1994; 49:115–121.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

164.

165.

166. 167.

168. 169.

170. 171.

172.

173.

174. 175. 176.

177.

178. 179.

180.

Belai A, Cooper S, Burnstock G. Effect of age on NADPH-diaphorase-containing myenteric neurones of rat ileum and proximal colon. Cell Tissue Res 1995; 279:379–387. Belai A, Burnstock G. Distribution and colocalisation of nitric oxide synthase and calretinin in the myenteric neurons of developing, aging and Crohn’s disease human small intestine. Dig Dis Sci 1999; 44:1579–1587. Belai A, Wheeler H, Burnstock G. Innervation of the rat gastrointestinal sphincters: changes during development and aging. Int J Dev Neurosci 1995; 13:81–95. Mudumbi RV, Olson RD, Hubler BE, Montamat SL, Vestal RE. Age-related effects in rabbit hearts of N6R-phenylisopropyladenosine, an adenosine A1 receptor agonist. J Geront A Biol Sci Med Sci 1995; 50:B351–B357. Bhat SG, Wilson M, Ramkumar V. Age-dependant reductions in A1 adenosine receptor expression in rat testes. Am J Physiol 1998; 274:C1057–C1064. Sjöberg N-O. Consideration of the cause of disappearance of the adrenergic neurotransmitter in the uterine nerves during pregnancy. Acta Physiol Scand 1968; 72:510–517. Bell C, Malcolm SJ. Neurochemistry of the sympathetic innervation to the uterus. Clin Exp Pharmacol Physiol 1988; 15:667–674. Alm P, Lundberg LM, Wharton J, Polak JM. Organization of the guinea-pig uterine innervation. Distribution of immunoreactivities for different neuronal markers. Effects of chemical and pregnancy-induced sympathectomy. Histochem J 1988; 20:290–300. Amira S, Morrison JF, Rayfield KM. The effects of pregnancy and parturition on the substance P content of the rat uterus : uterine growth is accompanied by hypertrophy of its afferent innervation. Exp Physiol 1995; 80:645–650. Anouar A, Schirar A, Germain G. Relaxant effect of the calcitonin gene-related peptide (CGRP) on the nonpregnant and pregnant rat uterus. Naunyn Schmeiderbergs Arch Pharmacol 1998; 357:446–453. Haase EB, Buchman J, Tietz AE, Schramm LP. Pregnancy-induced uterine neuronal degeneration in the rat. Cell Tissue Res 1997; 288:293–306. Mione MC, Gabella G. Nerve fibres in the uterine artery increase in number in pregnant guinea-pigs. NeuroReport 1991; 2:537–540. Mione MC, Cavanagh JFR, Lincoln J, Milner P, Burnstock G. Pregnancy reduces noradrenaline but not neuropeptide Y levels in the uterine artery of the guinea-pig. Cell Tissue Res 1990; 259:503–509. Mione MC, Cavanagh JFR, Burnstock G. Uptake of 5-hydroxydopamine into nonsympathetic nerves of guinea-pig uterine artery in late pregnancy. J Neurocytol 1993; 22:164–175. Bell, C. Dual vasoconstrictor and vasodilator innervation of the uterine arterial supply in guinea-pig. Circ Res 1968; 23:269–279. Nelson SH, Steinsland OS, Johnson RL, Suresh MS, Gifford A, Ehardt JS. Pregnancy-induced alterations of neurogenic constriction and dilation of human uterine artery. Am J Physiol 1995; 268:H1694–H1701. Ralevic V, Burnstock G. Mesenteric arterial function in the rat in pregnancy: role of sympathetic and sensory-motor perivascular nerves, endothelium, smooth muscle, nitric oxide and prostaglandins. Br J Pharmacol 1996; 117:1463–1470.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

181. 182.

183. 184.

185. 186.

187.

188.

189.

190.

191.

192.

193.

194.

195.

196.

Appenzeller O, Martignoni E. The autonomic nervous system and hypoxia: mountain medicine. J Auton Nerv Syst 1996; 57:1–12. Asano K, Yanagidaira Y, Yoshimura K, Sakai A. The cGMP pathway is not responsible for the blunted hypoxic vasoconstriction in rat lungs after altitude exposure. Acta Physiol Scand 1997; 160:393–400. Meyrick B, Reid L. The effect of continued hypoxia on rat pulmonary arterial circulation. An ultrastructural study. Lab Invest 1978; 38:188–200. Hung, KS, McKenzie JC, Mattioli L, Klein RM, Menon CD, Poulose AK. Scanning electron microscopy of pulmonary vascular endothelium in rats with hypoxia-induced hypertension. Acta Anat 1986; 126:13–20. Farinelli CC, Kayser B, Binzone T, Cerretelli P, Girardier L. Autonomic nervous control of heart rate at altitude (5050m). Eur J Appl Physiol 1994; 69:502–507. Ponchia A, Noventa D, Bertaglia M, Carretta R, Zaccaria M, Miraglia G, Pascotto P, Buja G. Cardiovascular neural regulation during and after prolonged high altitude. Eur Heart J 1994; 15:1463–1469. Cheng, JT, Chen CF, Shum AY, Wang JY, Chen HI. Increase in plasma neuropeptide Y-like immunoreactivity following chronic hypoxia in the rat. Neurosci Lett 1992; 140:211–214. Scherrer-Crosbie M, Mardon K, Cayla J, Syrota A, Merlet P. Alterations of myocardial sympathetic innervation in response to hypoxia. J Nucl Med 1997; 38:954–957. Burnstock G, Evans B, Gannon BJ, Heath JW, James V. A new method of destroying adrenergic nerves in adult animals using guanethidine. Br J Pharmacol 1971; 43:295–301. Johnson EM, O’Brien F, Werbit R. Modification and characterization of the permanent sympathectomy produced by the administration of guanethidine to newborn rats. Eur J Pharmacol 1976; 37:45–54. Kåhrström J, Hardebo JE, Nordberg C, Owman C. Experiments on cerebrovascular nerve plasticity and trophic vascular adaption in young and adult rats. In: Owman C, Hardebo JE, eds. Neural Regulation of Brain Circulation. Amsterdam: Elsevier, 1986:589–606. Gibbins IL, Morris JL. Co-existence of immunoreactivity to neuropeptide Y and vasoactive intestinal polypeptide in nonadrenergic axons innervating guinea-pig cerebral arteries after sympathectomy. Brain Res 1988; 444:402–406. Björklund H, Hökfelt T, Goldstein M, Terenius L, Olson L. Appearance of the noradrenergic markers tyrosine hydroxylase and neuropeptide Y in cholinergic nerves of the iris following sympathectomy. J Neurosci 1985; 5:1633–1643. Fan Q, Smith PG. Decreased vasoactive intestinal polypeptide-immunoreactivity of parasympathetic neurons and target innervation following long-term sympathectomy. Regul Pept 1993; 48:337–343. Morris JL, Gibbins IL, Furness JB. Increased dopamine--hydroxylase-like immunoreactivity in non-noradrenergic axons supplying the guinea-pig uterine artery after 6-hydroxydopamine treatment. J Auton Nerv Syst 1987; 21:15–27. Mione MC, Sancesario G, D’Angelo V, Bernardi G. Increase in dopamine--hydroxylase immunoreactivity in non-sympathetic nerves of rat cerebral arteries following long-term sympathectomy. Neurosci Lett 1991; 123:167–171.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

197. 198.

199.

200.

201.

202.

203. 204.

205. 206.

207.

208.

209.

210.

211.

Kessler JA, Bell WO, Black IB. Interactions between the sympathetic and sensory innervation of the iris. J Neurosci 1987; 3:1301–1307. Cole DF, Bloom SR, Burnstock G, Butler JM, McGregor GP, Saffrey MJ, Unger WG, Zhang SQ. Increase in SP-like immunoreactivity in nerve fibres of rabbit iris and ciliary body one to four months following sympathetic denervation. Exp Eye Res 1987; 37:191–197. Schon F, Ghatei M, Allen JM, Mulderry PK, Kelly JS, Bloom SR. The effect of sympathectomy on calcitonin gene-related peptide levels in the rat trigeminovascular system. Brain Res 1985; 348:197–200. Aberdeen J, Corr L, Milner P, Lincoln J, Burnstock G. Marked increases in calcitonin gene-related peptide-containing nerves in the developing rat following longterm sympathectomy with guanethidine. Neuroscience 1990; 35:175–184. Aberdeen J, Milner P, Lincoln J, Burnstock G. Guanethidine sympathectomy of mature rats leads to increases in calcitonin gene-related peptide and vasoactive intestinal polypeptide-containing nerves. Neuroscience 1992; 47:453–461. Van Ranst L, Lauweryns JM. Effects of long-term sensory vs sympathetic denervation on the distribution of calcitonin gene-related peptide and tyrosine hydroxylase immunoreactivities in the rat lung. J Neuroimmunol 1990; 29:131–138. Milner P, Lincoln J, Belai A, Burnstock G. Plasticity in the myenteric plexus of the rat ileum after long-term sympathectomy. Int J Dev Neurosci 1995; 13:385–392. Jonakait GM, Markey KA, Goldstein M, Dreyfus CF, Black IB. Selective expression of high affinity uptake of catecholamines by transiently catecholaminergic cells of the rat embryo: studies in vivo and in vitro. Dev Biol 1985; 108:6–17. Belai A, Aberdeen J, Burnstock G. Differential effect of immunosympathectomy on the expression of rat enteric neurotransmitters. Neurosci Lett 1992; 139:157–160. Ralevic V, Karoon P, Burnstock G. Long-term sensory denervation by neonatal capsaicin treatment augments sympathetic neurotransmission in rat mesenteric arteries by increasing levels of norepinephrine and selectively enhancing postjunctional actions. J Pharmacol Exp Ther 1995; 274:64–71. Rubino A, Ralevic V, Burnstock G. Sympathetic neurotransmission in isolated rat atria after sensory-motor denervation by neonatal treatment with capsaicin. J Pharmacol Exp Ther 1997; 282:671–675. Brauer MM, Lincoln J, Milner P, Sarner S, Blundell D, Passaro M, Corbacho A, Burnstock G. Plasticity of autonomic nerves: differential effects of long-term guanethidine sympathectomy on the sensory innervation of the rat uterus during maturation. Int J Dev Neurosci 1994; 12:579–586. Ventura S, Bavetta S, Milner P, Ralevic V, Burnstock G. Nitric oxide synthase is colocalized with vasoactive intestinal polypeptide in postganglionic parasympathetic nerves innervating the rat vas deferens. Neuroscience 1998; 83:607–616. Mione MC, Cavanagh JFR, Kirkpatrick KA, Burnstock G. Plasticity in expression of calcitonin gene-related peptide and substance P immunoreactivity in ganglia and fibres following guanethidine and/or capsaicin denervation. Cell Tissue Res 1992; 268:491–504. Yunker AM, Galligan JJ. Extrinsic denervation increases myenteric oxide synthasecontaining neurons and inhibitory neuromuscular transmission in guinea-pig. J Auton Nerv Syst 1998; 71:148–158.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

212.

213.

214.

215. 216. 217.

218. 219.

220. 221.

222.

223. 224.

225.

226.

227.

Stadelmann AM, Telford GL, Appel DA, Walgenbach-Telford S, Hopp K, Meier DA, Koch TR. Expression of mRNA for vasoactive intestinal peptide in rat small intestine. Mol Cell Endocrinol 1996; 116:31–67. Springall DR, Polak JM, Howard L, Power RF, Krausz T, Manickam S, Banner NR, Khagani A, Rose M, Yacoub M. Persistence of intrinsic neurones and possible phenotypic changes after extrinsic denervation of human respiratory tract by heart-lung transplantation. Am Rev Respir Dis 1990; 141:1538–1546. Tio RA, Reyners AKL, van Veldhuisen DJ, van den Berg MP, Brouwer RMHJ, Haaksma J, Smit AJ, Crijns HJGM. Evidence for differential sympathetic and parasympathetic reinnervation after heart transplantation in humans. J Auton Nerv Syst 1997; 67:176–183. de Groat WC. Anatomy and physiology of the lower urinary tract. Urol Clin North Am 1993; 20:383–401. de Groat WC. A neurologic basis for the overactive bladder. Urology 1997; 50(suppl 6A):36–53. Lincoln J, Burnstock G. Autonomic innervation of the urinary bladder and urethra. In: Maggi CA, ed. Nervous Control of the Urogenital System. Chur, Switzerland: Harwood Academic, 1993:33–68. Hoyle CHV, Lincoln J, Burnstock G. Neural control of pelvic organs. In: Rushton DN, ed. Handbook of Neuro-Urology. New York: Marcel Dekker 1994:1–54. de Groat WC, Kruse MN, Vizzard MA, Cheng C, Araki I, Yoshimura N. Modification of urinary bladder function after spinal cord injury. Adv Neurol 1997; 72:347–364. de Groat WC. Mechanisms underlying the recovery of lower urinary tract function following spinal cord injury. Paraplegia 1995; 33:493–505. Yoshimura N, de Groat WC. Plasticity of Na channels in afferent neurones innervating rat urinary bladder following spinal cord injury. J Physiol 1997; 503:269–276. Yoshimura N, Erdman SL, Snider MW, de Groat WC. Effects of spinal cord injury on neurofilament immunoreactivity and capsaicin sensitivity in rat dorsal root ganglion neurons innervating the urinary bladder. Neuroscience 1998; 83:633–643. Tuttle JB, Steers WD, Albo M, Nataluk E. Neural input regulates NGF and growth of the adult rat urinary bladder. J Auton Nerv Syst 1994; 49:147–158. Fowler CJ, Beck RO, Gerrard S, Betts CD, Fowler CG. Intravesical capsaicin for treatment of detrusor hyperreflexia. J Neurol Neurosurg Psychiatry 1994; 57:169–173. De Ridder D, Chandiramani V, Dasgupta P, Van Poppel H, Baert L, Fowler CJ. Intravesical capsaicin as a treatment for refractory detrusor hyperreflexia: a dual center study with long-term follow up. J Urol 1997; 158:2087–2092. Sundin T, Dahlström A. The sympathetic innervation of the urinary bladder and urethra in the normal state and after parasympathetic denervation at the spinal root level. Scand J Urol Nephrol 1973; 7:131–149. Norlén L, Dahlström A, Sundin T, Svedmyr N. The adrenergic innervation and adrenergic receptor activity of the feline urinary bladder and urethra in the normal state and after hypogastric and/or parasympathetic denervation. Scand J Urol Nephrol 1976; 10:177–184.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

228.

229.

230. 231.

232.

233.

234.

235. 236. 237. 238. 239.

240. 241.

242.

243. 244.

245.

de Groat WC, Kawatani M. Reorganization of sympathetic preganglionic connections in cat bladder ganglia following parasympathetic denervation. J Physiol 1989; 409:431–449. Sundin T, Dahlström A, Norlén L, Svedmyr N. The sympathetic innervation and adrenoceptor function of the human lower urinary tract in the normal state and after parasympathetic denervation. Invest Urol 1977; 14:322–328. Polak JM, Bloom SR. Localisation and measurement of VIP in the genitourinary system of man and animals. Peptides 1984; 5:225–230. Crowe R, Burnstock G, Light JK. Adrenergic innervation of the striated muscle of the intrinsic external urethral sphincter from patients with lower motor spinal cord lesion. J Urol 1989; 141:47–49. Elbadawi A, Atta MA. Intrinsic neuromuscular defects in the neurogenic bladder: V. Autonomic re-innervation of the male feline rhabdosphincter following somatic denervation by bilateral sacral ventral rhizotomy. Neurourol Urodyn 1986; 5:65–85. Flood HD, Downie JW, Awad SA. Urethral function after chronic cauda equina lesion in cats. II. the role of autonomically-innervated smooth and striated muscle in distal sphincter dysfunction. J Urol 1990; 144:1029–1035. Dail WG, Walton G, Olmsted MP. Penile erection in the rat: stimulation of the hypogastric nerve elicits increases in penile pressure after chronic interruption of the sacral parasympathetic outflow. J Auton Nerv Syst 1989; 28:251–258. Kihara K, Kakizaki H, de Groat WC. Reorganization of the innervation of the vas deferens after sympathetic decentralization. Am J Physiol 1996; 271:R1481–R1488. Weiss HD. The physiology of human penile erection. Ann Intern Med 1972; 76:793–799. Nickell K, Boone TB. Peripheral neuropathy and peripheral nerve injury. Urol Clin North Am 1996; 23:491–500. Dupont MC, Persson K, Spitsbergen J, Tuttle JB, Steers WD. The neuronal response to bladder outlet obstruction, a role for NGF. Adv Exp Med Biol 1995; 385:41–54. Gabella G, Uvelius B. Effect of decentralization or contralateral ganglionectomy on obstruction-induced hypertrophy of rat urinary bladder muscle and pelvic ganglion. J Neurocytol 1993; 22:827–834. Steers WD, Creedon DJ, Tuttle JB. Immunity to nerve growth factor prevents afferent plasticity following urinary bladder hypertrophy. J Urol 1996; 155:379–385. Steers, WD, J Ciambotti, S Erdman, de Groat WC. Morphological plasticity in efferent pathways to the urinary bladder of the rat following urethral obstruction. J Neurosci 1990; 10:1943–1951. Steers WD, Kolbeck S, Creedon D, Tuttle JB. Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. J Clin Invest 1991; 88:1709–1715. Perlberg S, Caine M. Adrenergic response of bladder muscle in prostatic obstruction. Urology 1982; 20:524–527. Sjögren C, Andersson K-E, Husted S, Mattiasson A, Möller-Madsen B. Atropine resistance of transmurally stimulated isolated human bladder muscle. J Urol 1982; 128:1368–1371. Smith DJ, Chapple CR. In vitro response of human bladder smooth muscle in unstable obstructed male bladders: a study of pathophysiological causes. Neurourol Urodyn 1994; 13:411–415.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

246. Burnstock G. Purinergic signalling in lower urinary tract. In: Abbracchio MP, Williams M, eds. Handbook of Experimental Pharmacology, Vol. 151/I. Purinergic and Pyrimidinergic Signalling I—Molecular, Nervous and Urinogenitary System Function. Berlin: Springer-Verlag, 2001:423–514. 247. Palea S, Artibani W, Ostardo E, Trist DG, Pietra C. Evidence for purinergic neurotransmission in human urinary bladder affected by interstitial cystitis. J Urol 1993; 150:2007–2012. 248. Wammack R, Weihe E, Dienes H-P, Hohenfeller R. Die neurogene Blase in vitro. Akt Urol 1995; 26:16–18. 249. Elbadawi A. Interstitial cystitis: a critique of current concepts with a new proposal for pathologic diagnosis and pathogenesis. Urology 1997; 49(suppl 5A):14–40. 250. Lowe EM, Anand P, Terenghi G, Williams-Chestnut RE, Sinicropi DV. Increased nerve growth factor levels in the urinary bladder of women with idiopathic sensory urgency and interstitial cystitis. Br J Urol 1997; 79:572–577. 251. Elbadawi A, Light JK. Distinctive ultrastructural pathology of nonulcerative interstitial cystitis. Urol Int 1996; 56:137–162. 252. Christmas TJ, Rode J, Chapple CR, Milroy EJG, Turner-Warwick RT. Nerve fibre proliferation in interstitial cystitis. Virchows Arch A 1990; 416:447–451. 253. Hofmeister MA, Fang H, Ratliff TL, Mahoney T, Becich MJ. Mast cells and nerve fibers in interstitial cystitis (IC): an algorithm for histologic diagnosis via quantitative image analysis and morphometry (QIAM). Urology 1997; 49(suppl 5A):41–47. 254. Vizzard MA, Erdman SL, de Groat WC. Increased expression of neuronal nitric oxide synthase in bladder afferent pathways following chronic bladder irritation. J Comp Neurol 1996; 370:191–202. 255. Dmitrieva N, Shelton D, Rice ASC, McMahon SB. The role of nerve growth factor in a model of visceral inflammation. Neuroscience 1997; 78:449–459. 256. Dmitrieva N, McMahon SB. Sensitisation of visceral afferents by nerve growth factor in the adult rat. Pain 1996; 66:87–97. 257. Patra PB, Westfall DP. Potentiation of purinergic neurotransmission in guinea pig urinary bladder by histamine. J Urol 1994; 151:787–790. 258. Patra PB, Westfall DP. Potentiation by bradykinin and substance P of purinergic neurotransmission in urinary bladder. J Urol 1996; 156:532–535. 259. Burnstock G. A unifying purinergic hypothesis for the initiation of pain. Lancet 1996; 347:1064–1065. 260. Burnstock G. P2X receptors in sensory neurones. Br J Anaesth 2000; 84:476–488. 261. Cockayne DA, Hamilton SG, Zhu Q-M, Dunn PM, Zhong Y, Novakovic S, Malmberg AB, Cain G, Berson A, Kassotakis L, Hedley L, Lachnit WG, Burnstock G, McMahon SB, Ford APDW. Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice. Nature 2000; 407:1011–1015. 262. Lincoln J, Crowe R, Burnstock G. Neuropeptides and impotence. In: Kirby RS, Carson C, Webster GD, eds. Impotence: Diagnosis and Management of Male Erectile Dysfunction. Oxford: Butterworth-Heinemann Ltd., 1991:3–18. 263. Saenz de Tejada I, Goldstein I, Azadzoi K, Krane RJ, Cohen RA. Impaired neurogenic and endothelium-mediated relaxation of penile smooth muscle from diabetic men with impotence. N Engl J Med 1989; 320:1025–1030.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

264.

265.

266.

267.

268.

269. 270.

271. 272.

273.

274.

275.

276. 277.

278.

279.

Azadzoi KM, Saenz de Tejada I. Diabetes mellitus impairs neurogenic and endothelium-dependent relaxation of rabbit corpus cavernosum smooth muscle. J Urol 1992; 148:1587–1591. Lincoln J, Crowe R, Blacklay PF, Pryor JP, Lumley JSP, Burnstock G. Changes in VIPergic, cholinergic and adrenergic innervation of human penile tissue in diabetic and non-diabetic impotent males. J Urol 1987; 137:1053–1059. Pickard RS, Powell PH, Zar MA. Nitric oxide and cyclic GMP formation following relaxant nerve stimulation in isolated human corpus cavernosum. Br J Urol 1995; 75:516–522. Choi YD, Mah SY, Xin ZC, Choi HK. The distribution of nitric oxide synthase in human corpus cavernosum on various impotent patients. Yonsei Med J 1997; 38:125–132. Boolell M, Allen MJ, Ballard SA, Gepi-Attee S, Muirhead GJ, Naylor AM, Osterloh IH, Gingell C. Sildenafil an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int J Impotence Res 1996; 8:47–52. Lazzeri, M, Barbanti G, Beneforti P, Turini D. Intraurethrally infused capsaicin induces penile erection in humans. Scand J Urol Nephrol 1994; 28:409–412. Stief CG, Wetterauer U, Schaebsdau FH, Jonas U. Calcitonin-gene-related peptide: a possible role in human penile erection and its therapeutic application in impotent patients. J Urol 1991; 146:1010–1014. Koch TR, Carney JA, Go L, Go VL. Idiopathic chronic constipation is associated with decreased colonic vasointestinal peptide. Gastroenterology 1988; 94:300–310. Milner P, Crowe R, Kamm MA, Lennard-Jones JE, Burnstock G. Vasoactive intestinal polypeptide levels in sigmoid colon in idiopathic constipation and diverticular disease. Gastroenterology 1990; 99:666–675. Lincoln J, Crowe R, Kamm MA, Burnstock G, Lennard-Jones JE. Serotonin and 5hydroxyindoleacetic acid are increased in the sigmoid colon in severe idiopathic constipation. Gastroenterology 1990; 98:1219–1225. Hutson JM, Chow CW, Borg J. Intractable constipation with a decrease in substance P immunoreactive fibres: is it a variant of neuronal intestinal dysplasia? J Ped Surg 1996; 31:580–583. Tzavella K, Riepl RL, Klauser AG, Voderholzer WA, Schindlbeck NE, Lissner SA. Decreased substance P levels in rectal biopsies from patients with slow transit constipation. Eur J Gastroenterol Hepatol 1996; 8:1207–1211. Gattuso JM, Hoyle CHV, Milner P, Kamm MA, Burnstock G. Enteric innervation in idiopathic megarectum and megacolon. Int J Colorect Dis 1996; 11:264–271. Ralevic V, Burnstock G. Neuropeptides in blood pressure control In: Laragh JH, Bremmer BM, eds. Hypertension: Pathophysiology, Diagnosis, and Management. 2d ed. New York: Raven Press, 1995:801–831. Brock JA, Van Helden DF, Dosen P, Rush R. Prevention of high blood pressure by reducing sympathetic innervation in the spontaneously hypertensive rat. J Auton Nerv Syst 1996; 61:97–102. Vidal M, Hicks PE, Langer SZ. Differential effects of ,-methylene ATP on responses to nerve stimulation in SHR and WKY tail arteries. Naunyn Schmeiderbergs Arch Pharmacol 1986; 332:384–390.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

280.

281. 282.

283.

284. 285. 286.

287.

288.

289.

290. 291.

292.

293. 294.

295. 296.

Brock JA, Van Helden DF. Enhanced excitatory junction potentials in mesenteric arteries from spontaneously hypertensive rats. Pflügers Arch Eur J Physiol 1995; 430:901–908. Thulin T, Erlinge D. Neuropeptide Y and hypertension. Nutrition 1995; 11:495–497. Lind H, Erlinge D, Brunkwall J, Edvinsson L. Attenuation of contractile responses to sympathetic co-transmitters in veins from subjects with essential hypertension. Clin Auton Res 1997; 7:69–76. Harper S, Charlton SJ, Ng LL, Boarder MR. Evidence that ATP and UTP regulate spontaneously hypertensive rat aorta smooth muscle cell proliferation by acting on P2Y4 receptors (abstr). Br J Pharmacol 1998; 123:59P. Schriffin EL. Endothelin: potential role in hypertension and vascular hypertrophy. Hypertension 1995; 25:1135–1143. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362:801–809. Burnstock G, Stewart-Lee A, Brizzolara A, Tomlinson A, Corr A. Dual control of nerves and endothelial cells of arterial blood flow in atherosclerosis. In: Wissler RW, Bond MG, Mercuri M, Tanganelli P, eds. Atherosclerotic Plaques. New York: Plenum Press, 1991:285–292. Verbeuren TJ, Simonet S, Herman AG. Diet-induced atherosclerosis inhibit release of noradrenaline from sympathetic nerves in rabbit arteries. Eur J Pharmacol 1994; 270:27–34. Stewart-Lee A, Aberdeen J, Burnstock G. The effect of atherosclerosis on neuromodulation of sympathetic neurotransmission by neuropeptide Y and calcitonin gene-related peptide in the rabbit mesenteric artery. Eur J Pharmacol 1992; 216:167–174. Matucci-Cerinic M, Peitrini U, Marabini S. Local venomotor response to intravenous infusion of substance P and glyceryl trinitrate in systemic sclerosis. Clin Exp Rheumatol 1990; 8:561–565. Klimiuk PS, Taylor L, Baker RD, Jayson MIV. Autonomic neuropathy in systemic sclerosis. Ann Rheum Dis 1988; 47:542–545. Dessein P, Joffe B, Metz R, Millar D, Lawson M, Stenwicks A. Autonomic dysfunction in systemic sclerosis: sympathetic overactivity and instability. Am J Med 1992; 93:143–150. Bunker CB, Terenghi G, Springall DR, Polak JM, Dowd PM. Deficiency of calcitonin gene-related peptide in Raynaud’s phenomenon. Lancet 1993; 336:1530–1533. Dowd P, Goldsmith P, Bull H, Burnstock G, Foreman J, Marshall I. Grand round: Raynaud’s phenomenon. Lancet 1995; 346:283–290. Luft D, Pagallies OB, Konz K, Mann K, Renn W, Eggstein M. Differential diagnosis of neuropathic lesions in diabetic and alcoholic patients. Clin Invest 1994; 72:200–205. McDougall AJ, McLeod JG. Autonomic neuropathy I: clinical features, investigation, pathophysiology and treatment. J Neurol Sci 1996; 137:79–88. Ewing J, Clarke BF. Diabetic autonomic neuropathy: present insights and future prospects. Diabetes Care 1986; 9:648–665.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

297.

298.

299.

300.

301.

302.

303.

304.

305.

306.

307. 308. 309.

310. 311.

Crowe R, Lincoln J, Blacklay PF, Pryor JP, Lumley JSP, Burnstock G. VIP-like-immunoreactive nerves in diabetic penis: a comparison between human and streptozotocin-diabetes in rats. Diabetes 1983; 32:1075–1077. Belai A, Lincoln J, Milner P, Burnstock G. Progressive changes in adrenergic, serotonergic and peptidergic nerves in proximal colon of streptozotocin-diabetic rats. Gastroenterology 1988; 95:1234–1241. Properzi G, Francavilla S, Poccia G, Aloisi P, Gu X-H, Terenghi G, Polak JM. Early increase precedes a depletion of VIP and PGP9.5 in the skin of insulin-dependent diabetics—correlation between quantitative immunohistochemistry and clinical assessment of peripheral neuropathy. J Pathol 1993; 169:269–277. Belai A, Lincoln J, Burnstock G. Lack of release of vasoactive intestinal polypeptide and calcitonin gene-related peptide during electrical stimulation of enteric nerves in streptozotocin-diabetic rats. Gastroenterology 1987; 93:1034–1040. Lagnado MLJ, Crowe R, Lincoln J, Burnstock G. Reduction of nerves containing vasoactive intestinal polypeptide and serotonin, but not neuropeptide Y and catecholamine, in cerebral blood vessels of the 8 week streptozotocin-induced diabetic rat. Blood Vessels 1987; 24:169–180. Lincoln J, Bokor J, Crowe R, Griffith SG, Haven AJ, Burnstock G. The myenteric plexus in streptozotocin-treated rats: neurochemical and histochemical evidence for diabetic neuropathy in the gut. Gastroenterology 1984; 86:654–661. Belai A, Lincoln J, Milner P, Crowe R, Loesch A, Burnstock G. Enteric nerves in diabetic rats: increase in vasoactive intestinal polypeptide but not substance P Gastroenterology 1985; 89:967–976. Belai A, Lincoln J, Milner P, Burnstock G. Differential effect of streptozotocin-induced diabetes on the innervation of the ileum and distal colon. Gastroenterology 1991; 100:1024–1032. Horrobin DF, Carmichael HA. Essential fatty acids in relation to diabetes. In: Horrobin DF, ed. Treatment of Diabetic Neuropathy: A New Approach. London: Churchill Livingstone, 1992:21–39. Cameron NE, Cotter MA. Potential therapeutic approaches to the treatment or prevention of diabetic neuropathy: evidence from experimental studies. Diabet Med 1993; 10:593–605. Cameron NE, Cotter MA. Metabolic and vascular factors in the pathogenesis of diabetic neuropathy. Diabetes 1997; 46(suppl 2):S31–S37. Stevens MJ, Feldman EL, Greene DA. The aetiology of diabetic neuropathy: the combined roles of metabolic and vascular defects. Diabet Med 1995; 12:566–579. Rösen P, Ballhausen T, Bloch W, Addicks K. Endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant. Diabetologia 1995; 38:1157–1168. Giugliano D, Ceriello A, Paolisso G. Oxidative stress and diabetic vascular complications. Diabetes Care 1996; 19:257–267. Ziegler D, Gries FA. -Lipoic acid in the treatment of diabetic peripheral and cardiac autonomic neuropathy. Diabetes 1997; 46(suppl 2):S62–S66.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

2 Immune Function and Regulation of the Autonomic Nervous System A Molecular Point of View Serge Rivest CHUL Research Center and Laval University, Quebec, Quebec, Canada

The immune system has a profound impact on the central nervous system (CNS). This is mediated via soluble molecules that circulate in the bloodstream and act on specific populations of supportive cells. The neurophysiological responses that are triggered and/or inhibited by these bloodborne molecules play in return crucial roles in restoring the homeostasis and regulating the innate immunity. Such fine regulation involves the endocrine and autonomic systems, especially the circuits that control the plasma release of glucocorticoids and those that engage and maintain the increase in body temperature (fever). There is also compelling evidence that the brain has its own innate immune system, which is believed to act as sentinel during both viral and bacterial infections. This chapter describes in depth the fundamental mechanisms underlying the influence of circulating inflammatory molecules on the cerebral tissue and the ability of the latter to mount an innate immune response. The physiological relevance and pathologies associated with a deviance of such interplay are also described.

I. INTRODUCTION Although long considered to be isolated from the immune system and its secreted molecules, the brain is now recognized to be a key organ in its ability to engage the physiological responses that play determinant roles in organizing, adapting, and restraining inflammatory events. The increase in body temperature (fever), se-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

cretion of plasma glucocorticoids by the adrenal glands, adjustment of the cardiovascular system in general and to the inflammation site, loss of appetite, nausea, and vomiting are some of the features that can be observed when the organism is challenged by foreign materials, including bacteria, viruses, and other antigens or injury. Obviously, several of these changes will occur only during quite severe immune stimuli, but activation of selective circuits involving the autonomic and endocrine functions clearly occurs during systemic and localized inflammatory insults. For instance, activation of the hypothalamic-pituitary-adrenal (HPA) axis reflected by an elevation in plasma glucocorticoid levels is one of the most powerful endogenous and well-controlled feedback effects on the proinflammatory signal transduction events taking place across organisms. There is now convincing evidence that an inappropriate control of this neuroendocrine system during innate immunity may lead to exaggerated responses and be associated with detrimental consequences for the integrity of the organism. Glucocorticoids, specifically cortisol in humans and corticosterone in rodents, have long been recognized as being among the most powerful anti-inflammatory agents known, and their use in restraining excessive immune responses, such as in individuals afflicted with rheumatoid arthritis, has been common in clinical practice for more than 50 years. Inappropriate plasma levels of glucocorticoids may therefore play a crucial role in contributing to deviant regulation of the immune response, indicating the importance of identifying and characterizing the mechanisms through which local inflammatory processes interact with, and depend on, the neuroendocrine system. The main center that controls the autonomic motor/neuroendocrine system is the hypothalamus which receives neuronal projections from numerous structures of the brain. During the past decade, our view of how inflammatory molecules send signals to the brain has much improved, and the objective of this chapter is to present the fundamental bases underlying the effect of the immune system on cerebral tissue. II. INFLAMMATORY RESPONSE AND SECRETED CYTOKINES A. NF- B Signaling in Response to Systemic Inflammation During Innate Immunity An essential feature of the early innate immune response to viruses and bacteria is the secretion of proinflammatory cytokines through exogenous ligands that bind to high-affinity receptors and trigger the proinflammatory signal transduction pathways. Indeed, host organisms detect the presence of infection by recognizing specific elements produced by microorganisms, such as gram-negative bacteria, gram-positive bacteria, and mannans of fungi (1). These elements are called pathogen-associated molecular patterns (PAMPs), which are recognized by specific cells of the immune system as innate mechanisms to mount a rapid response Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

to bacterial infection. The endotoxin lipopolysaccharide (LPS) is a major component of the outer membranes of gram-negative bacteria, which is the best characterized example of innate recognition associated with a robust inflammatory response by phagocytic cells (2). Secretion of cytokines by circulating monocytes/neutrophils and tissue macrophages by LPS requires a series of mechanisms in cascade, the first step being the binding of the endotoxin with the serum proteins LPS-binding protein (LBP) or septins. The newly formed complex may then activate different populations of cells in binding to its CD14 receptor (3). Two forms of the CD14 receptor can be found, the first being present at the surface of myeloid cells (mCD14) and acting as a glycosylphosphatidylinositol (GPI)–anchored membrane glycoprotein (Fig. 1). The other form is soluble in the serum (sCD14) and lacks the GPI properties, though it can bind LPS to activate cells devoid of mCD14, such as endothelial cells (4). LBP is not essential for LPS signaling, but the LPS/LPB complex is particularly powerful in activating cells of myeloid origin, including monocytes, macrophages, neutrophils, and microglia. One well-known consequence of such activation is the production of proinflammatory cytokines, chemokines, and prostaglandins (see below). It is not well known how cell activation is triggered after binding between the LBP/LPS complex and the GPI-anchored mCD14, although there is now evidence that activation of tyrosine kinase leads to transduction signal and cytokine gene transcription through nuclear factor kappa B (NF- B). NF- B is normally present in the cytoplasm forming an inactive complex with an inhibitor known as I B (Fig. 2). Following extracellular stimulation by growth factors, mitogens and cytokines that activate mitogen-activated protein (MAP) kinases, I B is phosphorylated by NF- B–inducible kinase (NIK)/I B kinases (IKK), ubiquitinated and degraded by cytoplasmic proteasome (5,6). Free active NF- B (the most common complex is the p50/p65 heterodimer) is then translocated into the nucleus, where it is able to regulate transcription of various genes in binding to its B consensus sequence. Following its degradation, I B is rapidly resynthesized to act as an endogenous inhibitory signal for NF- B, and monitoring its de novo expression is a powerful tool to investigate the activity of the transcription factor within the central nervous system (CNS) (for reviews, see Refs. 7,8). NF- B/Rel proteins are dimeric and sequence-specific transcription factors that control the biosynthesis of a wide variety of molecules that are involved in orchestrating the innate immune response (9,10). The intracellular cascade of events that lead to NF- B translocation and the receptors involved in engaging these signaling steps have now been unraveled. B. Toll-like Receptors Until recently, the exact mechanisms involved in the activation of the proinflammatory signal transduction pathways after binding between the LBP/LPS complex Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 1 Innate immune response engaged by the endotoxin lipopolysaccharide (LPS). LPS is a major component of the outer membranes of gram-negative bacteria, which is the best characterized example of innate recognition associated with a robust inflammatory response by phagocytic cells. Secretion of cytokines by circulating monocytes/neutrophils and tissue macrophages by LPS requires a series of mechanisms in cascade, the first step being the binding of the endotoxin with the serum proteins LPS-binding protein (LBP) or septins. The newly formed complex may then activate different populations of cells in binding to its CD14 receptor and the toll-like receptor 4 (TLR4). Two forms of the CD14 receptor can be found; the first one is present at the surface of myeloid cells (CD14) and acts as a glycosyl-phosphatidylinositol (GPI)–anchored membrane glycoprotein. The other form is soluble in the serum (sCD14) and lacks the GPI properties, though it can bind LPS to activate cells devoid of mCD14, such as endothelial cells. LBP is not essential for the LPS signaling, but the LPS/LPB complex is particularly powerful to activate cells of myeloid origin including monocytes, macrophages, neutrophils, and microglia. One of the well-known consequences of such activation is the production of proinflammatory cytokines, such as interleukin-1 (IL-1), tumor necrosis factor alpha (TNF-), and IL-6.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 The proinflammatory signal transduction pathways evolving the nuclear factor kappa B (NF- B). p50 and p65 are the two most common DNA-binding subunits of the NF B dimer that have the ability to trigger the transcription of numerous target genes. See text for details and abbreviations.

and the GPI-anchored mCD14 was unknown. Indeed, studies in CD14-deficient mice suggested the existence of a coreceptor to mediate LPS-induced NF- B activity and cytokine gene transcription (11,12). The recent characterization of human homologs of toll may be the missing link for the transduction pathways leading to NF- B activity and cytokine production in response to bacterial cell wall components. A large family of toll-like receptors (TLRs) has already been characterized, which share similar extracellular and cytoplasmic domains (1). The extracellular domains include 18–31 leucine-rich repeats (LRRs), whereas the cytoplasmic domains are similar to the cytoplasmic portion of the IL-1 receptor and is named the toll/IL-1-receptor homologous (TIR) region (1,13). Distinct TLRs have now been proposed as the key molecules to recognize quite selectively one of the major PAMPs produced by either gram-negative or gram-positive bacteria. The data that mutation of the mouse Lps locus abolishes the LPS response and that Lps encodes the TLR4 provided the first evidence that this particular receptor may play a key role in the innate immune response to gram-negative bacteria (for reCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

view, see Ref. 14). Further supporting this concept is the the fact that TLR4-deficient mice are unresponsive to LPS, whereas TLR2-deficient mice exhibit a normal inflammatory response to the endotoxin (15). These results demonstrate that while TLR2 makes no contribution to LPS signaling, TLR4 is critical to recognize the PAMP produced by gram-negative bacterial cell wall components. On the other hand, TLR2 recognizes various fungal, gram-positive, and mycobacterial elements. The broad spectrum of components recognized by TLR2, together with the existence of an additional eight TLRs, suggests that these receptors participate in a complex pattern of recognition complex (16). In this regard, dimerization of the cytoplasmic domain of TLR2 does not induce cytokine production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain is indeed associated with proinflammatory signaling (16). It was recently found that the cytoplasmic domain of TLR2 can form functional pairs with TLR6 and TLR1 to lead to signal transduction and cytokine gene expression (16). This is quite interesting, because these data provide evidence that while TLR4 homodimer is essentially responsible for triggering NF- B in response to cell wall components of gram-negative bacteria, a more complex combinatorial repertoire is needed to discriminate among the other PAMPs found in nature. In the case of gram-negative bacteria, it is not yet known how LBP, CD14, and TLR4 interact together to function as the LPS signal transducer leading to activation of NF- B and MAP kinases. It is possible that CD14 acts as the principal LPS-binding protein on the surface of monocytic cells and the newly formed complex reaches adjacent TLR4 receptors, which transduce the LPS signal via the general intracellular adaptor protein MyD88 (myeloid differentiation factor 88), which has the ability to interact with TLRs through its own carboxyl-terminal TIR domain (14,17). MyD88 recruits the serine kinase IRAK to engage the proinflammatory signal transduction pathways via its amino-terminal death domain. It has recently been shown that MD-2, a secreted protein with affinity for TLR4, can complement the LPS response if co-expressed with TLR4 in 293 cells (18,19). Hence, MD-2 may be required alongside TLR4 for reconstitution of the authentic LPS signal transduction pathway (see Fig. 3). C. Proinflammatory Cytokines Secreted During the Innate Immune Response The rapid production of cytokines, chemokines, and prostaglandins is an essential feature of the innate immune response. These soluble mediators belong to the family of proinflammatory molecules that are largely responsible for most of the autonomic changes that occur during the acute-phase response of all types of aggression. We will see in the following section the cytokines that are involved in adaptive immunity, and despite some reports showing that they may be involved in modulating specific neurophysiogical responses, their roles are largely re-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3 Hypothetical mechanisms of interaction between LBP, CD14, and TLR4 to function as the LPS signal transducer leading to activation of NF- B and MAP kinases by gram-negative bacteria. It is possible that CD14 acts as the principal LPS-binding protein on the surface of monocytic cells and the newly formed complex reaches adjacent TLR4 receptors, which transduce the LPS signal via the general intracellular adaptor protein MyD88 (myeloid differentiation factor 88), which has the ability to interact with TLRs through its own carboxyl-terminal TIR domain. MyD88 recruits the serine kinase IRAK to engage the proinflammatory signal transduction pathways via its amino-terminal death domain. It has recently been shown that MD-2, a secreted protein with affinity for TLR4, can complement the LPS response if coexpressed with TLR4 in 293 cells. Hence, MD-2 may be required alongside TLR4 for reconstitution of the authentic LPS signal transduction pathway (Adapted from Ref. 14.)

stricted to cells of the immune system and do not circulate in large quantity in the bloodstream. In contrast, most of the cytokines that are rapidly induced in response to foreign material can circulate in the plasma and act on distant organs. Tumor necrosis factor alpha (TNF-), interleukin-1 (IL-1 and ), IL-6, IL-10, IL-12, IL-15, IL-18, and type I interferons (IFN- and IFN-) are cytokines that belong to the innate immune system, although they are not all proinflammatory (IL-10 is anti-inflammatory) and only a few of them are generally recognized to be potent modulators of the autonomic/neuroendocrine system.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

1.

TNF-

TNF- is a pleiotropic cytokine originally recognized for its antitumor activity (20), but now referred to as a proinflammatory factor that plays a central role in initiating the cascade of other cytokines involved in a timely controlled immune response to infection. This cytokine is produced by a variety of cell types, including neutrophils, activated monocyte/macrophages (21), T and B lymphocytes (22), natural killer (NK) cells, astrocytes (23), mast cells, endothelial cells (24), smooth muscle cells, synovial cells (25), brain ependymal cells (26), and microglia (27,28). Macrophages and NK cells remain nevertheless the major cell source, at least in the periphery. The endotoxin LPS and other inflammatory agents cause a rapid production of TNF- from both in vitro and in vivo phagocytic cells and a subsequent increase of other proinflammatory cytokines, such as IL-1 and IL-6. TNF plays an important role in a wide variety of events, namely septic shock, cell proliferation, and apoptosis, which can be mediated by either one of the TNF receptors (p55 and p75), both of which belong to the TNF receptor superfamily. The binding of TNF- to its cognate receptors leads to the formation of the TNF-R1–associated death domain (TRADD)/TNF receptor–associated factor 2 (TRAF2) complex, which activates the NF- B signaling events. TNF- is actually one of the most potent effectors of NF- B activity through the 55 kDa TNF type I receptor in most types of cells in the systemic immune system as well as in the CNS. FAS-associated death domain protein (FADD)/MORT1, TRAF2, and the dead domain kinase receptor interacting protein (RIP) are recruited and may also interact directly with TRADD (see Fig. 4). While FADD/MORT1 is essential for TNF-induced apoptosis, RIP and TRAF2 seem to be the key molecules for activating both NF- B and c-Jun N-terminal kinase (JNK). This recruitment leads to activation of the protein kinase IKK, composed of two catalytic subunits, IKK and IKK, and one regulatory subunit, IKK/NEMO. IKK/NEMO is required for the activation of NF- B by many stimuli, including TNF, although only the IKK catalytic subunit is essential to trigger NF- B in response to the cytokine. Indeed, IKK was recently found to be the target for upstream signals generated by proinflammatory stimuli resulting in I B phosphorylation at serine 32/36 and degradation by proteasome after polyubiquitination (31,32). This frees NF- B and allows its translocation into the nucleus and the subsequent activation of target genes, as already mentioned. One of the best known immune functions of TNF is stimulating the recruitment of neutrophils and monocytes to the site of infection and activation of these cells in a paracrine manner to eliminate the foreign material. This mechanism involves members of the chemokine family that play a primary role in the recruitment of inflammatory cells in different tissues during acute and chronic inflammation. The monocyte chemoattractant protein-1 (MCP-1) is one of the principal CC chemokines (see below) that is rapidly synthesized by endothelial cells in reCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 4 Proximal components of the TNF receptor type I signal transduction pathways leading to apoptosis, activation of the nuclear factor kappa B (NF- B) cascade, or induction of the mitogen-activated protein (MAP) kinases. See text for details and abbreviations.

sponse to leukocyte-derived TNF and IL-1 for allowing the monocyte recruitment and rolling prior to transmigration and arrival at the site of inflammation. This transcriptional process is under the direct control of the previously described cascade of events, e.g., TRADD/TRAF2/RIP/IKK/I B/NF- B. In severe infections, TNF is produced in large concentrations and has been found to be involved in systemic clinical and pathological abnormalities, such as fall in blood pressure and shock. Septic shock is a complication of severe gram-negative bacterial sepsis associated with vascular collapse, disseminated intravascular coagulation, and profound metabolic disturbances. Although TNF was believed to be responsible for LPS-induced septic shock, numerous experiments failed to provide evidence that this syndrome could be entirely attributed to the high circulating levels of this proinflammatory cytokine. However, circulating levels of TNF may be predictive of the outcomes of severe gram-negative infection. At lower levels this cytokine was found to be involved in various autonomic and endocrine changes, including fever and increase in the HPA axis. The exact mechanisms and target cells mediating the effect of circulating TNF on the regions involved in autonomic control will be detailed later in the chapter. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

2.

Interleukin-1

This proinflammatory cytokine (especially the  form) is probably the most important molecule capable of modulating cerebral functions during systemic and localized inflammatory insults. There are two forms of IL-1, IL-1 and IL-1, that share less than 30% homology and bind to the same receptors. This explains the original thought that both had similar biological activities, although the  form is no longer believed to be a potent player in the neural-immune interaction. Actually, the IL-1 family consists of three different genes located in humans on the long arm chromosome 2 (33), which encode three distinct proteins with structural homologies. IL-1 and IL-1 act as agonist molecules as opposed to the third member of the family, IL-1 receptor antagonist (IL-1ra), which binds to the same receptors as IL-1 and IL-1 but does not induce any intracellular signal and therefore acts as an endogenous inhibitor of the IL-1 activity. The physiological relevance of IL-1ra in modulating the biological activity of IL-1 was confirmed in transgenic mice that either produce no IL-1ra or overproduce the protein (34). Mice lacking IL-1ra have a decreased body mass compared with wild-type controls. They were more susceptible than controls to lethal endotoxemia but were less susceptible to infection with Listeria monocytogenes. Conversely, IL-1ra overproducers were protected from the lethal effects of endotoxin but were more susceptible to listeriosis. Serum levels of IL-1 following an endotoxin challenge were decreased in IL-1ra-null mice and increased in IL-1ra overproducers in comparison to controls. These data were nevertheless quite surprising, because the absence of the antagonist was expected to lead to more IL-1 production. In most situations IL-1ra is secreted in the extracellular environment in the form of a 22 kDa glycosylated protein whose biological activity is analogous to that of the 17 kDa nonglycosylated protein. Unlike IL1, which has two binding sites for its receptor, IL-1ra has only one, which may explain the absence of signal transmission and inhibition of IL-1 activity. IL-1 and IL-1 have different mechanisms of expression, synthesis, and secretion. The IL-1 gene does not contain sequences corresponding to the classical transcription initiation motif known as “TATA box,” whereas this motif is found in the IL-1 gene (35). The transcription of the IL-1 gene seems to be controlled in a more complex way, and the promoter region contains cAMP-response element (CRE), an element responding to LPS called NF-IL6, a site analogous to the NF- B transcription factor, and numerous other binding sites (36). These observations are quite important for explaining the ability of peripheral blood monocytes to synthesize IL-1 in response to very low concentrations of LPS, although the IL-1 gene is not constitutively expressed in monocytes or other cell types. The regulation of that gene is under a sophisticated transcriptional control that is triggered by a number of immune insults, a phenomenon that is generally observed for most NF- B–responsive genes. In the presence of LPS, mRNA encoding of both IL-1 and IL-1 can be detected in monocytic cells in as quickly as 15 minutes, accumulates over 4 hours, and rapidly disappears thereafter (37). IL-1 Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

and IL-1 also differ substantially in relation to localization, maturation, and secretion; IL-1 remains mainly intracellular, whereas IL-1 is secreted after cleaving by a specific protease. Unlike pro-IL-1, pro-IL-1 is just as active as the mature form and remains intracellular, which explains why IL-1 is rarely found in the circulation or extracellular liquids, only in cases such as serious illnesses when it may have its origin from the lysed cells. Pro-IL-1 also remains in the cytosol until it is cleaved and transported in the extracellular environment. The specific enzyme responsible for this cleaving is a member of the caspase family called caspase-1 or IL-1–converting enzyme (ICE). ICE is constitutively present in most cells in the form of a 45 kDa inactive precursor that requires endo-cleaving before becoming an active heterodimer, composed of a 10 kDa and a 20 kDa chain containing the enzyme site. Following cleavage, IL-1 has the ability to circulate in the bloodstream and act on distant organs to trigger different physiological responses in binding to its type I receptor. IL-1, IL-1, and IL-1ra share the same receptors, called IL-1 receptor type I (IL-1RI), IL-1RII, and IL-1 receptor accessory protein (IL-1R-AcP). IL-1RI is a glycoprotein expressed at the surface of numerous types of cells, especially on endothelial cells where it mediates the rapid induction of the chemokine MCP-1 to engage the emigration process at the site of infection. The binding of IL-1 to its cognate type I receptor leads to the formation of the IL-1 receptor–associated kinase (IRAK)/TNF receptor–associated factor 6 (TRAF6) complex, which recruits the general adaptor protein MyD88 and activates NIK/IKK kinases involved in the phosphorylation and degradation of I B (5). NF- B is then translocated into the nucleus and may bind to its B consensus sequence on target genes (6). IL-1RAcP presents some homology with IL-1R1 in respect to both its extracellular domain and its cytoplasmic fragment and seems to be required for allowing or potentiating signal transduction in response to IL-1/IL-1RI interaction. Its exact contribution in IL-1 signaling is nevertheless not fully understood. IL-1RII is a 68 kDa glycoprotein whose intracytoplasmic portion has only 29 amino acids, consistent with the fact that it cannot transmit any signal (36). This receptor, however, is capable of binding IL-1 with great affinity and has the ability to compete with IL-1RI to decrease IL-1 biological activity, explaining the term “decoy receptor.” This inhibitory mechanism is quite different from the IL-1ra that may bind to both receptors without inducing signal transduction, whereas IL-1 is trapped by IL1RII to prevent its binding to IL-1RI. Both the IL-1ra and IL-1RII are nevertheless potent endogenous inhibitory molecules that prevent an exaggerated response in presence of high circulating levels of IL-1. It is therefore possible to conclude at this time that the binding of IL-1 to its type I receptor is the only potential trigger of the IL-1 family for rapidly engaging the signal transduction in distant organs during systemic immune stimuli. IL-18 is a recently found new member of the IL-1 family, although its action in mediating NF- B activity is quite different from IL-1 and does not seem to be involved in the early events occurring during the acute-phase response. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

3.

Interleukin-6 and gp130-Related Cytokines

Interleukin-6 (IL-6) is one of the most pleiotropic cytokines known that is involved in regulating a wide variety of immune functions, such as B- and cytotoxic T-cell differentiation, induction of IL-2 production and IL-2 receptor expression in T cells, T-cell growth, as well as the acute-phase reactions and hematopoiesis (38,39). A critical role of the proinflammatory cytokine in the acute-phase response was reported in IL-6–deficient mice that exhibit a lower acute-phase protein (APP) synthesis than wild-type mice during systemic inflammatory insults (40). Although the biosynthesis of APP by hepatocytes is regulated by several factors, including TNF and IL-1, it was found that IL-6 can function as the key hepatocyte-stimulating factor to induce, at least in rodents, fibrinogen, cysteine proteinase inhibitor, 2-macroglobulin and 1-acid glycoprotein (39). In addition to being produced by both systemic lymphoid and nonlymphoid cells, IL-6 protein and mRNA have also been found in specific populations of cells in the CNS during different experimental conditions. One of these stimuli is intraperitoneal (i.p.) or intravenous (i.v.) administration of the endotoxin LPS that caused a profound transcriptional activation of the gene encoding the cytokine in the choroid plexus (chp) and the circumventricular organs (CVOs), structures devoid of blood-brain barrier (41). This phenomenon is of particular interest, as it provides evidence that IL-6 may be secreted in the cerebrospinal fluid (CSF) and reaches its receptor subunits, widely distributed throughout the neural tissue (41), to influence different neurophysiological functions (see below). As shown in Figure 5, the first step in the induction of the transduction signals by IL-6 is the binding of the ligand to its IL-6 receptor subunit (IL-6R), which is either located at the cell surface or present in soluble form in the liquids of the organism. The association of these two molecules with the membrane subunit gp130 forms a high-affinity complex that triggers specific transduction signals (42). The gp130 protein serves as a signal transducer not only for IL-6, but also for the ciliary neurotrophic factor (CNTF), leukemia-inhibitory factor (LIF), oncostatin M (OSM), CT-1, and IL-11 (38,43). However, the actions of these proinflammatory cytokines are limited by the mechanisms that control their synthesis, as they are produced in a tissue-specific manner in response to different immunogenic stimuli. Three members of the janus kinase family, JAK1, JAK2, and TYK2, are closely related to gp130 and rapidly activated in the presence of IL-6 (44–46). These kinases phosphorylate the tyrosine residues of the gp130 cytoplasmic domain, which allows the recruitment and phosphorylation of at least two transcription factors of the signal transducers and activators of transcription family (STAT1, STAT3) and one tyrosine phosphatase (SHP-2) (45,47–51). Once activated, the STAT proteins may activate different genes in combining their SH2 domains and forming homodimers (52,53). Moreover, SHP-2 is able to activate the membrane protein Ras, which leads to the induction of the MAP kinase ERK1 and ERK-2 (54–57). Two pathways exist relating SHP-2 and Ras, one using the Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 5 Interleukin-6–induced transduction signals leading to the transcription of acute-phase protein (APP). The first step in the induction of the transduction signals by IL6 is the binding of the ligand to its IL-6 receptor subunit (IL-6R), which is either located at the cell surface or present as soluble form in the liquids of the organism. The association of these two molecules with the membrane subunit gp130 forms a high-affinity complex that triggers specific transduction signals. Three members of the janus kinase family, JAK1, JAK2, and TYK2, are closely related to gp130 and rapidly activated in the presence of IL6. These kinases phosphorylate the tyrosine residues of the gp130 cytoplasmic domain, which allows the recruitment and phosphorylation of at least two transcription factors of the signal transducers and activators of transcription family (STAT1, STAT3) and one tyrosine phosphatase (SHP-2). Once activated, the STAT proteins may activate different genes in combining their SH2 domains and forming homodimers. Moreover, SHP-2 is able to activate the membrane protein Ras, which leads to the induction of the MAP kinase ERK-1 and ERK-2. Two pathways exist relating SHP-2 and Ras, one using the adapter protein Gab1 that is associated with phosphatidylinositol-3-kinase and another via the Grb2Sos complex. The activated MAP kinases may in turn induce nuclear proteins, such as NFIL6. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

adapter protein Gab1, which is associated with phosphatidylinositol-3 kinase, and another via the Grb2-Sos complex (47,58). The activated MAP kinases may in turn induce nuclear proteins, such as NF-IL6. This short description of the signaling events that take place in response to IL-6 is quite simplistic, because gp130 may also be coupled to several other kinases, including Shc, Hck, Btk, and Tec (59–62) or the receptor ErbB2 to stimulate other MAP kinases (63). Like the NF- B signaling that is inhibited by I B, the JAK/STAT pathway is inhibited by at least two intracellular systems to avoid exaggerated responses. The first is the internalization of the IL-6/IL-6R/gp130 complex and its degradation by specific enzymes (64–66). This process does not require activation of the JAK kinases and is transduced by a degradation of the receptor at the level of the cell surface (67). The second involves activation of the transduction signals and the de novo production of inhibitory proteins that prevent phosphorylation of the transcription factor STAT and activation of the MAP kinases by interacting with the catalytic domain of the JAK kinases (68). These cytokine-inducible proteins, the suppressors of cytokine signaling (SOCS-1 to 7), JAK-binding protein (JAB), STAT-induced STAT inhibitors (SSI), and cytokine-inducible SH2 protein (CIS), are rapidly induced by IL-6 and other members of the gp130 cytokine family (69,70). All these different names are now being grouped under SOCS-1 to 7 and CIS, although there is still some controversy about this unique terminology. The SOCS proteins are characterized by a highly conserved carboxyl-terminal SOCS box motif, which is preceded by an SH2 domain (70). Although SOCS-1 and SOCS-3 have potent activity for the inhibition of IL-6 signaling, the other members of the SOCS family and CIS have little or no impact (for a review, see Ref. 71). Both SOCS-1 and SOCS-3 act by preventing cytokine-dependent activation of the JAK/STAT pathway, but the intracellular mechanisms involved in these effects are quite different. SOCS-1 inhibits intrinsic kinase activity by interacting with the catalytic domain of the JAK kinases (especially JAK2 kinase), whereas SOCS-3 prevents IL-6 signaling at steps distal to JAK activation, e.g., recruitment of STAT factors and/or via binding to the tyrosine-phosphorylated receptor (72). Indeed, recent studies have shown that SOCS-3 is unable to inhibit directly either JAK1 or JAK2 kinase activity (73). Elegant studies have shown that circulating leptin has the ability to induce specific expression of SOCS-3 mRNA in hypothalamic nuclei and that this inhibitory protein acts as an endogenous blocker for leptin receptor–mediated signal transduction in mammalian cell lines (74). Interestingly, the leptin receptor acts in concert with gp130, the signal-transducing subunit of the IL-6 cytokine receptor family (75,76). The fine distribution of the gene encoding SOCS-3 protein was also found in specific cellular populations of the brain that express both IL-6 receptor subunits during systemic inflammation (77). The endogenous production of the cytokine appears to be an essential prerequisite, as LPS-induced SOCS-3 transcription is prevented in microvascular-associated elements in IL-6–deficient mice (77). This latter study provided evidence that expression of this inhibitor of the JAK/STAT signaling molecules follows the necessary preinduction of IL-6 reCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ceptors during the time-related events that take place during the acute-phase reaction. Indeed, preimmune-treated animals were very sensitive to the proinflammatory cytokine, a phenomenon that can be explained by the necessary induction of IL-6 receptors in cells reachable by the systemic circulation (77). These events seem to take place in a logical order during the acute-phase response, because IL6 is released after IL-1 and TNF-, which can activate synthesis of the IL-6 receptors in cells of the blood-brain barrier (BBB) (41). The stimulation of IL-6R and gp130 in the endothelium of brain capillaries and the circumventricular organs (CVOs) is likely to be part of the mechanisms that prepare the cellular response when IL-6 is released into circulation. The profound induction of SOCS-3 may in return be a determinant intracellular signal to inhibit the IL-6–induced JAK/STAT signaling within selective structures of the brain (see below). 4.

Other Cytokines of Innate Immunity

Although TNF, IL-1, and IL-6 are by far the most relevant molecules of the immune system having the ability to interact with cerebral tissue and to influence different autonomic functions, numerous other cytokines and chemokines are released during the innate immune response and may somehow modulate and alter the action of the three previously described cytokines. These include IL-12, type I interferons (IFN- and ), IL-10, IL-15, IL-18, and small proteins of the chemokine family. Chemokines belong to a large family of small cytokines that can be divided into two subfamilies based on their genetic, structural, and functional characteristics. One amino acid separates the two cysteines in the CXC (or ) chemokines, whereas the two cysteines are adjacent in the CC (or ) family. In general, the primary structure predicts the role of chemokines in the recruitment of distinct subsets of leukocytes to the site of inflammation and injury. The CXC members were originally identified as potent activators and chemoattractant for neutrophils, whereas those of the CC family were believed to be attracting essentially monocytes (78). However, the activity of members of both families in attracting immune cells overlaps, because CC and CXC chemokines are chemotactic for lymphocytes (79,80). A nonexhaustive list of the CC chemokines include MIP-1, MIP-1, MIP-3, RANTES, MCP-1, 2, 3, 4, eotaxin, TARC, LARC, ELC, and 1309, whereas those of the CXC family include IL-8, Gro-/, IP10, Mig, SDF-1, and BLC/BCA-1. These molecules are involved in recruiting the cells of host defenses to sites of infection, regulating the traffic of lymphocytes and other leukocytes through peripheral lymphoid tissues, and the development of diverse organs. D. Cytokines Released During Adaptive Immunity and Hematopoiesis The present chapter focuses on proinflammatory cytokines that are rapidly produced during systemic inflammatory insults, because most of the autonomic and endocrine changes monitored in response to an immune challenge are closely reCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

lated to their secretion patterns, and single or combined administrations of these early cytokines have the ability to cause profound autonomic outcomes (see Sec. V). However, few data exist on the role of cytokines responsible for adaptive immunity, and a short overview of these molecules will therefore be presented. These include IL-2, IL-4, IL-5, IL-13, IL-16, IL-17, IFN-, transforming growth factor- (TGF-), and lymphotoxin (LT). The amounts produced are generally low and are usually undetectable in the serum, which explains the moderate interest in these cytokines as being potentially important players in mediating physiological responses at distant organs. Their principal functions consist in regulating lymphocyte growth and differentiation and activating effector cells, such as macrophages, eosinophils, and mast cells. INF- can play key roles in both innate and adaptive immunity. As opposed to cytokines of innate immunity, which are essentially produced by monocytic cells and polymorphonuclear cells, most cytokines of adaptive immunity are produced by CD4 T lymphocytes and, in lesser amounts, by CD8 T cells. Although IL-2 has been shown to be able to modulate the neuronal activity, neuroendocrine functions, and the corticotrophic axis in the rodent, these data are somewhat surprising due to the general role of this cytokine as an autocrine growth factor. Indeed, IL-2 acts mainly on the same cells that produce it for antigen-stimulated T lymphocytes and is responsible for T-cell clonal expansion after antigen recognition. A single bolus of cytokines not normally found in the circulation may not be very useful to clarify the contribution of the immune system in modulating brain circuits as they do or do not relate to autonomic functions. Cytokines are also necessary for normal hematopoiesis in the bone marrow and elicit production of new leukocytes. Mature leukocytes arise from pluripotent stem cells by commitment to a particular lineage and progressive expansion of the progeny. Colony-stimulating factors (CSFs) stimulate the differentiation and expansion of the bone marrow progenitor cells into cells of specific lineages, namely granulocytes, monocytes, or lymphocytes. Interleukin-7 (IL-7), which activates JAK/STAT signaling through its receptor of the type I cytokine family, is responsible for the survival and expansion of immune precursors committed to the Band T-lymphocyte lineages. Interleukin-3 (IL-3) is a member of the four -helical family of cytokines, known as multi-CSF, which acts on immature marrow progenitors to promote their expansion. Granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), IL-9, and IL-11 are the other hematopoietic cytokines that all trigger JAK/STAT signaling for engaging the cellular response of the bone marrow progenitors to increase production of inflammatory leukocytes. Some of them have also been used successfully for clinical applications, such as to stimulate bone marrow recovery after cancer chemotherapy and bone marrow transplantation. Their roles in cerebral tissue and the autonomic nervous system are largely unknown, and as for the cytokines involved in adaptive immunity, they most likely play very little role in the neuro-immune interface. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

III. CYTOKINES IN THE CNS There are accumulating data that the brain itself is capable of synthesizing and releasing proinflammatory cytokines. Recent developments in molecular biology have indeed provided new tools to investigate the genetic regulation of different immune-related factors within specific cellular populations of the CNS using both in vitro and in vivo models. However, this largely depends on the experimental models that are either associated or not with neurodegenerative features. Whether these molecules are constitutively expressed in the cerebral tissue remains a controversial issue. Although it is clear that transcriptional activation of proinflammatory cytokines occurs in the CNS during a wide variety of models and diseases, extremely low mRNA levels are generally found under basal conditions. This is actually the case not only in the CNS, but in all tissues. Therefore, one would seriously question the physiological relevance of constitutively found protein levels for molecules that are under sophisticated transcriptional processes triggered only in case of emergency, such as endotoxemia. A. Bacterial Endotoxins and Signaling Events in the Brain The LPS receptor CD14 mRNA is constitutively expressed in the circumventricular organs, brain regions that contain a rich vascular plexus with specialized arrangements of the blood vessels (81). The tight junctions normally present between the endothelial cells are shifted in part to the ventricular surface and partly to the boundary between the CVOs and the adjacent structures, explaining the diffusion of large molecules into the perivascular region (82). The mRNA encoding TLR4 is also found in structures that can be reached by the bloodstream, namely the CVOs, choroid plexus (chp), and leptomeninges (83). In contrast to the profound transcriptional activation of the LPS receptor CD14 and the indicator of NF- B activity I B, the endotoxin and circulating IL-1 caused a significant decrease of TLR4 transcript in most of the constitutively expressing parenchymal and nonparenchymal regions of the brain (83). The basal expression of CD14 and TLR4 in the CVOs is likely to be a key mechanism in the proinflammatory signal transduction events that originate from these structures during innate immune response. Indeed, cell wall components of gram-negative bacteria may be selectively recognized by the TLR4/CD14-bearing cells of the CVOs, which allows LPS signaling and then rapid transcription of proinflammatory cytokines first within these organs and thereafter across the brain parenchyma during severe endotoxemia. The rapid induction of IL-1, IL-6, and TNF- mRNA in the CVOs, chp, and leptomeninges by systemic LPS treatment clearly indicates that such events take place in these specific populations of cells in the brain (27,28,41,84). Microscopic analysis of emulsion-dipped slides revealed that TNF-positive cells spread over the anatomical boundaries of the CVOs in a migratory-like pattern during the Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

course of the endotoxemia (28). A similar pattern of de novo expression was also observed for the gene encoding CD14, but not TLR4 in response to circulating LPS. The LPS-induced CD14 transcription in parenchymal microglia is dependent on the centrally produced TNF-, which actually plays an essential autocrine/paracrine role in triggering parenchymal microglial cells during severe endotoxemia (27). The co-existence of both TLR4 and CD14 receptors in the CVOs may enable the endotoxin to trigger the proinflammatory signal transduction events in structures that can be reached from the systemic circulation, whereas the subsequent microglial activation in the brain parenchyma is dependent on TNF- (see Fig. 6). Therefore, TLR4 may be essential in this innate immune reaction that originates from the CVOs in response to cell wall components of gram-negative bacteria.

Figure 6 Autocrine and paracrine roles of TNF- in mediating the synthesis of the LPS receptor CD14 in the brain microglial cells during blood endotoxemia. Cell wall components of gram-negative bacteria may be selectively recognized by the TLR4/CD14-bearing cells of the CVOs, which allows the LPS signaling and then the rapid transcription of proinflammatory cytokines, first within these organs and thereafter across the brain parenchyma during severe endotoxemia. It is suggested here that circulating LPS targets its transmembrane receptors in CVOs/chp macrophages and microglia, which may stimulate the NF B–signaling events and trigger TNF transcription. The cytokine may in return take the relay in binding to its cognate p55 receptor and lead to the formation of the TRADD/TRAF2/RIP complex, which may activate the NF- B–signaling kinases in adjacent microglia. Such events are likely to contribute to the transcriptional activation of both CD14 and TNF genes in the brain of endotoxin-treated animals.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Although a strong increase in CD14 transcription is generally detected after systemic LPS injection, the endotoxin failed to stimulate the gene encoding TLR4. CD14-expressing cells were clearly devoid of TLR4 transcript in microglia across the brain parenchyma during moderate and severe endotoxemia. It is possible that TLR4 is the recognizing molecule for gram-negative bacterial components only in response to systemic infection, whereas CD14 has a more complex role in the proinflammatory signal transduction events in the brain parenchyma. These events may be determinant in orchestrating the neuroinflammatory responses that take place in a well-coordinated manner to activate the resident phagocytic population of cells in the brain. The physiological outcomes of this innate immune response of the CNS are likely to include a rapid elimination of LPS particles via increased opsonic activity of the transmembrane CD14 receptor to prevent potential detrimental effects on neuronal elements during blood sepsis. Nomura and colleagues have recently reported that TLR4 mRNA expression in mouse peritoneal macrophages significantly decreased within a few hours of LPS treatment and returned to the original level at 24 hours (85). A rapid decrease in surface TLR4 expression was seen as early as after 1 hour and remained suppressed over 24 hours in cells preexposed to LPS. These authors suggested that the downregulation of surface TLR4 expression may be responsible for the decrease in inflammatory cytokine production in tolerant macrophages, which may explain one of the mechanisms for LPS tolerance (85). These data obtained from systemic macrophages are in complete agreement with the previously described study that showed convincing downregulation of cerebral TLR4 genes in response to a single LPS bolus. Because the endotoxin has the ability to increase CD14 mRNA in the CVOs, it was possible to perform dual-labeling for the LPS receptor, and numerous resident macrophages were positive for the transcript (81). Although both transcripts (TLR4 and CD14) may not be expressed in the same cells, we speculate here that TLR4 is located at the surface of the phagocytic population of cells of the CVOs, chp, as well as the leptomeninges. TLR4 transcript levels were low in cerebral tissue under basal conditions. The signal was nevertheless specific, as we performed numerous controls to ensure that what was being seen could not be related to an artifact of the in situ hybridization procedure (83). Actually, we had to adjust and maximize the hybridization conditions to detect this transcript in situ by generating the riboprobe just after the prehybridization step on freshly mounted brain sections (83). This very low level in the brain fits, however, quite well with the fact that the copy number of TLR4 is extremely low in systemic phagocytes compared to the more abundant membrane protein CD14 (14). It is nevertheless remarkable that so few TLR4 receptors (perhaps 1000 or fewer per cell), residing on macrophages alone, have such an important influence in LPS signaling and the coordination of the biological responses to gram-negative infections (14). It is expected that CVO

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

TLR4 acts as a sensor for engaging the cerebral innate immune response in cases of invasion during systemic bacterial infection that may have detrimental consequences for the neuronal material. Of interest is the constitutive expression of TLR4 in different nuclei and areas of the brain suggesting a potential role for this membrane-spanning component in the parenchymal elements of the brain (83). However, TLR4 seems quite specific to cell wall components of gram-negative bacteria; what then will a receptor do without ligand? Obviously, this question is difficult to answer, as we are just at the embryonic stage of mammalian Toll biology, although it is proposed here that TLR4 may be a key element of the well-organized innate immune response that takes place in the CNS. B. TNF as Key Autocrine/Paracrine Mediator Within Brain Parenchyma Like systemic phagocytes (86), CD14 synthesis in parenchymal cells of the brain may be dependent on the production of proinflammatory cytokines. Of interest is data indicating that systemic injection of bacterial endotoxin induced strong expression of CD14 (81) mRNA in a pattern closely related to the induction of TNF (28) transcript with a rapid and delayed response. Although there is a large body of evidence that CD14 is necessary for the role of LPS in the induction of cytokine transcription from different myeloid cells, the possibility remains that the cytokine itself acts as an autocrine and paracrine factor to upregulate the LPS receptor. Indeed, TNF- is able to induce a transient increase in plasma CD14 levels with a peak at 6–8 hours, and this elevation in CD14 antigen levels was shown to be accompanied by increased levels of CD14 mRNA in lung, liver, and kidney (87). Pretreatment of mice with an anti-TNF- antibody significantly prevented LPSinduced mCD14 transcription (87,88). Whether such a mechanism is operating in the brain was recently investigated, and it was indeed found that the cytokine is a key paracrine factor contributing to activation of adjacent cells within the brain parenchyma (27). To ascertain that TNF was capable of triggering its own production and CD14 transcription in specific cellular populations of the brain, animals received a single intracerebroventricular (i.c.v.) bolus of recombinant rat TNF- and were killed at different times thereafter. A second experiment consisted of neutralizing the biological activity of the cytokine but infusing an antirat TNF- antibody in the lateral ventricle 10 hours before the systemic challenge with the endotoxin LPS. The results confirmed the hypothesis that TNF- has the ability to trigger I B, TNF-, and CD14 transcripts in microglia across the brain parenchyma and that endogenous release of the proinflammatory molecule is responsible for the paracrine autostimulation of the phagocytic population of cells in the brain during blood sepsis (27).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The direct action of LPS on myeloid-derived cells expressing mCD14/TLR4 and present in structures accessible from the systemic circulation may allow a rapid production of proinflammatory cytokines within these organs (see Fig. 6). Like CD14, scattered small TNF-expressing cells can be found across the brain parenchyma in response to LPS, although this depends on the dose of the endotoxin and the route of administration (28). This clearly indicates that the endotoxin first reaches available targets devoid of BBB, which in return, depending on the severity of the challenge, causes prime adjacent cells within the parenchymal brain to stimulate TNF- transcription. The bacterial endotoxins are among the most powerful agents known to stimulate circulating monocytes and tissue macrophages, which leads to the synthesis and release of a variety of proinflammatory cytokines (4). The most important target of macrophage-derived secretory products is the macrophage itself (89). The early production of TNF may be an essential step in these autocrine and paracrine loops, as this cytokine is able to induce its own production by an autocrine stimulation, followed by the synthesis of other proinflammatory cytokines, such as IL-1 and IL-6 (4). It is suggested here that circulating LPS targets its transmembrane receptors in CVOs/chp macrophages and microglia, which may stimulate the NF- B-signaling events and trigger TNF transcription (see Fig. 6). The cytokine may in return take the relay in binding to its cognate p55 receptor and lead to the formation of the TRADD/TRAF2/RIP complex, which may activate the NF- B–signaling kinases in adjacent microglia. Such events are likely to contribute to the transcriptional activation of both CD14 and TNF genes in the brain of endotoxin-treated animals. IL-1 has also been reported to stimulate CD14 expression in different organs, and anti-IL-1 antibody attenuated the induction of the LPS receptor in response to the endotoxin (87,88). IL-1 and TNF- are known to have numerous overlapping activities, and inhibiting one cytokine may frequently be associated with redundant mechanisms because of the presence of the other cytokine (90). Although IL-1 may have the ability to stimulate CD14 in the brain microglia, its involvement most likely depends on the prior production of TNF as the anti-TNF completely inhibited LPS-induced CD14 transcription (27). On the other hand, IL-1 is the key inflammatory signal in the brain to stimulate the production of growth factors by astrocytes during brain trauma, whereas TNF is not essential for such a response. Indeed, we have recently observed rapid production of numerous proinflammatory molecules in cells lining the lesion site, followed by a robust increase in ciliary neurotrophic factor (CNTF) biosynthesis (91). The release of CNTF was completely inhibited in IL-1–deficient mice notwithstanding ongoing TNF- production by microglial cells lining the cortical lesion (91). These data support the concept that despite the recognized overlapping activities of both NF- B–signaling cytokines in the systemic immune system, IL-1 and TNF-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

seem to have a distinct role in orchestrating the inflammatory events that take place in the brain. C. The Complement System as Part of the Cerebral Innate Immune Response The complement system consists of nearly 30 proteins that can be activated via three different pathways: the classical, alternative, and mannan-binding lectin (MBL) circuits (for review, see Ref. 92). All three pathways lead to the formation of C3 and C5 convertases and result in the formation of several products aimed to promote inflammation, phagocytosis, and cytolysis (92,93). The liver was initially considered as the major site for producing elements of the complement system, but extrahepatic cellular sources of C3a and C5a are now known to be essential in the initiation and regulation of the inflammatory response (94). Activated monocytes with pathogens, LPS, cytokines alone or together with the endotoxin result in biosynthesis of the complement proteins. Actually, cells of monocytic lineage have the ability to synthesize most of the complement ligands and their receptors with appropriate stimuli. The complement system is an essential modulator of the acute and chronic inflammatory responses in allowing appropriate release of cytokines, which can act as an autocrine loop on monocytes/macrophages to further increase complement proteins from specific pathways. C3a and C5a are considered the most powerful molecules generated by the complement system; they trigger signal transduction by interacting with their seven transmembrane receptors, which are members of the rhodopsin family of Gprotein–coupled receptors (95–97). As mentioned, the CNS is isolated from the circulation by the BBB, and proteins of the complement system produced by systemic immune cells may not reach the cerebral tissue, at least when the BBB is intact. However, the presence of complement proteins were found during CNS inflammation and neurodegenerative diseases, such as multiple sclerosis, Guillain-Barré syndrome, Huntington’s disease, bacterial meningitis, Alzheimer’s disease, stroke, and brain injuries (97–101). However, the exact cellular source of the complement components in the CNS during acute or chronic inflammatory responses associated or not with neurodegeneration remains largely unknown, although astrocytes and microglia represent good candidates for the local production of the complement proteins in the brain. Indeed, these cells are considered as immunocompetent, as they express the class II MHC, adhesion molecules, and numerous proinflammatory genes in response to cerebral insults and endotoxemia (27,28,81,83,99,101–103). Astrocytes in culture synthesize C3 and factor B in presence of LPS and cytokines (104,105), whereas microglia and astrocytes bear the complement anaphylatoxin receptors (100).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

These studies indicate that proteins of the complement system may be produced in different populations of cells during neuropathological conditions and cerebral insults, but whether the CNS can mount such responses normally or during systemic immune challenges not associated with neurodegenerative disorders or alteration of the BBB is still an open question. Recent data provided evidence of a time-related regulation of different components of the complement system in the CNS and further supported the existence of an elegant innate immune response that takes place within specific populations of cells in the cerebral tissue during blood endotoxemia (106). C3 and C5 are two major complement transcripts that are expressed in the CNS under basal conditions, although their patterns of expression are highly different. Indeed, the mRNA encoding C3 was found in very limited nonneuronal structures, whereas C5 mRNA was widely distributed across the brain parenchyma in both neuronal and nonneuronal elements (106). Such strong hybridization signals remained unchanged in response to circulating LPS, which differed from the gene encoding C5aR, which was upregulated in the brain of LPS-challenged mice. A single bolus of endotoxin also caused profound transcriptional activation of C3, C3aR, and factor B in numerous nonneuronal structures, and the induction wave supported the concept of an integrated response of the complement system during endotoxemia. These genes are under a sophisticated transcriptional process in endothelial and myeloid-derived cells, because all induced transcripts returned to basal levels after the insult. An alternative pathway therefore exists in the brain, and this highly organized innate immune response may be essential for eliminating pathogens and orchestrating the inflammatory response to prevent neuronal damage and restore body homeostasis during systemic bacterial infection. Such systemic infection is censored by a group of supporting cells in regions devoid of BBB and the cerebral microvasculature to engage a timely innate immune reaction necessary for preventing the neuronal damage, although it may have detrimental consequences in case of exaggeration. A low but positive C3 mRNA signal was found along the leptomeninges of the ventrolateral medulla in cells lining the ventral third ventricle and the epithelial ependyma of the chp at the edge of the ventral hippocampal commissure (106). All these structures are strategically well positioned for releasing the C3 protein in the CSF and thereafter diffuse throughout the brain parenchyma. The structures devoid of tight junctions also exhibited a very strong transcriptional activation of the C3 gene in response to circulating LPS, which may contribute to raising, quite significantly, C3 protein concentrations in the CSF. Due to the critical role of C3 protein in the complement cascade, we propose here that its presence in structures devoid of BBB is a potential mechanism in the defense of cerebral tissue against invading pathogens circulating in the bloodstream and across the brain capillaries. The CVOs represent a route of entry for pathogens in the CNS, and C3 may tag the pathogen surface to increase the phagocytic activity of the resident

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

macrophages and microglia. C3 contains an unstable thioester bound and is subject to spontaneous hydrolysis upon contact with yeast, gram-positive or -negative cell wall components, a process that results in the deposition of C3b at the surface of the pathogen. The C3b (C3b-tagged pathogen) would then promote phagocytosis of the foreign pathogen by the binding of C3b to its receptor CR1 (CD35), expressed mainly on the surface of leukocytes (see Fig. 7).

Figure 7 Schematic and hypothetical representation of the role of the complement system in the central nervous system in the elimination of pathogens to prevent potential detrimental consequences on the neuronal material during severe endotoxemia caused by infection with gram-negative bacteria. The alternative pathway therefore exists in the brain, and this highly organized innate immune response may be essential for orchestrating the cerebral inflammatory response during systemic bacterial infection. Such systemic infection is censored by a group of supporting cells in regions devoid of BBB and the cerebral microvasculature to engage a timely innate immune reaction necessary for preventing neuronal damage, although it may have detrimental consequences in case of exaggeration. See the text for details.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

While LPS may be directly responsible for the increase in C3 transcription in available structures from the systemic circulation when interacting with mCD14 and TLR4 (83), such a mechanism is unlikely to occur in the brain parenchyma that is protected by the BBB. Indeed, upregulation of C3 mRNA by the ependymal lining cells of the ventral third ventricle probably results from the production of soluble mediators secreted by either chp or other cells inside the BBB. Proinflammatory cytokines are presumably such mediators, because they are rapidly synthesized by the CVOs, chp, and leptomeninges in response to circulating LPS (27,28,41,83,84) and they are potent regulators of the complement system in the periphery. The identity of such local mediators has yet to be clarified, although TNF- is a potential candidate for triggering the biosynthesis of different members of the complement system in the cerebral parenchyma (see below). A constitutive expression of the C3aR has previously been reported in human and rat lung, spleen, heart, liver, and brain, although in the mouse, basal C3aR levels were observed only in the spleen and the brain (107,108). A modest increase in C3aR levels was also found in the rat brain after LPS administration (107). Using in situ hybridization, our laboratory failed to detect constitutive C3aR mRNA levels in the mouse brain, though a strong transcriptional activation occurred in response to a single i.p. LPS bolus (106). The C3aR mRNA signal was detected first in the CVOs, chp, the meninges, and thereafter across the brain parenchyma in a migratory-like pattern similar to the proinflammatory cytokine TNF- (28) and the LPS receptor mCD14 (81). As mentioned, centrally produced TNF- was also found to play an essential autocrine/paracrine role in triggering parenchymal microglial cells during severe endotoxemia (27). The diffusion of the C3aR message across the cerebral parenchyma took place 3 hours after the induction of the gene encoding TNF- in response to similar treatment. This delay fits quite well with the hypothesis that centrally produced TNF- is an endogenous prerequisite factor in the activation of cerebral innate immune response. These data, together with the fact that i.c.v. TNF- infusion increased C3aR mRNA levels in the rat brain (S. Nadeau and S. Rivest, unpublished data), support the role of this cytokine in triggering C3aR gene expression within microglial cells across the brain parenchyma. De novo induction of the C3aR in the CNS may be part of the mechanisms that control the proinflammatory events to prepare specific populations of cells to act immediately in the case of pathogen invasion into the brain parenchyma via damaged or altered blood vessels (see Fig. 7). The integrity and physical characteristics of the BBB are compromised during severe endotoxemia, allowing diffusion of molecules that normally have no access to the cerebral tissue, which is detrimental for the neuronal elements (109). Increasing the phagocytic activity of the CVO macrophages/microglia and adjacent regions by the binding of C3a fragment to its receptor, which are both upregulated in response to circulating LPS,

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

may therefore be crucial in such a situation to prevent pathogen-induced neuronal damage. Complement factor B is a serine protease that plays a pivotal role in the alternative pathway in the formation of C3 convertase. As for C3aR, the mRNA encoding factor B was only detectable after a systemic LPS challenge in different nonneuronal structures, namely the meninges, CVOs, chp, and along the endothelium of the brain capillaries (106). The formation of C3 convertase at the surface of C3b-tagged invading pathogens may then be possible by the presence of factor B in the regions devoid of BBB (see Fig. 7). On the other hand, the positive FB mRNA signal in the cerebral capillaries is quite intriguing, because such induction within the endothelium of the brain microvasculature was not found for the mRNA encoding the C3 and C5 proteins. The cerebral endothelium is very sensitive to very low circulating levels of bacterial endotoxin despite the lack of membrane CD14 at the surface of the endothelial cells (110). However, the endotoxin has the ability to directly activate endothelial cells via its soluble receptor CD14 (sCD14), which triggers the proinflammatory signal transduction pathways (NF B) in cells of the BBB (29,102,111). These data, together with the presence of a specific NF- B–binding site on the factor B promoter (112), indicate that these signaling events are likely to be involved in the transcriptional activation of that particular gene in the endothelium of the brain blood vessels. The physiological relevance of LPS-induced factor B biosynthesis in cells of the BBB has yet to be determined. It may be secreted into the circulation or diffuse within a limited surface surrounding the capillaries to engage the alternative pathway in case of a tight junction breakdown. This is of particular interest, because the mRNAs encoding factor B and C5aR were not found across the entire microvasculature and the positive blood vessels may either be more susceptible to the infection and circulating pathogens or be the primary sentinels and gatekeepers for cerebral innate immune recognition. Also conceivable is the idea that uncontrolled expression of these molecules changes the BBB properties featuring pathologies such as endotoxemia and cerebral bacterial infections. Alteration of the BBB during severe endotoxemia would open the door for immunological substances that have to be recognized and processed. Activation of the microglial cells across the CNS may rapidly eliminate this foreign material, although sustained activity of these cells is not suitable. There is indeed accumulating evidence that chronic microglial reactivity is associated with neurodegenerative disorders. Better understanding this innate immune response in the cerebral tissue may therefore lead us to the fundamental mechanisms underlying how the brain is capable of mounting inflammatory responses that either protect or contribute to damaged neurons. Cerebral innate immunity is likely to be an essential player in the etiology of inflammatory CNS disorders resulting from infection as well as those assumed to have an immune etiology, e.g., multiple sclerosis. Future studies may well unravel unexpected findings support-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ing the concept that the complement system directs and adapts the bilateral interaction between the cerebral endothelium and microglia. D. Monocyte Chemoattractant Protein-1 MCP-1 is one of the principal CC chemokines that plays a primary role in the recruitment of inflammatory cells in different tissues during acute and chronic inflammation. This chemokine is rapidly synthesized by endothelial cells in response to leukocyte-derived IL-1 to allow monocyte recruitment and rolling prior to transmigration and arrival at the site of inflammation. Although the early events of monocyte rolling and attachment may occur along cells of the BBB under systemic immune challenges, the subsequent step of emigration is unlikely to take place because of the tight junctions that characterize the cerebrovascular system. Cellular trafficking remains nevertheless possible when there is alteration of the BBB in inflammatory CNS disorders (for a review, see Ref. 113). Of interest is that all models that use NF- B as a signal-transducing system (LPS, IL-1, and TNF-) caused a profound and transient induction of MCP-1 mRNA in vascular-associated elements and few isolated parenchymal microglia in both mouse and rat brains, whereas high doses of IL-6 (inducer of JAK/STAT signaling) remained without effect (114). An elegant pattern of MCP-1–expressing cells was indeed found across the CNS in response to NF- B–signaling molecules, a phenomenon that may have may have a leading role in allowing cellular trafficking within the cerebral tissue during chronic inflammatory diseases. Although early secreted cytokines are the main stimuli for triggering MCP-1 expression and synthesis, products of Th1 and Th2 cells have also been shown to induce MCP-1 production and synergize with IL-1 and TNF. Interaction between lymphocyte-derived cytokines and MCP-1 gene regulation is somewhat complex and may involve a self-amplifying loop whereby they may enhance their mutual expression during T-cell emigration and differentiation. In this regard, MCP-1 and its receptor CCR2 have been implicated in a number of inflammatory diseases, and the recent report that mice lacking CCR2 are resistant to experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, provided decisive evidence that this chemokine has a leading role in the cerebral infiltrating process (115). These authors propose that induction of MCP-1 in the CNS by activated neuroantigen-specific T cells is necessary for the recruitment of peripheral blood monocytes into the brain and the spinal cord, which may serve to amplify the initial local T-cell–mediated response into an active lesion (115). This concept is further supported by the total absence of mononulear cell inflammatory infiltrates within the cerebral tissue of CCR2-deficient mice and immunized the myelin oligodendrocyte glycoprotein peptide 35–55 (115) and the fact that monocyte recruitment to the CNS is a prerequisite step for the development of inflammatory lesions in EAE (116). Differentiated macrophages into the brain

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

parenchyma are believed to be directly responsible for the demyelination and axonal dysfunction, as a consequence of myelin sheath destruction by these activated phagocytes. Anyhow, the early events involved in this emigration process of leukocytes across the BBB are far from being understood, and why such a phenomenon suddenly occurs is an open question that may not soon be answered. A recent study provided data on the regions and cell types responsible for producing MCP-1 within the CNS in response to systemic insults not associated with neurodegenerative disorders (114). A single systemic injection with either LPS, IL-1, or TNF caused robust and transient transcriptional activation of the gene encoding this CC chemokine across the endothelium of the brain-penetrating arterioles and capillaries and a mixed population of cells in the CVOs, chp, and leptomeninges. These regions would therefore have the ability to rapidly attract cells that bear CCR2, such as circulating monocytes and lymphocytes. This chemoattraction may explain the cells of monocytic lineage that are attached to the cerebral endothelium in animals that received systemic injection with recombinant IL-1 or LPS. The so-called perivascular microglia may actually be circulating monocytes that are attached to the BBB in response to systemic immune challenges. Although these attached cells may be activated by the release of MCP-1 from the cerebral endothelium, the subsequent step of transmigration across the brain parenchyma does not occur in animals that received a single dose of recombinant cytokines or LPS. Besides, we speculate here that these MCP-1–expressing regions, a newly discovered feature of the cerebral innate immune system, are crucial early targets that may engage the inflammatory response characterizing neurodegenerative disorders that have an immune etiology. Of interest is that mononuclear cell infiltrate appears in the brain parenchyma at times of maximal chemokine synthesis in MCP-1–transgenic mice in which expression is driven by myelin basic protein promoter, and this infiltrate is enhanced by systemic LPS administration (117). The strong induction of MCP-1 in leaky organs of LPS-injected animals was expected, because receptors of the innate immune system are constitutively expressed in these structures, and CD14- and TLR4-expressing cells are believed to act as the immune sentinels for engaging the cerebral inflammatory response in the presence of gram-negative bacterial cell wall components (see above). However, IL-1 and TNF were as effective as LPS in triggering MCP-1 transcription in the CVOs and other vascular-associated elements of the brain, suggesting a more general mechanism evolving NF- B signaling. In agreement with this idea is the strong NF- B activity in cells that exhibited MCP-1 induction in response to LPS, IL-1, and TNF, but not IL-6 (29). While these circulating molecules may be directly responsible for the increase in MCP-1 transcription in available structures from the systemic circulation in interacting with their specific receptors (81,83,118,119), such a mechanism is unlikely to occur in the brain parenchyma protected by the BBB. Indeed, scattered small cells (not always associated with

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

blood vessels) were found across the brain parenchyma of immune-challenged animals, which may result from the production of soluble mediators secreted by either the chp or other cells inside the BBB. Although centrally produced cytokines may be such mediators during endotoxemia, they are unlikely to contribute to MCP-1 expression in the brain parenchyma of IL-1–treated animals. In contrast to the recognized ability of LPS to induce cytokine production in the CNS, high doses of IL-1 injected either i.v. or i.p. usually failed to activate TNF, IL-1, and IL-6 gene expression within the CNS (S. Rivest and colleagues, unpublished data). The chemokine itself may be responsible for inducing parenchymal MCP1, because it is induced in chp within less than 30 minutes in response to systemic IL-1 injection, and its low molecular weight may facilitate its diffusion across the cerebral parenchyma. Unlike CD14 (81), TNF (27,28), and different members of the complement system (106), and except for the area postrema and its surrounding tissue in the medulla, there was no induced wave of MCP-1–expressing cells from the leaky organs to deeper brain parenchymal regions in response to systemic LPS injection (114). The gene encoding MCP-1 is therefore regulated in a very unique manner in vascular-associated cells of the CNS in response to circulating molecules that trigger NF- B as a signal transduction system and is an integrated part of the cerebral innate immune system. Better understanding this system will provide essential clues underlying the “good” and the “bad” roles of the inflammatory response in the brain that either protect neurons or is the direct cause of the neurodegenerative disorders.

IV. HOW CIRCULATING MOLECULES TALK WITH THE BRAIN PARENCHYMA The previous sections described the different players that may be involved in the neural immune interface, and while immune molecules can be produced by both systemic and cerebral cells, their contributions and mechanisms of action in altering the autonomic response are quite distinct. It is indeed very difficult to compare studies in which cytokines were injected systemically or centrally, because the receptors involved and the pathways are obviously very different despite a general outcome that may be similar, e.g., increase in body temperature, cardiovascular changes, and stimulation of the HPA axis. The main reason for this is that cytokines produced by the systemic immune system are unlikely to diffuse in concentrations high enough to the cerebral tissue and must target cells that can be reachable from the systemic circulation. The following section will present an overview of these likely regions and how they may relay the information to those involved in the autonomic functions.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

A. Via CVOs and Other Leaky Structures As mentioned, sensorial CVOs contain a rich vascular plexus with specialized arrangements of the blood vessels. The tight junctions normally present between the endothelial cells are shifted in part to the ventricular surface and partly to the boundary between the CVOs and the adjacent structures, explaining the diffusion of large molecules into the perivascular region. Such structural organization makes them privileged target sites not only for proinflammatory cytokines, but also for infectious agents that may have a rapid access to these regions consisting of four organs, namely the vascular organ of the lamina terminalis (OVLT), subfomical organ (SFO), median eminence (ME), and area postrema (AP). The subcommissural organ is another CVO located in the posterior wall of the third ventricle that actually forms the dorsal roof of the entrance to the aquaduct of Sylvius. In contrast to the other CVOs, there are no neuronal cell bodies in the subcommissural organ, and its functions remain largely unknown. The chp and leptomeninges are also recognized as being highly vascularized regions, very sensitive to infectious agents in exhibiting a rapid transcriptional activation of different inflammatory molecules (see above). These structures are also devoid of neurons, and they are not considered CVOs. In the OVLT, the external zone and the pia matter enveloping the microvessel loop extend to the core of the organ and spread out to the ependymal lining cells. Circulating inflammatory molecules may reach specific compartments through the fenestrated capillaries originating from the anterior communicating artery. On the other hand, blood-derived molecules may target specific population(s) of cells of the external lamina via the capillaries of the primary plexus that irrigates the basement membrane of the lower palisade layer of the ME. Both the SFO and AP display an extensive network of capillary loops, and the entire organs are exposed to hema milieu. These anatomical features and the fact that neurons innervating the regions that integrate the autonomic outputs are found in these CVOs support the concept that they are key structures in mediating cerebral responses to circulating immunogenic agents. The constitutive expression of immune receptors together with a rapid induction of functional indices of cellular activity in response to systemic boluses of cytokines and LPS provide supporting evidence that CVOs act as a route of entry for numerous inflammatory agents that circulate in the bloodstream. The particular distribution of IL-6 mRNA in the CVOs may also offer some clues to understanding the role of this cytokine within the CNS. The increase of IL-6 synthesis in the OVLT might be a central mechanism participating in the thermogenic effects of the bacterial endotoxin; the OVLT/MPOA is a recognized region involved in the appropriate control of thermoregulation (120,121); IL-6 is a component of the fever response to LPS (122), and the firing rate of thermosensitive neurons of the preoptic area can be influenced by IL-6 (123). Neurons of the OVLT and SFO have direct projections to the paraventricular nucleus (PVN), and those of the AP connect with the nucleus of the NTS, which in turn sends catecholaminergic proCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

jections (A2/C2) to the hypothalamic PVN. As seen in this book, the PVN is the key integrative nucleus for autonomic functions and also contains the corticotropin-releasing hormone (CRF) neurons responsible for the control of the ACTH release from the adenohypophysis. The CVOs could therefore be possible sites of action through which cytokines may exert their actions on the HPA axis. In this regard, recent data suggested that the AP and adjacent nucleus of the solitrary tract (NTS) play a pivotal role in transducing a circulating IL-1 signal into HPA axis activation by a pathway that may be comprised of known anatomical links between the AP, NTS, and CRF neurons of the PVN (124). Moreover, it has been suggested that the neuroendocrine effects of cytokines may occur by increasing prostaglandin synthesis at the level of the ME (125). This issue will be further developed in the section on cells of the BBB and cyclooxygenase pathways. Finally, the wave of cytokine induction from the CVOs to deeper parenchymal regions is also likely to contribute to the neuronal signaling, although this phenomenon is believed to be an autocrine loop for microglial activation that may have very little to do with the neuronal activity, at least as it relates to autonomic functions (83). B. Via the BBB and Synthesis of Soluble Mediators Cells forming the BBB are in a privileged position to transfer information from the circulation to the brain parenchyma, and there are exciting new developments regarding the molecular events taking place in the endothelium of the cerebral arterioles, small capillaries, and venules during systemic immune challenges. Cytokines, when secreted by cells of myeloid origin in the circulation, trigger a series of events in cascade leading to the MAP kinases/NF- B or the JAK/STAT transduction pathways in vascular-associated cells of the CNS. The blood vessels of the brain exhibit both constitutive and induced expression of receptors for different proinflammatory ligands that can stimulate these signaling molecules. Depending on the challenges and the cytokines involved, the transduction signal(s) solicited in cells of the BBB may orient the neuronal activity in a very specific manner in activating the transcription and production of soluble factors, such as prostaglandins (PGs) and nitric oxide (NO). 1.

Prostaglandin Synthesis

The formation of PGs is initiated by the action of cyclooxygenase (COX, also known as prostaglandin endoperoxide H-synthase, or PGHS), which catalyzes two separate reactions, the first being oxygenation of arachidonic acid to the unstable PGG2 by a cyclooxygenase function, and the second, the subsequent reduction of PGG2 leading to a more stable PGH2 by peroxidase reaction. Although constitutive expression of the isoform COX-1 was found in various cell types, mRNA and protein levels remain somewhat unchanged during inflammatory chalCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

lenges and therefore are believed to play housekeeping roles (126,127). In contrast, the COX-2 isoform is undetectable in most tissues under basal conditions, but marked transcriptional activation can be observed in macrophages and other cell types by the endotoxin LPS and proinflammatory cytokines (128–130). Interestingly, systemic inflammatory insults cause a robust transcriptional activation of COX-2 along cells of the mouse and rat microvasculature, although the intensity of the signal and the pattern of expression depend on the challenge and the dose of the inflammatory agents (102,119,131–133). Intravenous and intraperitoneal injections of low to moderate doses of the entodoxin LPS generally induce COX2 mRNA and protein along blood vessels of the entire brain microvasculature, choroid plexus, and leptomeninges (see Fig. 8). The stimulation of PGs by cells of the BBB is observed not only during endotoxemia, but also in other experimental models of systemic inflammation; intramuscular (i.m.) turpentine insult stimulates transcription of COX-2 gene within the microvessels, chp, and leptomeninges, and the signal of this transcript paralleled the inflammation of the left hind limb (132). This experimental model induces, local tissue damage provoking sterile inflammation, e.g., an inflammatory response that develops in the absence of any microbial stimulus provoking a specific induction of IL-1 and IL-6 without any detectable IL-1 or TNF- production (134). This suggests the existence of a common cascade of cytokine release, characteristic of sterile inflammation, where IL-1 and IL-6 might play a critical role (134). A robust COX-2 mRNA signal is also rapidly detected in the cerebral microvessels in response to i.v. IL1 and TNF- injection, but not following high doses of IL-6 (132). These results indicate that specific populations of cells, in particular vascular- and/or perivascular-associated cells, are responsible for the central production of PGs during systemic inflammation, and circulating IL-1 and TNF- are likely to be potent mediators of this response. The cellular source of PG production by cells lining the cerebral blood vessels is currently a matter of debate and controversy. In our original report, we faced a number of problems in the identification of the cell type(s) in the brain microvasculature expressing COX-2 transcript in response to immunogenic stimuli (132). Indeed, COX-2 mRNA is very sensitive to the immunocytochemistry procedure and essentially vanished, rendering evaluation of double-labeled cells barely possible. We then worked on the conditions of the immunocytochemistry procedure to prevent mRNA degradation and have successfully determined that the large majority of COX-2–expressing cells were positive for a marker of the cerebral endothelium, e.g., von Willebrand factor (vWF). Figure 9 (top and middle panels) shows representative examples of such dual labeling in a long blood vessel of the caudal medulla 45 minutes after i.v. injection of recombinant rat TNF-. Numerous other reports have shown similar data that the endothelium of the cerebral blood vessels was the main source of PG production in response to systemic LPS and IL-1 treatments (133,135–139). However, two other well-per-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 8 High-power darkfield (top panels) and brightfield (bottom panels) photomicrographs showing the expression of cyclooxygenase2 (COX-2) mRNA in blood vessels (b.v.), choroid plexus (chp), and leptomeninges of rats sacrificed 1 hour after intravenous (i.v.) injection of the bacterial endotoxin lipopolysaccharide (LPS, 10
g/100 g b.w.). Note the robust hybridization signal within nonparenchymal cells surrounding the b.v., chp and leptomeninges 1 hour following the i.v. LPS challenge. Magnification top panels, 10; bottom panels, 100. (From Ref. 132.) Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 9 Phenotype of COX-2- and I B-expressing cells in a rat cerebral blood vessels (bv) during systemic inflammatory insults. Endothelial cells were labeled by immunohistochemistry using antisera against the von Willebrand factor (vWF-ir), whereas a COX2 antibody (COX-2-ir) was used to perform the dual labeling with I B-expressing cells (bottom panels). COX-2 or I B mRNA was thereafter hybridized on the same sections by means of a radioactive in situ hybridization technique (silver grains). Arrowheads, dual-labeled cells (endothelial/COX-2 mRNA, top and middle panels; COX-2-ir/I B mRNA, bottom panels). (From Ref. 8.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

formed studies have shown evidence that COX-2-immunoreactive (ir) cells were not endothelial, but perivascular microglia and meningeal macrophages (140,141). However, these authors observed, at the edge of the blood vessels, several COX-2-ir cells alone and not colocalized within microglia (140), indicating that perivascular microglia was not the only cell type responsible for the production of PGs within the brain. In our hands, the large majority of COX-2–expressing cells were positive for vWF in response to circulating LPS, IL-1, and TNF, and very few cells were positive for markers of microglia. Figure 10 shows the pattern of ED2-ir cells and those expressing COX-2 mRNA 3 hours after a high dose of LPS (250
g/100 g b.w.) injected i.p. It is interesting to note that there is very little overlap between these perivascular microglia and COX-2 cells lining the large blood vessels, although we have found on some occasions some doublelabeled cells. It is difficult to explain such discrepancies between studies, but the dose, time course, the experimental models of systemic inflammation and the antisera used to reveal the specific cell types (perivascular microglia vs. the endothelium) are essential elements to take into consideration. In this regard, endothelial cells and perivascular cells in the rodent brain are not simple to label and mistakes can be made in the interpretation of the exact cell type forming the BBB. Moreover, the presence of the so-called perivascular microglia may actually be circulating monocytes that are attached to the cerebral endothelium in response to systemic immune challenges. Although the exact proportion of endothelial versus perivascular microglial cells may remain a matter of debate, COX-2 is produced by cells of the BBB during the acute-phase response, and there is no doubt that the endothelium has the biosynthetic machinery to produce PG. Robust activation of NF- B is also detected in the endothelium of the brain capillaries in response to different systemic inflammatory stimuli (29,102). Endotoxin LPS injected either i.v. or i.p. provoked a rapid and prolonged expression of I B mRNA in the endothelium of the brain blood vessels and parenchymal microglia (29,102) (Fig. 11). The effects of i.v. IL-1 and TNF- also take place rapidly (within 30–60 min), but vanished 3 hours after the injection (29) (Fig. 12). On the other hand, selective expression of I B was detected along the cerebral endothelium of i.m. turpentine-injected rats (29) (Fig. 13). Figure 14 (bottom right) shows that the mRNA encoding I B is essentially expressed within endothelial cells of the brain-irrigating system. The rapid and transient induction of I B indicates strong NF- B activity in cells of the BBB by circulating proinflammatory cytokines, which may lead to the transcription of target genes. One potential candidate is the gene encoding COX-2; the nuclear factor binding to the COX-2 promoter is able to influence the enzyme transcription in response to different immunogenic ligands, including IL-1 (142,143). The binding of IL-1 to its cognate type I receptor leads to the formation of the IRAK/TRAF6/MyD88 complex, which activates NIK/IKK kinases involved in the phosphorylation and degradation of I B (5). NF- B is then translocated into

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 10 Expression pattern of perivascular microglia (ED2-ir) and COX-2 mRNA in the rat blood vessels 3 hours after an intraperitoneal injection of the endotoxin lipopolysaccharide (LPS). Perivascular microglial cells were labeled by immunohistochemistry using an antisera directed against ED2. COX-2 mRNA was thereafter hybridized on the same sections via a radioactive in situ hybridization technique (silver grains). Please note that ED2-ir cells and COX-2 hybridization signal do not overlap along the large blood vessel. White arrowheads, ED2-ir cells; black arrowheads, COX-2-expressing cells. (From Ref. 8.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 11 Effects of the bacterial endotoxin LPS injected either intravenously (20
g/kg) or within the peritoneal cavity (250
g/kg) on the expression of I B mRNA in different brain structures. These darkfield photomicrographs of dipped NTB-2 emulsion slides (7 days exposure) depict induction of I B mRNA in the circumventricular organ (CVOs), the brain microvasculature and parenchymal microglia at 1 hour postinjection. The bottom right panel exhibits dispersed small positive cells within the brain parenchyma 6 hours after i.p. treatment. AP, area postrema; b.v., blood vessels; ME, median eminence; OVLT, organum vasculosum of the lamina terminalis; SFO, subfornical organ. Magnification: 25. (From Ref. 29.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 12 Representative examples of the influence of proinflammatory cytokines on the expression of mRNA encoding I B in the rat brain. Animals were killed 1 hour after intravenous (i.v.) injection of TNF- (4.1
g/kg of b.w.), IL-1 (1.8
g/kg of b.w), and IL6 (12
g/kg of b.w.). These darkfield photomicrographs show in situ hybridization signals for I B mRNA throughout the entire brain microvasculature and within small cells of the brain parenchyma in both TNF-– and IL-1–treated rats, but not following IL-6 injection. b.v., blood vessels; chp, choroid plexus; OVLT, organum vasculosum of the lamina terminalis; SFO, subfornical organ. Magnifications: 25 and 50 (bottom panels). (From Ref. 29.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 13 Darkfield photomicrographs showing the expression of I B mRNA along the microvasculature of rats during a systemic localized inflammatory insult. Animals were killed 3, 6, and 24 hours after a single intramuscular injection of turpentine (500
l/kg of b.w.) into the left hind limb, and coronal brain slices (30
m) were hybridized with a full-length cRNA antisense probe labeled with 35S. These darkfield photomicrographs of NTB-2–dipped emulsion sections exhibit positive signal quite exclusively along the blood vessels (b.v.) 6–24 hours after the challenge, whereas the transcript was undetectable at times earlier and in vehicle-injected rats. This clearly suggests activation of the nuclear factor kappa B (NF- B) within the brain microvasculature in response to a localized systemic inflammation. Magnifications: 25 (top panels) and 50 (bottom panels). (From Ref. 29.) Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 14 Activation of nuclear factor kappa B (NF- B) and cyclooxygenase-2 (COX2) genes in the cerebral blood vessels in response to a systemic inflammation. Top panels depict COX-2 gene expression in a large blood vessel (bv) of a rat that received the endotoxin lipopolysaccharide (LPS) into the peritoneal cavity. The bottom panels are examples of I B mRNA signal within the endothelium of brain microvasculature. Left panels are darkfield photomicrographs, whereas right panels are the same samples but taken under brightfield illumination. Endothelial cells were labeled by immunohistochemistry using antisera against the von Willebrand factor (vWF-ir), whereas I B mRNA was thereafter hybridized on the same sections by means of a radioactive in situ hybridization technique (silver grains). COX-2 mRNA was visualized by single radioactive in situ hybridization histochemistry (top panels, silver grains). (From Ref. 8.)

the nucleus and may bind to its B consensus sequence on target genes (6). Two putative NF- B motifs from the COX-2 promoter were found to bind p50/p65 NF B heterodimers in an IL-1–dependent manner, and the two NF- B subunits synergistically activate a 917/49 COX-2 promoter construct (142,143). Like IL-1, i.v. TNF- injection causes a robust and transient transcriptional activation of I B and COX-2 in endothelial cells lining the CNS vascular system (29,119,132). TNF- is actually one of the most potent effectors of NF- B activity through the 55 kDa TNF type I receptor that we found along the BBB (29,119). Activation of the cerebral endothelium also occurs in response to circulating LPS (29,102,132), but endothelial cells do not express membrane CD14. However, these cells respond to LPS/LBP in a soluble (s)CD14-dependent manner in stimCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ulating the tyrosine phosphorylation of MAP kinases (110), which engages the NF- B signaling. The NF- B site (223 to 214) is involved in LPS-induced expression of COX-2 in monocytic cells (144), whereas hypoxia induces COX-2 transcription via p65 binding to 3 NF- B consensus element in the enzyme upstream promoter region in endothelial cells (145). We recently found colocalization between I B transcript and COX-2 protein, indicating a potential interaction between the transcription factor and COX-2 in the brain vascular cells of immune-challenged animals (Fig. 9, bottom panels). Obviously, this remains speculative from these anatomical data, and the signaling pathways that lead to the NF- B nuclear translocation and COX-2 transcription have yet to be determined in the cells of the BBB. In vivo approaches are quite limited in the investigation of the intracellular events occurring within specific cellular populations of the CNS, but they provide an essential integration of the systems that interact during the acute-phase response. These molecular events occurring in the cerebral microvasculature lead to the formation of specific PGs that have the ability to talk with neurons in cognate with their transmembrane receptors. This issue will be discussed in detail later in the chapter. 2.

NO and the BBB

Another potential candidate involved in the bilateral communication between immune and nervous systems is nitric oxide (NO), a short-lived free radical gas. NO is produced from the oxidation of L-arginine, a reaction catalyzed by the enzyme nitric oxide synthase (NOS). To date, three distinct isoforms of NOS have been cloned and characterized: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). nNOS and eNOS are both constitutively expressed, but nNOS is found mainly in neuronal populations and to a lesser extent in endothelial cells of large blood vessels, while eNOS is present in the endothelium of all types of blood vessels and also in several nonendothelial cells (for a review, see Ref. 146). These isoforms have been involved principally in neurotransmission (147) and in regulation of vascular tone and blood pressure (148), respectively. On the other hand, iNOS is inducible by many types of stimuli, including inflammatory insults, and seems almost ubiquitous (146). iNOS is responsible for the cytotoxic actions of macrophages and neutrophils against viruses, bacteria, and tumor cells (149). A fine regulation of all three forms of NOS is necessary for NO to exert its physiological functions, because dysregulation has been associated with pathological states including cardiovascular diseases, neurodegeneration, and chronic inflammation (for a review, see Ref. 150). The role of NO in inflammation has been extensively studied, although its exact influence on the gene encoding COX-2 and PG synthesis remains controversial. In murine macrophages treated with LPS alone or combined with IFN-, NO was shown to stimulate the activity of COX-2 and PGE2 and PGD2 synthesis (151,152). NO was also found to enhance the enzymatic activity of COX-2 and Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

PGE2 production in rat islets of Langerhans treated with IL-1 (153), whereas NO donors increased PGI2 synthesis in rat endothelial cells (154) and it stimulated PGF2 production in human embryonic microglial cells incubated with IL-1 and IFN- (155). In contrast, NO suppressed COX-2 gene transcription (156) as well as its protein product and PGE2 synthesis (157) in LPS-treated macrophages. NO inhibition was also found to enhance the influence of circulating TNF- on NF B activity and COX-2 transcription in cells of the microvasculature (158). A dual-labeling procedure provided the anatomical evidence that such molecular events are taking place within the endothelium of the cerebral blood vessels and capillaries and suggests that NO plays a key role in modulating the proinflammatory signal transduction pathways within a specific population of cells forming the BBB. NO may therefore be an important modulator of the signaling events that take place in cells of the BBB to moderate and adjust the production of inflammatory molecules within the brain parenchyma. In agreement with this concept are the data that NO inhibition increases the neuronal response to systemic inflammation and circulating cytokines (159), whereas L-NAME enhances LPS-induced hyperthermia (160) and augments the stimulatory action of the endotoxin and cytokines on the different neuroendocrine functions, such as the HPA axis (161). In contrast, other results indicate that NO could be an important second signal for the stimulation of PGE2 production induced by immune complexes in peripheral blood mononuclear cells from human schistosomiasis patients (162). Cytokine-induced PGE2 synthesis and COX-2 activity was also found to be influenced by both a NO-dependent and -independent mechanism in rat osteoblast in vitro (163). The proinflammatory action of NO in the systemic immune response that is derived from the activity of the iNOS expressed in a variety of myeloid-derived cells may regulate COX-2 expression at both transcriptional and posttranscriptional levels quite differently from the anti-inflammatory influence of NO in the endothelium of the brain capillaries. The antiatherogenic and anti-inflammatory properties of NO to limit cytokine-induced endothelial activation and inhibit monocyte adhesion within the vessel wall have indeed been reported (164). NO also has the ability to attenuate cytokine-induced adhesion molecule expression primarily by inhibiting NF- B activity in smooth muscle cells (165), and NF- B inhibition prevented proinflammatory gene transcription, namely COX-2 (166). As discussed, a single i.v. bolus of recombinant TNF- activated I B and COX-2 across the cerebral microvasculature, and this effect was clearly exacerbated by inhibiting the L-arginine–NO pathway (158). NF- B may also be activated by oxidative stresses, because the production of reactive oxygen species (ROS), like hydrogen peroxyde (H2O2) and superoxide anion (O2-), are potent inducers of NF- B activation (167,168). The suppression of NF- B activation by various antioxidant treatments further supports this hypothesis (166,169–171). ROS are produced following several proinflammatory and prooxidant stimuli, e.g., TNF- (172), which supports a potential

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

role of ROS in NF- B activation by the cytokine. The phosphorylation and degradation of I B and NF- B translocation may be involved in these effects, as antioxidants prevented NF- B nuclear translocation possibly by preventing IKK activity and I B phosphorylation (167,168), although antioxidants also failed to influence NF- B translocation, DNA binding, and I B proteolysis (172). Moreover, I B degradation by proteasome was found to take place before ROS generation, indicating that ROS may exert their actions directly on NF- B transcriptional activity and not via the nuclear translocation (172). Ras and MAP kinase have recently been implicated in NF- B activation by oxidants, and this event seems independent of I B degradation (173). These data are of particular interest, because TNF-induced NF- B transcriptional activity was increased in the cerebral endothelial cells of animals that were pretreated with L-NAME (158). This inhibitory action of NO may be explained by an antioxidant function; NO can act as a scavenger of H2O2 and O2produced in presence of the cytokine, which may repress the oxidative effects of ROS (174). Considering the fact that L-NAME and TNF- act in synergy, two different signaling pathways are likely to participate in these effects associated with a profound increase in COX-2 gene expression. In this regard, the reaction between NO and H2O2 or O2- yields in both cases to the generation of peroxynitrite (OONO) (152,174), a potent oxidant implicated in the enzymatic activation of COX-2 and the biosynthesis of PGE2 and PGD2 (152). Because NO may exert its effect on transcriptional activity by altering the redox state of the cell, its influence may be different depending on the cellular type and on the balance between oxidants and antioxidants. The inhibitory action of NO on TNF-induced NF- B activity and COX-2 transcription in cells of the BBB may be an essential mechanism to prevent exaggerated responses and overproduction of proinflammatory molecules in the brain parenchyma by microvascular-associated cells. NO therefore has a different role from that of PGs and may not be considered as a potential candidate to trigger neuronal activity during the acute-phase response. C. The Vagus Nerve Vagal afferents originating in the periphery were proposed by numerous studies to monitor circulating immune molecules, because neurons within the NTS are primary recipients of sensory information from the vagus nerve and this region of the CNS is quite sensitive to circulating proinflammatory agents. However, a recent study investigated whether vagal connections with the brain stem were necessary for LPS-induced activation of dorsal vagal complex (DVC) neurons and found that systemic exposure to LPS elicited a significant activation of c-fos in neurons in the NTS and area postrema regardless of the integrity of the vagal nerve (175). Another group reported that low doses of LPS stimulated expression of CRF mRNA in rats subjected to axotomy of the gastric or celiac branches of the

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

vagus nerve but did not change the intensity of autoradiographic labeling in animals with transected hepatic branches (176). High doses of endotoxin, on the other hand, enhanced expression of the neuropeptide in vagotomized rats of all groups (176). The role of the vagus nerve of the parasympathetic system in mediating the effects of LPS and inflammatory cytokines would then occur only when endotoxin is injected i.p. and at a very low dose. Indeed, vagotomy can prevent neuronal activation in response to fairly low doses of LPS and IL-1 injected i.p., but not following moderate to high doses and not when the endotoxin and proinflammatory cytokines are injected directly into the circulation (for a review, see Ref. 177). The exact contribution of the vagus nerve has also been challenged by numerous studies that failed to prevent cerebral responses even in the presence of fairly low doses of cytokines injected i.p.. Other groups have found that behavioral responses to immune stimuli can be attenuated by selective transection of the gastric, celiac, and hepatic branches of the vagus nerve without altering significantly other parameters, such as increase in the HPA axis, CRF, and c-fos transcription in the PVN as well as in other regions of the brain. It can be concluded that the participation of vagal sensory mechanisms in mediating the neuronal response to immune challenge is therefore limited to local peritoneal inflammation and may not be considered as a general feature by which circulating proinflammatory molecules trigger the cascade of events taking place in the CNS during systemic innate immunogenic stimuli.

V. PROINFLAMMATORY SIGNAL TRANSDUCTION AND THE ANS An increase in different autonomic functions is obviously the result of bilateral talk between immune molecules and the neurons. Although not fully understood, notable progress has been made over the past decade in identifying the mechanisms and receptors involved in triggering key physiological responses, such as fever and increase in plasma glucocorticoid levels. A. Prostaglandins as Sophisticated Intermediates Activation of PG synthesis by cyclooxygenase pathways is believed to play a key role in the cascade of events mediating the effects of circulating cytokines on numerous functions, including fever, activation of neuroendocrine CRF neurons and the HPA axis as well as the sympathetic nervous system of immune-challenged animals. Indeed, blockage of the eicosanoid cyclooxygenase pathways can prevent the stimulation of CRF release by proinflammatory cytokines from in vitro hypothalamic explants (125) and ME (178), and IL-1–(179,180) and TNF–(181) induced ACTH release in vivo. Inhibition of PG production has been reported to prevent IL-1–induced alteration of other neuroendocrine functions, such

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

as LHRH and LH release (182), as well as hypothalamic AVP and OT release (183). The role of PGs in mediating fever is also well accepted and involves neurons of the ventromedial preoptic area (VmPO) that project to the autonomic division of the PVN. Indeed, injection of ketorolac into the anteroventral POA markedly decreased the fever produced by LPS, compared with injections into more rostral, caudal, or dorsal locations (184). As mentioned, the microvasculature is the source of PG formation into the brain during systemic inflammatory challenges (LPS, IL-1, and TNF) that trigger NF- B–signaling pathways and COX-2 transcription within the cerebral endothelium. It is interesting to note that inflammation-induced NF- B activity and COX-2 transcription is rather unspecific across the cerebral blood vessels and small capillaries, while neuronal activity is limited to selective nuclei. PG synthesis via COX-2 activity along with sitespecific expression of PG receptors within parenchymal cells adjacent to the site of production determine the action of these molecules in the brain. B. PGE2 as Key Endogenous Ligand in the CNS The exact PG subtype(s) and the site(s) of action within the brain involved in these effects still remain unclear, but a large body of evidence indicates that E2-type PG might be involved in several changes observed during immune challenge and treatment with cytokines. IL-1 increases the release of PGE2 from rat hypothalamic explants in vitro (185), medial preoptic area (MPOA)/OVLT, PVN, dorsal hippocampus, lateral ventricle in vivo (186), rat astrocyte cultures (187), isolated pancreatic islets (188), and papillary collecting duct (189). Mapping of PGE 2 binding sites in the rat brain using quantitative autoradiography revealed binding sites in numerous brain structures including the hypothalamus (190,191). Moreover, intracerebroventricular (i.c.v.) administration of PGE2 (192) or direct administration into the MPOA/OVLT (193) elevated plasma ACTH and corticosterone in rats, an effect most likely mediated through neuroendocrine CRF neurons. Central treatment with PGE2 is not only associated with an increase of the HPA axis activity, but also known to produce many other physiological responses such as the alteration of the cardiovascular and sympathetic nervous system functions (194,195) and hyperthermia in rats (196). The most pyrogenic preoptic sites are clustered along the ventromedial aspect of the POA, surrounding and just anterior to the OVLT (197). When injected into the brain, PGE2 causes a selective cellular activation, as indicated by the rapid and transient expression of the IEG c-fos mRNA and protein within multiple regions of the brain recognized to be activated during the acute-phase response of an immune challenge (197,198). In a similar manner, the PG triggered transcription of CRF and its type 1 receptor essentially in the hypothalamic PVN (198). Local production of PGE2 might therefore be a crucial step within the CNS to mediate the effects of cytokines and other immune-related systemic factors on the neuronal circuitry in-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

volved in the activation of the fever, HPA axis, and other autonomic functions. The PG is produced by vascular-associated elements, as indicated by the robust and transient expression of COX-2 within the microvessels in response to systemic inflammation (132), and the selectivity of the neuronal responses may depend on the expression of specific PGE2 receptor subtypes or isoforms within defined parenchymal structures of the brain. Classic PG receptors comprise a family of eight genes encoding transmembrane G-protein–coupled receptors. These receptors are classified on the basis of selective affinities for naturally occurring prostanoids. There are distinct receptors for thromboxane A2, prostacyclins (PGI2), PGF2, PGD2 and four different receptors for PGE2: EP1, EP2, EP3, and EP4 (199,200). Multiple alternatively spliced isoforms exist for one of the PGE receptors (EP3) (201). Each receptor is associated with a unique G-protein and consequently a unique second messenger system, namely elevation of intracellular Ca2+ (EP1) and stimulation (EP2, EP4) or inhibition (EP3) of adenylyl cyclase. We have recently reported an interesting distribution pattern for EP2 and EP4 in the rat brain and proposed the EP4 subtype as the possible functional receptor for mediating the action of PGE 2 on specific groups of neurons in response to circulating proinflammatory cytokines, such as IL-1 (202). Indeed, EP4-expressing neurons of the PVN, nucleus of the solitary tract (NTS), and the caudal ventrolateral medulla (cVLM) are activated by circulating IL-1 (202), and it is possible that such activation depends on the local synthesis of PGE2 by cells of the microvasculature penetrating these regions. C. PGE2 Receptors The distribution of the EP2 and EP4 receptor subtypes throughout the brain under both basal and immune-challenged conditions was recently reported (202–204). The expression pattern was highly distinct among transcripts, which suggests a different role for each receptor in numerous populations of neurons. It is interesting to note that EP4 receptors were located in regions that are likely involved in the control of neuroendocrine and autonomic activities. These include the PVN, SON, parabrachial nucleus (PB), NTS, and cVLM, and change in expression levels during systemic immunogenic challenges indicates that specific compartments of these nuclei may participate in the circuits triggered by the PG. The most dramatic change was the profound transcriptional activation of the gene encoding the EP4 receptor over the parvocellular CRF neurons of the hypothalamic PVN in response to different experimental models of systemic inflammation. The presence of Fos-ir proteins in EP4-expressing neurons of the endocrine hypothalamus also confirmed that these cells were activated during the immunogenic insults. Another interesting result is the expression of the EP4 receptor in the A2/C2 and A1 cell groups, which were positive for Fos-ir labeling 3 hours after i.v. IL-1 injection. The proinflammatory cytokine also stimulated the EP4 biosynthesis within the A1 noradrenergic cells, whereas the A2/C2 group remained without significant effects. These studies provided anatomical evidence that Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

both EP2 and EP4 subtypes are widely distributed across different neuronal populations of the brain and that systemic immunogenic stimuli modulate expression of these transcripts in a site- and cell-dependent manner. The activation of CRF and A1 groups of neurons along with the robust hybridization signal for the EP4 receptor clearly indicate that PGE2 may have a direct effect on these cells, which may lead to the appropriate physiological responses necessary to preserve the integrity of the organism when challenged by foreign materials. Once produced, PGE2 has the ability to diffuse through the brain parenchyma and activate different populations of cells, and it is possible that the selectivity of the response depends on the expression of specific EP receptors. The distribution and regulation of the EP2/4 receptor subtypes clearly support this concept and shed light on the previous binding studies using radiolabeled PGE2 as ligand (190,191). Indeed, high densities were found in regions that exhibited hybridization signal for either EP2 or EP4 receptor mRNA, such as the anterior wall of the third ventricle (191). This region that corresponds to the VSA/anterior preoptic area displayed strong EP4 mRNA labeling and may be crucial in the control of body temperature during the acute-phase response (see below). High densities of PGE2-binding sites were also reported in the PB and NTS in a pattern closely related to the compartments showing constitutive expression of EP4 mRNA. Surprisingly, PGE2-binding sites were low to undetectable in PVN, SON, and VLM, while the gene encoding EP4 receptor subtype was abundant in these regions. The fact that systemic immune challenge caused a strong increase in EP4 mRNA levels in parvocellular PVN and cVLM may help to explain the lack of binding in these regions under basal conditions and the fact that rapid and dynamic mechanisms take place in these groups of neurons during systemic inflammatory processes. Constitutive hybridization signal was detected in magnocellular PVN and SON, but whether these transcripts are translated into functional proteins has yet to be determined. The inconsistency between PGE2 binding and receptor mRNA may be attributed to the different affinity of EP receptor subtypes. For example, the Kd value of EP3 is 2.9 nM, whereas the Kd of EP4 is 11 nM. Therefore, the receptor binding study detected EP3 receptor more preferentially than others. It is also possible that receptor proteins are transported into neuronal processes, but their levels may remain relatively low into the cell soma. Be that as it may, the gene encoding PGE2 EP4 receptor was highly expressed within the neurohypophyseal component of the hypothalamus under basal conditions. Along the same lines, blockage of the eicosanoid cyclooxygenase pathways can prevent the stimulation of vasopressin (AVP) and oxytocin (OT) by IL-1 from in vitro hypothalamic explants (183), and i.v. IL-1 (41,205), i.p. LPS (206), and i.c.v. PGE2 (198) injections cause strong activation of magnocellular PVN and SON, especially within OT-ir cells. The role played by these neurons during immune challenge has yet to be investigated, but OT belongs to a family of neuropeptides able to potentiate the action of neuroendocrine CRF on the secretion of

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ACTH from corticotrophic cells of the adenohypophysis during various types of challenges. Secretion of OT into the infundibular process and in the circulation would therefore be another possible mechanism stimulating the HPA axis during immune challenge. It is also possible that some of these cells, in particular those localized in the compartment parvocellular parts of the nucleus that project to dorsal vagal complex and the intermedio-lateral column of the spinal cord, participate to directly trigger sympathetic nervous system (SNS) activity. Central injection of PGE2 increases splenic sympathetic nerve activity in rats (195), and involvement of this system in immunosuppression has been suggested (207), whereas the stimulatory influence of IL-1 on SNS activity can be prevented by cyclooxygenase inhibitors (208). PGE2 of central origin could therefore be involved in the activation of OT neurons projecting to the spinal cord to stimulate the SNS in immunechallenged animals, although this hypothesis still remains to be fully investigated. We have also detected EP4 transcripts over the neurons of the mediolateral magnocellular component of the PVN that were also positive for IEG Fos following IL-1 treatment, and these neurons, like those of the SON, send their projections to the posterior pituitary. The physiological relevance of these results remains unclear, but involvement of AVP system in response to cytokines and immune challenge has been suggested (209–211), and the antidiuretic action of arginine vasopressin (AVP) is obviously an integrated physiological process solicited during systemic inflammation. The neuropeptide is also believed to act as a potent neurosecretague for the regulation of the corticotroph axis, in particular during prolonged and sustained stressful conditions, such as inflammation. EP4 mRNA-positive neurons were found in many structures implicated in the central regulation of autonomic functions, and PGs have been reported to modulate blood pressure, heart rate (212), and respiration (213). At the level of medulla, the EP4 mRNA was prominent within the caudal NTS, a nucleus considered as an entry point for visceral sensory information arising from the vagal, glossopharyngeal, and facial nerves—the taste afferents occupying the most rostral portion and gastrointestinal afferents synapsing in the intermediate portion of the nucleus. Cardiovascular afferents terminate in the caudal half of the nucleus (dorsomedial, medial, parvocellular, and commissural subnuclei) and in the AP, while respiratory afferents end mainly in the ventrolateral, intermediate, and commissural subnuclei (for review, see 214). The distribution of EP4 transcript in different subdivisions of the NTS (notably in its commissural and medial parts) suggests that EP4 may be the PGE2 receptor subtype mediating the central responses to changes in cardiorespiratory status, which is an important component of the acute-phase reaction. One of the NTS projections is into the medullary reticular formation, including the rostral and caudal ventrolateral areas (215,216) that regulate the cardiovascular and respiratory reflexes. EP4-expressing cells are prominent more caudally in the region of ventrolateral medulla and systemic immune challenge caused upregulation of the mRNA in the A1 cell group. Immunohistochemical la-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

beling of medullary catecholamine-containing cells with an antiserum against the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH), combined with in situ hybridization for EP4 transcript indicated the presence of EP4 mRNA-positive cells in the noradrenergic (A1, A2) and adrenergic (C2) neurons of the NTS and cVLM (202,204). These neurons were also activated in response to circulating recombinant rat IL-1, although only the A1 cell group exhibited upregulation of the mRNA encoding the PGE2 receptor (202,204). This indicates that the noradrenergic neurons of the lateral reticular formation are key targets of PGE2 during systemic inflammatory processes. The primary destination of the A1 cell group of cVLM is the magnocellular neurosecretory AVP-containing neurons (217), although these medullary neurons have also been shown to participate in the control of neuroendocrine CRF neurons (205,218). Ericsson and colleagues have provided evidence that the stimulatory effects of i.v. recombinant human IL-1 on CRF neurosecretory neurons depend specifically on the integrity of catecholaminergic projections originating in caudal medulla (205) and that microinjections of PGE2 in the VLM provoked activation of the parvocellular PVN (218). Although these elegant studies suggested that expression of the EP3 receptor subtype in the C1 cell group may be responsible for the intramedullary action of PGE2 (218), recent data support the role of the EP4 in the A1 group of TH neurons (202,204). However, the EP2 receptor subtype may also contribute to the effects of the PG on neuroendocrine functions, as low EP2 mRNA levels were detected in the rostral VLM in response to i.v. LPS administration (202). The NTS provides a rich innervation to the PB, which occupies a key position in the central autonomic network, as in interface between the medullary reflex control, forebrain behavioral and integrative regulation of the autonomic system. Superior, dorsal, and central lateral subnuclei (PBsl, PBdl, PBcl) give their outputs to the hypothalamic PVN, whereas the external and extreme lateral nuclei of PB (PBel, PBexl) project mainly to the amygdala and associated portions of the substantia innominata and the bed nucleus of the stria terminalis (BnST). Some descending projections originate from the Kölliker-Fuse subnucleus (KF) that innervates the sympathetic preganglionic column of the spinal cord, the nucleus ambiguus, the ventrolateral and intermediate NTS, and the ventrolateral medullary reticular formation. The distribution of EP4 in PBsl, PBcl, PBel, PBexl, and KF assumes the PB bodywide regulatory considerations, such as control of body fluids, energy metabolism, and blood oxygenation, rather than specific organs or autonomic reflexes or behavioral contexts. Moreover, IL-1 induced Fosir nuclei into EP4-positive neurons located in different compartments of the PB, suggesting activation of these neurons during a systemic stressor (202). EP2 and EP4 mRNA-expressing cells were observed in the VSA, including diagonal band nucleus and medial septal nucleus, and in the anterodorsal part of third ventricle, especially over the MPOA and MePO (202,203). The MePO receives chemosensory input from the neighboring circumventricular organs, vis-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ceral sensory information from NTS, PB, and neighboring hypothalamic nuclei (219). The presence of PGE2 receptors in this region is particularly intriguing, because it contains high PGE2-binding sites in the rat brain (190) and is the most sensitive site in the hypothalamus for the antipyretic effects of salicylate (220). Microinjection of PGE2 into the ventromedial aspect of the POA is one of the most effective sites for causing fever and its related efferent pathways (197), whereas selective PG inhibition into this region markedly decreased the fever produced by systemic LPS, compared with injections into more rostral, caudal, or dorsal locations (184). It is then proposed that circulating cytokines and LPS stimulate local synthesis and release of PGE2, which in turn may trigger the EP4 receptors expressed in neurons of the VSA/anterior POA to induce fever. Dual-labeling data (Fos-ir/EP4 mRNA) in this region following i.v. IL-1 and LPS injection strongly support this cascade of events (202–204), although EP4-deficient mice seem to show a normal febrile response to PGE2 and to either IL-1 or LPS (221). Indeed, only mice lacking the EP3 receptor failed to generate fever to both exogenous and endogenous pyrogens (221). Due to the general inhibitory role of the EP3 receptor subtype, these results are somewhat surprising, and it is possible that the fine interplay between PGE2 and EP4 receptors within the preoptic area participates in thermoregulatory processes. Such essential physiological activity may therefore not be adequately addressed by homologous recombination but rather by localized inhibition within specific regions of the brain. The robust signals over the neuroendocrine population of CRF neurons are quite fascinating, as they provide direct anatomical evidence that the release of PGE2 within the PVN may have direct access to the neurons controlling the corticotroph axis (202). A robust hybridization signal for COX-2 mRNA along the microvessels of the parvocellular PVN occurs in response to systemic inflammatory insults (132), and systemic injection of IL-1 provokes sharply increased levels of PGE2 in the PVN (211). Central PGE2 injection also induces c-fos mRNA in CRF neurons, triggers the transcription of the neuropeptide specifically within the parvocellular PVN (198), and increases plasma corticosterone levels (192). Like the EP2 subtype, PGE2 EP4 receptors activate Gs proteins and adenylate cyclase (222), which is the most effective second messenger system taking place in activated CRF neurons and activates gene transcription through cAMP-responsive elements located on the CRF promoter (223). Together these data clearly suggest that PGE2 produced by PVN blood vessels may directly target the EP4 receptor onto neuroendocrine CRF cells, which may lead to cAMP signaling and infundibular release as well as transcription of stress-related neuropeptide during systemic inflammatory processes (see Fig. 15). D. IL-6 and Autonomic Functions Although IL-6 can stimulate the HPA axis directly at the level of both pituitary and adrenal glands, the cytokine is also believed to trigger infundibular CRF secretion Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 15 Intracellular mechanisms mediating the influence of circulating interleukin1 (IL-1) on the transcription of the cyclooxygenase-2 (COX-2) within an endothelial cell of the blood-brain barrier. Although simplistic, both the MAP kinases and NF- B pathways may be transduction/transcription signals in these processes. The production of the prostaglandin of E2 type is believed to be a key mediator to diffuse through the parenchymal brain and the neurons that control fever and the hypothalamic-pituitary-adrenal axis. The subsequent release of glucocorticoids is determinant for the immunosuppression of the systemic inflammation and the downregulation COX-2 transcription. Glucocorticoids may increase I B transcription and/or interfere with NF- B–binding ability on COX-2 promoter in cerebral vascular cells.

originating from parvocellular PVN neurons. It is therefore surprising that a single injection of IL-6 is insufficient to induce hypothalamic transcription of c-fos and CRF (224), a phenomenon contrasting with the profound stimulation of these genes in the PVN of IL-1– and LPS-injected rats (7,206,225–228). This lack of effect of IL-6 could be explained by the absence of IL-6 receptor (IL-6R) in the PVN neurons under basal conditions. Induction of the acute-phase response stimulated the expression of IL-6R mRNA in this hypothalamic nucleus, and this event could either permit or potentiate the action of IL-6 on neuroendocrine CRF cells and the Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

HPA axis. However, the fact that blood vessels express the receptor for IL-6 in response to LPS and IL-1 suggests that such a phenomenon may occur in a nonselective manner through brain microvasculature. The physiological relevance of the IL-6R expression over blood vessels of LPS- and IL-1–injected rats remains to be clearly established, although it is highly possible that endothelial cells are target cells of circulating IL-6, which in turn may transduce specific signals to parenchymal cells (including neurons) during a complex systemic immune challenge. As for the previously discussed cytokines, it is conceivable that IL-6 stimulates intermediate cells, such as endothelial or microglial cells, to release paracrine factors, which in turn could influence neuronal activity. As potential candidates, prostaglandins and nitric oxide are key mediators in neuroimmune communication, and their production can be controlled by IL-1 and TNF- (161,218,229,230). Unfortunately, because of differences in experimental procedure and tissues studied, their involvement in mediating the central effects of IL6 has not been demonstrated. For example, it has been demonstrated that prostaglandins mediate IL-6–induced fever (123,231,232) and HPA axis stimulation (125), but IL-6 is unable to activate prostaglandin formation in cerebral microvessels (233) or to induce COX-2 mRNA synthesis in rat brains (234) or in cultured microglial cells (235). It is clear, however, that IL-6 does not stimulate the production of prostaglandins in peripheral organs and that, conversely, its own synthesis is induced by them (236,237). In agreement with the latter, it has recently been shown that IL-6 expression in astrocytes can be induced in vitro by prostaglandin E2 (238). Taken together, these observations suggest that IL-6 does not stimulate COX-2 gene transcription, but the possibility that IL-6 may influence prostaglandin synthesis at posttranscriptional levels or may cooperate with them to activate CRF neurons cannot be ruled out. On the other hand, much less information is available regarding the effect of IL-6 on nitric oxide production. It has been shown that IL-6 stimulates nitrite formation in cultured hippocampal slices (239). However, IL-6–induced glial cell growth (240) and neuroblastoma cell differentiation (230) are not mediated by nitric oxide, and transgenic mice overexpressing IL-6 in the brain do not manifest any increase of inducible NOS gene expression (241). In the light of the above observations, the role of prostaglandins and NO in mediating the effects of LPS, IL-1, and TNF- cannot be generalized to IL-6, and more detailed studies will be needed to clarify the mechanisms by which this cytokine may affect the neuroendocrine response. Recent work suggested that neural cells become more sensitive to IL-6 during systemic immune challenges by increasing the number of receptor molecules on their surface (41,242). Although this modification seems to be essential, additional physiological changes might be required to allow such a permissive effect. The shedding of soluble IL-6R, the alteration of BBB, and the activation of transduction pathways cooperating with those solicited by IL-6 are examples of mechanisms that may amplify, widen, and prolong the IL-6 activities. On the one

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

hand, these changes may be initiated by the direct action of LPS on endothelial and parenchymal cells after binding to its specific receptor CD14 and by proinflammatory cytokines released by macrophages and glial cells in response to endotoxin. It has been shown that systematically injected IL-1 is capable of stimulating IL-6R synthesis in cerebral blood vessels (41) and that TNF- can modulate the expression of both IL-6R and gp130 mRNAs in cultured neurons (243). IL-1 and IL-6 are strongly synergistic in various immunological processes, including induction of cell proliferation and acute-phase protein synthesis by hepatocytes (244,245), while IL-1 is an important stimulator of IL-6 production in peripheral organs (246). IL-6 has been defined as one of the principal endogenous pyrogens, although this conclusion is quite surprising due to the inability of the cytokine to stimulate COX-2 transcription, which seems an essential prerequisite for engaging fever during immune stimuli. Recent observations suggest that IL-6 signaling is enhanced during endotoxemia and IL-6 modulates PVN functions only after pre-induction of its receptor in immune-challenged animals (242). Using a dual-labeling technique, we also found that some CRF neurons express IL-6R, suggesting that IL-6 may directly target these cells to trigger neuronal activation and CRF secretion (242). In this regard, systemic LPS insult caused a profound transcriptional activation of the gene encoding IL-6 selectively in the sensorial CVOs and the choroid plexus (41), which provides solid evidence that the cytokine is produced in the brain and may act on neurons or other parenchymal elements of the CNS. In addition, our latest data obtained from the experiment performed in wild-type and IL-6–deficient mice support the concept that IL-6, although not involved during the initial phases of endotoxemia, is necessary during the later phases for maintaining the stimulation of CRF neurons controlling the HPA axis and prolonging the activation of neural cells throughout the brain (242). This phenomenon might be of great importance to protect the brain and to restore homeostasis during bacterial septic shock. The increase in circulating corticosterone levels has recently been found to be lower in IL-6/ than IL-6/ mice in response to i.p. LPS injection, but not during restraint stress (247). This suggests that the involvement of IL-6 in the control of the HPA axis is quite specific to the immune stimuli and not neurogenic stresses. The participation of IL-6 was further suggested by the fact that pretreatment with anti-IL-6 antibody abrogates ACTH secretion both 2 and 4 hours after LPS administration, but not at 1 hour (248), while anti-IL-6 antibody significantly prevents the IL-1–induced increase in plasma ACTH (249). Interestingly, this determining role has been confirmed in models using inflammatory agents lacking the intrinsic capacity to stimulate the HPA axis but able to induce cytokine production. It was concluded that IL-6 is an obligate factor in increasing glucocorticoid production during cytomegalovirus infection or after the injection of a synthetic analog of viral nucleic acid (247).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The exact mechanisms by which IL-6 stimulates PVN neurons remain to be elucidated. The presence of IL-6R in some CRF neurons offers the possibility that IL-6 may directly target these cells. Alternatively, IL-6 may act on neurons devoid of IL-6R, but expressing the signal transducer gp130, after binding its soluble receptor. At first sight these possibilities seem to conflict with the current view that circulating cytokines cannot cross the BBB, but a number of mechanisms may explain this discrepancy. First, PVN neurons project to the median eminence and to the neurohypophysis, thereby exposing their axon terminals to molecules circulating in blood or secreted locally from microglia or macrophages. It has been proposed that IL-6 may act within the median eminence to trigger the release of CRF into the hypophyseal portal system (250). This is supported by our recent observations that median eminence expresses the IL-6 receptor subunits and that intravenously injected IL-6 induces c-fos expression in this region (41,224). Second, excessive NO and IL-6 production caused by endotoxin can alter cerebral endothelium functions and disrupt the BBB (251). The resulting increase in permeability may then allow high molecular weight molecules, including cytokines, to reach sites behind the BBB. Third, it has been demonstrated that a small quantity of IL-6 can penetrate across the intact BBB via a transport system distinct from those for IL-1 and TNF- (252). Some brain areas may therefore be equipped with an as yet uncharacterized active transport for IL-6, which might be activated under stressful circumstances. Fourth, the choroid plexus is a cerebrospinal fluid–synthesizing structure that has been pointed out as one of the principal sources of central IL-6 during endotoxemia (41). After being released into the ventricular system, centrally produced IL-6 may circulate throughout the brain and reach PVN neurons through passive diffusion. Assuming that LPS acts directly in the brain, its effect on the HPA axis may mask those of the proinflammatory cytokines, principally during the early phases. It can be presumed that the influence of IL-6 and other cytokines may be amplified during the later phases, when they become available in the circulation and when LPS is progressively eliminated from blood. The importance of macrophage products in LPS-induced HPA axis stimulation is illustrated by the observation that selective depletion of macrophages completely inhibits the increase of plasma ACTH and corticosterone in response to subpyrogenic doses of endotoxin (253). The participation of IL-6 in this process is suggested by the fact that pretreatment with anti-IL-6 antibody abrogates the ACTH secretion both 2 and 4 hours after LPS administration, but not at 1 hour (248). It is interesting to mention that IL-6 can also induce the secretion of AVP (254,255), an important ACTH and corticosteroid secretagogue, and that chronic cerebral expression of IL-6 modulates the stress-induced increase of plasma corticosterone via a mechanism involving AVP (256). Given that the activation of neural cells located in the PVN magnocellular division takes place after cotreatment with LPS and IL-6 (242), it is plausible that IL-6 stimulates the vasopressin neuronal system during endotoxemia, and, if that

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

is the case, the mechanisms underlying this phenomenon will be an intriguing question to address in future investigations taking advantage of IL-6–deficient mice. Besides controlling the HPA axis, IL-6 induces a series of other brain-mediated responses and participates to the modulation of local inflammatory events. Over the past years, we have used the proto-oncogene c-fos to identify within the brain the sites of IL-6 action. We found that CVOs, the meninges, the ependymal cell layer covering the ventricular spaces, and the portion of parenchyma surrounding the ventricles are profoundly activated after an intracerebroventricular injection of IL-6 (224). More recently we reported that IL-6 is not synthesized in the brain under basal conditions, but is rapidly expressed by the choroid plexus and other CVOs in response to systematically injected endotoxin. Furthermore, LPS-induced c-fos expression is markedly reduced in the brain of IL-6 knockout animals, especially in the above-mentioned regions, several hours after LPS administration (242). Taken together, these observations suggest that IL-6 is produced during endotoxemia in structures devoid of BBB, progressively secreted by the choroid plexus into cerebrospinal fluid, diffuses across the brain through the ventricles, and then stimulates various neural cells, principally those associated with or close to the ventricular system. Although accumulating data support the evidence that IL-6 is necessary during endotoxemia to trigger cellular activation throughout the brain, its exact roles remain obscure. Most of them have been deduced from experiments performed either in peripheral organs, in degenerated nervous tissue, or in cell cultures. For example, it is likely that IL-6 could be produced in the CVOs to activate the immune response by stimulating B- and T-cell growth and differentiation and by inducing gliosis. These effects may serve to protect the brain against the invasion of microorganisms or deleterious molecules. Because overactivation of the immune response can be as injurious as the intruding agent itself, IL-6 may, one the other hand, play anti-inflammatory roles by stimulating the production of IL-1 receptor antagonist, soluble TNF receptor (257,258), and acute-phase proteins, such as 2-macroglobulin (259), 1-antichymotrypsin (260), and metallothionein-I and -III (261,262). In addition, IL-6 may promote neuronal survival during inflammation by cooperating with the receptor trkA (263) and by inducing nerve growth factor secretion (264). In summary, the following sequence of events presumably takes place in the central nervous system during endotoxemia: Bacterial endotoxin reaches the brain through the circulation and directly acts on nonparenchymal and parenchymal structures accessible to blood leading to the activation of endothelial and microglial cells and to the development of the inflammatory response. PVN neurons are also activated in the presence of LPS and respond by increasing neuroendocrine secretion. A variety of neural cells become gradually more sensitive to IL6, mainly by expressing more receptor molecules on their surface. When it becomes available, circulating IL-6 interacts with other proinflammatory cytokines

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

to maintain the activation state of the hypothalamic CRF neurons and to induce specific cellular responses necessary for eliminating the endotoxin. Centrally produced IL-6 may play a local role in regulating inflammatory processes and can serve as a neuroprotective signal after being released into cerebrospinal fluid. The inflammation can be further attenuated by the inhibitory effects of glucocorticoids on cytokine expression. E. IL-6 Signaling Cascade in the CNS The bacterial endotoxin caused a profound stimulation of suppressor of cytokine signaling 3 (SOCS-3) gene in all the CVOs and their adjacent structures, chp, the ependymal lining cells of the cerebroventricular system, and along the endothelium of the cerebral capillaries (77). This indicates that IL-6 can trigger the JAK/STAT signaling in these groups of cells that may allow the transcription of the target genes involved in the neuro-immune interface. The exact JAK and STAT molecules involved in these events have yet to be determined, although investigating the transduction pathways is technically limited in vivo. Nevertheless, such an approach is essential to study the complex systems that interact together and the key populations of cells involved during the acute-phase response. In this regard, the essential role of IL-6 in mediating SOCS-3 expression in the endothelium and the ependymal lining cells of the brain is quite intriguing, as the molecules of the SOCS family are rather redundant and can be triggered by numerous other cytokines (for a review, see Ref. 265). These data contrast with our recent report that NF- B signaling in the brain is independent on the release of proinflammatory cytokines during endotoxemia (102). However, in the presence of the CD14 soluble form (sCD14), circulating LPS has the ability to activate directly the NF- B pathway in endothelial cells (110). The NF- B pathway is actually the main transduction signal for the endotoxin, and there is very little convincing evidence that LPS can trigger, at least by itself, the JAK/STAT molecules in endothelial cells. Induction of this signal transduction pathway must therefore be dependent on the production of circulating inflammatory cytokines in response to LPS. The late expression of SOCS-3 in the cerebral blood vessels and brain ependymal cells support this concept. This is quite different from the rapid transcription of the inhibitor of the NF- B I B (77), which is stimulated in the microvessels as early as 30 minutes post–i.p. LPS injection (102). A single systemic injection of LPS caused a rapid increase of TNF- and IL1 in the bloodstream, followed by a gradual elevation of plasma IL-6 (266). Despite the possible overlap with the JAK/STAT/SOCS pathways, TNF and IL-1 are well recognized NF- B–inducible cytokines (5,6), and their contribution may be minimal in regulating SOCS-3. Supporting this idea is the fact that the inhibitory factor was no longer expressed in the cerebral microvasculature and the ventricle lining walls of IL-6–deficient mice during endotoxemia (77). Such a phenomenon Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

was, however, not generalized to all responsive structures, as the CVOs and chp of these animals still exhibited a positive signal for SOCS-3 mRNA following the LPS challenge. This result suggests that IL-6 is a key ligand for activating SOCS-3 transcription only in barrier-associated groups of cells, but not in CVOs or chp. Other cytokines produced locally within these organs are likely to be responsible for residual SOCS-3 production in the brain of IL-6–deficient mice. Indeed, systemic LPS administration caused a rapid biosynthesis of different proinflammatory cytokines in all CVOs and chp (28,84,267), and these molecules are likely to induce the JAK/STAT signaling within the cells that bear the receptors for these ligands. Besides, IL-6 remains a potential inducer of SOCS-3 expression in the CVOs, because the hybridization signal for this transcript was higher in wild-type mice than IL6–deficient animals 4 hours after the LPS administration (77). Without being essential, this cytokine-dependent gp130 receptor subunit may be an important player to intensify SOCS-3 activity in the CVOs and chp at key moments during endotoxemia. On the other hand, the obligatory role of IL-6 in triggering the JAK/STAT signaling pathways that lead to SOCS-3 transcription seems only specific to two cell groups, namely the cerebral capillaries and the ventricle ependyma (77). Despite the fact that SOCS-3 transcription is dependent on the endogenous release of IL-6 in cells lining the BBB and the ventricles, a single administration of cytokine failed to activate the signaling events leading to SOCS-3 production in vivo. This result may be explained by the lack of constitutive expression of both IL-6 receptors, at least in the endothelium of the brain microvessels (41,242). As mentioned, systemic LPS challenge induced both IL-6R and gp130 transcripts in endothelial cells, and this phenomenon is a prerequisite for the IL-6 signaling during the acute-phase response. Whether IL-6R and gp130 expression in the brain microvasculature depends on the release of IL-1 and/or TNF- or is a direct action of the endotoxin onto the cerebral endothelium is still an open question. Besides, IL-1 is capable of activating IL-6 receptors in cells that can be reached from the systemic circulation (41) and IL-1 precedes the release of IL-6 during systemic inflammation. This potential mechanism is quite interesting as it follows the time-related events that occur during the acute-phase reaction of an immune challenge. Further supporting this concept is the late expression of SOCS-3 in the brain microvasculature of animals injected with turpentine in the left hind paw (77), an experimental model of sterile and localized inflammatory insult associated with a specific induction of IL-1 and IL-6 (134). It is therefore tempting to propose the following sequence of events: 1. IL-1 and TNF- are released early in the bloodstream during the acute-phase reaction. 2. These circulating cytokines have the ability to reach the large arterioles, small capillaries, and venules, which triggers NF- B nuclear translocation and stimulate IL-6R and gp130 transcription in endothelial cells. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

3.

These receptors may then respond when the ligand becomes available in the circulation that activates selective JAK/STAT molecules. 4. Phosphorylated homodimers of STATs (most likely STAT1 and/or STAT3) target SOCS-3 promoter and stimulate its transcription. 5. Increase in SOCS-3 protein inhibits this cytokine signaling and the proinflammatory signal transduction pathways. Similar events may also take place directly in the brain with the chp resident macrophages as being the cellular source of IL-1, TNF-, and IL-6 and the ependymal cells responding to these ligands of central origin. Altogether these data suggest that the preinduction of the IL-6 receptors is a prerequisite allowing IL-6 to trigger the transducing events and then SOCS-3 production in the cerebral endothelium and a specific group of supportive cells. Cytokine, when present in the circulation, may then act as the subsequent step to maintain the neuronal activity involved in the adequate control of the homeostatic balance during systemic inflammation. Moreover, IL-6–induced SOCS-3 is likely to be part of the anti-inflammatory mechanisms that take place in a very organized manner within the CNS. The molecules mediating the action of circulating IL-6 that are targeted by the JAK/STAT signaling and diffuse across the brain parenchyma to stimulate neurons involved in the autonomic functions have yet to be determined. As mentioned, the JAK/STAT transduction pathways do not activate COX-2 transcription and are unlikely to increase PG synthesis, which are quite selectively induced by cytokines and agents that activate the NF- B signaling cascade. Therefore, although the molecular events mediating the action of LPS, IL-1, and TNF are well characterized (see above), those completing the IL-6 transduction pathways in the cerebral tissue still await full documentation. Of interest is the reported role of SOCS-3 as an intracellular regulator of proopiomelanocortin (POMC) gene expression and ACTH secretion within AtT20 corticotroph cells (268). The idea that SOCS-3 acts as a potential negative feedback for the synthesis and production of ACTH from the adenohypophysis in response to locally produced gp130 cytokines is very interesting, although such a mechanism is likely to take place only during chronic and prolonged systemic immune challenges or induction of proinflammatory molecules locally within the anterior pituitary gland.

VI. IMPORTANT POINTS TO RETAIN 1.

Circulating molecules produced by systemic inflamed sites target cells of the BBB to release intermediates in the brain parenchyma. 2. Induction of the NF- B pathway leads to COX-2 gene transcription and brain PGE2 production by microvascular-associated cells.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

3. 4.

5. 6.

7.

8. 9.

VII.

IL-1 is the most likely candidate to mediate these effects during systemic and localized insults. The binding of IL-1 to its type I receptor and IL-1R-AcP engages the IRAK/TRAF6/MyD88/NIK/IKK pathway responsible to trigger COX2 gene transcription in the cerebral endothelium. Activation of the COX-2 enzyme leads to PGE2 synthesis. PGE2 may therefore diffuse through the parenchymal elements of the brain and bind to its EP4 receptor expressed at the surface of CRF dendrites, which induces cAMP/CREB transduction pathway. This PG may also cognate to its receptor expressed in key innervating neurons of the VmPO and medulla to induce/maintain fever and the corticotroph axis, respectively. Together, these circuits are responsible for engaging the autonomic functions during the acute-phase response of an immune-challenge. IL-6, in activating the JAK/STAT/SOCS signaling, takes the relay to prolong neuronal activity and autonomic functions.

PHYSIOPATHOLOGIES AND CONCLUDING REMARKS

Because of the essential metabolic role of glucocorticoids, a proper response of CRF neuronal activity during exogenous or endogenous challenges is crucial for the homeostasis of the organism. One of the best studied animal models, Lewis (LEW) rats exhibit a deficient hypothalamic CRF response to multiple inflammatory and noninflammatory stimuli, whereas histocompatible Fisher-344 (F344) rats display a robust increase in hypothalamic CRF (269–272). These two strains also exhibit differences in ACTH and corticosterone responses to various challenges (269–272). This led scientists to postulate that these differences in HPA axis responses play a role in the differential susceptibility of LEW and F344 rats to experimentally induced inflammatory arthritis and other experimental autoimmune diseases (269–272). LEW rats also exhibit a marked increase in CRF expression in the joints and surrounding tissues with streptococcal cell wall–and adjuvant-induced arthritis, whereas the levels of this neuropeptide are not increased in similarly treated F344 rats (273). This suggests that the mechanisms involved in the control of peripheral CRF secretion might also be defective in the LEW strain and participate, along with an altered CRF neuronal activity, in the high susceptibility of LEW strain to inflammation-induced arthritis. Although the LEW strain may provide a useful model for studying the relationship between neuroendocrine and inflammatory responses, it is important to realize that all the studies described above were performed in immature female rats, and one must be careful in generalizing these conclusions to mature animals of both genders (228). Be that as it may, these data provide solid evidence that alteration of the HPA axis

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

during inflammatory stimuli may lead to an exaggerated inflammatory response and be associated with chronic disorders, such as arthritis. An impaired immune system is also reported in Cushing’s syndrome (high cortisol levels) and Addison’s disease (low cortisol levels). Glucocorticoids have in fact long been recognized as being among of the most powerful anti-inflammatory agents known, and their use in restraining excessive immune responses, such as in individuals afflicted with rheumatoid arthritis, has been common in clinical practice for more than 50 years. The importance of a timely release of glucocorticoids is indicated by the high mortality observed in untreated Addisonian patients (274) and in experimentally adrenalectomized animals (269,275,276). We know a little more about the site(s) of action and the neuronal pathways participating in the influence of circulating cytokines on the brain functions fundamental to restoring homeostasis during immunogenic stimuli. Obviously, investigating the transduction signals involved in these events is somewhat limited in vivo, though the time-related induction of specific signaling molecules within the same population(s) of cells will be crucial information for future studies. The participation of the endothelium, microglia, or other cell types lining the BBB may depend on numerous factors, including the severity and duration of the inflammatory insults as well as the model of the systemic immunogenic challenges. Although the intracellular events may largely differ among stimuli and endogenous ligands (LPS, TNF, IL-1, vs. IL-6), the resulting production of PGE2 is likely to be the critical link between the circulating immune molecules and parenchymal elements of the brain to activate the neuronal circuits controlling different neurophysiological and neuroendocrine functions. The HPA axis is one of these functions having a profound impact on regulating inflammation (see Fig. 16). Inappropriate plasma levels of glucocorticoids may play a crucial role in contributing to deviant regulation of the immune response, indicating the importance of identifying and characterizing the mechanisms through which inflammatory molecules interact with, and depend on, the neuroendocrine system. Disorders of this fine interplay may indeed contribute to the onset and progression of various pathological states characterized by an exaggerated inflammatory response in the periphery as well as in the CNS. The recent evidence that corticoids may inhibit proinflammatory molecules via direct genomic effects in stimulating the transcription of the inhibitory factor I B (277,278) or in interfering with the transactivation potential of NF- B p65 subunit (279,280) are exciting new developments, and we believe that such events are essential for signal feedback to the brain. Because COX-2 is the key molecule for producing PGE2 in the CNS and plays a determinant role in controlling the autonomic system, the negative feedback of glucocorticoids on NF- B signaling and COX-2 transcription is likely to be the mechanism by which corticoids suppress the previously engaged responses, namely fever, HPA axis, and other autonomic functions. A defect in such fine signaling is not only associated with an aberrant immune

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 16 Schematic illustration of the possible circuits mediating the activation of PVN and the hypothalamic-pituitary-adrenal (HPA) axis during systemic innate immune response. The endotoxin LPS and cytokines use several pathways and sites of entry to communicate with the brain and neuroendocrine functions. It is suggested that circumventricular organs (organs devoid of blood-brain barrier) and the blood vessels (bv) are crucial target sites of LPS and proinflammatory cytokines of systemic origin, whereas activated regions involved in the autonomic control play a determinant role in the integration of information received from the periphery. Among these integrative structures, the PVN may be central to the appropriate control of homeostasis during immune challenge in directly controlling the autonomic outputs and the activity of the HPA axis. AP, area postrema; ARC, arcuate nucleus; BnST, bed nucleus of the stria terminalis; bv, blood vessels; chp, choroid plexus; CeA, central nucleus of the amygdala; DMH, dorsomedial nucleus of the hypothalamus; ME, median eminence; LC, locus ceruleus; LDT, laterodorsal tegmental nucleus; LPS, lipopolysaccharide; LRNm, lateral reticular nucleus medial; MPOA, medial preoptic area; NTS, nucleus of the solitary tract; OVLT, organum vasculosum of the lamina terminalis; PB, parabrachial nucleus; PP, posterior pituitary; PVN, paraventricular nucleus of the hypothalamus [parvocellular (pc) and magnocellular divisions (mc)]; SFO, subfornical organ; SON, supraoptic nucleus; VLM, ventrolateral medulla.

response, but also with exaggerated autonomic outcomes that can be detrimental for the organism and lead to disease and, in more severe cases, death (e.g., septic shock). ACKNOWLEDGMENTS This work is supported by the Canadian Institutes of Health Research [CIHR; the former Medical Research Council of Canada (MRCC)]. The author is an MRCC Scientist and holds a Canadian Research Chair in Neuroimmunology. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

REFERENCES 1. 2. 3.

4.

5. 6. 7.

8. 9. 10. 11.

12.

13.

14. 15.

16.

17.

Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol 2000; 12:13–19. Wright SD. Toll, a new piece in the puzzle of innate immunity. J Exp Med 1999; 189:605–609. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipolysaccharide (LPS) and LPS binding protein. Science 1990; 249:1431–1433. Andersson J, Nagy S, Björk L, Abrams J, Holm S, Andersson U. Bacterial toxin-induced cytokine production studied at the single-cell level. Immunological Rev 1992; 127:69–96. Baeuerle PA. Pro-inflammatory signaling: last pieces in the NF-kappa B puzzle. Curr Biol 1998; 8:R19–R22. Baeuerle PA, Baltimore D. NF kappa B: ten years after. Cell 1996; 87:13–20. Rivest S, Lacroix S, Vallières L, Nadeau S, Zhang J, Laflamme N. How the blood talks to the brain parenchyma and the paraventricular nucleus of the hypothalamus during systemic inflammatory and infectious stimuli. Proc Soc Exp Biol Med 2000; 223:22–38. Rivest S. What is the cellular source of prostaglandins in the brain in response to systemic inflammation? Facts and controversies. Mol Psychiatry 1999; 4:501–507. Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF[kappa]B activity. Annu Rev Immunol 2000; 18:621–663. Karin M. The beginning of the end: IkappaB kinase (IKK) and NF-kappaB activation. J Biol Chem 1999; 274:27339–42. Perera PY, Vogel SN, Detore GR, Haziot A, Goyert SM. CD14-dependent and CD14-independent signaling pathways in murine macrophages from normal and CD14 knockout mice stimulated with lipopolysaccharide or taxol. J Immunol 1997; 158:4422–4429. Haziot A, Ferrero E, Kontgen F, Hijiya N, Yamamoto S, Silver J, Stewart CL, Goyert SM. Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice. Immunity 1996; 4:407–414. Muzio M, Polentarutti N, Bosisio D, Prahladan MK, Mantovani A. Toll-like receptors: a growing family of immune receptors that are differentially expressed and regulated by different leukocytes. J Leukoc Biol 2000; 67:450–456. Beutler B. Tlr4: central component of the sole mammalian LPS sensor. Curr Opin Immunol 2000; 12:20–26. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, Akira S. Differential roles of TLR2 and TLR4 in recognition of gram-negative and grampositive bacterial cell wall components. Immunity 1999; 11:443–451. Ozinsky A, Underhill DM, Fontenot JD, Hajjar AM, Smith KD, Wilson CB, Schroeder L, Aderem A. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc Natl Acad Sci USA 2000; 97:13766–71. Kawai T, Adachi O, Ogawa T, Takeda K, Akira S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 1999; 11:115–122.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

18. Yang H, Young DW, Gusovsky F, Chow JC. Cellular events mediated by lipopolysaccharide-stimulated toll-like receptor 4. MD-2 is required for activation of mitogen-activated protein kinases and Elk-1. J Biol Chem 2000; 275:20861–20866. 19. Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, Kimoto M. MD2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 1999; 189:1777–1782. 20. Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. An endotoxininduced serum factor that causes necrosis of tumors. Proc Natl Acad Sci USA 1975; 72:3666–3670. 21. Miller VE, Rogers K, Muirden KD. Detection of tumour necrosis factor alpha and interleukin-1 beta in the rheumatoid osteoarthritic cartilage-pannus junction by immunohistochemical methods. Rheumatol Int 1993; 13:77–82. 22. Goldfeld AE, Strominger JL, Doyle C. Human tumor necrosis factor alpha gene regulation in phorbol ester stimulated T and B cell lines. J Exp Med 1991; 174:73–81. 23. Chung IY, Benveniste EN. Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1beta. J Immunol 1990; 144:2999–3007. 24. Wilkinson LS, Edwards JC. Binding of antibodies raised against tumour necrosis factor alpha (TNF alpha) to blood vessels and macrophages in inflamed synovial tissue. Rheumatol Int 1991; 11:19–25. 25. Brennan FM, Maini RN, Feldmann M. TNF alpha—a pivotal role in rheumatoid arthritis? Br J Rheumatol 1992; 31:293–298. 26. Liu L, Kita T, Tanaka N, Kinoshita Y. The expression of tumour necrosis factor in the hypothalamus after treatment with lipopolysaccharide. Int J Exp Pathol 1996; 77:37–44. 27. Nadeau S, Rivest S. Role of microglial-derived tumor necrosis factor in mediating CD14 transcription and NF- B activity in the brain during endotoxemia. J Neurosci 2000; 20:3456–3468. 28. Nadeau S, Rivest S. Regulation of the gene encoding tumor necrosis factor alpha in the rat brain and pituitary in response to different models of systemic immune challenge. J Neuropathol Exp Neurol 1999; 58:61–77. 29. Laflamme N, Rivest S. Effects of systemic immunogenic insults and circulating proinflammatory cytokines on the transcription of the inhibitory factor kappa B alpha within specific cellular populations of the rat brain. J Neurochem 1999; 73:309–321. 30. Chu WM, Ostertag D, Li ZW, Chang L, Chen Y, Hu Y, Williams B, Perrault J, Karin M. JNK2 and IKKbeta are required for activating the innate response to viral infection. Immunity 1999; 11:721–731. 31. Delhase M, Hayakawa M, Chen Y, Karin M. Positive and negative regulation of IkappaB kinase activity through IKKbeta subunit phosphorylation. Science 1999; 284:309–313. 32. Hu Y, Baud V, Delhase M, Zhang P, Deerinck T, Ellisman M, Johnson R, Karin M. Abnormal morphogenesis but intact IKK activation in mice lacking the IKKalpha subunit of IkappaB kinase. Science 1999; 284:316–320. 33. Webb AC, Collins KL, Auron PE, Eddy RL, Nakai H, Byers MG, Haley LL, Henry

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

34.

35.

36. 37.

38. 39. 40.

41.

42.

43. 44.

45.

46.

47.

48.

WM, Shows TB. Interleukin-1 gene (IL1) assigned to long arm of human chromosome 2. Lymphokine Res 1986; 5:77–85. Hirsch E, Irikura VM, Paul SM, Hirsh D. Functions of interleukin 1 receptor antagonist in gene knockout and overproducing mice. Proc Natl Acad Sci USA 1996; 93:11008–11013. Shirakawa F, Saito K, Bonagura CA, Galson DL, Fenton MJ, Webb AC, Auron PE. The human prointerleukin 1 beta gene requires DNA sequences both proximal and distal to the transcription start site for tissue-specific induction. Mol Cell Biol 1993; 13:1332–44. Roux-Lombard P. The interleukin-1 family. Eur Cytokine Netw 1998; 9:565–76. Fenton MJ, Vermeulen MW, Clark BD, Webb AC, Auron PE. Human pro-IL-1 beta gene expression in monocytic cells is regulated by two distinct pathways. J Immunol 1988; 140:2267–2273. Taga T, Kishimoto T. Gp130 and the interleukin-6 family of cytokines. Annu Rev Immunol 1997; 15:797–819. Hirano T. Interleukin 6 and its receptor: ten years later. Int Rev Immunol 1998; 16:249–284. Kopf M, Baumann H, Freer G, Freudenberg M, Lamers M, Kishimoto T, Zinkernagel R, Bluethmann H, Köhler G. Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 1994; 368:339–342. Vallières L, Rivest S. Regulation of the genes encoding interleukin-6, its receptor, and gp130 in the rat brain in response to the immune activator lipopolysaccharide and the proinflammatory cytokine interleukin-1. J Neurochem 1997; 69:1668–1683. Taga T, Hibi M, Hirata Y, Yamasaki K, Yasukawa K, Matsuda T, Hirano T, Kishimoto T. Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp130. Cell 1989; 58:573–581. Taga T. The signal transducer gp130 is shared by interleukin-6 family of haematopoietic and neurotrophic cytokines. Ann Med 1997; 29:63–72. Narazaki M, Witthuhn BA, Yoshida K, Silvennoinen O, Yasukawa K, Ihle JN, Kishimoto T, Taga T. Activation of JAK2 kinase mediated by the interleukin 6 signal transducer gp130. Proc Natl Acad Sci USA 1994; 91:2285–2289. Stahl N, Farruggella TJ, Boulton TG, Zhong Z, Darnell JE, Jr., Yancopoulos GD. Choice of STATs and other substrates specified by modular tyrosine- based motifs in cytokine receptors. Science 1995; 267:1349–1353. Lütticken C, Wegenka UM, Yuan J, Buschmann J, Schindler C, Ziemiecki A, Harpur AG, Wilks AF, Yasukawa K, Taga T, et al. Association of transcription factor APRF and protein kinase Jak1 with the interleukin-6 signal transducer gp130. Science 1994; 263:89–92. Fukada T, Hibi M, Yamanaka Y, Takahashi-Tezuka M, Fujitani Y, Yamaguchi T, Nakajima K, Hirano T. Two signals are necessary for cell proliferation induced by a cytokine receptor gp130: involvement of STAT3 in anti-apoptosis. Immunity 1996; 5:449–460. Zhong Z, Wen Z, Darnell JE, Jr. Stat3 and Stat4: members of the family of signal transducers and activators of transcription. Proc Natl Acad Sci USA 1994; 91:4806–4810.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

49.

50.

51.

52. 53. 54.

55.

56.

57.

58.

59.

60.

61.

62.

Zhong Z, Wen Z, Darnell JE, Jr. Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 1994; 264:95–98. Akira S, Nishio Y, Inoue M, Wang XJ, Wei S, Matsusaka T, Yoshida K, Sudo T, Naruto M, Kishimoto T. Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway. Cell 1994; 77:63–71. Sadowski HB, Shuai K, Darnell JE, Jr., Gilman MZ. A common nuclear signal transduction pathway activated by growth factor and cytokine receptors. Science 1993; 261:1739–1744. Watanabe S, Arai K. Roles of the JAK-STAT system in signal transduction via cytokine receptors. Curr Opin Genet Dev 1996; 6:587–596. Leaman DW, Leung S, Li X, Stark GR. Regulation of STAT-dependent pathways by growth factors and cytokines. FASEB J 1996; 10:1578–1588. Daeipour M, Kumar G, Amaral MC, Nel AE. Recombinant IL-6 activates p42 and p44 mitogen-activated protein kinases in the IL-6 responsive B cell line, AF-10. J Immunol 1993; 150:4743–4753. Boulton TG, Stahl N, Yancopoulos GD. Ciliary neurotrophic factor/leukemia inhibitory factor/interleukin 6/oncostatin M family of cytokines induces tyrosine phosphorylation of a common set of proteins overlapping those induced by other cytokines and growth factors. J Biol Chem 1994; 269:11648–11655. Kumar G, Gupta S, Wang S, Nel AE. Involvement of Janus kinases, p52shc, Raf-1, and MEK-1 in the IL-6-induced mitogen-activated protein kinase cascade of a growth-responsive B cell line. J Immunol 1994; 153:4436–4447. Ogata A, Chauhan D, Teoh G, Treon SP, Urashima M, Schlossman RL, Anderson KC. IL-6 triggers cell growth via the Ras-dependent mitogen-activated protein kinase cascade. J Immunol 1997; 159:2212–2221. Takahashi-Tezuka M, Yoshida Y, Fukada T, Ohtani T, Yamanaka Y, Nishida K, Nakajima K, Hibi M, Hirano T. Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase. Mol Cell Biol 1998; 18:4109–4117. Ernst M, Gearing DP, Dunn AR. Functional and biochemical association of Hck with the LIF/IL-6 receptor signal transducing subunit gp130 in embryonic stem cells. EMBO J 1994; 13:1574–1584. Matsuda T, Takahashi-Tezuka M, Fukada T, Okuyama Y, Fujitani Y, Tsukada S, Mano H, Hirai H, Witte ON, Hirano T. Association and activation of Btk and Tec tyrosine kinases by gp130, a signal transducer of the interleukin-6 family of cytokines. Blood 1995; 85:627–633. Neumann C, Zehentmaier G, Danhauser-Riedl S, Emmerich B, Hallek M. Interleukin-6 induces tyrosine phosphorylation of the Ras activating protein Shc, and its complex formation with Grb2 in the human multiple myeloma cell line LP-1. Eur J Immunol 1996; 26:379–384. Giordano V, De Falco G, Chiari R, Quinto I, Pelicci PG, Bartholomew L, Delmastro P, Gadina M, Scala G. Shc mediates IL-6 signaling by interacting with gp130 and Jak2 kinase. J Immunol 1997; 158:4097–4103.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

63. 64.

65. 66.

67.

68. 69. 70.

71.

72.

73.

74. 75.

76.

Qiu Y, Ravi L, Kung HJ. Requirement of ErbB2 for signalling by interleukin-6 in prostate carcinoma cells. Nature 1998; 393:83–85. Zohlnhofer D, Graeve L, Rose-John S, Schooltink H, Dittrich E, Heinrich PC. The hepatic interleukin-6 receptor. Down-regulation of the interleukin- 6 binding subunit (gp80) by its ligand. FEBS Lett 1992; 306:219–222. Nesbitt JE, Fuller GM. Dynamics of interleukin-6 internalization and degradation in rat hepatocytes. J Biol Chem 1992; 267:5739–5742. Rose-John S, Hipp E, Lenz D, Legres LG, Korr H, Hirano T, Kishimoto T, Heinrich PC. Structural and functional studies on the human interleukin-6 receptor. Binding, cross-linking, internalization, and degradation of interleukin- 6 by fibroblasts transfected with human interleukin-6-receptor cDNA. J Biol Chem 1991; 266:3841–3846. Thiel S, Behrmann I, Dittrich E, Muys L, Tavernier J, Wijdenes J, Heinrich PC, Graeve L. Internalization of the interleukin 6 signal transducer gp130 does not require activation of the Jak/STAT pathway. Biochem J 1998; 330:47–54. Aman MJ, Leonard WJ. Cytokine signaling: cytokine-inducible signaling inhibitors. Curr Biol 1997; 7:R784–788. Nicholson SE, Hilton DJ. The SOCS proteins: a new family of negative regulators of signal transduction. J Leukoc Biol 1998; 63:665–668. Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA, Sprigg NS, Starr R, Nicholson SE, Metcalf D, Nicola NA. Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci USA 1998; 95:114–119. Alexander WS, Starr R, Metcalf D, Nicholson SE, Farley A, Elefanty AG, Brysha M, Kile BT, Richardson R, Baca M, Zhang JG, Willson TA, Viney EM, Sprigg NS, Rakar S, Corbin J, Mifsud S, DiRago L, Cary D, Nicola NA, Hilton DJ. Suppressors of cytokine signaling (SOCS): negative regulators of signal transduction. J Leukoc Biol 1999; 66:588–592. Hansen JA, Lindberg K, Hilton DJ, Nielsen JH, Billestrup N. Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins. Mol Endocrinol 1999; 13:1832–1843. Nicholson SE, Willson TA, Farley A, Starr R, Zhang JG, Baca M, Alexander WS, Metcalf D, Hilton DJ, Nicola NA. Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL-6 signal transduction. Embo J 1999; 18:375–385. Bjorbaek C, Elmquist JK, Frantz JD, Shoelson SE, Flier JS. Identification of SOCS3 as a potential mediator of central leptin resistance. Mol Cell 1998; 1:619–625. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 1996; 84:491–495. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, Sanker S, Moriarty A, Moore KJ, Smtko JS, Mays GG, Woolf EA, Monroe CA, Tepper RI. Identification and expression cloning of a leptin receptor, OB-R. Cell 1995; 83:1263–1271.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

77.

78.

79.

80. 81.

82. 83.

84.

85.

86.

87. 88. 89. 90.

91.

92. 93.

Lebel E, Vallieres L, Rivest S. Selective involvement of interleukin-6 in the transcriptional activation of the suppressor of cytokine signaling-3 in the brain during systemic immune challenges. Endocrinology 2000; 141:3749–3763. Cross AK, Richardson V, Ali SA, Palmer I, Taub DD, Rees RC. Migration responses of human monocytic cell lines to alpha- and beta-chemokines. Cytokine 1997; 9:521–8. Strieter RM, Standiford TJ, Huffnagle GB, Colletti LM, Lukacs NW, Kunkel SL. The good, the bad, and the ugly; The role of chemokines in models of human disease. J Immunol 1996; 156:3583–3586. Baggiolini M, Dewald B, Moser B. Human chemokines: an update. Annu Rev Immunol 1997; 15:675–705. Lacroix S, Feinstein D, Rivest S. The bacterial endotoxin lipopolysaccharide has the ability to target the brain in upregulating its membrane CD14 receptor within specific cellular populations. Brain Pathol 1998; 8:625–640. Oldfield BJ, Mckinley MJ. Circumventricular organs. In: Paxinos G, ed. The Rat Nervous System. San Diego: Academic Press, 1995:391–403. Laflamme N, Rivest S. Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components. FASEB J 2001; 15:155–163. Quan N, Whiteside M, Herkenham M. Time course and localization patterns of interleukin-1 mRNA expression in the brain and pituitary after peripheral administration of lipopolysaccharide. Neuroscience 1997; 83:281–293. Nomura F, Akashi S, Sakao Y, Sato S, Kawai T, Matsumoto M, Nakanishi K, Kimoto M, Miyake K, Takeda K, Akira S. Cutting edge: endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface toll-like receptor 4 expression. J Immunol 2000; 164:3476–3479. Fearns C, Kravchenko VV, Ulevitch RJ, Loskutoff DJ. Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide. J Exp Med 1995; 181:857–866. Fearns C, Loskutoff DJ. Role of tumor necrosis factor alpha in induction of murine CD14 gene expression by lipopolysaccharide. Infect Immun 1997; 65:4822–4831. Fearns C, Ulevitch RJ. Effect of recombinant interleukin-1beta on murine CD14 gene expression in vivo. Shock 1998; 9:157–163. Sweet MJ, Hume DA. Endotoxin signal transduction in macrophages. J Leukoc Biol 1996; 60:8–26. Baumann H, Morella KK, Wong GH. TNF-alpha, IL-1 beta, and hepatocyte growth factor cooperate in stimulating specific acute phase plasma protein genes in rat hepatoma cells. J Immunol 1993; 151:4248–4257. Herx LM, Rivest S, Yong VW. Central nervous system-initiated inflammation and neurotrophism in trauma: IL-1beta is required for the production of ciliary neurotrophic factor. J Immunol 2000; 165:2232–2239. Taylor P, Botto M, Walport M. The complement system. Curr Biol 1998; 8:R259–261. Merino S, Nogueras MM, Aguilar A, Rubires X, Alberti S, Benedi VJ, Tomas JM. Activation of the complement classical pathway (C1q binding) by mesophilic

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

94. 95. 96. 97. 98.

99.

100. 101.

102.

103. 104.

105.

106. 107.

108.

109.

110.

Aeromonas hydrophila outer membrane protein. Infect Immun 1998; 66:3825–3831. Colten HR. Immunology. Drawing a double-edged sword. Nature 1994; 371:474–475. Gerard C, Gerard NP. C5A anaphylatoxin and its seven transmembrane-segment receptor. Annu Rev Immunol 1994; 12:775–808. Wetsel RA. Structure, function and cellular expression of complement anaphylatoxin receptors. Curr Opin Immunol 1995; 7:48–53. Nataf S, Stahel PF, Davoust N, Barnum SR. Complement anaphylatoxin receptors on neurons: new tricks for old receptors? Trends Neurosci 1999; 22:397–402. Gasque P, Dean YD, McGreal EP, VanBeek J, Morgan BP. Complement components of the innate immune system in health and disease in the CNS. Immunopharmacology 2000; 49:171–186. Gasque P, Singhrao SK, Neal JW, Wang P, Sayah S, Fontaine M, Morgan BP. The receptor for complement anaphylatoxin C3a is expressed by myeloid cells and nonmyeloid cells in inflamed human central nervous system: analysis in multiple sclerosis and bacterial meningitis. J Immunol 1998; 160:3543–3554. Morgan BP, Gasque P. Expression of complement in the brain: role in health and disease. Immunol Today 1996; 17:461–466. Gasque P, Fontaine M, Morgan BP. Complement expression in human brain. Biosynthesis of terminal pathway components and regulators in human glial cells and cell lines. J Immunol 1995; 154:4726–4733. Laflamme N, Lacroix S, Rivest S. An essential role of interleukin-1 in mediating NF- B activity and COX-2 transcription in cells of the blood-brain barrier in response to systemic and localized inflammation, but not during endotoxemia. J Neurosci 1999; 19:10923–10930. Fabry Z, Raine CS, Hart MN. Nervous tissue as an immune compartment: the dialect of the immune response in the CNS. Immunol Today 1994; 15:218–224. Levi-Strauss M, Mallat M. Primary cultures of murine astrocytes produce C3 and factor B, two components of the alternative pathway of complement activation. J Immunol 1987; 139:2361–2366. Rus HG, Kim LM, Niculescu FI, Shin ML. Induction of C3 expression in astrocytes is regulated by cytokines and Newcastle disease virus. J Immunol 1992; 148:928–933. Nadeau S, Rivest S. The complement system is an integrated part of the natural innate immune response in the brain. FASEB J 2001; 15:1410–1412. Fukuoka Y, Ember JA, Hugli TE. Cloning and characterization of rat C3a receptor: differential expression of rat C3a and C5a receptors by LPS stimulation. Biochem Biophys Res Commun 1998; 242:663–668. Tornetta MA, Foley JJ, Sarau HM, Ames RS. The mouse anaphylatoxin C3a receptor: molecular cloning, genomic organization, and functional expression. J Immunol 1997; 158:5277–5282. Mayhan WG. Effect of lipopolysaccharide on the permeability and reactivity of the cerebral microcirculation: role of inducible nitric oxide synthase. Brain Res 1998; 792:353–357. Arditi M, Zhou J, Torres M, Durden DL, Stins M, Kim KS. Lipopolysaccharide

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

111.

112.

113. 114.

115.

116.

117.

118.

119.

120.

121. 122.

123. 124.

stimulates the tyrosine phosphorylation of mitogen-activated protein kinases p44, p42, and p41 in vascular endothelial cells in a soluble CD14-dependent manner. Role of protein tyrosine phosphorylation in lipopolysaccharide-induced stimulation of endothelial cells. J Immunol 1995; 155:3994–4003. Quan N, Whiteside M, Kim L, Herkenham M. Induction of inhibitory factor B mRNA in the central nervous system after peripheral lipopolysaccharide administration: an in situ hybridization histochemistry study in the rat. Proc Natl Acad Sci USA 1997; 94:10985–10990. Nonaka M, Huang ZM. Interleukin-1-mediated enhancement of mouse factor B gene expression via NF kappa B-like hepatoma nuclear factor. Mol Cell Biol 1990; 10:6283–6289. Antel JP, Owens T. Immune regulation and CNS autoimmune disease. J Neuroimmunol 1999; 100:181–189. Thibeault I, Laflamme N, Rivest S. Regulation of the gene encoding the monocyte chemoattractant protein 1 (MCP-1) in the rat brain in response to circulating LPS and proinflammatory cytokines. J Comp Neurol 2001; 434:461–477. Izikson L, Klein RS, Charo IF, Weiner HL, Luster AD. Resistance to experimental autoimmune encephalomyelitis in mice lacking the CC chemokine receptor (CCR)2. J Exp Med 2000; 192:1075–1080. Tran EH, Hoekstra K, van Rooijen N, Dijkstra CD, Owens T. Immune invasion of the central nervous system parenchyma and experimental allergic encephalomyelitis, but not leukocyte extravasation from blood, are prevented in macrophage-depleted mice. J Immunol 1998; 161:3767–3775. Fuentes ME, Durham SK, Swerdel MR, Lewin AC, Barton DS, Megill JR, Bravo R, Lira SA. Controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1. J Immunol 1995; 155:5769–5776. Ericsson A, Liu C, Hart RP, Sawchenko PE. Type-1 interleukin-1 receptor in the rat brain: distribution, regulation, and relationship to sites of IL-1-induced cellular activation. J Comp Neurol 1995; 361:681–698. Nadeau S, Rivest S. Effects of circulating tumor necrosis factor (TNF) on the neuronal activity and expression of the genes encoding the TNF receptors (p55 and p75) in the rat brain: a view from the blood-brain barrier. Neuroscience 1999; 93:1449–1464. Blatteis CM, Bealer SL, Hunter WS, Llanos QJ, Ahokas RA, Mashburn Jr. TA. Suppression of fever after lesions of the anteroventral third ventricle in guinea pigs. Brain Research Bulletin 1983; 11:519–526. Blatteis CM, Hales JRS, McKinley MJ, Fawcett AA. Role of the anteroventral third ventricle in fever in sheep. Can J Physiol Pharmac 1987; 65:1255–1260. Chai Z, Gatti S, Toniatti C, Poli V, Bartfai T. Interleukin (IL)-6 gene expression in the central nervous system is necessary for fever response to lipopolysaccharide or IL-1 beta: a study on IL-6-deficient mice. J Exp Med 1996; 183:311–316. Xin L, Blatteis CM. Hypothalamic neuronal responses to interleukin-6 in tissue slices: effects of indomethacin and naloxone. Brain Res Bull 1992; 29:27–35. Lee HY, Whiteside MB, Herkenham M. Area postrema removal abolishes stimulatory effects of intravenous interleukin-1beta on hypothalamic-pituitary-adrenal axis

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

125.

126.

127.

128.

129.

130.

131.

132.

133.

134.

135.

136.

137.

138.

activity and c-fos mRNA in the hypothalamic paraventricular nucleus. Brain Res Bull 1998; 46:495–503. Lyson K, McCann SM. Involvement of arachidonic acid cascade pathways in interleukin-6-stimulated corticotropin-releasing factor release in vitro. Neuroendocrinology 1992; 55:708–713. DeWitt DL, Smith WL. Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc Natl Acad Sci USA 1988; 85:1412–1416. DeWitt DL, El-Harith EA, Kraemer SA, Andrews MJ, Yao EF, Armstrong RL, Smith WL. The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases. J Biol Chem 1990; 265:5192–5198. Hempel SL, Monick MM, Hunninghake GW. Lipopolysaccharide induces prostaglandin H synthase-2 protein and mRNA in human alveolar macrophages and blood monocytes. J Clin Invest 1994; 93:391–396. Jones DA, Carlton DP, McIntyre TM, Zimmerman GA, Prescott SM. Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines. J Biol Chem 1993; 268:9049–9054. Lee SH, Soyoola E, Chanmugam P, Hart S, Sun W, Zhong H, Liou S, Simmons D, Hwang D. Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J Biol Chem 1992; 267:25934–25938. Cao C, Matsumura K, Yamagata K, Watanabe Y. Induction by lipopolysaccharide of cyclooxygenase-2 mRNA in the rat brain; its possible role in the febrile response. Brain Res 1995; 697:187–196. Lacroix S, Rivest S. Effect of acute systemic inflammatory response and cytokines on the transcription of the genes encoding cyclooxygenase enzymes (COX-1 and COX-2) in the rat brain. J Neurochem 1998; 70:452–466. Quan N, Whiteside M, Herkenham M. Cyclooxygenase 2 mRNA expression in rat brain after peripheral injection of lipopolysaccharide. Brain Res 1998; 802:189–197. Fantuzzi G, Dinarello CA. The inflammatory response in interleukin-1-deficient mice: comparison with other cytokine-related knock-out mice. J Leukoc Biol 1996; 59:489–493. Cao CY, Matsumura K, Yamagata K, Watanabe Y. Endothelial-cells of the rat-brain vasculature express cyclooxygenase-2 messenger-RNA in response to systemic interleukin-1: a possible site of prostaglandin synthesis responsible for fever. Brain Res 1996; 733:263–272. Cao C, Matsumura K, Ozaki M, Watanabe Y. Lipopolysaccharide injected into the cerebral ventricle evokes fever through induction of cyclooxygenase-2 in brain endothelial cells. J Neurosci 1999; 19:716–725. Matsumura K, Cao C, Watanabe Y. Prostaglandin system in the brain: sites of biosynthesis and sites of action under normal and hyperthermic states. Prog Brain Res 1998; 115:275–295. Matsumura K, Cao C, Ozaki M, Morii H, Nakadate K, Watanabe Y. Electron microscopic evidence for induction of cyclooxygenase-2 in brain endothelial cells. Ann NY Acad Sci 1998; 856:278–280.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

139.

140.

141. 142.

143.

144.

145.

146. 147. 148. 149. 150. 151.

152.

153.

154.

155.

Matsumura K, Cao C, Ozaki M, Morii H, Nakadate K, Watanabe Y. Brain endothelial cells express cyclooxygenase-2 during lipopolysaccharide-induced fever: light and electron microscopic immunocytochemical studies. J Neurosci 1998; 18:6279–6289. Elmquist JK, Breder CD, Sherin JE, Scammell TE, Hickey WF, Dewitt D, Saper CB. Intravenous lipopolysaccharide induces cyclooxygenase 2-like immunoreactivity in rat brain perivascular microglia and meningeal macrophages. J Comp Neurol 1997; 381:119–129. Schiltz JC, Sawchenko PE. Interleukin-1 induction of cyclooxygenase-2 expression in ED2-positive perivascular microglia. Soc Neurosci Abstr 1998; 24:1615. Crofford LJ, Tan B, McCarthy CJ, Hla T. Involvement of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression by interleukin-1 in rheumatoid synoviocytes. Arthritis Rheum 1997; 40:226–236. Sorli CH, Zhang HJ, Armstrong MB, Rajotte RV, Maclouf J, Robertson RP. Basal expression of cyclooxygenase-2 and nuclear factor-interleukin 6 are dominant and coordinately regulated by interleukin 1 in the pancreatic islet. Proc Natl Acad Sci USA 1998; 95:1788–1793. Inoue H, Tanabe T. Transcriptional role of the nuclear factor kappa B site in the induction by lipopolysaccharide and suppression by dexamethasone of cyclooxygenase-2 in U937 cells. Biochem Biophys Res Commun 1998; 244:143–148. Schmedtje JF, Jr., Ji YS, Liu WL, DuBois RN, Runge MS. Hypoxia induces cyclooxygenase-2 via the NF-kappaB p65 transcription factor in human vascular endothelial cells. J Biol Chem 1997; 272:601–608. Griffith OW, Stuehr DJ. Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol 1995; 57:707–736. Snyder SH. Nitric oxide: first in a new class of neurotransmitters. Science 1992; 257:494–496. Umans JG, Levi R. Nitric oxide in the regulation of blood flow and arterial pressure. Annu Rev Physiol 1995; 57:771–790. MacMicking J, Xie QW, Nathan C. Nitric oxide and macrophage function. Annu Rev Immunol 1997; 15:323–350. Gross SS, Wolin MS. Nitric oxide: pathophysiological mechanisms. Annu Rev Physiol 1995; 57:737–769. Salvemini D, Misko TP, Masferrer JL, Seibert K, Currie MG, Needleman P. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci USA 1993; 90:7240–7244. Landino LM, Crews BC, Timmons MD, Morrow JD, Marnett LJ. Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis. Proc Natl Acad Sci USA 1996; 93:15069–15074. Corbett JA, Kwon G, Turk J, McDaniel ML. IL-1 beta induces the coexpression of both nitric oxide synthase and cyclooxygenase by islets of Langerhans: activation of cyclooxygenase by nitric oxide. Biochemistry 1993; 32:13767–13770. Salvemini D, Currie MG, Mollace V. Nitric oxide-mediated cyclooxygenase activation. A key event in the antiplatelet effects of nitrovasodilators. J Clin Invest 1996; 97:2562–2568. Janabi N, Chabrier S, Tardieu M. Endogenous nitric oxide activates prostaglandin

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

156.

157.

158.

159. 160.

161.

162.

163.

164.

165.

166.

167. 168. 169.

170.

F2 alpha production in human microglial cells but not in astrocytes: a study of interactions between eicosanoids, nitric oxide, and superoxide anion (O2-) regulatory pathways. J Immunol 1996; 157:2129–2135. Habib A, Bernard C, Lebret M, Creminon C, Esposito B, Tedgui A, Maclouf J. Regulation of the expression of cyclooxygenase-2 by nitric oxide in rat peritoneal macrophages. J Immunol 1997; 158:3845–3851. Patel R, Attur MG, Dave M, Abramson SB, Amin AR. Regulation of cytosolic COX-2 and prostaglandin E2 production by nitric oxide in activated murine macrophages. J Immunol 1999; 162:4191–4197. Blais V, Rivest S. Inhibitory action of nitric oxide on circulating tumor necrosis factor-induced NF- B activity and COX-2 transcription in the endothelium of the brain capillaries. J Neuropathol Exp Neurol 2001; 60:893–905. Krukoff TL. Central actions of nitric oxide in regulation of autonomic functions. Brain Res Brain Res Rev 1999; 30:52–65. Raghavendra V, Agrewala JN, Kulkarni SK. Role of centrally administered melatonin and inhibitors of COX and NOS in LPS-induced hyperthermia and adipsia. Prostaglandins Leukot Essent Fatty Acids 1999; 60:249–253. Rivier C, Shen GH. In the rat, endogenous nitric oxide modulates the response of the hypothalamic-pituitary-adrenal axis to interleukin-1 beta, vasopressin, and oxytocin. J Neurosci 1994; 14:1985–1993. Neves SM, Rezende SA, Goes AM. Nitric oxide-mediated immune complex-induced prostaglandin E(2) production by peripheral blood mononuclear cells of humans infected with Schistosoma mansoni. Cell Immunol 1999; 195:37–42. Hughes FJ, Buttery LD, Hukkanen MV, O’Donnell A, Maclouf J, Polak JM. Cytokine-induced prostaglandin E2 synthesis and cyclooxygenase-2 activity are regulated both by a nitric oxide-dependent and -independent mechanism in rat osteoblasts in vitro. J Biol Chem 1999; 274:1776=–n1782. De Caterina R, Libby P, Peng HB, Thannickal VJ, Rajavashisth TB, Gimbrone MA, Jr., Shin WS, Liao JK. Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest 1995; 96:60–8. Shin WS, Hong YH, Peng HB, De Caterina R, Libby P, Liao JK. Nitric oxide attenuates vascular smooth muscle cell activation by interferon-gamma. The role of constitutive NF-kappa B activity. J Biol Chem 1996; 271:11317–11324. Liu SF, Ye X, Malik AB. Inhibition of NF-kappaB activation by pyrrolidine dithiocarbamate prevents In vivo expression of proinflammatory genes. Circulation 1999; 100:1330–1337. Kunsch C, Medford RM. Oxidative stress as a regulator of gene expression in the vasculature. Circ Res 1999; 85:753–766. Li N, Karin M. Is NF-kappaB the sensor of oxidative stress? FASEB J 1999; 13:1137–1143. Schreck R, Meier B, Mannel DN, Droge W, Baeuerle PA. Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. J Exp Med 1992; 175:1181–1194. Ziegler-Heitbrock HW, Sternsdorf T, Liese J, Belohradsky B, Weber C, Wedel A, Schreck R, Bauerle P, Strobel M. Pyrrolidine dithiocarbamate inhibits NF-kappa B

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

171.

172.

173.

174. 175.

176.

177. 178.

179.

180. 181. 182.

183.

184.

185.

mobilization and TNF production in human monocytes. J Immunol 1993; 151:6986–6993. Munoz C, Pascual-Salcedo D, Castellanos MC, Alfranca A, Aragones J, Vara A, Redondo MJ, de Landazuri MO. Pyrrolidine dithiocarbamate inhibits the production of interleukin-6, interleukin-8, and granulocyte-macrophage colony-stimulating factor by human endothelial cells in response to inflammatory mediators: modulation of NF-kappa B and AP-1 transcription factors activity. Blood 1996; 88:3482–3490. Vlahopoulos S, Boldogh I, Casola A, Brasier AR. Nuclear factor-kappaB-dependent induction of interleukin-8 gene expression by tumor necrosis factor alpha: evidence for an antioxidant sensitive activating pathway distinct from nuclear translocation. Blood 1999; 94:1878–1889. Janssen-Heininger YM, Macara I, Mossman BT. Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-kappaB: requirement of Ras/mitogen- activated protein kinases in the activation of NF-kappaB by oxidants. Am J Respir Cell Mol Biol 1999; 20:942–952. Stamler JS, Singel DJ, Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Science 1992; 258:1898–1902. Hermann GE, Emch GS, Tovar CA, Rogers RC. c-Fos generation in the dorsal vagal complex after systemic endotoxin is not dependent on the vagus nerve. Am J Physiol Reg Integr Comp Physiol 2001; 280:R289–R299. Sergeev VG, Akmaev IG. Effect of transection of various branches of the vagus nerve on lipopolysaccharide-induced synthesis of corticotropin-releasing hormone mRNA in the paraventricular nuclei of rat hypothalamus. Bull Exp Biol Med 2000; 130:705–708. Elmquist JK, Scammell TE, Saper CB. Mechanisms of CNS response to systemic immune challenge: the febrile response. Trends Neurosci 1997; 20:565–570. McCoy JG, Matta SG, Sharp BM. Prostaglandins mediate the ACTH response to interleukin-1-beta instilled into the hypothalamic median eminence. Neuroendocrinology 1994; 60:426–435. Katsuura G, Gottschall PE, Dahl RR, Arimura A. Adrenocorticotropin release induced by intracerebroventricular injection of recombinant human interleukin-1 in rats: Possible involvement of prostaglandin. Endocrinology 1988; 122:1773–1779. Rivier C. Neuroendocrine effects of cytokines in the rat. Rev Neurosci 1993; 4:223–237. Sharp BM, Matta SG. Prostaglandins mediate the adrenocorticotropin response to tumor necrosis factor in rats. Endocrinology 1993; 132:269–274. Rivest S, Rivier C. Centrally injected interleukin-1 inhibits the hypothalamic LHRH secretion and circulating LH levels via prostaglandins in rats. J Neuroendocrinol 1993; 5:445–450. Yasin SA, Costa A, Forsling ML, Grossman A. Interleukin-1 and interleukin-6 stimulate neurohypophysial hormone release in vitro. J Neuroendocrinol 1994; 6:179–184. Scammell TE, Griffin JD, Elmquist JK, Saper CB. Microinjection of a cyclooxygenase inhibitor into the anteroventral preoptic region attenuates LPS fever. Am J Physiol 1998; 274:R783–789. Navarra P, Pozzoli G, Brunetti L, Ragazzoni E, Besser M, Grossman A. Interleukin-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

186.

187.

188.

189. 190.

191.

192. 193.

194.

195.

196. 197.

198.

199. 200.

201.

1 and interleukin-6 specifically increase the release of prostaglandin E2 from rat hypothalamic explants in vitro. Neuroendocrinology 1992; 56:61–68. Komaki G, Arimura A, Koves K. Effect of intravenous injection of IL-1 on PGE2 levels in several brain areas as determined by microdialysis. Am J Physiol 1992; 262 (Endocrinol Metab 25):E246–E251. Katsuura G, Gottschall PE, Dahl RR, Arimura A. Interleukin-1 increases prostaglandin E2 in rat astrocyte cultures: modulating effect of neuropeptides. Endocrinology 1989; 124:3125–3127. Hughes JH, Easom RA, Wolf BA, Turk J, McDaniel ML. Interleukin-1-induced prostaglandin E2 accumulation by isolated pancreatic islets. Diabetes 1989; 38:1251–1257. Kohan DE. Interleukin-1 regulation of prostaglandin E2 synthesis by the papillary collecting duct. J Lab Clin Med 1989; 114:717–723. Matsumura K, Watanabe Y, Onoe H, Watanabe Y, Hayaishi O. High density of prostaglandin E2 binding sites in the anterior wall of the 3rd ventricle: a possible site of its hyperthermic action. Brain Res 1990; 533:147–151. Matsumura K, Watanabe Y, Imai-Matsumura K, Connolly M, Koyama Y, Onoe H, Watanabe Y. Mapping of prostaglandin E2 binding sites in rat brain using quantitative autoradiography. Brain Res 1992; 581:292–298. Rassnick S, Zhou DH, Rabin BS. Central administration of prostaglandin E (2) suppresses in vitro cellular immune responses. Am J Physiol 1995; 269:R92–97. Katsuura G, Arimura A, Koves K, Gottschall PE. Involvement of organum vasculosum of the lamina terminalis and preoptic area in interleukin-1-induced ACTH release. Am J Physiol 1990; 258:E163–E171. Feuerstein G, Adelberg SA, Kopin IJ, Jacubowitz DM. Hypothalamic sites for cardiovascular and sympathetic modulation by prostaglandin E2. Brain Res 1982; 231:335–342. Ando T, Ichijo T, Katafuchi T, Hori T. Intracerebroventricular injection of prostaglandin E2 increases splenic sympathetic nerve activity in rats. Am J Physiol 1995; 269:R662–R668. Oka T, Hori T. EP1-receptor mediation of prostaglandin E2-induced hyperthermia in rats. Am J Physiol 1994; 267:R289–R294. Scammell TE, Elmquist JK, Griffin JD, Saper CB. Ventromedial preoptic prostaglandin E2 activates fever-producing autonomic pathways. J Neurosci 1996; 16:6246–6254. Lacroix S, Vallières L, Rivest S. C-fos mRNA pattern and CRF neuronal activity throughout the brain of rats injected centrally with a prostaglandin of E2 type. J Neuroimmunol 1996; 70:163–179. Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 1999; 79:1193–1226. Coleman RA, Smith WL, Narumiya S. International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 1994; 46:205–229. Namba T, Sugimoto Y, Negishi M, Irie A, Ushikubi F, Kakizuka A, Ito S, Ichikawa A, Narumiya S. Alternate splicing of C-terminal tail of prostaglandin E receptor subtype EP3 determine G-protein specificity. Nature 1993; 365:166–170.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

202.

203.

204.

205.

206. 207.

208. 209.

210.

211.

212. 213. 214. 215.

216.

Zhang J, Rivest S. Distribution, regulation and colocalization of the genes encoding the EP2- and EP4-PGE2 receptors in the rat brain and neuronal responses to systemic inflammation. Eur J Neurosci 1999; 11:2651–2668. Oka T, Oka K, Scammell TE, Lee C, Kelly JF, Nantel F, Elmquist JK, Saper CB. Relationship of EP(1–4) prostaglandin receptors with rat hypothalamic cell groups involved in lipopolysaccharide fever responses. J Comp Neurol 2000; 428:20–32. Zhang J, Rivest S. A functional analysis of EP4 receptor-expressing neurons in mediating the action of PGE2 within specific nuclei of the brain in response to circulating interleukin-1. J Neurochem 2000; 74:2134–2145. Ericsson A, Kovács KJ, Sawchenko PE. A functional anatomical analysis of central pathways subserving the effects of interleukin-1 on stress-related neuroendocrine neurons. J Neurosci 1994; 14:897–913. Rivest S, Laflamme N. Neuronal activity and neuropeptide gene transcription in the brain of immune-challenged rats. J Neuroendocrinol 1995; 7:501–525. Sundar SK, Cierpial MA, Kilts C, Ritchie JC, Weiss JM. Brain IL-1-induced immunosuppression occurs through activation of both pituitary-adrenal axis and sympathetic nervous system by corticotropin-releasing factor. J Neurosci 1990; 10:3701–3706. Ichijo T, Katafuchi T, Hori T. Central interleukin-1 enhances splenic sympathetic nerve activity in rats. Brain Res Bull 1994; 34:547–553. Berkenbosch F, Oers JV, Rey AD, Tilders F, Besedovsky H. Corticotropin-releasing factor producing neurons in the rat activated by interleukin-1. Science 1987; 238:524–526. Harbuz MS, Rees RG, Eckland D, Jessop DS, Brewerton D, Lightman SL. Paradoxical responses of hypothalamic corticotropin-releasing factor (CRF) messenger ribonucleic acid (mRNA) and CRF-41 peptide and adenohypophysial proopiomelanocortin mRNA during chronic inflammatory stress. Endocrinology 1992; 130:1394–1400. Watanobe H, Takebe K. Effects of intravenous administration of interleukin-1-beta on the release of prostaglandin E2, corticotropin-releasing factor, and arginine vasopressin in several hypothalamic areas of freely moving rats: estimation by pushpull perfusion. Neuroendocrinology 1994; 60:8–15. Forstermann U, Neufang B. Endothelium-dependent vasodilation by melittin: are lipoxygenase products involved? Am J Physiol 1985; 249:H14–19. Koos BJ. Central stimulation of breathing movements in fetal lambs by prostaglandin synthetase inhibitors. J Physiol (Lond) 1985; 362:455–466. Saper CB. The spinoparabrachial pathway: shedding new light on an old path. J Comp Neurol 1995; 353:477–479. Cunningham ET, Jr., Sawchenko PE. A circumscribed projection from the nucleus of the solitary tract to the nucleus ambiguus in the rat: anatomical evidence for somatostatin- 28-immunoreactive interneurons subserving reflex control of esophageal motility. J Neurosci 1989; 9:1668–1682. Cunningham ET, Miselis RR, Sawchenko PE. The relationship of efferent projections from the area postrema to vagal motor and brain stem catecholamine-containing cell groups: an axonal transport and immunohistochemical study in the rat. Neuroscience 1994; 58:635–648.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

217.

218.

219.

220. 221.

222.

223.

224.

225.

226.

227. 228.

229.

230.

231.

Cunningham ET, Sawchenko PE. Anatomical specificity of noradrenergic inputs to the paraventricular and supraoptic nuclei of the rat hypothalamus. J Comp Neurol 1988; 274:60–76. Ericsson A, Arias C, Sawchenko PE. Evidence for an intramedullary prostaglandindependent mechanism in the activation of stress-related neuroendocrine circuitry by intravenous interleukin-1. J Neurosci 1997; 17:7166–7179. Swanson LW, Mogenson GJ, Simerly RB, Wu M. Anatomical and electrophysiological evidence for a projection from the medial preoptic area to the ‘mesencephalic and subthalamic locomotor regions’ in the rat. Brain Res 1987; 405:108–122. Stitt JT. Passage of immunomodulators across the blood-brain barrier. Yale J Biol Med 1990; 63:121–131. Ushikubi F, Segi E, Sugimoto Y, Murata T, Matsuoka T, Kobayashi T, Hizaki H, Tuboi K, Katsuyama M, Ichikawa A, Tanaka T, Yoshida N, Narumiya S. Impaired febrile response in mice lacking the prostaglandin E receptor subtype EP3. Nature 1998; 395:281–284. Fedyk ER, Ripper JM, Brown DM, Phipps RP. A molecular analysis of PGE receptor (EP) expression on normal and transformed B lymphocytes: coexpression of EP1, EP2, EP3beta and EP4. Mol Immunol 1996; 33:33–45. Guardiola-Diaz HM, Boswell C, Seasholtz AF. The cAMP-responsive element in the corticotropin-releasing hormone gene mediates transcriptional regulation by depolarization. J Biol Chem 1994; 269:14784–14791. Vallières L, Lacroix S, Rivest S. Influence of interleukin-6 on neural activity and transcription of the gene encoding corticotropin-releasing factor in the rat brain: an effect depending upon the route of administration. Eur J Neurosci 1997; 9:1461–1472. Rivest S, Laflamme N, Nappi RE. Immune challenge and immobilization stress induce transcription of the gene encoding the CRF receptor in selective nuclei of the rat hypothalamus. J Neurosci 1995; 15:2680–2695. Rivest S. Molecular mechanisms and neural pathways mediating the influence of interleukin-1 on the activity of neuroendocrine CRF motoneurons in the rat. Int J Dev Neurosci 1995; 13:135–146. Rivest S, Rivier C. The role of CRF and interleukin-1 in the regulation of neurons controlling reproductive functions. Endocr Rev 1995; 16:177–199. Rivest S, Rivier C. Stress and interleukin-1-induced activation of c-fos, NGFI-B and CRF gene expression in the hypothalamic PVN: comparison between SpragueDawley, Fisher-344 and Lewis rats. J Neuroendocrinol 1994; 6:101–117. Dayer JM, Beutler B, Cerami A. Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts. J Exp Med 1985; 162:2163–2168. Munoz-Fernandez MA, Cano E, O’Donnell CA, Doyle J, Liew FY, Fresno M. Tumor necrosis factor-alpha (TNF-alpha), interferon-gamma, and interleukin-6 but not TNF-beta induce differentiation of neuroblastoma cells: the role of nitric oxide. J Neurochem 1994; 62:1330–1336. Dinarello CA, Cannon JG, Mancilla J, Bishai I, Lees J, Coceani F. Interleukin-6 as an endogenous pyrogen: induction of prostaglandin E2 in brain but not in peripheral blood mononuclear cells. Brain Res 1991; 562:199–206.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

232.

233.

234.

235.

236.

237.

238.

239. 240. 241.

242.

243.

244.

245.

246. 247.

Fernandez-Alonso A, Benamar K, Sancibrian M, Lopez-Valpuesta FJ, Minano FJ. Role of interleukin-1 beta, interleukin-6 and macrophage inflammatory protein-1 beta in prostaglandin-E2-induced hyperthermia in rats. Life Sci 1996; 59:L185–190. Bishai I, Coceani F. Differential effects of endotoxin and cytokines on prostaglandin E2 formation in cerebral microvessels and brain parenchyma: implications for the pathogenesis of fever. Cytokine 1996; 8:371–376. Lacroix S, Rivest S. Effect of acute systemic inflammatory response and cytokines on the transcription of the genes encoding cyclooxygenase enzymes (COX-1 and COX-2) in the rat brain. J Neurochem 1998; 70:452–466. Bauer MK, Lieb K, Schulze-Osthoff K, Berger M, Gebicke-Haerter PJ, Bauer J, Fiebich BL. Expression and regulation of cyclooxygenase-2 in rat microglia. Eur J Biochem 1997; 243:726–731. Hinson RM, Williams JA, Shacter E. Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation: possible role of cyclooxygenase-2. Proc Natl Acad Sci USA 1996; 93:4885–4890. Williams JA, Shacter E. Regulation of macrophage cytokine production by prostaglandin E2. Distinct roles of cyclooxygenase-1 and -2. J Biol Chem 1997; 272:25693–25699. Fiebich BL, Hull M, Lieb K, Gyufko K, Berger M, Bauer J. Prostaglandin E2 induces interleukin-6 synthesis in human astrocytoma cells. J Neurochem 1997; 68:704–709. Ma T, Zhu X. Interleukin-6 increases the levels of cyclic GMP and nitrite in rat hippocampal slices. Eur J Pharmacol 1997; 321:343–347. Munoz-Fernandez MA, Fresno M. Involvement of nitric oxide on the cytokine induced growth of glial cell. Biochem Biophys Res Commun 1993; 194:319–325. Chiang CS, Stalder A, Samimi A, Campbell IL. Reactive gliosis as a consequence of interleukin-6 expression in the brain: studies in transgenic mice. Dev Neurosci 1994; 16:212–221. Vallières L, Rivest S. Interleukin-6 is a needed proinflammatory cytokine in the prolonged neural activity and transcriptional activation of corticotropin-releasing factor during endotoxemia. Endocrinology 1999; 140:3890–3903. Marz P, Gadient RA, Otten U. Expression of interleukin-6 receptor (IL-6R) and gp130 mRNA in PC12 cells and sympathetic neurons: modulation by tumor necrosis factor alpha (TNF-alpha). Brain Res 1996; 706:71–79. Tritarelli E, Greco G, Testa U, Belardelli F, Peschle C, Proietti E. Combined interleukin-1 beta/interleukin-6 treatment in mice: synergistic myelostimulatory activity and myelorestorative effect after cyclophosphamide-induced myelosuppression. Cancer Res 1994; 54:6469–6476. Conti P, Bartle L, Barbacane RC, Reale M, Placido FC, Sipe J. Synergistic activation of serum amyloid A (SAA) by IL-6 and IL-1 in combination on human Hep 3B hepatoma cell line. Role of PGE2 and IL-1 receptor antagonist. Immunol Invest 1995; 24:523–535. Akira S, Hirano T, Taga T, Kishimoto T. Biology of multifunctional cytokines: IL6 and related molecules (IL-1 and TNF). FASEB J 1990; 4:2860–2867. Ruzek MC, Miller AH, Opal SM, Pearce BD, Biron CA. Characterization of early cytokine responses and an interleukin (IL)-6- dependent pathway of endogenous

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

248.

249.

250.

251.

252. 253.

254.

255.

256.

257.

258.

259.

260.

261.

glucocorticoid induction during murine cytomegalovirus infection. J Exp Med 1997; 185:1185–1192. Perlstein RS, Whitnall MH, Abrams JS, Mougey EH, Neta R. Synergistic roles of interleukin-6, interleukin-1, and tumor necrosis factor in the adrenocorticotropin response to bacterial lipopolysaccharide in vivo. Endocrinology 1993; 132:946–952. Neta R, Perlstein R, Vogel SN, Ledney GD, Abrams J. Role of interleukin 6 (IL-6) in protection from lethal irradiation and in endocrine responses to IL-1 and tumor necrosis factor. J Exp Med 1992; 175:689–694. Spinedi E, Hadid R, Daneva T, Gaillard RC. Cytokines stimulate the CRH but not the vasopressin neuronal system: evidence for a median eminence site of interleukin-6 action. Neuroendocrinology 1992; 56:46–53. Brett FM, Mizisin AP, Powell HC, Campbell IL. Evolution of neuropathologic abnormalities associated with blood-brain barrier breakdown in transgenic mice expressing interleukin-6 in astrocytes. J Neuropathol Exp Neurol 1995; 54:766–775. Banks WA, Kastin AJ, Gutierrez EG. Penetration of interleukin-6 across the murine blood-brain barrier. Neurosci Lett 1994; 179:53–56. Derijk R, Van Rooijen N, Tilders FJ, Besedovsky HO, Del Rey A, Berkenbosch F. Selective depletion of macrophages prevents pituitary-adrenal activation in response to subpyrogenic, but not to pyrogenic, doses of bacterial endotoxin in rats. Endocrinology 1991; 129:330–338. Mastorakos G, Weber JS, Magiakou MA, Gunn H, Chrousos GP. Hypothalamic-pituitary-adrenal axis activation and stimulation of systemic vasopressin secretion by recombinant interleukin-6 in humans: potential implications for the syndrome of inappropriate vasopressin secretion. J Clin Endocrinol Metab 1994; 79:934–939. Kageyama K, Watanobe H, Takebe K. In vivo evidence that arginine vasopressin is involved in the adrenocorticotropin response induced by interleukin-6 but not by tumor necrosis factor-alpha in the rat. Neuroimmunomodulation 1995; 2:137–140. Raber J, O’Shea RD, Bloom FE, Campbell IL. Modulation of hypothalamic-pituitary-adrenal function by transgenic expression of interleukin-6 in the CNS of mice. J Neurosci 1997; 17:9473–9480. Tilg H, Trehu E, Atkins MB, Dinarello CA, Mier JW. Interleukin-6 (IL-6) as an antiinflammatory cytokine: induction of circulating IL-1 receptor antagonist and soluble tumor necrosis factor receptor p55. Blood 1994; 83:113–118. Tilg H, Vannier E, Vachino G, Dinarello CA, Mier JW. Antiinflammatory properties of hepatic acute phase proteins: preferential induction of interleukin 1 (IL-1) receptor antagonist over IL-1 beta synthesis by human peripheral blood mononuclear cells. J Exp Med 1993; 178:1629–1636. Ganter U, Strauss S, Jonas U, Weidemann A, Beyreuther K, Volk B, Berger M, Bauer J. Alpha 2-macroglobulin synthesis in interleukin-6-stimulated human neuronal (SH-SY5Y neuroblastoma) cells. Potential significance for the processing of Alzheimer beta-amyloid precursor protein. FEBS Lett 1991; 282:127–131. Kordula T, Rydel RE, Brigham EF, Horn F, Heinrich PC, Travis J. Oncostatin M and the interleukin-6 and soluble interleukin-6 receptor complex regulate alpha1-antichymotrypsin expression in human cortical astrocytes. J Biol Chem 1998; 273:4112–4118. Hernandez J, Molinero A, Campbell IL, Hidalgo J. Transgenic expression of inter-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

262. 263.

264.

265. 266.

267.

268.

269.

270.

271.

272.

273.

274.

leukin 6 in the central nervous system regulates brain metallothionein-I and -III expression in mice. Brain Res Mol Brain Res 1997; 48:125–131. Hernandez J, Hidalgo J. Endotoxin and intracerebroventricular injection of IL-1 and IL-6 induce rat brain metallothionein-I and -II. Neurochem Int 1998; 32:369–373. Sterneck E, Kaplan DR, Johnson PF. Interleukin-6 induces expression of peripherin and cooperates with Trk receptor signaling to promote neuronal differentiation in PC12 cells. J Neurochem 1996; 67:1365–1374. Kossmann T, Hans V, Imhof HG, Trentz O, Morganti-Kossmann MC. Interleukin6 released in human cerebrospinal fluid following traumatic brain injury may trigger nerve growth factor production in astrocytes. Brain Res 1996; 713:143–152. Kovanen PE, Leonard WJ. Cytokine signaling: Inhibitors keep cytokines in check. Cur Biol 1999; 9:R899–R902. Givalois L, Dornand J, Mekaouche M, Solier MD, Bristow AF, Ixart G, Siaud P, Assenmacher I, Barbanel G. The temporal cascade of plasma level surges in ACTH, corticosterone and cytokines in endotoxin-challenged rats. Am J Physiol 1994; 266:R164–R170. Breder CD, Hazuka C, Ghayur T, Klug C, Huginin M, Yasuda K, Teng M, Saper CB. Regional induction of tumor-necrosis factor-alpha expression in the mouse brain after systemic lipopolysaccharide administration. Proc Natl Acad Sci USA 1994; 22:11393–11397. Auernhammer CJ, Chesnokova V, Bousquet C, Melmed S. Pituitary corticotroph SOCS-3: novel intracellular regulation of leukemia-inhibitory factor-mediated proopiomelanocortin gene expression and adrenocorticotropin secretion. Mol Endocrinol 1998; 12:954–961. Sternberg EM, Hill JM, Chrousos GP, Kamilaris T, Listwak SJ, Gold PW, Wilder RL. Inflammatory mediator-induced hypothalamic-pituitary-adrenal axis activation is defective in streptoccocal cell wall arthritis-susceptible Lewis rats. Proc Natl Acad Sci USA 1989; 86:2374–2378. Sternberg EM, III WSY, Bernardini R, Calogero AE, Chrousos GP, Gold PW, Wilder RL. A central nervous system defect in biosynthesis of corticotropin-releasing hormone is associated with susceptibility to streptoccocal cell wall-induced arthritis in Lewis rats. Proc Natl Acad Sci USA 1989; 86:4771–4775. Sternberg EM, Glowa JR, Smith MA, Calogero AE, Listwak SJ, Aksentijevich S, Chrousos GP, Wilder RL, Gold PW. Corticotropin releasing hormone related behavioral and neuroendocrine responses to stress in Lewis and Fisher rats. Brain Res 1992; 570:54–60. Calogero AE, Sternberg EM, Bagdy G, Smith C, Bernardini R, Aksentijevich S, Wilder RL, Gold PW, Chrousos GP. Neurotransmitter-induced hypothalamic-pituitary-adrenal axis responsiveness in inflammatory disease-susceptible Lewis rats: in vivo and vitro studies suggesting a global defect in CRH secretion. Neuroendocrinology 1992; 55:600–608. Crofford LJ, Sano H, Karalis K, Webster EL, Goldmuntz EA, Chrousos GP, Wilder RL. Local secretion of corticotropin-releasing hormone in the joints of Lewis rats with inflammatory arthritis. J Clin Invest 1992; 90:2555–2564. Orth DN, Kovacs WJ, DeBold CR. The adrenal cortex. In: Wilson JD, DW Foster, eds. Textbook of Endocrinology. Philadelphia: W.B. Saunders, 1992:489–619.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

275.

276.

277.

278.

279.

280.

Mason D, MacPhee I, Antoni F. The role of the neuroendocrine system in determining genetic susceptibility to experimental allergic encephalomyelitis. Immunology 1990; 70:1–5. Edwards CK, Yunger LM, Lorence RM, Dantzer R, Kelley KW. The pituitary gland is required for protection against lethal effects of Salmonella typhimurium. Proc Natl Acad Sci USA 1991; 88:2274–2277. Scheinman RI, Cogswell PC, Lofquist AK, Baldwin AS, Jr. Role of transcriptional activation of I kappa B alpha in mediation of immunosuppression by glucocorticoids. Science 1995; 270:283–286. Auphan N, DiDonato JA, Rosette C, Helmberg A, Karin M. Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis. Science 1995; 270:286–290. De Bosscher K, Schmitz ML, Vanden Berghe W, Plaisance S, Fiers W, Haegeman G. Glucocorticoid-mediated repression of nuclear factor-kappaB-dependent transcription involves direct interference with transactivation. Proc Natl Acad Sci USA 1997; 94:13504–13509. Wissink S, van Heerde EC, vand der Burg B, van der Saag PT. A dual mechanism mediates repression of NF-kappaB activity by glucocorticoids. Mol Endocrinol 1998; 12:355–363.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

3 Organ Specificity of Autonomic Nervous System Responses Shaun F. Morrison Neurological Sciences Institute, Oregon Health and Sciences University, Beaverton, Oregon, U.S.A.

The rapid, aggressive, and widespread autonomic response required to cope with life-threatening danger was the model on which early views of the sympathetic nervous system as a monolithic, undifferentiated effector were based. With advances in experimental techniques and a greater appreciation for the autonomic influences on the function of noncardiovascular tissues, this preliminary construct has been eclipsed by an organizational model featuring an extensive array of functionally specific output channels, which can be simultaneously activated or inhibited in combinations that result in the patterns of autonomic activity that support behavior, mediate homeostatic reflexes, and cope with injury and disease. With this perspective, the defense response is but one of the many activational states of the central autonomic network. Considerable evidence supports the existence of tissue-specific sympathetic output pathways, which are likely to include distinct populations of premotor neurons whose target specificity could be assessed using the functional fingerprints developed from characterizations of postganglionic efferents to known targets. The organ-specific responses of sympathetic outflows to stimulation of a variety of reflex inputs suggests that the circuits regulating the activity of sympathetic premotor neurons must have parallel access to groups of premotor neurons controlling different functions but that these connections vary in the strength of their influence on different sympathetic outputs. Understanding the structural and physiological substrates antecedent to premotor neurons that mediate the differential control of sympathetic outflows, including those to noncardiovascular targets, represents a challenge to our current technical and analytical approaches.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

I. INTRODUCTION The autonomic nervous system (ANS) can respond to central stimuli and to activation of reflex sensory inputs with changes in sympathetic and parasympathetic outflows that occupy a continuum from those that appear globally directed to alter the level of activity in many target tissues, through those that represent defined patterns of ANS responses in a limited set of effectors, to changes that are discretely targeted to a single tissue. In the earliest concepts of the regulation of sympathetic outflow, the sympathetic nervous system was viewed as a monolithic effector that was activated to cope with stressful challenges to homeostasis by globally enhancing organ function and substrate availability. In contrast, the parasympathetic component of the ANS is engaged during recovery from such challenges to coordinate a reduction in energy utilization and a replenishment of energy stores. While these general views appropriately described the important role of the ANS in coping with life-threatening challenges, they ignored the contribution of basal levels of ANS activity to the maintenance of homeostasis and they failed to reflect the complex autonomic networks within the central nervous system that allow for the organ specificity of ANS responses in individual tissues and for the patterning of autonomic outflows necessary for the support of behavior. As experimental techniques have become more precise, the earlier view has been eclipsed by an organizational model that emphasizes the differential central control of the sympathetic outflows to functionally specific targets and the hierarchical interactions among populations of neurons that produce the patterns of autonomic efferent activity supporting a wide variety of behaviors and reflex responses. Investigation of the central neural mechanisms underlying differential autonomic responses is an ongoing area of autonomic neuroscience that has led to several hypotheses contributing to our understanding of the basis for organ specificity of autonomic responses. The foundation for these models comes from the results of both anatomical and physiological studies. Neuronal tracing techniques and immunocytochemical approaches have identified characteristics of the central and peripheral pathways to different tissues that provide a structural basis for differential regulation of these tissues. Comparisons of the centrally evoked and reflex responses of multiple autonomic effectors, in some cases recorded simultaneously, have demonstrated heterogeneity among the responses of functionally distinct outputs. Patterns emerge from these data that support a hierarchical organization, including neurons in the hypothalamus, midbrain, and medulla to the preganglionic neurons and ganglion cells, that is capable of either selective or more widespread activation of autonomic inputs to different tissues. This chapter summarizes the evidence for the foundation of this organizational structure, which is comprised of numerous populations of tissue-specific output pathways including sympathetic premotor neurons [and their associated oscillator circuits (1)], their preganglionic targets and the ganglion cells that innervate individual tissues.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The circuits underlying the characteristic autonomic components of behavior, emotion, and homeostasis must also include neurons whose axonal projections allow them to simultaneously influence multiple outputs as a mechanism for organizing patterns of organ-specific responses.

II. ANATOMICAL BASIS FOR TISSUE-SPECIFIC AUTONOMIC RESPONSES Langley divided the autonomic outflow from the central nervous system to the cardiovascular and visceral tissues into parasympathetic and sympathetic components based on their spinal origins as well as the differential effects on various tissues of nerve stimulation and application of adrenergic and cholinergic agents (2). The results of Cannon’s functional studies led him to propose that parasympathetic efferents mediate more precise, target-focused responses than did sympathetic efferents, which were considered to have more widespread effects (3). The tissue specificity of parasympathetic ganglion cells and thus the ability to provide selective regulation of various autonomic functions would appear manifest by the general anatomical arrangement in which parasympathetic ganglion cells are located within the innervated tissue and are excited by long preganglionic axons projecting from the central nervous system. Improved anatomical approaches have complemented physiological studies indicating the fine detail to which functionally specific responses can be evoked by parasympathetic efferents. In examining the vagal innervation of the heart, chronotropic and dromotropic effects were found to be mediated by distinct ganglia within the cardiac fat pads (4), and this arrangement has been extended to the inotropic effects as well (5). Retrograde tracing and immunohistochemistry have provided evidence that these ganglia are, in turn, innervated by populations of vagal cardiac preganglionic neurons that can be distinguished on the basis of location and neurochemical inputs (6–8). Target specificity has also been demonstrated for cranial parasympathetic ganglia innervating the lacrimal and parotid glands and the iris and ciliary body (9). A. Do Sympathetic Ganglion Cells Innervate Only One Target Tissue? Although the axons of sympathetic ganglion cells are, as a rule, markedly longer than those of their parasympathetic counterparts and thus would have more opportunity for branching, the results of many studies support a model in which sympathetic ganglion cells innervate a single tissue type and thus provide the minimal structural basis necessary for generation of organ-specific sympathetic responses. Most organs are made up of multiple tissues, and it seems most reasonable to ex-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

pect ganglion cells (and their antecedent preganglionic inputs) to be tissue-specific. In the pancreas, for example, although both -cells and -cells receive sympathetic and parasympathetic innervation to regulate their respective secretions of glucagon and insulin, the autonomic outflows to each target are activated under different circumstances (10) and must, therefore originate from separate populations of ganglion cells. Using retrograde transport from separate tissues, different distributions were found for ganglion cells innervating the kidney and the spleen (11) and for superior cervical ganglion neurons that project to the submandibular salivary glands, eyes, and pineal gland (12). Using immunohistochemical techniques to identify afferent terminals to sympathetic ganglion cells, superior cervical ganglion cells likely to innervate the secretory cells of the submandibular gland were uniquely surrounded by calretinin-positive terminals (13). Ganglion cells containing tyrosine hydroxylase and neuropeptide Y (NPY) are likely to innervate blood vessels, while stellate cells containing calbindin and NPY innervate cardiac tissue (14). B. Are Sympathetic Preganglionic Neurons Target Specific? Recent experiments in which the transynaptic, retrograde tracer, pseudorabies virus, has been injected into either the rat pinna or the eye resulted in specific segmental distributions of labeled preganglionic neurons (15). Similarly, sympathetic preganglionic neurons (SPNs) innervating stellate ganglion cells or the adrenal medulla were intermixed in the mid-thoracic spinal cord, but were never doublelabeled following injections of two retrograde tracers in the same animal (16). Neurochemical coding, describing the unique or selective associations of histochemically labeled terminal fields and transmitter or receptor-containing neurons, has provided evidence for functionally specific populations of SPNs as well as ganglion cells (see above). For instance, NK1 receptor is localized on a relatively higher percentage of adrenal SPNs than those projecting to either the superior cervical or the L5 chain ganglion (17,18). In contrast, calbindin-containing SPNs project to superior cervical and stellate ganglia, but not to the adrenal gland (19). Calretinin-positive SPNs send terminals to the vicinity of noradrenergic adrenal medullary chromaffin cells, but not those synthesizing epinephrine (20). Similar analysis has been used to indicate a unique identification of SPNs controlling cardiac function in the rat (14). C. Anatomical Differentiation of Inputs to Preganglionic Neurons Autonomic preganglionic neurons have both excitatory and inhibitory inputs, arising from spinal neurons or from brainstem, hypothalamic, and cortical sites. “Sympathetic premotor neuron” is the term that has generally been used to describe those neurons, particularly in the rostral ventrolateral medulla (RVLM), that synapse directly on SPNs to provide the excitatory input that maintains their basal discharge

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

and through which reflex and evoked responses in SPNs are effected. Regarding the central anatomical substrate for functionally specific autonomic responses, the question arises whether populations of tissue-selective premotor neurons provide unique driving inputs to their respective target groups of tissue-specific preganglionic neurons. Although the physiological data described below strongly suggest that this is the case, several complicating factors have prevented a direct anatomical answer to the question. Due to the intermixing of preganglionic neurons within the intermediolateral column of the spinal cord (16), it is not possible to apply classical retrograde tracers to populations of functionally homogeneous preganglionic neurons. The situation is improved by the recent advent of pseudorabies viral tracing approaches, which have be used, in separate experiments, to identify the premotor neurons regulating the autonomic efferents to peripheral tissues with different functions (21–23). While the target-specific differences that have been observed in the intensity and the temporal and spatial qualities of the labeling at different central sites (23) are suggestive of underlying distinctions in the relative importance of the inputs from these areas to SPNs controlling different targets, inherent limitations on the interpretation of the data obtained with this technique and the relatively few tissues that have been injected have thus far limited the identification of tissue-specific premotor neurons. Application of two histochemically distinct viruses at different sites in the same experiment has the potential to reveal individual neurons that regulate the sympathetic outflow to more than one target tissue. Although this approach has resulted in double-labeled neurons in hypothalamic and brainstem sites (24), it is not possible to determine from a single time point whether such labeling resulted from direct infection of the neuron by viruses from two populations of preganglionic neurons or whether one of the infections may have resulted, for instance, from a virus in the axonal branches of a different population of premotor neurons in the brainstem.

III. PHYSIOLOGICAL BASIS FOR DIFFERENTIAL AUTONOMIC REGULATION Characterization of the physiological and reflex response properties of individual pre- and postganglionic axons or nerve fascicles in human nerve recordings has been used to segregate sympathetic efferents into distinct categories, which have been related to the potential target tissues they regulate on the basis of unique endorgan behaviors. These are important data. The ability to establish a different characteristic pattern of reflex responses (see examples below) that define a functional fingerprint for the autonomic innervation of each target tissue is strong evidence that its autonomic regulation occurs via a unique, tissue-specific population of preganglionic and ganglion neurons (reviewed in Refs. 25, 26). In addition, with the exception of the preganglionic innervation of the adrenal medullary chromaffin

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

cells, the target tissue of a central neuron in a sympathetic pathway can only be inferred from the correspondence between its behavior and one of the functional fingerprints that has been established for the peripheral innervations of a variety of tissues. Finally, since the majority of reflex- and behaviorally evoked responses are mediated via supraspinal networks and sympathetic premotor pathways, these results are consistent with a model of central autonomic regulation, which includes tissue-specific populations of premotor neurons, and it is the detailed descriptions of the response characteristics of functionally defined sympathetic postganglionic or parasympathetic preganglionic axons that will provide the principal source of information for the physiological identification of such antecedent (sympathetic preganglionic, premotor, etc.) neurons in target-specific pathways. The meticulous studies carried out over many years in the laboratory of Wilfrid Janig have provided a significant portion of the information available on the behavior of a variety of peripheral sympathetic efferents in the cat and rat lumbar and cervical sympathetic outflows. They have characterized sympathetic efferent channels as 1) vasoconstrictors to muscle and viscera, 2) cutaneous vasoconstrictors, 3) sudomotor, 4) pilomotor, 5) “inspiratory” potentially innervating vessels of the nasal mucosa, 6) pupillomotor, and 7) motility regulating for the gastrointestinal system and for the reproductive system (25,27). The extensive studies of Gunnar Wallin and colleagues have provided considerable data indicating a strong correlation between the functional fingerprints of muscle and skin sympathetic outflow in experimental animals and those determined from recordings of human sympathetic nerve activity. While there are many tissues, such as the heart, adipose, pancreas, spleen, thymus, thyroid, pineal, gastrointestinal, reproductive, etc., whose autonomic functional fingerprints cannot be determined in humans and for which only limited data are available from experimental preparations, the growing appreciation of the role of alterations in autonomic regulation in a variety of disease states may stimulate research that will expand the characterization of these autonomic outflows as well. The sympathetic innervation of the adrenal medullary chromaffin cells, which secrete either epinephrine or norepinephrine (28,29), provides an example of target specificity at the level of the preganglionic neuron. Anatomically, terminals of calretinin-containing, cat adrenal SPNs terminate in the vicinity of noradrenergic chromaffin cells, while those adrenal SPNs without calretinin are located preferentially among adrenergic chromaffin cells (20). Physiologically, adrenal SPNs can be segregated into two populations. Those with long-latency responses to stimulation of the RVLM and little sensitivity to the baroreceptor reflex are strongly stimulated by the glucopenic agent 2-deoxyglucose (2-DG), indicating their role in regulation of adrenal epinephrine secretion, which is markedly increased by 2-DG (Fig. 1). Conversely, adrenal SPNs with short-latency responses to RVLM stimulation and which can be completely inhibited by baroreceptor reflex activation are unaffected by 2-DG and are presumed to con-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

trol adrenal norepinephrine release (30). These data provide direct evidence for the distinct functional organization of the central networks governing the release of the two catecholamines from the adrenal medulla that had been suggested by differences in the catecholamine secretion previously observed in response to physiological challenges and experimental stimuli (31–36).

IV. TISSUE-SPECIFIC AUTONOMIC RESPONSES TO REFLEX INPUTS A. Baroreceptor Reflex Baroreceptor reflex–mediated inhibition is a hallmark of sympathetic outflows regulating muscle and visceral vasoconstrictor targets, the kidney and the heart, as would be expected since the performance of these tissues contributes directly to determining the level of arterial pressure. Strong stimulation of vagal parasympathetic input to the heart accompanies the sympathoinhibitory responses to pressor stimuli (37,38). One criterion often used to assess the sensitivity of a particular sympathetic outflow to modulation by the baroreceptor reflex is the synchrony between the bursts in nerve activity and the arterial pressure wave (cardiac cycle). This analysis is based on the premise that the baroreceptor reflex pathway is briefly stimulated during the systolic pressure rise, which often exceeds 40 mmHg, resulting in a reduced probability of sympathetic discharge during a portion of the cardiac cycle and an entrainment of the sympathetic bursts to the frequency of the heart rate. Pulse-synchronous discharges have been observed in the muscle vasoconstrictor nerves of the cat and human (39–41), renal nerve of the cat and rat (42–46), cardiac and splenic nerves of the cat (44,45), and the lumbar and splanchnic nerves of the rat (47,48). Similarly, the discharge of adrenal SPNs controlling the chromaffin cells that secrete norepinephrine is strongly modulated by baroreceptor input (30). However, as illustrated in Figure 2, averages of sympathetic nerve activity triggered with the R-wave of the ECG indicate that there is relatively little pulse-synchronous modulation of the activity of most cutaneous vasoconstrictor nerves in cat, rat, or human (39,40,42,49,50) or of sympathetic sudomotor fibers in humans (51). This relative absence of a baroreceptor influence on sympathetic outflows related to thermoregulation is also supported by the lack of a cardiac frequency–related component in the autospectrum of the sympathetic outflow to rat brown adipose tissue (52). Similarly, the spontaneous discharge of epinephrine-regulating adrenal SPNs is relatively insensitive to arterial baroreceptor input (Fig. 1) (30). Testing the ability of increases (or decreases) in arterial or carotid sinus pressure to induce an inhibition (or excitation) of sympathetic discharge has yielded qualitatively similar results: skeletal muscle vasoconstrictor nerve activ-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Differential baroreceptor reflex influences on human muscle sympathetic activity (MSA) and skin sympathetic activity (SSA). Records of ECG and sympathetic nerve activity (SYMP) from human peroneal nerve indicate an entrainment of the bursts in MSA to the cardiac cycle, also shown by the large amplitude deflections on the ECG-triggered average of MSA (lower left trace). In contrast, there is no synchrony of the bursts in SSA to the heart frequency, and the ECG-triggered average of SSA is flat. (From Ref. 50.)

ity in cat and human (53–56), pelvic visceral vasoconstrictor nerve activity in cat (54), splenic, renal, and cardiac nerve discharge in the cat (42,57); lumbar, renal, and splanchnic nerve activity in the rat (48,58) and adrenal nerve activity and adrenal norepinephrine-controlling preganglionic neurons in the rat (30,58–61)

Figure 1 Differential responses of adrenal (ADR) sympathetic preganglionic neurons (SPNs) regulating epinephrine (EPI) and norepinephrine (NE) secretion. Left panel: Mean (SEM) peri-stimulus time histograms of the responses of EPI ADR SPNs (n  71, upper trace) and NE ADR SPNs (n  68, lower trace) to twin-pulse stimuli applied to the ipsilateral RVLM. Each bin represents the mean (SEM) of the values (normalized for number of RVLM stimuli delivered) for that bin from all of the individual peri-stimulus time histograms. Note the marked difference in peak latency. Bin width is 4 msec. Middle panel: Mean (SEM) discharge frequency (expressed as % control) histogram (bin width: 1 sec) obtained by averaging the responses of EPI ADR SPNs (n  20, upper trace) and NE ADR SPNs (n  17, lower trace) to glucopenia evoked by administration of 2-deoxyglucose (2DG, 250 mg/kg, time 0). Note that only those ADR SPNs with the long-latency response to RVLM stimulation are excited by 2-DG. Right panel: Discharges of an EPI ADR SPN (top trace) and a NE ADR SPN (lower mid-trace) during stimulation (bar, 3 pulses) of the baroreceptor afferents in the aortic depressor nerve (ADN) and the mean (SEM) peri-stimulus time interval histograms for EPI ADR SPN (n  37, upper mid-trace) and NE ADR SPN (n  32, bottom trace) responses to ADN stimulation. Bin width is 10 msec. Counts from stimulus artifacts were eliminated by zeroing the two corresponding bins. Note the relative absence of a baroreceptor-mediated inhibition in EPI ADR SPN. (From Ref. 30.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

are very sensitive to baroreceptor-mediated inhibition, while most of the sympathetic fibers innervating the skin exhibit a markedly weaker baroreceptor responsiveness (42,53,54), as do the sympathetic outflow to brown adipose tissue and the epinephrine-regulating adrenal SPNs (Fig. 1) (30,62). Tissue-selective responses are prominent when the hypotensive stimulus is extended into the decompensatory phase of hemorrhage. The latter elicits a dramatic inhibition in renal sympathetic outflow and a large increase in adrenal sympathetic nerve activity and epinephrine secretion (61,63–66). These responses are significantly dependent on cardiac vagal afferent input to the nucleus of the solitary tract, from which differential responses paralleling those in the decompensatory phase of hemorrhage can be elicited through activation of purinergic receptors (67,68). B. Chemoreceptor Reflex The immediate cardiovascular response to stimulation of arterial chemoreceptors with hypoxia or reduced blood flow through the carotid or aortic bodies is an increase in sympathetically mediated vasoconstriction to reduce tissue oxygen consumption and to increase arterial pressure and augment cerebral oxygen availability. Cardiac vagal activation is also present (69), possibly reducing coronary oxygen consumption through bradycardia. Differential responses to chemoreceptor reflex stimulation have been demonstrated in the sympathetic nerves controlling muscle and visceral vasoconstriction versus those to the skin. Stimulation of the arterial chemoreceptor reflex with systemic hypoxia, CO2-saturated saline, or sodium cyanide excited the preganglionic cervical sympathetic nerve and the postganglionic sympathetic innervation of skeletal muscle and pulmonary vasculature and to the kidney in the cat (53,69–72), the splanchnic nerve in the rabbit (Fig. 3) (73,74), and the renal and splanchnic sympathetic outflows in the rat (46,75). In contrast, cutaneous postganglionic neurons are inhibited by arterial chemoreceptor reflex stimulation in the cat, dog, and rabbit (Fig. 3) (53,73,74,76). The influence of central respiratory generating networks on those controlling sympathetic outflow is significantly stronger in muscle and visceral vasoconstrictor sympathetic discharge than in that of cutaneous sympathetic efferents, although this differential respiratory modulation is expressed more in the cat than in the rat or human. Using the phrenic nerve discharge to monitor the central respiratory cycle, a strong respiratory modulation has been observed in the cardiac vagal outflow (77) and in the amplitude of the bursts in the sympathetic nerves to the kidney in the cat and rat (42,44,78,79), to the skeletal muscle in the cat and human (40,77,80,81), to the cardiac nerve in the cat and rat (44,78,79), and to the lumbar, adrenal, cervical, and splanchnic nerves in the rat (79,82,83). Although such modulation is absent in the majority of cutaneous sympathetic fibers recorded in the cat (40,42), it is present in the rat (49,84) and human (39,85) cutaneous sympathetic outflow.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3. Arterial chemoreceptor reflex activation evokes differential responses in cutaneous sympathetic outflow and visceral vasoconstrictor efferents. Arterial hypoxia elicited a simultaneous fall in sympathetic nerve activity to vessels in the rabbit ear (Sympath. ear) and an increase in splanchnic sympathetic nerve activity (N. splanchn.) to mesenteric blood vessels. Cutaneous vasodilatation is reflected in the increase in ear temperature (Tleft ear). The reduction in heart rate (HF) suggests a reduction in cardiac sympathetic nerve activity. Arterial pressure (Pmar) also fell slightly. (From Ref. 74.)

C. Thermal Stimuli Autonomic thermoregulatory responses are directed towards changes in the level of 1) heat conservation through circulatory adjustments focused on modifying cutaneous blood flow and through changes in sudomotor and pilomotor activity and 2) heat production through influences on thermogenic mechanisms. Secondary effects, from arterial and cardiopulmonary baroreceptor reflexes, for instance, would be expected during some thermoregulatory responses, as in those requiring a large increase in cutaneous blood flow. Thus, direct thermoregulatory responses are mediated by differential changes in the sympathetic outflows to the skin and tissues involved in thermogenesis, relative to muscle and visceral vasoconstrictor nerve activity.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Exposure to a warm (43°C) environment activated sudomotor sympathetic activity in humans, while suppressing cutaneous vasoconstrictor fiber discharge (86). Similarly, heating the spinal cord in anesthetized rabbits and dogs produced an inhibition of cutaneous sympathetic nerve activity, accompanied by an excitation of the cardiac and splanchnic nerve activities (87,88). Peripheral thermal receptor stimulation (heat) elicits an increase in rat renal sympathetic nerve activity (46), resulting in a fall in renal blood flow. Conversely, a cold (15°C) environment stimulates activity in human cutaneous vasoconstrictor efferents, while inhibiting sudomotor sympathetic outflow (86). Exposure to cold stimulates sympathetic nerve activity to rat brown adipose tissue, but has only a minor effect on splanchnic sympathetic discharge, at least some of which may arise from sympathetic axons regulating thermogenesis in mesenteric brown adipose tissue depots (52). Spinal cooling activated cutaneous, as well as splanchnic sympathetic discharge in rabbits and dogs, but evoked little change in cardiac sympathetic activity or heart rate (87,88). Although cold-stimulated increases in plasma norepinephrine could arise from both sympathetic terminals and adrenal chromaffin cells, the absence of a change in plasma epinephrine during cold exposure (89,90) and a fall in adrenal norepinephrine content (89) are consistent with the differential stimulation of adrenal norepinephrine release in response to cold exposure (36). D. Nociceptor Stimulation Peripheral nociceptor stimulation, such as that produced by pinching the skin, evokes a strong excitation of muscle and visceral vasoconstrictor sympathetic outflow, but a prompt decrease in cutaneous vasoconstrictor efferent discharge in cat (39,91). An equally strong differential response, but in the opposite direction, is evoked by arousal stimuli and brief superficial painful stimuli in man (Fig. 4) (39), and cutaneous vasoconstriction has also been demonstrated in anesthetized human during surgical incision (92,93). The “trigeminal depressor response” evoked by stimulation of the trigeminal nerve or pinching the facial skin produces a reduction in splanchnic, renal, and skeletal muscle sympathetic outflow and a simultaneous increase in that to the cutaneous vessels in anesthetized rabbit (94,95). Recent experiments have suggested an involvement of sympathetic premotor neurons in the rostral medullary raphe in the control of cutaneous blood flow as a potential neural substrate contributing to the differential responses in cutaneous vs. muscle and visceral vasoconstrictor sympathetic outflow. Activation of raphe neurons, independent of those in the RVLM, produced a large increase in the sympathetic outflow to the cutaneous vascular bed in the rat tail and the rabbit ear, with little effect on the visceral vasomotor outflow to the kidney or mesentery (96,97). Inhibition of neurons in the raphe selectively prevented the cutaneous constriction in the rabbit ear during trigeminal stimulation, while inhibition of RVLM neurons was necessary to block the mesenteric vasoconstriction from stimulation of abdominal vagal afferents (98). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 4. Nociceptive and alerting stimuli elicit cutaneous vasoconstriction, but vasodilatation in skeletal muscle vasculature of humans. Simultaneous recording from human muscle (MVC) and cutaneous (CVC) vasoconstrictor efferents in peroneal nerve during an alerting stimulus (loud sound, arrow) and during a brief, painful electric shock to the skin (electr. stim., arrow). Note that both stimuli evoked a dramatic increase in skin vasoconstrictor discharge, but either no response or a decrease in MVC. (From Ref. 39.)

E. Hypoglycemia A fall in blood glucose, sensed by central and hepatic glucoreceptors, stimulates an autonomic response directed primarily at restoring levels of circulating glucose through stimulation of hepatic glycogenolysis and inhibition of insulin-sensitive glucose uptake. Secondary effects include a reduction in body temperature and a restriction of muscle blood flow, both of which would reduce glucose utilization. Experimentally, hypoglycemia is achieved with injections of insulin or 2-deoxyglucose (2-DG), which blocks cellular glucose metabolism. Since insulin can have direct effects on sympathetic nerve activity, euglycemic control studies are required to interpret the results of insulin-induced hypoglycemia. In humans, muscle sympathetic nerve activity and the sudomotor component of skin sympathetic activity are stimulated by acute hypoglycemia, whereas the vasoconstrictor component of skin sympathetic activity is reduced (99–101). The increase in sweating and cutaneous vasodilation would contribute to a fall in body temperature, as would the inhibition of sympathetic outflow and thermogenesis in rat brown adipose tissue evoked by administration of 2-DG (102,103). Indirect evidence has also been presented in humans and dogs for an increased sympathetic outflow to the liver during hypoglycemia (104,105), which would contribute to glycogenolysis and an elevation in blood glucose. Hypoglycemia stimulates a large increase in adrenal sympathetic nerve activity and epinephrine Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

secretion in rats and humans, but decreases or has little effect on renal and cardiac sympathetic outflows (35,100,101,104,106–112). The release of adrenal catecholamines is differentially affected by hypoglycemia, which is a much stronger stimulus for the secretion of epinephrine than of norepinephrine (Fig. 1) (30,89).

V. TISSUE-SPECIFIC SYMPATHETIC PREMOTOR NEURONS The prominent role of supraspinal circuits, in particular the premotor neurons, in the functional organization of the autonomic reflexes described above gives strong support to the view that the differential reflex responses observed in sympathetic nerves controlling different target tissues is a reflection of the existence of tissue-specific populations of premotor neurons. As alluded to above, the characterization of these neuronal populations will be dependent on the precision with which functional fingerprints of the autonomic outflow to specific tissues can be defined. The requirement for antidromic activation from the spinal intermediolateral nucleus and the likely necessity to determine the neuronal responses to a series of reflex or centrally evoked stimuli represent significant impediments to obtaining these data. Current evidence, albeit indirect, for functionally specific populations of sympathetic premotor neurons has been derived from two approaches: correlation of the activity of individual neurons with that in multiple, simultaneously recorded nerve bundles and monitoring the differential responses in multiple nerves during activation of subsets of premotor neurons. These studies have been performed in the RVLM and the raphe, which are among the brain regions identified anatomically as the principal sources of direct premotor input to SPNs (21). A. Correlation of Brainstem Unit Activity to Sympathetic Nerve Discharge If the discharge of a sympathetic premotor neuron contributes to the synchronous activation (i.e., burst behavior) of a single, homogeneous population of tissue-specific sympathetic ganglion cells, then the expectation would be for the discharge of that neuron to be more strongly correlated to the bursts of activity on the nerve that it influences than to those on nerves regulating other target tissues. Using this paradigm, Barman and colleagues (113) identified RVLM and raphe neurons with discharges that were more tightly coupled to either the cardiac, renal, or external carotid nerves. This analysis is inherently complicated by the attempt to correlate unit activity with a group of sympathetic efferents that each regulate a target with a cardiovascular function: the potential for a variable degree of coupling among the supraspinal circuits that generate the activity in each sympathetic channel to cardiovascular target tissues (1) would make it more difficult to observe differ-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ences among the unit to nerve activity correlations. Additionally, sympathetic nerve bundles, such as the renal and cardiac, are likely to contain multiple populations of functionally specific axons (114). The use of partial coherence analysis (115) has been an improvement on the original approach, but the conclusions are still limited by the array of efferent nerves one has available in any particular experiment. B. Differential Responses from Altering the Activity of RVLM Neurons With the discovery of cardiovascular sympathetic premotor neurons in the RVLM, several labs began to address the question of whether there was a viscerotopic organization of premotor neuron populations within the RVLM. Inherent in finding a distinct tissue topography in the sympathetic responses elicited by microstimulation within the RVLM would also be evidence for functionally specific populations of premotor neurons, thereby establishing that the “private communication lines” through which the ANS influences the myriad of tissue functions could be traced centrally from the sympathetic ganglion cells and preganglionic neurons to their antecedent premotor neurons. The focus of microstimulation experiments within RVLM has been on differentiating subregions that regulate different cardiovascular tissues. Activation of neurons in restricted regions of the RVLM in the cat with microinjections of an excitatory amino acid have produced the following general results: neurons in pathways controlling the vasoconstrictor sympathetic outflow to muscle are located more laterally and caudally in the RVLM, those capable of increasing cutaneous sympathetic nerve activity were located more medially in the RVLM, and those activating renal, cardiac, and lumbar splanchnic nerves are found rostromedially (116–120). Although there were relatively large areas from which responses on more than one nerve were evoked, the finding of restricted regions of the RVLM from which responses in only one tissue could be recorded (Fig. 5) is consistent with the existence of sympathetic premotor neurons directed solely to the regulation of the SPNs for that tissue. In this scenario, responses evoked in multiple nerves would arise from the anatomical intermixing of populations of functionally specific premotor neurons in the RVLM. This and the relatively smaller anatomical size of the RVLM are likely to be the explanations for the failure to find similar results in the rat (121,122). Importantly, simultaneous changes in blood flow to hindlimb and forelimb could be produced independently of those to the kidney, suggesting that the variable being regulated at the level of the premotor neuron is related to the target tissue (skeletal muscle blood flow vs renal resistance, in this example) rather than the anatomical location within the body (123). Additionally, as with cardiac vagal preganglionic neurons in the nucleus ambiguous (6), the data indicating selective regulation by different popula-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 5. Stimulation in different regions within the RVLM activates organ-specific sympathetic outflows. Traces in each panel are the arterial blood pressure (BP); paired records of the discharges of a single axon and the ratemeter count of its action potential frequency recorded simultaneously from a muscle vasoconstrictor nerve (MVC), a visceral vasoconstrictor nerve (lumbar splanchnic, VVC), and the renal nerve (RSN); and CUSUM traces showing cumulative excess spikes above those expected from control firing rate (stars indicate significant increases). Arrows indicate microinjections of glutamate at three sites (panels A, B, C) in the RVLM. Note that activation of local neurons at three different sites produces selective increases in MVC, VVC, and RSN discharge, suggesting a degree of spatial separation of populations of cardiovascular sympathetic premotor neurons in the RVLM. (From Ref. 120.)

tions of RVLM neurons of cardiac contractility, rate, and conduction (124) emphasize the degree of functional specificity residing within the autonomic premotor and preganglionic efferents. Although relatively few noncardiovascular efferent pathways have been examined, the failure to demonstrate an excitatory effect of RVLM neurons on sympathetic regulation of pupillary dilation, retraction of the nictitating membrane, piloerection, sweat glands, rat tail blood flow, and rat brown adipose tissue sympathetic activity (52,96,125–127) is consistent with a selective role for premotor neurons in the RVLM in regulating sympathetic functions related to the maintenance of tissue perfusion pressure. Experimental designs involving the stimulation or inactivation of large numbers of neurons cannot, however, rule out the possibility that individual RVLM neurons branch to innervate multiple populations of SPNs controlling different tissues. Thus, while the available evidence favors tissue-specific populations of sympathetic premotor Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

neurons within RVLM, we do not yet have direct evidence for their existence. Indeed, although sympathetic premotor neurons displaying the functional fingerprint indicating a role in the regulation of vasoconstriction have been extensively studied (48,128–130), the same rudimentary characteristics would be expected of those controlling venous compliance, cardiac performance, and renal glomerular filtration. C. Effects of Altering the Activity of Neurons in Rostral Medullary Raphe The responses evoked from activation of the rostral medullary raphe, another locus of sympathetic premotor neurons (131,132), provide evidence of a broader functional specificity: the populations of SPNs driven by raphe-spinal neurons are involved in thermoregulation, and possibly energy balance, rather than the control of organ perfusion pressure that is the purview of RVLM premotor neurons. In studies on the central regulation of the sympathetic outflow to brown adipose tissue in the rat, disinhibition of neurons in the rostral raphe pallidus region produced large increases in brown adipose tissue sympathetic nerve activity, with only a small increment in splanchnic sympathetic nerve activity (52). The activation of brown adipose sympathetic outflow from raphe was independent of the activity of neurons in the RVLM (Fig. 6). Considering the significance of brown adipose tis-

Figure 6 Activation of brown adipose sympathetic nerve activity (BAT SNA) from raphe pallidus (RPa) after inhibition of neurons in rostral ventrolateral medulla (RVLM). Left panel: Integrated and raw phrenic nerve activity (PHR), arterial pressure (AP), splanchnic nerve activity (SPL SNA), and BAT SNA under normothermic, control conditions. Middle panel: Same variables following bilateral microinjections of muscimol into the RVLM to inhibit local neuronal discharge. Note the marked reduction in SPL SNA and AP. Right panel: Same variables following disinhibition of RPa neurons. The large increase in BAT SNA suggests that RPa, rather than RVLM, contains sympathetic premotor neurons regulating BAT SNA. (From Ref. 52.) Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

sue thermogenesis in the overall energy expenditure in small mammals, it seems likely that raphe neurons also play a role in regulating sympathetic outflows involved in metabolism and energy balance. Neurons in the rostral raphe were also found to be a source of sympathoexcitatory drive to the cutaneous circulation in the rabbit ear (97) and in the rat tail (96), both of which contribute significantly to thermoregulation through their role in heat dissipation. In these cases as well, RVLM neurons were found to have little influence on the sympathetic control of these thermoregulatory circulations (96,98). While it seems likely that target-specific populations of sympathetic premotor neurons exist within the raphe-spinal projection, the identification of sympathetic premotor neurons controlling different thermoregulatory and metabolic tissues remains to be accomplished. D. Summary Current physiological data provide strong support for the existence of tissue-specific populations of sympathetic premotor neurons as an important structural and functional basis for the ability of the central nervous system to generate autonomic responses at selective sites in the body and to orchestrate complex autonomic patterns involving differential changes in the amplitudes of the sympathetic outflows to relevant targets. Although only two of the several anatomically segregated groups of sympathetic premotor neurons have been studied from a functional perspective, the results suggest an organizational model in which the basis for the anatomical clustering of different subpopulations of premotor neurons resides in the larger homeostatic function to which the regulation of their target tissue contributes. Thus, RVLM premotor neurons regulate cardiovascular target tissues to control arterial pressure at an appropriate level to maintain nutritive tissue blood flow, while raphe-spinal neurons control sympathetic outputs selectively related to thermoregulation and metabolism. Within this model, it is expected not only that certain supraspinal loci will comprise unique sites for premotor neurons regulating particular tissues, but also that some populations of SPNs will receive premotor inputs from more than one source.

VI. HIERARCHICAL ORGANIZATION OF DIFFERENTIAL AUTONOMIC RESPONSES Given the evidence described above that sympathetic premotor neurons are functionally “dedicated” by virtue of their axonal projection pattern, one might ask whether the selective control of tissue-specific autonomic outflow is maintained at any organizational level that is central to the premotor neurons? Specifically, are neurons antecedent to premotor neurons functionally specific or are their terminals distributed to provide mechanisms for linking sympathetic premotor out-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

put channels to effect simultaneous changes in different target tissues? While these questions do pose a potential, albeit simplistic, framework for the neural substrates underlying patterned autonomic responses, a number of factors combine to complicate the experimental testing of such hypotheses. As with all such studies, there are significant limitations set by the number of outputs that can be simultaneously monitored and by the difficulty in distinguishing whether simultaneous activation of multiple outputs arises from stimulating 1) separate populations of neurons controlling each output or 2) a single group of neurons with axons that branch to innervate multiple, functionally specific efferent pathways. Regions containing sympathetic premotor neurons receive inputs from multiple sources, which may, themselves, be interconnected. Additionally, considerable evidence supports the existence of individual “oscillator” circuits that drive targetspecific populations of premotor neurons (1) to generate the bursting activity characteristic of each sympathetic outflow. The interaction of inputs from more rostral sites with such brainstem oscillator circuits, the potential for coupling among oscillators for different outputs, and the presence of a significant inhibitory input to premotor neurons (62,133) represent further complexities of the central autonomic networks. A. Stimulation of the Hypothalamus A variety of differential autonomic responses have been documented from activation of various regions of the hypothalamus. The varied use of electrical vs. chemical stimuli, the activation of relatively large populations of neurons, and the existence of intrahypothalamic connections as well as descending pathways to the periaqueductal gray, the RVLM, and the spinal cord allow few conclusions on the pathways or, in some cases, the functional significance for these responses. Nonetheless, in the best light, their differential nature does indicate a degree of separate regulation that may be reflecting only a portion of an organized homeostatic response involving a constellation of outputs beyond those being monitored. Along these lines, stimulation at various sites in the cat hypothalamus elicited markedly different ratios of adrenal epinephrine and norepinephrine secretion (34,134), consistent with the activation of pathways with differential access to adrenal sympathetic premotor neurons controlling epinephrine- vs. norepinephrine-secreting chromaffin cells (30). In this regard, different hypothalamic regions contribute to the autonomic response to hypoglycemia and to hypothermia, which elicit differential release of adrenal catecholamines (89). Opposite responses can also be evoked in cardiac sympathetic nerve activity and muscle vasoconstrictor outflow in cat and in cardiac and renal sympathetic nerve activities in the rabbit by stimulation at certain hypothalamic sites (135,136). Stimulation within neighboring sites in the lateral hypothalamus evoked differential responses in cardiac and in vasoconstrictor efferents to neck or to pharyngeal muscles (137).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Although these responses resemble aspects of the sympathetic response to arterial chemoreceptor activation, the behavioral or reflex context for these data have not been determined. Historically, the hypothalamically evoked defense response, also referred to as the fight-or-flight or visceral alerting response, has often been described as a behavior involving differential control of sympathetic outflow (138–140). This view seems to have arisen from the finding that the cardiac stimulation, widespread visceral vasoconstriction, piloerection, and pupillary dilatation observed during the defense response is accompanied by a characteristic increase in skeletal muscle blood flow (141). However, the evoked increase in muscle blood flow is mediated by activation of a cholinergic (atropine-sensitive) vasodilator pathway, with little evidence for a marked inhibition of adrenergic muscle vasoconstrictors (141,142). While the utility of studies examining the defense response in anesthetized animals has been questioned (143) and the prominence of a cholinergic vasodilator pathway is unclear in species other than the cat, experiments to identify the descending pathways for the hypothalamic defense response were a significant motivation for examining the role of the periaqueductal gray (PAG) in the organization of patterned autonomic responses (144). B. Stimulation of the Periaqueductal Gray Detailed stimulation studies have delineated an organizational structure based on longitudinal columns within the PAG for circuits that are capable of coordinating a variety of complex autonomic, motor, and sensory-modulating commands mimicking the fight-or-flight response (dorsal and lateral columns; Fig. 7A) as well as those seen during recovery from stress, activation of antinociception, or the vasovagal syncope that can accompany deep pain (ventrolateral columns) (145–147). The portions of the PAG columns from which autonomic responses are evoked have extensive, viscerotopically organized, descending projections to sympathetic premotor neurons in the RVLM (148–150). Differential activation of these pathways is likely to mediate the vasoconstrictor and tachycardic components of “active emotional coping” evoked from the dorsal and lateral columns of PAG and the vasodepressor and bradycardic responses of “passive coping” strategies mimicked by stimulation of ventrolateral PAG (151,152). These response patterns suggest that PAG neurons, by virtue of the topography of their inputs to the RVLM, provide a substrate for the parallel activation or inhibition of premotor neurons controlling an extensive array of cardiovascular targets. Recent studies have also provided evidence that activation of certain PAG sites can evoke differential changes in visceral vs. skeletal muscle sympathetic outflow (153). Under conditions allowing 10 Hz rhythms in these nerves, PAG stimuli that elicited an increase in cardiac sympathetic nerve activity produced a prompt inhibition of vertebral nerve activity (Fig. 7B) and a prolongation of the

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 7 Differential blood flow and sympathetic nerve responses elicited from activation of the defense region in lateral periaqueductal gray (PAG). (A) Activation of neurons in PAG with microinjection of D,L-homocysteic acid (DLH, arrow) produces a simultaneous increase in iliac flow and a reduction in renal blood flow, as well as an increase in arterial pressure and defensive behaviors. (From Ref. 155.) (B) Electrical stimulation in PAG evokes a simultaneous decrease in vertebral nerve (VN) activity to muscle and increases in renal (RN) and cardiac (CN) nerve activities. (From Ref. 156.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

phase angle between them. These finding have led to the proposal of a novel mechanism by which differential responses may be produced in functionally specific sympathetic outflows: if an input (such as that from PAG) to a network of coupled oscillators (in this case involving populations of premotor neurons in RVLM) shifts the phase relationship between the oscillators generating the bursts in different sympathetic nerves, then, by virtue of their coupling, the reduced synchrony could result in a decrease in the burst amplitude of one output relative to the other, including a complete inhibition if the shift in phase is of sufficient magnitude (154).

VII.

CONCLUSIONS

The demonstration that sympathetic outflows to different targets can be differentially affected by central stimulation and during reflex and behavioral responses reflects a much more complex structure to the central autonomic network than that necessary to effect the relatively simple global changes in sympathetic discharge that were the focus of early investigators. Anatomical and functional studies have indicated a structural foundation for organ-specific autonomic responses in the projection patterns and response characteristics of ganglion cell axons and those of their preganglionic inputs. Although it has been more difficult to obtain direct evidence for dedicated, tissue-specific populations of sympathetic premotor neurons, the ability to elicit differential, organ-specific responses in different autonomic outflows from stimulations at stereotyped sites within a single locus of premotor neurons (such as RVLM) or at sites within anatomically separate loci of premotor neurons (such as RVLM and raphe) is strongly supportive of such a model. In addition, differential autonomic responses to activation of a number of reflexes that are mediated through changes in premotor neuronal discharge are most easily accounted for through differential inputs to populations of functionally dedicated premotor neurons. The functional fingerprints of organ-specific autonomic outflows could provide a basis for distinguishing among premotor neurons controlling different tissues. Although current evidence is still sparse, the clustering of different populations of tissue-specific premotor neurons at a particular anatomical site may relate to a similarity in the overall function of the outputs being regulated by those groups of premotor neurons. Within the framework of this hypothesis, the premotor neurons located in the RVLM appear to be primarily involved in regulating cardiovascular target tissues involved in maintaining organ perfusion pressure and nutritive blood flow, in contrast to those in the rostral raphe nuclei, which influence targets with thermoregulatory and metabolic functions. Determining the substrate for the organ specificity of ANS responses represents not only a significant advance in our understanding of the functional orga-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

nization of autonomic control networks, but also a stepping stone to identifying the mechanisms through which multiple, tissue-dedicated output systems are engaged to produce the constellation of changes in individual effector outflows that comprises a complete autonomic response or behavior component. Insights into the underlying organizational structure and operating principles of the central autonomic network will be gained as the functional contexts are identified for patterns of differential, stimulation-evoked responses.

ACKNOWLEDGMENTS This work was supported by grants from the National Heart, Lung and Blood Institute (HL-56365) and the National Institute of Diabetes and Digestive and Kidney Diseases (DK20378).

REFERENCES 1.

2. 3. 4.

5.

6.

7.

8.

9. 10.

Gebber GL, Zhong S, Barman SM. Synchronization of cardiac-related discharges of sympathetic nerves with inputs from widely separated spinal segments. Am J Physiol 1995; 268:R1472–1483. Langley JN. The Autonomic Nervous System, Part I. Cambridge: Heffer and Sons, 1921. Cannon WB. Bodily Changes in Pain, Hunger, Fear, and Rage. New York: Appleton and Company, 1915:22–39. Gatti PJ, Johnson TA, Phan P, Jordan IK, 3rd, Coleman W, Massari VJ. The physiological and anatomical demonstration of functionally selective parasympathetic ganglia located in discrete fat pads on the feline myocardium. J Auton Nerv Syst 1995; 51:255–259. Gatti PJ, Johnson TA, McKenzie J, Lauenstein JM, Gray A, Massari VJ. Vagal control of left ventricular contractility is selectively mediated by a cranioventricular intracardiac ganglion in the cat. J Auton Nerv Syst 1997; 66:138–44. Gatti PJ, Johnson TA, Massari VJ. Can neurons in the nucleus ambiguus selectively regulate cardiac rate and atrio-ventricular conduction? J Auton Nerv Syst 1996; 57:123–127. Massari VJ, Johnson TA, Llewellyn-Smith IJ, Gatti PJ. Substance P nerve terminals synapse upon negative chronotropic vagal motoneurons. Brain Res 1994; 660:275–287. Massari VJ, Johnson TA, Gatti PJ. Cardiotopic organization of the nucleus ambiguus? An anatomical and physiological analysis of neurons regulating atrioventricular conduction. Brain Res 1995; 679:227–240. Leblanc GG, Landis SC. Target specificity of neuropeptide Y-immunoreactive cranial parasympathetic neurons. J Neurosci 1988; 8:146–155. Edwards AV. Autonomic conrol of endocrine pancreatic and adrenal function. In:

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

11. 12.

13.

14. 15. 16.

17.

18.

19.

20.

21.

22.

23.

24.

25. 26. 27.

Loewy AD, Spyer KM, eds. Central Regulation of Autonomic Functions. New York: Oxford University Press, 1990:286–309. Meckler RL, Weaver LC. Comparison of the distributions of renal and splenic neurons in sympathetic ganglia. J Auton Nerv Syst 1984; 11:189–200. Luebke JI, Wright LL. Characterization of superior cervical ganglion neurons that project to the submandibular glands, the eyes, and the pineal gland in rats. Brain Res 1992; 589:1–14. Grkovic I, Anderson CR. Calretinin-containing preganglionic nerve terminals in the rat superior cervical ganglion surround neurons projecting to the submandibular salivary gland. Brain Res 1995; 684:127–135. Anderson CR. Identification of cardiovascular pathways in the sympathetic nervous system. Clin Exp Pharmacol Physiol 1998; 25:449–452. Strack AM, Loewy AD. Pseudorabies virus: a highly specific transneuronal cell body marker in the sympathetic nervous system. J Neurosci 1990; 10:2139–2147. Jansen AS, Farwell DG, Loewy AD. Specificity of pseudorabies virus as a retrograde marker of sympathetic preganglionic neurons: implications for transneuronal labeling studies. Brain Res 1993; 617:103–112. Grkovic I, Anderson CR. Distribution of immunoreactivity for the NK1 receptor on different subpopulations of sympathetic preganglionic neurons in the rat. J Comp Neurol 1996; 374:376–386. Llewellyn-Smith IJ, Martin CL, Minson JB, et al. Neurokinin-1 receptor-immunoreactive sympathetic preganglionic neurons: target specificity and ultrastructure. Neuroscience 1997; 77:1137–1149. Grkovic I, Anderson CR. Calbindin D28K-immunoreactivity identifies distinct subpopulations of sympathetic pre- and postganglionic neurons in the rat. J Comp Neurol 1997; 386:245–259. Edwards SL, Anderson CR, Southwell BR, McAllen RM. Distinct preganglionic neurons innervate noradrenaline and adrenaline cells in the cat adrenal medulla. Neuroscience 1996; 70:825–832. Strack AM, Sawyer WB, Hughes JH, Platt KB, Loewy AD. A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections. Brain Res 1989; 491:156–162. Schramm LP, Strack AM, Platt KB, Loewy AD. Peripheral and central pathways regulating the kidney: a study using pseudorabies virus. Brain Res 1993; 616:251–262. Sved AF, Cano G, Card JP. Neuroanatomical specificity of the circuits controlling sympathetic outflow to different targets. Clin Exp Pharmacol Physiol 2001; 28:115–119. Jansen AS, Nguyen XV, Karpitskiy V, Mettenleiter TC, Loewy AD. Central command neurons of the sympathetic nervous system: basis of the fight-or-flight response. Science 1995; 270:644–664. Janig W. Pre- and postganglionic vasoconstrictor neurons: differentiation, types, and discharge properties. Annu Rev Physiol 1988; 50:525–539. Janig W, McLachlan EM. Specialized functional pathways are the building blocks of the autonomic nervous system. J Auton Nerv Syst 1992; 41:3–13. Janig W, McLachlan EM. Characteristics of function-specific pathways in the sympathetic nervous system. Trends Neurosci 1992; 15:475–481.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

28.

29. 30.

31.

32. 33.

34. 35.

36. 37. 38. 39. 40.

41. 42. 43.

44.

45.

46.

Coupland RE, Pyper AS, Hopwood D. A method for differentiating between noradrenaline- and adrenaline-storing cells in the light and electron microscope. Nature 1964; 201:1240–1242. Hillarp NA, Hokfelt B. Evidence of adrenaline and noradrenaline in separate adrenal medullary cells. Acta Physiol Scand 1953; 25:1–134. Morrison SF, Cao WH. Different adrenal sympathetic preganglionic neurons regulate epinephrine and norepinephrine secretion. Am J Physiol 2000; 279:R1763–1775. Vollmer RR. Selective neural regulation of epinephrine and norepinephrine cells in the adrenal medulla—cardiovascular implications. Clin Exp Hypertens 1996; 18:731–751. Scheurink A, Ritter S. Sympathoadrenal responses to glucoprivation and lipoprivation in rats. J Neurochem 1993; 50:1302–1308. Sun CL, Thoa NB, Kopin IJ. Comparison of the effects of 2-deoxyglucose and immobilization on plasma levels of catecholamines and corticosterone in awake rats. J Endocrinol 1979; 105:305–311. Folkow B, von Euler US. Selective activation of noradrenaline and adrenaline producing cells in the cat’s adrenal gland by hypothalamic stimulation. Circ Res 1954; 2:191–195. Young JB, Landsberg L. Sympathoadrenal activity in fasting pregnant rats. Dissociation of adrenal medullary and sympathetic nervous system responses. J Clin Invest 1979; 64:109–116. Young JB, Landsberg L. Effect of concomitant fasting and cold exposure on sympathoadrenal activity in rats. Am J Physiol 1981; 240:E314–319. Fritsch JM, Smith ML, Simmons DT, Eckberg DL. Differential baroreflex modulation of human vagal and sympathetic activity. Am J Physiol 1991; 260:R635–641. Kollai M, Koizumi K. Cardiovascular reflexes and interrelationships between sympathetic and parasympathetic activity. J Auton Nerv Syst 1981; 4:135–148. Janig W, Sundlof G, Wallin BG. Discharge patterns of sympathetic neurons supplying skeletal muscle and skin in man and cat. J Auton Nerv Syst 1983; 7:239–256. Gregor M, Janig W, Wiprich L. Cardiac and respiratory rhythmicities in cutaneous and muscle vasoconstrictor neurones to the cat’s hindlimb. Pflugers Arch 1977; 370:299–302. Delius W, Hagbarth KE, Hongell A, Wallin BG. General characteristics of sympathetic activity in human muscle nerves. Acta Physiol Scand 1972; 84:65–81. Ninomiya I, Irisawa A, Nisimaru N. Nonuniformity of sympathetic nerve activity to the skin and kidney. Am J Physiol 1973; 224:256–264. Kunitake T, Kannan H. Discharge pattern of renal sympathetic nerve activity in the conscious rat: spectral analysis of integrated activity. J Neurophysiol 2000; 84:2859–2867. Koizumi K, Seller H, Kaufman A, Brooks CM. Pattern of sympathetic discharges and their relation to baroreceptor and respiratory activities. Brain Res 1971; 27:281–294. Meckler RL, Weaver LC. Characteristics of ongoing and reflex discharge of single splenic and renal sympathetic postganglionic fibres in cats. J Physiol 1988; 396:139–153. DiBona GF, Sawin LL, Jones SY. Differentiated sympathetic neural control of the kidney. Am J Physiol 1996; 271:R84–90.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

47. 48.

49. 50. 51. 52. 53.

54.

55.

56. 57. 58.

59.

60. 61.

62.

63. 64.

Guyenet PG, Brown DL. Nucleus paragigantocellularis lateralis and lumbar sympathetic discharge in the rat. Am J Physiol 1986; 250:R1081–1094. Morrison SF, Milner TA, Reis DJ. Reticulospinal vasomotor neurons of the rat rostral ventrolateral medulla: relationship to sympathetic nerve activity and the C1 adrenergic cell group. J Neurosci 1988; 8:1286–1301. Johnson CD, Gilbey MP. Sympathetic activity recorded from the rat caudal ventral artery in vivo. J Physiol (Lond) 1994; 476:437–442. Bini G, Hagbarth KE, Wallin BG. Cardiac rhythmicity of skin sympathetic activity recorded from peripheral nerves in man. J Auton Nerv Syst 1981; 4:17–24. Macefield VG, Wallin BG. The discharge behaviour of single sympathetic neurones supplying human sweat glands. J Auton Nerv Syst 1996; 61:277–286. Morrison SF. RVLM and raphe differentially regulate sympathetic outflows to splanchnic and brown adipose tissue. Am J Physiol 1999; 276:R962–973. Blumberg H, Janig W, Rieckmann C, Szulczyk P. Baroreceptor and chemoreceptor reflexes in postganglionic neurones supplying skeletal muscle and hairy skin. J Auton Nerv Syst 1980; 2:223–240. Michaelis M, Boczek-Funcke A, Habler HJ, Janig W. Responses of lumbar vasoconstrictor neurons supplying different vascular beds to graded baroreceptor stimuli in the cat. J Auton Nerv Syst 1993; 42:241–249. Eckberg DL, Rea RF, Andersson OK, et al. Baroreflex modulation of sympathetic activity and sympathetic neurotransmitters in humans. Acta Physiol Scand 1988; 133:221–231. Rea RF, Eckberg DL. Carotid baroreceptor-muscle sympathetic relation in humans. Am J Physiol 1987; 253:R929–934. Ninomiya I, Nisimaru N, Irisawa H. Sympathetic nerve activity to the spleen, kidney, and heart in response to baroceptor input. Am J Physiol 1971; 221:1346–1351. Scislo TJ, Augustyniak RA, O’Leary DS. Differential arterial baroreflex regulation of renal, lumbar, and adrenal sympathetic nerve activity in the rat. Am J Physiol 1998; 275:R995–R1002. Kurosawa M, Sato A, Sato Y, Suzuki H. Undiminished reflex responses of adrenal sympathetic nerve activity to stimulation of baroreceptors and cutaneous mechanoreceptors in aged rats. Neurosci Lett 1987; 77:193–198. Nijima A. Baroreceptor effects on renal and adrenal nerve activity. Am J Physiol 1976; 230:1733–1736. Carlsson S, Skarphedinsson JO, Delle M, Hoffman P, Thoren P. Reflex changes in post- and preganglionic sympathetic adrenal nerve activity and postganglionic sympathetic renal nerve activity upon arterial baroreceptor activation and during severe haemorrhage in the rat. Acta Physiol Scand 1992; 144:317–323. Morrison SF, Sved AF, Passerin AM. GABA-mediated inhibition of raphe pallidus neurons regulates sympathetic outflow to brown adipose tissue. Am J Physiol 1999; 276:R290–297. Skoog P, Mansson J, Thoren P. Changes in renal sympathetic outflow during hypotensive haemorrhage in rats. Acta Physiol Scand 1985; 125:655–660. Victor RG, Thoren P, Morgan DA, Mark AL. Differential control of adrenal and renal sympathetic nerve activity during hemorrhagic hypotension in rats. Circ Res 1989; 64:686–694.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

65. 66. 67. 68.

69. 70.

71.

72.

73. 74.

75. 76. 77. 78. 79.

80. 81. 82. 83. 84.

Koyama S, Sawano F, Matsuda Y, et al. Spatial and temporal differing control of sympathetic activities during hemorrhage. Am J Physiol 1992; 262:R579–585. Tochihara M. Reflex control of cardiac sympathetic nerve activity in anesthetized rats. Hokkaido Igaku Zasshi 1996; 71:247–258. Scislo TJ, O’Leary DS. Differential control of renal vs. adrenal sympathetic nerve activity by NTS A2a and P2x purinoceptors. Am J Physiol 1998; 275:H2130–2139. Scislo TJ, Kitchen AM, Augustyniak RA, O’Leary DS. Differential patterns of sympathetic responses to selective stimulation of nucleus tractus solitarius purinergic receptor subtypes. Clin Exp Pharmacol Physiol 2001; 28:120–124. Kollai M, Koizumi K, Brooks CM. Nature of differential sympathetic discharges in chemoreceptor reflexes. Proc Natl Acad Sci USA 1978; 75:5239–5243. Katona PG, Dembowsky K, Czachurski J, Seller H. Chemoreceptor stimulation on sympathetic activity: dependence on respiratory phase. Am J Physiol 1989; 257:R1027–1033. Huang W, Lahiri S, Mokashi A, Sherpa AK. Relationship between sympathetic and phrenic nerve responses to peripheral chemoreflex in the cat. J Auton Nerv Syst 1988; 25:95–105. Shirai M, Matsukawa K, Nishiura N, Kawaguchi AT, Ninomiya I. Changes in efferent pulmonary sympathetic nerve activity during systemic hypoxia in anesthetized cats. Am J Physiol 1995; 269:R1404–1409. Iriki M, Kozawa E. Factors controlling the regional differentiation of sympathetic outflow—influence of the chemoreceptor reflex. Brain Res 1975; 87:281–291. Iriki M, Simon E. Regional differentiation of sympathetic efferents. In: Ito M, ed. Integrative Control Functions of the Brain. Vol. 1. New York: Elsevier/North Holland Biomedical Press, 1978:221–238. Koshiya N, Guyenet PG. Role of the pons in the carotid sympathetic chemoreflex. Am J Physiol 1994; 267:R508–518. Britton SL. Cutaneous venodilation in response to systemic hypoxemia in dogs. J Surg Res 1984; 36:9–16. Eckberg DL, Nerhed C, Wallin BG. Respiratory modulation of muscle sympathetic and vagal cardiac outflow in man. J Physiol 1985; 365:181–196. Bainton CR, Richter DW, Seller H, Ballantyne D, Klein JP. Respiratory modulation of sympathetic activity. J Auton Nerv Syst 1985; 12:77–90. Numao Y, Koshiya N, Gilbey MP, Spyer KM. Central respiratory drive-related activity in sympathetic nerves of the rat: the regional differences. Neurosci Lett 1987; 81:279–284. Kocsis B, Lenkei Z. Coordination between cardiovascular and respiratory control systems during and after cerebral ischemia. J Appl Physiol 1992; 72:1595–1603. Barman SM, Gebber GL. Basis for synchronization of sympathetic and phrenic nerve discharges. Am J Physiol 1976; 231:1601–1607. Morrison SF. Respiratory modulation of sympathetic nerve activity: effect of MK801. Am J Physiol 1996; 270:R645–651. Haselton JR, Guyenet PG. Central respiratory modulation of medullary sympathoexcitatory neurons in rat. Am J Physiol 1989; 256:R739–750. Habler H, Bartsch T, Janig W. Rhythmicity in single fiber postganglionic activity supplying the rat tail. J Neurophysiol 1999; 81:2026–2036.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

85.

86.

87. 88.

89.

90.

91. 92.

93.

94.

95. 96. 97. 98.

99. 100. 101.

102.

Hagbarth KE, Hallin RG, Hongell A, Torebjork HE, Wallin BG. General characteristics of sympathetic activity in human skin nerves. Acta Physiol Scand 1972; 84:164–176. Bini G, Hagbarth KE, Hynninen p, Wallin BG. Thermoregulatory and rhythm-generating mechanisms governing the sudomotor and vasoconstrictor outflow in human cutaneous nerves. J Physiol 1980; 306:537–552. Simon E, Riedel W. Diversity of regional sympathetic outflow in integrative cardiovascular control: patterns and mechanisms. Brain Res 1975; 87:323–333. Conradt M, Kullmann R, Matsuzaki T, Simon E. Arterial baroreceptor function in differential cardiovascular adjustments induced by central thermal stimulation. Basic Res Cardiol 1975; 70:10–28. Vollmer RR, Baruchin A, Kolibal-Pegher SS, Corey SP, Stricker EM, Kaplan BB. Selective activation of norepinephrine- and epinephrine-secreting chromaffin cells in rat adrenal medulla. Am J Physiol 1992; 263:R716–721. Fukuhara K, Kvetnansky R, Weise VK, et al. Effects of continuous and intermittent cold (SART) stress on sympathoadrenal system activity in rats. J Neuroendocrinol 1996; 8:65–72. Janig W, Szulczyk P. The organization of lumbar preganglionic neurons. J Auton Nerv Syst 1981; 3:177–191. Mashimo T, Zhang P, Kamibayashi T, et al. Laser Doppler skin blood flow and sympathetic nervous responses to surgical incision during halothane and isoflurane anesthesia. Anesth Analg 1997; 85:291–298. Shimoda O, Ikuta Y, Nishi M, Uneda C. Magnitude of skin vasomotor reflex represents the intensity of nociception under general anesthesia. J Auton Nerv Syst 1998; 71:183–189. Kumada M, Dampney RA, Whitnall MH, Reis DJ. Hemodynamic similarities between the trigeminal and aortic vasodepressor responses. Am J Physiol 1978; 234:H67–73. Yu YH, Blessing WW. Constriction of the ear pinna vascular bed accompanies the trigeminal depressor response in rabbits. Neurosci Lett 1998; 255:172–174. Rathner JA, McAllen RM. Differential control of sympathetic drive to the rat tail artery and kidney by medullary premotor cell groups. Brain Res 1999; 834:196–199. Blessing WW, Yu YH, Nalivaiko E. Raphe pallidus and parapyramidal neurons regulate ear pinna vascular conductance in the rabbit. Neurosci Lett 1999; 270:33–36. Blessing WW, Nalivaiko E. Regional blood flow and nociceptive stimuli in rabbits: patterning by medullary raphe, not ventrolateral medulla. J Physiol (Lond) 2000; 524 Pt 1:279–292. Berne C, Fagius J. Skin nerve sympathetic activity during insulin-induced hypoglycaemia. Diabetologia 1986; 29:855–860. Fagius J, Berne C. Changes of sympathetic nerve activity induced by 2-deoxy-Dglucose infusion in humans. Am J Physiol 1989; 256:E714–720. Hoffman RP, Sinkey CA, Anderson EA. Hypoglycemia increases muscle sympathetic nerve activity in IDDM and control subjects. Diabetes Care 1994; 17:673–680. Holt SJ, York DA. Studies on the sympathetic efferent nerves of brown adipose tissue of lean and obese Zucker rats. Brain Res 1989; 481:106–112.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

103.

104. 105.

106.

107.

108.

109.

110. 111.

112.

113. 114. 115.

116. 117.

118. 119.

Egawa M, Yoshimatsu H, Bray GA. Effects of 2-deoxy-D-glucose on sympathetic nerve activity to interscapular brown adipose tissue. Am J Physiol 1989; 257:R1377–1385. Brodows RG, Pi S, Campbell RG. Sympathetic control of hepatic glycogenolysis during glucopenia in man. Metabolism 1975; 24:617–624. Mundinger TO, Boyle MR, Taborsky GJ. Activation of hepatic sympathetic nerves during hypoxic, hypotensive and glucopenic stress. J Auton Nerv Syst 1997; 63:153–160. Zhang W, Thoren P. Hyper-responsiveness of adrenal sympathetic nerve activity in spontaneously hypertensive rats to ganglionic blockade, mental stress and neuronglucopenia. Pflugers Arch 1998; 437:56–60. Vollmer RR, Balcita-Pedicino JJ, Debnam AJ, Edwards DJ. Adrenal medullary catecholamine secretion patterns in rats evoked by reflex and direct neural stimulation. Clin Exp Hypertens 2000; 22:705–715. Carlsson S, Skarphedinsson JO, Delle M, Hoffman P, Thoren P. Differential responses in post- and pre-ganglionic adrenal sympathetic nerve activity and renal sympathetic nerve activity after injection of 2-deoxy-D-glucose and insulin in rats. Acta Physiol Scand 1992; 145:169–175. Medvedev OS, Delle M, Thoren P. 2-Deoxy-D-glucose-induced central glycopenia differentially influences renal and adrenal nerve activity in awake SHR rats. Clin Exp Hypertens A 1988; 10:375–381. Nijima A. The effect of 2-deoxy-D-glucose and D-glucose on the efferent discharge rate of sympathetic nerves. J Physiol 1975; 251:231–243. Lu H, Duanmu Z, Scislo T, Dunbar JC. The co-existence of insulin-mediated decreased mean arterial pressure and increased sympathetic nerve activity is not mediated by the baroreceptor reflex and differentially by hypoglycemia. Clin Exp Hypertens 1998; 20:165–183. Rappaport EB, Young JB, Landsberg L. Effects of 2-deoxy-D-glucose on the cardiac sympathetic nerves and the adrenal medulla in the rat: further evidence for a dissociation of sympathetic nervous system and adrenal medullary responses. Endocrinology 1982; 110:650–656. Barman SM, Gebber GL, Calaresu FR. Differential control of sympathetic nerve discharge by the brain stem. Am J Physiol 1984; 247:R513–519. DiBona GF. Neural control of the kidney: functionally specific renal sympathetic nerve fibers. Am J Physiol Regul Integr Comp Physiol 2000; 279:R1517–1524. Cohen MI, Yu Q, Huang WX. Preferential correlations of a medullary neuron’s activity to different sympathetic outflows as revealed by partial coherence analysis. J Neurophysiol 1995; 74:474–478. Campos RR, McAllen RM. Cardiac sympathetic premotor neurons. Am J Physiol 1997; 272:R615–620. Dampney RA, McAllen RM. Differential control of sympathetic fibres supplying hindlimb skin and muscle by subretrofacial neurones in the cat. J Physiol (Lond) 1988; 395:41–56. Lovick TA. Differential control of cardiac and vasomotor activity by neurones in nucleus paragigantocellularis lateralis in the cat. J Physiol (Lond) 1987; 389:23–35. Dean C, Seagard JL, Hopp FA, Kampine JP. Differential control of sympathetic ac-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

120. 121. 122. 123.

124. 125.

126. 127. 128. 129. 130.

131. 132.

133. 134.

135. 136. 137. 138. 139.

tivity to kidney and skeletal muscle by ventral medullary neurons. J Auton Nerv Syst 1992; 37:1–10. McAllen RM, May CN. Differential drives from rostral ventrolateral medullary neurons to three identified sympathetic outflows. Am J Physiol 1994; 267:R935–944. Beluli DJ, Weaver LC. Areas of rostral medulla providing tonic control of renal and splenic nerves. Am J Physiol 1991; 261:H1687–1692. Beluli DJ, Weaver LC. Differential control of renal and splenic nerves without medullary topography. Am J Physiol 1991; 260:H1072–1079. McAllen RM, Dampney RA. Vasomotor neurons in the rostral ventrolateral medulla are organized topographically with respect to type of vascular bed but not body region. Neurosci Lett 1990; 110:91–96. Campos RR, McAllen RM. Cardiac inotropic, chronotropic, and dromotropic actions of subretrofacial neurons of cat RVLM. Am J Physiol 1999; 276:R1102–1111. McAllen RM, Neil JJ, Loewy AD. Effects of kainic acid applied to the ventral surface of the medulla oblongata on vasomotor tone, the baroreceptor reflex and hypothalamic autonomic responses. Brain Res 1982; 238:65–76. McAllen RM. Action and specificity of ventral medullary vasopressor neurones in the cat. Neuroscience 1986; 18:51–59. McAllen RM, May CN, Shafton AD. Functional anatomy of sympathetic premotor cell groups in the medulla. Clin Exp Hypertens 1995; 17:209–221. Barman SM, Gebber GL. Axonal projection patterns of ventrolateral medullospinal sympathoexcitatory neurons. J Neurophysiol 1985; 53:1551–1566. Brown DL, Guyenet PG. Cardiovascular neurons of brain stem with projections to spinal cord. Am J Physiol 1984; 247:R1009–1016. McAllen RM. Identification and properties of sub-retrofacial bulbospinal neurones: a descending cardiovascular pathway in the cat. J Auton Nerv Syst 1985; 17:151–164. Loewy AD. Raphe pallidus and raphe obscurus projections to the intermediolateral cell column in the rat. Brain Res 1981; 222:129–133. Bacon SJ, Zagon A, Smith AD. Electron microscopic evidence of a monosynaptic pathway between cells in the caudal raphe nuclei and sympathetic preganglionic neurons in the rat spinal cord. Exp Brain Res 1990; 79:589–602. Guyenet PG, Darnall RA, Riley TA. Rostral ventrolateral medulla and sympathorespiratory integration in rats. Am J Physiol 1990; 259:R1063–1074. Robinson RL, Culberson JL, Carmichael SW. Influence of hypothalamic stimulation on the secretion of adrenal medullary catecholamines. J Auton Nerv Syst 1983; 8:89–96. Kollai M, Koizumi K. The mechanisms of differential control in the sympathetic system studied by hypothalamic stimulation. J Auton Nerv Syst 1980; 2:377–89. Okada Y, Ninomiya I. Different cardiac and renal inhibitory and excitatory areas in rabbit hypothalamus. Am J Physiol 1983; 244:H832–838. Blum B, Israeli J, Hart O, Farchi M. Organ-localized hypothalamic-stimulated vasculature changes in the cat. Clin Auton Res 1994; 4:105–111. Hilton SM, Zbrozyna AW. Amygdaloid regions for defence reactions and its efferent pathway to the brain stem. J Physiol London 1963; 165:160–173. Abrahams VC, Hilton SM, Zbrozyna AW. The role of active muscle dilatation in the alerting stage of the defence reaction. J Physiol London 1964; 171:189–202.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

140. 141.

142.

143. 144.

145. 146. 147. 148.

149.

150.

151.

152. 153. 154. 155.

156.

Hilton SM, Spyer KM. Central nervous regulation of vascular resistance. Annu Rev Physiol 1980; 42:399–441. Coote JH, Hilton SM, Zbrozyna AW. The ponto-medullary area integrating the defence reaction in the cat and its influence on muscle blood flow. J Physiol 1973; 229:257–274. Horeyseck G, Janig W, Kirchner F, Thamer V. Activation and inhibition of muscle and cutaneous postganglionic neurones to hindlimb during hypothalamically induced vasoconstriction and atropine-sensitive vasodilation. Pflugers Arch 1976; 361:231–240. Blessing WW. The Lower Brainstem and Bodily Homeostasis. New York: Oxford University Press, 1997. Carrive P, Dampney RA, Bandler R. Excitation of neurones in a restricted portion of the midbrain periaqueductal grey elicits both behavioural and cardiovascular components of the defence reaction in the unanaesthetised decerebrate cat. Neurosci Lett 1987; 81:273–278. Lovick TA. Integrated activity of cardiovascular and pain regulatory systems: role in adaptive behavioural responses. Prog Neurobiol 1993; 40:631–644. Bandler R, Shipley MT. Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? Trends Neurosci 1994; 17:379–389. Carrive P. The periaqueductal gray and defensive behavior: functional representation and neuronal organization. Behav Brain Res 1993; 58:27–47. Carrive P, Bandler R. Viscerotopic organization of neurons subserving hypotensive reactions within the midbrain periaqueductal grey: a correlative functional and anatomical study. Brain Res 1991; 541:206–215. Carrive P, Bandler R, Dampney RA. Viscerotopic control of regional vascular beds by discrete groups of neurons within the midbrain periaqueductal gray. Brain Res 1989; 493:385–390. Carrive P, Bandler R, Dampney RA. Anatomical evidence that hypertension associated with the defence reaction in the cat is mediated by a direct projection from a restricted portion of the midbrain periaqueductal grey to the subretrofacial nucleus of the medulla. Brain Res 1988; 460:339–345. Bandler R, Keay KA, Floyd N, Price J. Central circuits mediating patterned autonomic activity during active vs. passive emotional coping. Brain Res Bull 2000; 53:95–104. Bandler R, Price JL, Keay KA. Brain mediation of active and passive emotional coping. Prog Brain Res 2000; 122:333–349. Gebber GL, Zhong S, Lewis C, Barman SM. Differential patterns of spinal sympathetic outflow involving a 10-Hz rhythm. J Neurophysiol 1999; 82:841–854. Gebber GL. A defence-like reaction: an emergent property of a system of coupled non-linear oscillators. Clin Exp Pharmacol Physiol 2001; 28:125–129. Carrive P, Bandler R, Dampney RA. Somatic and autonomic integration in the midbrain of the unanesthetized decerebrate cat: a distinctive pattern evoked by excitation of neurones in the subtentorial portion of the midbrain periaqueductal grey. Brain Res 1989; 483:251–258. Gebber GL, Zhong S, Lewis C, Barman SM. Defenselike patterns of spinal sympathetic outflow involving the 10-Hz and cardiac-related rhythms. Am J Physiol Regul Integr Comp Physiol 2000; 278:R1616–1626.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

4 Plasticity in the Autonomic Nervous System Responses of Adult Sympathetic Neurons to Injury Richard E. Zigmond Case Western Reserve University School of Medicine, Cleveland, Ohio, U.S.A.

Following peripheral nerve injury, there is a dramatic change in gene expression in axotomized peripheral neurons. While this fact has been recognized for some years, almost nothing has been known about the signals that trigger these changes. Evidence now indicates that at least two sets of factors are involved: factors induced in nonneuronal cells in response to axotomy and target factors, whose availability to the cell body is dramatically reduced by axotomy. The data also raise the possibility that simultaneous changes in these two factors might lead to a different pattern of gene expression than that seen when injury factors are induced without changes in target factors, for example, after preganglionic axotomy. The term “neural plasticity” is used by neuroscientists in two contexts: first, with regard to the developing nervous system and, second, with regard to the adult nervous system. As applied to neuronal development, the concept of plasticity is used in contrast to an opposing concept, sometimes referred to as “hard-wiring,” i.e., development according to an unalterable, and often cell-autonomous, mechanism. The best studied example of developmental plasticity in the autonomic nervous system is the transformation of certain sympathetic neurons from ones that use norepinephrine to ones that use acetylcholine as their neurotransmitter. The phenotypic “switch” depends on what target tissues the specific neurons come to innervate (for review, see Ref. 1). Thus, the transmitter phenotype of all sympathetic neurons is not predetermined, but can be altered depending on target-derived differentiation factors. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The use of the term plasticity with regard to the adult nervous system refers to changes in supposedly “fully developed” neurons in response to environmental changes. There are many examples of this type of plasticity, the most widely recognized being that of learning. More extreme changes in a neuron’s environment, but ones of important clinical significance, are those seen following injury (2–5). The subject of this chapter is the molecular responses of sympathetic neurons and their associated cells to injury. Much is known about plasticity after neuronal injury in the peripheral nervous system of the adult mammal. The term peripheral nervous system includes, among other things, the cell bodies and axons of autonomic postganglionic neurons, the cell bodies and peripheral axons of sensory neurons, and the axons of central motor neurons. These systems are of particular interest because regeneration is often successful in these neurons after injury in the adult. In contrast, axotomy of these same neurons during development typically leads to cell death (4,6). Large cytochemical and neurochemical changes occur in the cell bodies of axotomized neurons following peripheral axotomy (2–6). These changes have been referred to collectively as the “cell body response.” For many years, the cell body response has been conceptualized as underlying a functional shift in neurons from one adapted for synaptic transmission to one adapted for regeneration (3,5). In fact, however, as the number of changes that occur after axotomy have multiplied, the functional significance of many of these changes remain unexplored. In addition, the molecular signals underlying the cell body response to axotomy have remained unknown (5). The latter gap was recognized in a classic review article in 1975 by Cragg entitled “What is the signal for chromatolysis?” (7), chromatolysis being the original set of cytochemical changes documented to occur in axotomized neurons. A key part of the neuronal cell body response is a profound change in the pattern of expression of neuronal proteins. The emphasis of the present chapter is on the molecular and cellular mechanisms by which the expression of at least one class of proteins are regulated after axotomy, namely neuropeptides. While changes in other proteins will be mentioned, less is known of the molecular mechanisms underlying those changes. The studies to be discussed indicate that adult autonomic neurons retain considerable neurochemical plasticity and that injury appears to switch dramatically the pattern of gene expression in such neurons. While the studies to be cited in this chapter primarily concern experiments on postganglionic sympathetic neurons, there is no reason to suspect that comparable changes do not occur in parasympathetic neurons. Rather, due to the dispersed nature of such neuronal cell bodies and their generally very short axonal projections, they have rarely been studied in this context.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

I. NEUROPEPTIDE EXPRESSION IN NORMAL SYMPATHETIC NEURONS IN VIVO Peptides function as transmitters and modulators in the nervous systems (8). In addition, and especially relevant to this chapter, increasing evidence suggests that certain neuropeptides have trophic activities towards neurons and/or glial cells (9–11) and chemoattractant and activating activities towards macrophages (12). The latter circulating cells commonly enter an area of nerve injury and have been found to infiltrate into the superior cervical ganglion (SCG) after axonal damage (13). For reasons that will become more evident below, a striking example of an overall trophic action of a peptide in vivo is that of galanin, as demonstrated by studies on mice with a null mutation of the gene for this peptide. These animals show greatly retarded regeneration of axons in their sciatic nerve following injury to that nerve trunk (14). The distribution of specific neuropeptides in the peripheral nervous system has been examined in detail, and characteristic differences have been found between sympathetic, sensory, and motor neurons. To provide further complication, even within the sympathetic nervous system, reproducible differences in peptide content can be found between different ganglia within the same species and between different neurons within the same ganglion (15,16). Thus, while the vast majority of sympathetic neurons contain the enzymes involved in norepinephrine and synthesize and release this catecholamine as a transmitter, the distribution of peptide (putative) cotransmitters introduces neurochemical diversity into this system. The rat SCG is the sympathetic ganglion that has been examined most extensively with regard to its neuropeptide expression. This ganglion is the rostralmost ganglion in the paravertebral sympathetic chain. There is general agreement that three neuropeptides are expressed by principal neurons in this ganglion under normal (intact) conditions: neuropeptide Y (NPY), methionine-enkephalin, and the latter’s C-terminally extended form, methionine-enkephalin-arginine-glycineleucine. Interestingly, none of these peptides is detectable in all of the neurons within the SCG or even in all the noradrenergic neurons in this ganglion. NPY is estimated to be present in 60% of the principal neurons of the SCG (17–19). Immunoreactivities (IR) for methionine-enkephalin and its extended form are detectable in a much smaller group of neurons (about 10–20%) (20–22). Strikingly, in terms of the biochemical diversity of what has generally been considered a homogeneous population of neurons, the enkephalin-positive neurons in the SCG are TH-positive but NPY-negative (22,23). Differences in neuropeptide content of specific sympathetic neurons correlates in part with differences in the target cells the neurons innervate (24,25), although other influences must also contribute to this diversity. The functional importance of these differences remains to be elucidated. When examined at the mRNA level by in situ hybridization, heterogeneity is also found concerning the localization of NPY and enkephalin in SCG neurons (19,26,27). Also, higher proportions of neurons contain detectable mRNA for

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

these peptides than contain detectable peptide, raising the possibility that the translation of these mRNAs is a step that is under molecular regulation in addition to their transcription. Alternatively, such apparent discrepancies may simply reflect a higher sensitivity for detecting these molecules with available methods at the mRNA than at the peptide level.

II. CHANGES IN NEUROPEPTIDE EXPRESSION IN SYMPATHETIC NEURONS AFTER AXOTOMY A striking aspect of the changes in peptide expression in postganglionic sympathetic neurons that occur after axotomy is that those peptides that increase their expression are either absent or present at very low concentrations under normal conditions (28). In general, no neuronal cell bodies in the rat SCG exhibit substance P (SP) (29–31), and only a few exhibit vasoactive intestinal peptide (VIP) (32–34), pituitary adenylate cyclase activating peptide (PACAP) (35–38), or galanin IR (39–42). Within 48 hours after transection of the major postganglionic trunks of the SCG—the internal and external carotid nerves—however, substantial numbers of principal neurons can be found containing these IR (31,35,41,43). By radioimmunoassay, the levels of SP, VIP, and galanin IR increase 10-fold,

20-fold, and 200-fold, respectively (31,41,43). As noted, cellular heterogeneity exists normally in the peptide phenotypes of neurons in the SCG. There is also heterogeneity in the changes in peptide expression of these neurons in response to axotomy, though again one must consider the possibility that this apparent heterogeneity is in part a reflection of the sensitivities of the techniques utilized. For example, cutting the two major postganglionic trunks of this ganglion leads to the axotomy of 80% of the neurons in this ganglion (44); however, only about half of these neurons express detectable galanin IR after axotomy (41). Another example of heterogeneity of response to axotomy is that, while both galanin and VIP IR increase after axotomy, these peptides are colocalized in only a subpopulation of axotomized neurons (41). In early immunohistochemical studies on the distribution of neuropeptides in neuronal cell bodies, people often did not see detectable peptides until they blocked axonal transport in some manner, usually by ligating the nerve or by giving the axonal transport blocker colchicine. The common interpretation of such studies was that these peptides were expressed by these neurons at all times, but that they were rapidly transported out of the cell body under normal conditions to the nerve terminals, where they were stored and from which they could be released. Obviously, a similar explanation could account for the build-up of certain neuropeptides after nerve transection. To determine whether this somewhat trivial phenomena was at play after axotomy, the levels of the respective mRNAs for the induced peptides were examined. As with the peptides themselves, the mRNAs for SP, PACAP, galanin, and VIP (31,35,41–43) all increased following axCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

onal transection. In retrospect, it is quite likely that in many cases, a similar increase in mRNA was occurring in the earlier studies, rather than there being a simple build-up of a peptide that was synthesized at a similar rate in control situations and after blockade of transport. During the period when VIP, PACAP, galanin, and SP mRNA expression are increased, the expression of the mRNA for NPY, the peptide normally most prominent in the SCG, decreases (45). As will be discussed later, NPY is only one of a number of other proteins whose expression decreases in axotomized sympathetic neurons.

III. CHANGES IN NEUROPEPTIDE EXPRESSION AFTER AXOTOMY IN SENSORY AND MOTOR NEURONS Changes in peptide phenotype have also been examined in other neurons whose axons project into the peripheral nervous system (Table 1). First of all, the distribution of peptide phenotypes differs among intact sympathetic, sensory, and motor neurons. Second, these neurons differ in the changes in peptide expression they exhibit after axotomy. For example, while NPY expression decreases in SCG neurons after axotomy (45), it increases in neurons in dorsal root ganglia (DRG) (Table 1) (46). On the other hand, while SP expression increases in SCG neurons Table 1 Changes in Neuropeptide Expression in Axotomized Sympathetic, Sensory, and Motor Neurons Neuropeptide

Sympathetic neurons

Sensory neurons

Motor neurons

NPY Substance P CGRP a Somatostatin CCKb Galanin VIP PACAP

↓ 45 ↑ 31  up nd nd ↑ 41,42 ↑ 42 ↑ 35–38

↑ 46 ↓ 47,48 ↓ 49–51 ↓ 50,51 ↓ 53↑ 54,55 ↑ 56 ↑ 47,48,52,53 ↑ 57

↑ 58 ↑ 59 ↑ 49, 59–62 ↑ 59 ↑ 62↓ 63 ↑ 58,59,62 ↑ 62 ↑ 64

This table cites studies on neuropeptide expression at either the peptide or mRNA. The numbers refer to works cited in the reference list. The techniques utilized to obtain the data were either biochemical or histochemical. Peripheral nerves were either transected, crushed, or ligated. CCK, cholecystokinin; CGRP, calcitonin gene–related peptide; up, unpublished observations from the author’s laboratory; nd, not determined. a Both  and  CGRP mRNAs exist, differing by only one amino acid in the rat, though they are distinguishable by making use of cDNA probes that include part of the divergent 3 untranslated regions of these mRNAs. After axotomy of motor neurons, -CGRP mRNA increases and -CGRP mRNA decrease. After axotomy of sensory neurons, both - and -CGRP mRNA decrease. b Some apparent discrepancies exist in studies on CCK expression after axotomy in sensory and motor neurons, though these may simply reflect that different tissues were used for the different studies. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

after axotomy (30), it decreases after sciatic nerve section in those small-diameter neurons in lumbar DRG that normally express the peptide (47,48). The heterogeneity in the decreases in expression of different peptides in different classes of neurons in response to axotomy is consistent with the concept referred to previously that axotomized neurons tend to decrease expression of molecules involved in normal synaptic transmission. Nevertheless, the heterogeneity in the increases in expression of neuropeptides in different peripheral neurons raises questions as to the involvement of these substances in central aspects of nerve regeneration. It is of particular interest, therefore, that three neuropeptides have been found to exhibit similar increases in sympathetic, sensory, and motor neurons, namely, galanin, VIP, and the PACAP. This uniformity of response supports the hypothesis that these particular peptides might have common roles after injury in the three neuronal cell types (Table 1).

IV. CHANGES IN PEPTIDE EXPRESSION AFTER AXOTOMY VIEWED IN THE CONTEXT OF CHANGES OF GROWTHASSOCIATED PROTEINS Chromatolysis in axotomized sympathetic neurons involves dissolution of the stacks of rough endoplasmic reticulum normally seen in these neurons and the appearance of free polyribosomes (65). Early studies on the changes in protein expression after axotomy led to the idea that these morphological changes reflect a change in whether the proteins that are synthesized are primarily for secretion or for intracellular use (65,66). Interestingly, the expression of proteins (and their mRNA) associated with regeneration, such as tubulin and actin (66–68) and GAP43 (unpublished data), increase in axotomized sympathetic neurons, while proteins associated with transmitter synthesis, degradation, and postsynaptic actions [e.g., TH (45,66), nicotinic (69,70), and muscarinic receptors (71) and acetylcholinesterase (72)] decreased. The decreases in neuropeptide expression seen after axotomy can be viewed from this same perspective. As noted, in general the expression of those peptides involved in transmission or modulation in peripheral neurons is decreased—for example, NPY in sympathetic neurons and SP in small-diameter sensory neurons. Whether the increases in peptide expression after axotomy can be understood under this same general view still remains a matter of speculation, although the evidence is growing. According to such a view, an increase in the expression of certain peptides would indicate their involvement in neuronal survival and regeneration after axotomy. Suggestive support for such a proposal comes in part from a report that VIP prevents neurite retraction and cell death in cultured SCG neurons after withdrawal of NGF (73). VIP (74) and PACAP (75) also increase proliferation, survival, and neurite extension in sympathetic neuroblasts in cul-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ture, apparently by a direct action on the neuroblasts. In dissociated embryonic spinal cord cultures, VIP has mitogenic effects on astrocytes (76) and causes them to release a neuronal survival factor (77). VIP also appears to function as an autocrine growth factor for neuroblastoma cells (78), and PACAP causes neurite outgrowth in PC12 cells (79). While all of these studies are restricted to actions of peptides in cell culture, there is very strong evidence for an in vivo trophic action of galanin. As already mentioned, following lesioning of the sciatic nerve in galanin homozygous null mutants, the time course of restoration of normal sciatic nerve function is substantially slowed (14). The site of action of neuropeptides, following their induction by axotomy in vivo, is at present unknown. VIP and galanin, once induced, are not restricted to sympathetic ganglia but are rapidly transported into regenerating axons (80). Thus, in principle such peptides might act locally within the ganglion or at the site of axonal elongation or at both locations. Furthermore, the target cell type through which these induced peptides act remains to be determined and could certainly include autocrine effects on the neurons themselves or paracrine effects on glial cells or macrophages. Saturable binding sites for VIP, galanin, and SP have been identified within the normal SCG (81), and PACAP type 1 receptors have been identified in SCG neurons (36,82). VIP has been shown to increase cAMP levels and phosphoinositol turnover in these ganglia (83–85), although in neither case have the cell types involved been identified. It is known that VIP increases cAMP levels in Schwann cells isolated from sciatic nerve and that it can also increase laminin synthesis in these cells (86,87). Interestingly, the expression of peptide receptors may be coregulated with that of the peptide itself. In the SCG in vivo after axotomy and in explant culture, mRNA for both SP and its receptor increase (88). Somewhat surprising, however, is the finding that when mRNA for PACAP increases in this ganglion after axotomy, mRNA for the PACAP type 1 receptor decreases substantially (36). Whether axotomized sympathetic neurons remain sensitive to PACAP remains a question. Maintained responsiveness might result from the fact that the receptor downregulation is incomplete or because a different PACAP receptor subtype is induced.

V. ROLE OF LEUKEMIA INHIBITORY FACTOR IN TRIGGERING INDUCTION OF NEUROPEPTIDES AFTER AXOTOMY Interestingly, changes in expression of SP, VIP, PACAP, and galanin IR, similar to those seen in vivo after axotomy, occur in vitro in explanted adult SCG even though explantation involves considerably more than simply axotomizing sympathetic neurons. As in vivo, in vitro the increases in peptide levels are accompanied by increases in the corresponding mRNA (31,37,41,89,90).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

When neonatal SCG are dissociated and maintained in cultures containing both neuronal and nonneuronal cells for 48 hours, VIP IR increases; however, when the vast majority of nonneuronal cells are removed from these cultures, a much smaller increase in VIP IR expression is seen (91). In addition, the preplating step by which the nonneuronal cells are removed takes 4 hours, indicating that even the residual stimulation of VIP levels may depend on interactions between neurons and nonneuronal cells. Conditioning culture medium with nonneuronal cells for 48 hour prior to transferring the medium to neuron-enriched cultures leads to stimulation of VIP levels (91). In trying to determine the identity of the neuropeptide-inducing activity released by nonneuronal cells from the SCG into the medium, two well-characterized factors were chosen as the first candidates to examine: leukemia inhibitory factor (LIF) (92) and ciliary neurotrophic factor (CNTF) (93,94). Both of these factors belong to a larger family of proteins that also includes interleukins 6 and 11, oncostatin M, and cardiotropin 1(95), share a common three-dimensional configuration (96), activate a related set of receptors (97), and act through the JAK/STAT signal transduction pathway (98). These factors were of particular interest because, as exogenously added agents, they had been shown to increase the expression of VIP and SP in dissociated cultures of neonatal SCG neurons (99,100). Interestingly, LIF had already been proposed as the target-derived differentiation factor responsible for VIP induction and choline acetyltransferase induction in those noradrenergic sympathetic neurons that become cholinergic/VIPergic after reaching their synaptic targets (95,101), and, indeed, the level of LIF mRNA is high in rodent footpads, where the sweat glands, a primary target of cholinergic sympathetic neurons, are located (102). This interpretation of these experiments turned out to be incorrect, however, for several reasons. First of all, although exogenous LIF can induce cholinergic differentiation of neonatal sympathetic neurons, there is no evidence that LIF is the endogenous cholinergic differentiation factor. In fact, studies on LIF knockout mice and even LIF/CNTF double knockout mice suggest that neither cytokine is required for a normal cholinergic switch to occur (1,103). Furthermore, studies on Tabby mutant mice, in which the exocrine cells of the sweat glands do not develop, indicate that the foot pads of these animals have virtually the same LIF content as do those from wild-type mice (103). Nevertheless, when conditioned medium was pretreated with an antiserum that immunoprecipitated LIF, the active agent leading to the induction of VIP, galanin, and SP in neuron-enriched cultures was removed (91,104). More importantly, the requirement for LIF in neuropeptide induction in axotomized SCG neurons was also supported by experiments with transgenic mice in LIF null mutant mice. SCG from wild-type mice behaved similarly to SCG from rats following explantation or axotomy in vivo in that levels of VIP, galanin, and substance K (a peptide coregulated with SP) increased. These increases were significantly re-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

duced, however, and in some cases abolished when ganglia from homozygous LIF were examined (103). In contrast to the LIF antiserum, an anti-CNTF antiserum did not block the ability of medium conditioned by nonneuronal cells to induce neuropeptides, even though the antiserum did block the peptide induction produced by exogenous CNTF (91). Interestingly, histochemical studies on CNTF-IR indicate that the factor is easily detectable in myelinating Schwann cells in the sciatic nerve, but not in nonmyelinating satellite/Schwann cells in the SCG (105). Furthermore, when the conditioned medium from SCG nonneuronal cells was tested on ciliary neurons for CNTF bioactivity, none was detectable (91). Thus, although CNTF can mimic the neuropeptide induction, there is no evidence that it actually plays such a role in the SCG in response to axotomy. LIF mRNA was detectable in cultures of nonneuronal cells from the SCG by reverse transcriptase–polymerase chain reaction (RT-PCR); however, when SCG were taken from normal adult animals, the mRNA was undetectable (45,106). Maintaining these ganglia in organ culture for 2–24 hour led to a large increase in LIF mRNA that could be detected both by RT-PCR and by Northern blot analysis (45,106). In situ hybridization studies demonstrated that LIF mRNA in these cultures is present in non-neuronal cells, although the exact cell type(s) involved remains to be determined (107). An increase in LIF mRNA very similar to that seen in explants also occurs in the SCG in vivo after axotomy (45,106). In addition, after sciatic nerve transection, LIF mRNA is induced in the nerve trunk distal to and immediately proximal to the lesion (108,109). In the nerve trunk, LIF has been shown to be expressed in Schwann cells (108). Interestingly, under the same conditions, expression of CNTF in the distal nerve stump is significantly decreased (109–111). The fact that LIF induction occurs both within the ganglion and at the site of the lesion raises the question of whether LIF produces its effects via an action at one or both of these sites. Retrograde transport of 125I-LIF from the cut nerve ending was detected in sensory and motor neurons after sciatic nerve section, but was not detected in sympathetic neurons (108,112). On the other hand, sympathetic neurons in culture do transport LIF when the cytokine is placed on their distal processes (113). The extent of transport of LIF by sympathetic neurons is considerably less than that for NGF raising the possibility that the apparent lack of transport of LIF by these neurons in vivo may be a reflection of the sensitivity of the assay. One of the many missing links in the cascade of events that occur following axotomy is the signal that triggers LIF induction by nonneuronal cells. We have shown, in a spontaneously immortalized Schwann cell line, that stimulation of protein kinase C activity leads to an increase in LIF mRNA that is, at least, in part dependent on the MAP kinase pathway (114). In addition, we were able to identify a proteinaceous factor released from SCG after explanation that increases LIF mRNA in dissociated SCG cultures and may serve as a LIF-inducing signal (115).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

VI. ROLE OF REDUCED TARGET-DERIVED TROPHIC FACTOR AVAILABILITY IN TRIGGERING INDUCTION OF NEUROPEPTIDES AFTER AXOTOMY The fact that the LIF null mutant mice showed smaller, yet still significant increases in expression of galanin and VIP after axotomy, at both the peptide and the mRNA levels, suggested that part of the increase in these neuropeptides is LIFindependent. This residual effect could be due to the action of another cytokine induced by injury, and we have recently found such increases in IL-6, a member of the same cytokine family as LIF (unpublished data). Another interpretation is that in addition to being under the positive control of LIF after axotomy, peptide expression is normally under the negative control of target-derived NGF. To test the latter hypothesis, we injected intact rats systemically with antiserum raised against NGF and asked whether the reduction of NGF availability, in the absence of any surgery or nerve lesion, could alter peptide expression in the SCG. We found that anti-NGF by itself increased the expression of galanin and VIP both at the peptide and mRNA levels (116). In addition, these injections decreased NPY and NPY mRNA. In contrast, administration of NGF to explanted ganglia or to axotomized ganglia in vivo partially inhibited the increase in galanin and VIP expression seen in the absence of NGF. These findings of an inhibitory influence of NGF on peptide expression brought to mind an earlier result that Yi Sun in my laboratory had made but that we had not really understood. The finding was that placing a slow-release pellet of LIF near the SCG did not induce galanin expression in intact animals (unpublished results). Based on the effect of anti-NGF and of NGF itself, we later hypothesized that this target-derived trophic factor might be able to inhibit LIF’s ability to stimulate galanin expression. Animals were injected systemically with normal sheep serum or NGF antiserum and were at the same time implanted with a pellet that contained either a placebo or LIF. The results clearly indicated that, in the presence of antibodies to NGF, LIF induced galanin expression in intact animals. These results are intriguing because they raise the possibility that LIF only triggers a response relevant to regeneration when it is induced under conditions in which there is also a decrease in the availability of a target-derived growth factor. This synergy would allow LIF induction in response to other types of injury, e.g., deafferentation, while still only triggering a regenerative response in the sympathetic axons themselves when the injury involves axotomy.

ACKNOWLEDGMENTS The research discussed in this chapter was performed in many laboratories. The contributions from my laboratory were supported by grants NS17512 and

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

NS12651 from the National Institutes of Health. I particularly want to acknowledge the contributions of my students, postdoctoral fellows, and research assistants: T. Bennett, K. Boeshore, H. Hyatt-Sachs, R. P. Mohney, K. NakammotoCombs, R. C. Schreiber, A. M. Shadiack, Y. Sun, S. A. Vaccariello, U. Vaidyanathan, and Y. Zhou.

REFERENCES 1. 2.

3. 4.

5.

6. 7. 8. 9. 10. 11. 12.

13.

14.

Francis NJ, Landis SC. Cellular and molecular determinants of sympathetic neuron development. Annu Rev Neurosci 1999; 22:541–566. Lieberman AR. The axon reaction: a review of the principle features of perikaryal responses to axon injury. In: Pfeiffer CC, Smythies JR, eds. International Review of Neurobiology, Vol. 14. New York: Academic Press, 1971:49–124. Grafstein B, McQuarrie IG. Role of the nerve cell body in axonal regeneration. In: Cotman CW, ed. Neuronal Plasticity. New York: Raven Press, 1978:155–195. Hendry IA. Response of autonomic neurones to target deprivation: axotomy and regeneration. In: Hendry IA, Hill CE, eds. Development, Regeneration and Plasticity of the Autonomic Nervous System. Chur: Harwood Academic Publishers, 1992:415–462. Zigmond RE, Hyatt-Sachs H, Mohney RP, Schreiber RC, Shadiack AM, Sun Y, Vaccariello SA. Changes in neuropeptide phenotype after axotomy of adult peripheral neurons and the role of leukemia inhibitory factor. Perspect Dev Neurobiol 1996; 4:75–90. Hendry IA. The response of adrenergic neurones to axotomy and nerve growth factor. Brain Res 1975; 94(1):87–97. Cragg BG. What is the signal for chromatolysis? Brain Res 1970; 23:1–21. Hökfelt T, Johansson O, Ljungdahl Å, Lundberg JM, Schultzberg M. Peptidergic neurones. Nature 1980; 284:515–521. Gozes I, Brenneman DE. Neuropeptides as growth and differentiation factors in general and VIP in particular. J Mol Neurosci 1993; 4:1–9. Unsicker K. The trophic cocktail made by adrenal chromaffin cells. Exp Neurol 1980; 123:167–173. Schwartz JP, Taniwaki T. Heterogeneity of expression of neuropeptide genes by astrocytes: functional implications. Perspect Dev Neurobiol 1980; 2:251–257. Leceta J, Gomariz RP, Martinez C, Abad C, Ganea D, Delgado M. Receptors and transcriptional factors involved in the anti-inflammatory activity of VIP and PACAP. Ann NY Acad Sci. 2000; 921:92–102. Schreiber RC, Shadiack AM, Bennett TA, Sedwick CE, Zigmond RE. Changes in the macrophage population of the rat superior cervical ganglion after postganglionic nerve injury. J Neurobiol 1995; 27:141–153. Holmes FE, Mahoney S, King VR, Bacon A, Kerr NC, Pachnis V, Curtis R, Priestley JV, Wynick D. Targeted disruption of the galanin gene reduces the number of sensory neurons and their regenerative capacity. Proc Natl Acad Sci USA 2000; 97:11563–11568.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

15.

16. 17.

18.

19. 20.

21.

22.

23.

24.

25. 26. 27.

28. 29.

30.

Heym C, Lang R. Transmitters in sympathetic postganglionic neurons. In: Panula P, Päivärinta H, Soinila S, eds. Neurohistochemistry: Modern Methods and Applications. New York: Alan R. Liss, 1986:493–525. Elfvin L-G, Lindh B, Hökfelt T. The chemical neuroanatomy of sympathetic ganglia. Ann Rev Neurosci 1993; 16:471–507. Lundberg J, Terenius L, Hökfelt T, Tatemoto K. Comparative immunohistochemical and biochemical analysis of pancreatic polypeptide-like peptides with special reference to presence of neuropeptide Y in central and peripheral neurons. J Neurosci 1984; 4:2376–2386. Järvi R, Helén P, Pelto-Huikko M, Hervonen A. Neuropeptide Y (NPY)-like immunoreactivity in rat sympathetic neurons and small granule-containing cells. Neurosci Lett 1986; 67:223–227. Tyrrell S, Landis SC. The appearance of NPY and VIP in sympathetic neuroblasts and subsequent alterations in their expression. J Neurosci 1994; 14:4529–4547. Schultzberg M, Hökfelt T, Terenius L, Elfvin L-G, Lundberg JM, Brandt J, Elde RP, Goldstein M. Enkephalin immunoreactive nerve fibers and cell bodies in sympathetic ganglia of the guinea-pig and rat. Neuroscience 1979; 4:249–270. Häppölä O, Soinila S, Päivärinta H, Panula P. [Met 5]enkephalin-Arg6-Phe7 and [met5]enkephalin-Arg6-Gly7-Leu8 immunoreactive nerve fibers and neurons in the superior cervical ganglion of the rat. Neuroscience 1987; 21:283–295. Karhula T. Comparison of immunohistochemical localization of [Met5]enkephalinArg6-Gly7Leu8, [Met5]enkephalin, neuropeptide Y and vasoactive intestinal polypeptide in the superior cervical ganglion of the rat. J Auton Nerv Syst 1995; 51:9–18. Domeij S, Dahlqvist A, Forsgren S. Enkephalin-like immunoreactivity in ganglionic cells in the larynx and superior cervical ganglion of the rat. Regul Pept 1991; 32:95–107. Lundberg J, Hökfelt T, Änggård A, Terenius L, Elde R, Markey K, Goldstein M, Kimmel J. Organizational principles in the peripheral sympathetic nervous system: Subdivision by co-existing peptides (somatostatin-, avian pancreatic polypeptide-, and vasoactive intestinal polypeptide-like immunoreactive material). Proc Natl Acad Sci USA 1977; 79:1303–1307. Gibbons IL. Vasoconstrictor, vasodilator and pilomotor pathways in sympathetic ganglia of guinea-pigs. Neuroscience 1992; 47:657–672. Greif KF. Expression of preproenkephalin mRNA in rat superior cervical ganglion during postnatal development. Neurosci Lett 1994; 180:203–208. Tyrrell S, Landis SC. Disruption of target interactions prevents the development of enkephalin immunoreactivity in sympathetic neurons. J Neurosci 1994; 14:5708–5721. Zigmond RE. Neuropeptide action in sympathetic ganglia: Evidence for distinct functions in intact and axotomized ganglia. Ann NY Acad Sci 2000; 921:103–108. Hökfelt T, Elfvin LG, Schultzberg M, Goldstein M, Nilsson G. On the occurrence of substance P containing fibers in sympathetic ganglia: Immunohistochemical evidence. Brain Res 1977; 132:29–41. Gurusinghe CJ, Bell C. Substance P immunoreactivity in the superior cervical ganglia of normotensive and genetically hypertensive rats. J Auton Nerv Sys 1989; 27:249–256.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

31.

32.

33.

34.

35. 36.

37.

38.

39. 40.

41. 42.

43.

44.

45.

46.

Rao MS, Sun Y, Vaidyanathan U, Landis SC, Zigmond RE. Regulation of substance P is similar to that of vasoactive intestinal peptide after axotomy or explanation of the rat superior cervical ganglion. J Neurobiol 1993; 24:571–580. Hökfelt T, Elfvin L-G, Schultzberg M, Fuxe K, Said SI, Mutt V, Goldstein M. Immunohistochemical evidence of vasoactive intestinal polypeptide-containing neurons and nerve fibers in sympathetic ganglia. Neuroscience 1977; 2:885–896. Sasek CA, Zigmond RE. Localization of vasoactive intestinal peptide- and peptide histidine isoleucine amide-like immunoreactivities in the rat superior cervical ganglion and its nerve trunks. J Comp Neurol 1989; 280:522–532. Domeij S, Dahlqvist A, Forsgren S. Studies on colocalization of neuropeptide Y, vasoactive intestinal polypeptide, catecholamine-synthesizing enzymes and acetylcholinesterase in the larynx of the rat. Cell Tissue Res 1994; 263:495–505. Klimaschewski L, Hauser C, Heym C. PACAP immunoreactivity in the rat superior cervical ganglion in comparison to VIP. Neuroreport 1996; 7:2797–801. Moller K, Reimer M, Ekblad E, Hannibal J, Fahrenkrug J, Kanje M, Sundler F. The effects of axotomy and preganglionic denervation on the expression of pituitary adenylate cyclase activating peptide (PACAP), galanin and PACAP type 1 receptors in the rat superior cervical ganglion. Brain Res 1997; 775:166–182. Moller K, Reimer M, Hannibal J, Fahrenkrug J, Sundler F, Kanje M. Pituitary adenylate cyclase-activating peptide (PACAP) and PACAP type 1 receptor expression in regenerating adult mouse and rat superior cervical ganglia in vitro. Brain Res 1997; 775:156–165. Brandenburg CA, May V, Braas KM. Identification of endogenous sympathetic neuron pituitary adenylate cyclase-activating polypeptide (PACAP): depolarization regulates production and secretion through induction of multiple propeptide transcripts. J Neurosci 1997; 17:4045–4055. Kummer W. Galanin- and neuropeptide Y-like immunoreactivities coexist in paravertebral sympathetic neurones of the cat. Neurosci Lett 1987; 78:127–131. Strömberg I, Björklund H, Melander T, Rökaeus Å, Hökfelt T, Olson L. Galaninimmunoreactive nerves in the rat iris: alterations induced by denervations. Cell Tissue Res 1987; 250:267–275. Schreiber RC, Hyatt-Sachs H, Bennett TA, Zigmond RE. Galanin expression increases in adult rat sympathetic neurons after axotomy. Neuroscience 1994; 60:17–27. Mohney RP, Siegel RE, Zigmond RE. Galanin and vasoactive intestinal peptide messenger RNA expression increase following axotomy of the adult rat SCG. J Neurobiol 1994; 25:108–118. Hyatt-Sachs H, Schreiber RC, Bennett TA, Zigmond RE. Phenotypic plasticity in adult sympathetic ganglia in vivo: effects of deafferentation and axotomy on the expression of vasoactive intestinal peptide. J Neurosci 1993; 13:1642–1653. Bowers CW, Zigmond RE. Localization of neurons in the rat superior cervical ganglion that project into different postganglionic trunks. J Comp Neurol 1979; 185:381–392. Sun Y, Zigmond RE. Involvement of leukemia inhibitory factor in the increases in galanin and vasoactive intestinal peptide mRNA and the decreases in neuropeptide Y and tyrosine hydroxylase mRNA in sympathetic neurons after axotomy. J Neurochem 1996; 67:1751–1760. Wakisaka S, Kajander KC, Bennett GJ. Increased neuropeptide Y (NPY)-like im-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58. 59.

60.

munoreactivity in rat sensory neurons following peripheral axotomy. Neurosci Lett 1991; 124:200–203. Nielsch U, Keen P. Reciprocal regulation of tachykinin- and vasoactive intestinal peptide-gene expression in rat sensory neurones following cut and crush injury. Brain Res 1989; 481:25–30. Noguchi K, Senba E, Morita Y, Sato M, Tohyama, M. Prepro-VIP and preprotachykinin mRNAs in the rat dorsal root ganglion cells following peripheral axotomy. Mol Brain Res 1989; 6:327–330. Noguchi K, Senba E, Morita Y, Sato M, Tohyama M. Alpha-CGRP and beta-CGRP mRNAs are differentially regulated in the rat spinal cord and dorsal root ganglion. Mol Brain Res 1990; 7:299–304. Doughty SE, Atkinson ME, Shehab SAS. A quantitative study of neuropeptide immunoreactive cell bodies of primary afferent sensory neurons following rat sciatic nerve peripheral axotomy. Reg Pept 1991; 35:59–72. Noguchi K, De Leon M, Nahin RL, Senba E, Ruda MA. Quantification of axotomyinduced alteration of neuropeptide mRNAs in dorsal root ganglion neurons with special reference to neuropeptide Y mRNA and the effects of neonatal capsaicin treatment. J Neurosci Res 1993; 35:54–66. McGregor GP, Gibson SJ, Sabate IM, Blank, MA, Christofides ND, Wall PD, Polak JM, Bloom SR. Effect of peripheral nerve section and nerve crush on spinal cord neuropeptides in the rat; increased VIP and PHI in the dorsal horn. Neuroscience 1984; 13:207–216. Shehab SAS, Atkinson ME. Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons. Brain Res 1986; 372:37–44. Verge VMK, Wiesenfeld Hallin Z, Hökfelt T. Cholecystokinin in mammalian primary sensory neurons and spinal cord: In situ hybridization studies on rat and monkey spinal ganglia. Eur J Neurosci 1992; 5:240–250. Xu XJ, Puke MJ, Verge VM, Wiesenfeld Hallin Z, Hughes J, Hökfelt T. Up-regulation of cholecystokinin in primary sensory neurons is associated with morphine insensitivity in experimental neuropathic pain in the rat. Neurosci Lett 1993; 152:129–132. Hökfelt T, Wiesenfeld-Hallin Z, Villar M, Melander T. Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci Lett 1987; 83:217–220. Zhang Q, Shi TJ, Ji RR, Zhang YZ, Sundler F, Hannibal J, Fahrenkrug J, Hokfelt T, Zhang Y. Expression of pituitary adenylate cyclase-activating polypeptide in dorsal root ganglia following axotomy: time course and coexistence. Brain Res 1995; 705:149–58. Moore RY. Cranial motor neurons contain either galanin- or calcitonin gene-related peptide-like immunoreactivity. J Comp Neurol 1989; 282:512–522. Zhang X, Verge VMK, Wiesenfeld-Hallin Z, Piehl F, Hökfelt T. Expression of neuropeptides and neuropeptide mRNAs in spinal cord after axotomy in the rat, with special reference to motoneurons and galanin. Exp Brain Res 1993; 93:450–461. Streit WJ, Dumoulin FL, Raivich G, Kreutzberg GW. Calcitonin gene-related peptide increases in rat facial motoneurons after peripheral nerve transection. Neurosci Lett 1989; 101:131–148.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72. 73.

74.

75.

Haas CA, Streit WJ, Kreutzberg GW. Rat facial motoneurons express increased levels of calcitonin gene-related peptide mRNA in response to axotomy. J Neurosci Res 1990; 27:270–275. Saika T, Senba E, Noguchi K, Sato M, Kubo T, Matsunaga T, Tohyama M. Changes in expression of peptides in rat facial motoneurons after facial nerve crushing and resection. Mol Brain Res 1991; 11:187–196. Piehl F, Arvidsson U, Johnson H, Cullheim S, Dagerlind Å, Ulfhake B, Cao Y, Elde R, Pettersson RF, Terenius L, Hökfelt T. GAP-43, FGF, CCK and - and -CGRP in rat spinal motoneurons subjected to axotomy and/or dorsal root severance. Eur J Neurosci 1993; 5:1321–1333. Zhou X, Rodriguez WI, Casillas RA, Ma V, Tam J, Hu Z, Lelievre V, Chao A, Waschek JA. Axotomy-induced changes in pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP receptor gene expression in the adult rat facial motor nucleus. J Neurosci Res 1999; 57:953–961. Matthews MR, Raisman G. A light and electron microscopic study of the cellular response to axonal injury in the superior cervical ganglion of the rat. Proc R Soc Lond 1972; 181:43–79. Cheah TB, Geffen LB. Effects of axonal injury on norepinephrine, tyrosine hydroxylase, and monoamine oxidase levels in sympathetic ganglia. J Neurobiol 1973; 4:443–452. Koo EH, Hoffman RN, Price DL. Levels of neurotransmitter and cytoskeletal protein mRNAs during nerve regeneration in sympathetic ganglia. Brain Res 1988; 449:361–363. Hall ME, Wilson DL, Stone GC. Changes in synthesis of specific proteins following axotomy: detection with two-dimensional gel electrophoresis. J Neurobiol 1978; 9:353–366. Zhou Y, Deneris E, Zigmond RE. Differential regulation of levels of nicotinic receptor subunit transcripts in adult sympathetic neurons after axotomy. J Neurobiol 1998; 34:164–178. Zhou Y, Deneris E, Zigmond RE. Nicotinic acetylcholine receptor subunit proteins alpha7 and beta4 decrease in the superior cervical ganglion after axotomy. J Neurobiol 2001; 46:178–192. Sinicropi, DV, Kauffman FC, Burt DR. Axotomy in rat sympathetic ganglia: reciprocal effects on muscarinic receptor binding and 6-phosphogluconate dehydrogenase activity. Brain Res 1979; 161:560–565. Viana GB, Kauffman FC. Cholinesterase activity in the rat superior cervical ganglion: Effect of denervation and axotomy. Brain Res 1984; 304:37–45. Tanaka S, Koike T. Vasoactive intestinal peptide suppresses neuronal cell death induced by nerve growth factor deprivation in rat sympathetic ganglion cells in vitro. Neuropeptides 1994; 26:103–111. Pincus DW, DiCicco-Bloom EM, Black IB. Vasoactive intestinal peptide regulates mitosis, differentiation and survival of cultured sympathetic neuroblasts. Nature 1990; 343:564–567. DiCicco-Bloom E, Deutsch PJ, Maltzman J, Zhang J, Pintar JE, Zheng J, Friedman WF, Zhou X, Zaremba T. Autocrine expression and ontogenetic functions of the PACAP ligand/receptor system during sympathetic development. Dev Biol 2000; 219:197–213.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

76.

77.

78. 79.

80.

81.

82.

83. 84.

85.

86.

87.

88.

89.

90.

Brenneman DE, Nicol T, Warren D, Bowers LM. Vasoactive intestinal peptide: a neurotrophic releasing agent and an astroglial mitogen. J Neurosci Res 1990; 25:386–394. Brenneman DE, Neale EA, Foster GA, d’Autremont SW, Westbrook GL. Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide. J Cell Biol 1987; 104:1603–1610. O’Dorisio MS, Fleshman DJ, Qualman SJ, O’Dorisio TM. Vasoactive intestinal peptide: autocrine growth factor in neuroblastoma. Reg Pept 1992; 37:213–326. Deutsch PJ, Sun Y. The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth. J Biol Chem 1992; 267:5108–5113. Shadiack AM, Zigmond RE. Galanin induced in sympathetic neurons after axotomy is anterogradely transported toward regenerating nerve endings. Neuropeptides 1998; 32:257–264. Mantyh PW, Catton MD, Allen CJ, Labenski, ME, Maggio JE, Vigna, SR. Receptor binding sites for cholecystokinin, galanin, somatostatin, substance P and vasoactive intestinal polypeptide in sympathetic ganglia. Neuroscience 1992; 46:739–754. Nogi H, Hashimoto H, Hagihara N, Shimada S, Yamamoto K, Matsuda T, Tohyama M, Baba A. Distribution of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), PACAP receptor, vasoactive intestinal polypeptide (VIP), and VIP receptors in the rat superior cervical ganglion. Neurosci Lett 1997; 227:37–40. Volle RL, Quenzer LF, Patterson BA. The regulation of cyclic nucleotides in a sympathetic ganglion. J Auton Nerv Sys 1982; 6:65–72. Ip NY, Baldwin C, Zigmond RE. Regulation of the concentration of adenosine 3,5cyclic monophosphate the activity of tyrosine hydroxylase in the rat superior cervical ganglion by three neuropeptides of the secretin family. J Neurosci 1985; 5:1947–1954. Audigier S, Barberis C, Jard S. Vasoactive intestinal polypeptide increases inositol phospholipid breakdown in the rat superior cervical ganglion. Brain Res 1986; 376:363–367. Yasuda T, Sobue G, Mitsuma T, Takahasi A. Peptidergic and adrenergic regulation of the intracellular 3,5-cyclic adenosine monophosphate content in cultured rat Schwann cells. J Neurol Sci 1988; 88:315–325. Zhang QL, Lin PX, Shi D, Xian H, Webster HD. Vasoactive intestinal peptide: mediator of laminin synthesis in cultured Schwann cells. J Neurosci Res 1996; 43:496–502. Ludlam WH, Chandross KJ, Kessler JA. LIF- and IL-1-mediated increases in substance P receptor mRNA in axotomized, explanted or dissociated sympathetic ganglia. Brain Res 1995; 685:12–20. Zigmond RE, Hyatt-Sachs H, Baldwin C, Qu XM, Sun Y, McKeon TW, Schreiber RC, Vaidyanathan U. Phenotypic plasticity in adult sympathetic neurons: changes in neuropeptide expression in organ culture. Proc Natl Acad Sci USA 1992; 89:1507–1511. Sun Y, Rao MS, Landis SC, Zigmond RE. Depolarization increases vasoactive in-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

91.

92.

93.

94.

95. 96. 97. 98. 99.

100.

101.

102.

103.

104.

105.

testinal peptide- and substance P-like immunoreactivities in cultured neonatal and adult sympathetic neurons. J Neurosci 1992; 12:3717–3728. Sun Y, Rao MS, Zigmond RE, Landis SC. Regulation of vasoactive intestinal peptide expression in sympathetic neurons in culture: the role of cholinergic differentiation factor/leukemia inhibitory factor. J Neurobiol 1994; 25:415–430. Yamamori T, Fukada K, Aebersold R, Korsching S, Fann M-J, Patterson PH. The cholinergic neuronal differentiation factor from heart cells is identical to leukemia inhibitory factor. Science 1989; 246:1412–1416. Stockli KA, Lottspeich F, Sendtner M, Masiakowski P, Carroll P, Gotz R, Lindhom D, Thoenen H. Molecular cloning, expression and regional distribution of rat ciliary neurorophic factor. Nature 1989; 342:920–923. Lin LF, Mismer D, Lile JD, Armes LG, Butler ET, Vannice JL, Collins F. Purification, cloning, and expression of ciliary neurotrophic factor (CNTF) Science 1989; 246:1023–1025. Patterson PH. The emerging neuropoietic cytokine family: first CDF/LIF, CNTF and IL-6; next ONC, MGF, GCSF? Curr Opin Neurobiol 1992; 2:94–97. Bazan JF. Neuropoietic cytokines in the hematopoietic fold, Neuron 1992; 7:197–208 Ip NY, Yancopoulos GD. Ciliary neurotrophic factor and its receptor complex. Prog Growth Factor Res 1992; 4:139–155. Bonni A, Frank DA, Schindler C, Greenberg ME. Characterization of a pathway for ciliary neurotrophic factor signaling to the nucleus. Science 1992; 262:1575–1579. Nawa H, Nakanishi S, Patterson P. Recombinant cholinergic differentiation factor (leukemia inhibitory factor) regulates sympathetic neuron phenotype by alterations in the size and amounts of neuropeptide mRNAs. J Neurochem 1991; 56:2147–2150. Ernsberger U, Sendtner M, Rohrer H. Proliferation and differentiation of embryonic chick sympathetic neurons: effects of ciliary neurotrophic factor. Neuron 1989; 2:1275–1284. Patterson PH, Landis SC. Phenotype specifying factors and the control of neuronal differentiation decisions. In: Hendry IA, Hill CE, eds. Development, Regeneration and Plasticity of the Autonomic Nervous System. Chur: Harwood Academic Publishers, 1992:231–265. Yamamori T. Localization of cholinergic differentiation factor/leukemia inhibitory factor in the rat brain and peripheral tissues. Proc Natl Acad Sci USA 1991; 88:7298–7302. Rao MS, Sun Y, Escary JL, Perreau J, Tresser S, Patterson PH, Zigmond RE, Brulet P, Landis SC. Leukemia inhibitory factor mediates an injury response but not a target-directed developmental transmitter switch in sympathetic neurons. Neuron 1993; 11:1175–1185. Shadiack AM, Hart RP, Carlson CD, Jonakait GM. Interleukin-1 induces substance P in sympathetic ganglia through the induction of leukemia inhibitory factor (LIF). J Neurosci 1993; 13:2601–2609. Dobrea GM, Unnerstall JR, Rao MS. The expression of CNTF message and immunoreactivity in the central and peripheral nervous system of the rat. Dev Brain Res 1992; 66:209–219.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

106.

107.

108.

109.

110.

111. 112.

113.

114.

115.

116.

117. 118.

Sun Y, Landis SC, Zigmond RE. Signals triggering the induction of leukemia inhibitory factor in sympathetic superior cervical ganglia and their nerve trunks after axonal injury. Mol Cell Neurosci 1966; 7:152–163. Banner LR, Patterson PH. Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia. Proc Natl Acad Sci USA 1994; 91:7109–7113. Curtis R, Scherer SS, Somogyi R, Adryan KM, Ip NY, Zhu Y, Lindsay RM, Di Stefano PS. Retrograde axonal transport of LIF is increased by peripheral nerve injury: correlation with increased LIF expression in distal nerve. Neuron 1994; 12:191–204. Sendtner M, Stockli, KA, Thoenen H. Synthesis and localization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration. J Cell Biol 1992; 118:139–148. Rabinovsky ED, Smith GM, Browder DP, Shine HD, McManaman JL. Peripheral nerve injury down-regulates CNTF expression in adult rat sciatic nerves. J Neurosci Res 1992; 31:188–192. Seniuk N, Altares M, Dunn R, Richardson PM. Decreased synthesis of ciliary neurotrophic factor in degenerating peripheral nerves. Brain Res 1992; 572:300–302. Hendry IA, Murphy M, Hilton DJ, Nicola NA, Bartlett PF. Binding and retrograde transport of leukemia inhibitory factor by the sensory nervous system. J Neurosci 1992; 12:3427–3434. Ure DR, Campenot RB. Leukemia inhibitory factor and nerve growth factor are retrogradely transported and processed by cultured rat sympathetic neurons. Dev Biol 1994; 162:339–347. Nagamoto-Combs K, Vaccariello SA, Zigmond RE. The levels of LIF mRNA in a Schwann cell line are regulated by multiple second messenger pathways. J Neurochem 1999; 72:1871–1881. Shadiack AM, Sun Y, Zigmond RE. Nerve growth factor antiserum induces axotomy-like changes in neuropeptide expression in intact sympathetic and sensory neurons. Neuroscience 2001; 21:363–371. Shadiack AM, Vaccariello SA, Sun Y, Zigmond RE. Nerve growth factor inhibits sympathetic neuron’s response to an injury ctokine. Proc Natl Acad Sci USA 1998; 95:7727–7730. Mohney RP, Zigmond RE. Vasoactive intestinal peptide enhances its own expression in sympathetic neurons after injury. J Neurosci 1998; 18:5285–5293. Mohney RP, Zigmond RE. Galanin expression is decreased by cAMP-elevating agents in cultured sympathetic ganglia. NeuroReport 1999; 10:1221–1224.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

5 The Adrenal Medulla Physiology and Pathophysiology Graeme Eisenhofer National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, U.S.A.

Monika Ehrhart-Bornstein and Stefan R. Bornstein University of Düsseldorf, Düsseldorf, Germany

I. PHYSIOLOGY A. Functions of the Adrenal Medulla Epinephrine was first isolated and identified as the principal secretory product of the adrenal medulla at the turn of the twentieth century by Takamine (1). The concept of the sympathoadrenal system as a single functional unit stems largely from the studies by Walter Cannon of epinephrine as the primary mediator of the fightor-flight response (2). Although norepinephrine, not epinephrine, was subsequently identified as the principal transmitter secreted by sympathetic nerves (3), the concept of the sympathoadrenal system as a single functional unit persisted. It is now becoming increasingly clear, however, that the sympathoneural and adrenal medullary systems are regulated separately and often in divergent directions in response to different forms of stress (4). This and the many functional differences between the two systems make their separate consideration and comparison appropriate. Although both adrenal medullary and sympathoneural systems share some common neural pathways, these pathways are somewhat distinct from one another. Neural input to the adrenal medulla includes direct innervation by cholinergic fibers that pass through the sympathetic paravertebral chain from preganglionic sympathetic cell bodies of the spinal cord. In contrast, the innervation of

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

the sympathoneural system involves both preganglionic cholinergic and postganglionic noradrenergic components, with connections made in ganglia for the most part located in the sympathetic paravertebral chain. The human adrenal medulla produces mainly epinephrine, which, as a hormone, is secreted directly into the bloodstream to act on cells distant from the sites of release. The postganglionic sympathetic nerves that innervate blood vessels, the heart, and other tissues secrete norepinephrine, which as a neurotransmitter acts close to the sites of release. In contrast to the epinephrine released by the adrenals, most of the norepinephrine released by sympathetic nerves is removed back into nerves by neuronal uptake so that only small amounts of the transmitter escape into the bloodstream (5). Nevertheless, over 90% of the norepinephrine in the circulation is derived from sympathetic nerves, mostly those innervating mesenteric organs, kidneys, and skeletal muscle. Both epinephrine and norepinephrine have overlapping but also different effects on - and -adrenergic receptors (6,7). In particular, epinephrine has much more potent effects on 2-adrenoceptors than norepinephrine, while norepinephrine is a more potent 1-adrenoceptor agonist than epinephrine. Epinephrine is also a more potent -adrenoceptor agonist than norepinephrine. However, the proximity of sites of norepinephrine and epinephrine release to adrenoceptors and resulting concentrations at effector sites are also important determinants of adrenoceptor-mediated responses to the two catecholamines. Due to the above differences, epinephrine exerts its effects on different populations of adrenoceptors than norepinephrine. As a circulating hormone, epinephrine acts potently on 2-adrenergic receptors of the skeletal muscle vasculature causing vasodilation. In contrast, norepinephrine released locally within the vasculature causes 1-adrenoceptor–mediated vasoconstriction. This and the chronotrophic and inotrophic effects of neurally released norepinephrine mediated by way of cardiac 1-adrenergic receptors reflect a primary and critical function of the sympathoneural system in cardiovascular regulation, particularly maintenance of blood pressure. Increases in circulating epinephrine during mental stress or exercise may contribute to skeletal muscle vasodilatory responses, but appear to play little role in other cardiovascular changes, including increases in heart rate (8,9). Thus, despite the potent hemodynamic actions of epinephrine, the adrenal medulla has a minimal role in cardiovascular regulation compared to sympathetic nerves. Epinephrine is more important as a metabolic than as a hemodynamic regulatory hormone (10,11). In particular, epinephrine stimulates lipolysis, ketogenesis, thermogenesis, and glycolysis and raises plasma glucose levels by stimulating glycogenolysis and gluconeogenesis. Epinephrine also has potent effects on pulmonary function, causing 2-adrenoceptor–mediated dilation of airways (12). The above actions of epinephrine occur at threshold plasma concentrations well within the physiological range. Circulating norepinephrine, in part

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

derived from the adrenal medulla and functioning as a hormone, may have additional metabolic actions, but appears to have little importance for cardiovascular regulation compared to the higher concentrations of norepinephrine at neuroeffector sites. Despite the apparent importance of the adrenal medulla in homeostasis, particularly regulation of metabolism, the medulla in contrast to the adrenal cortex is not vital for survival. Studies in adrenalectomized subjects clearly show that both hemodynamic and glucose-counter-regulatory responses to insulin-hypoglycemia, exercise, and other manipulations remain intact despite absence of epinephrine responses (13–16). This contrasts with the severe disturbances of blood pressure regulation accompanying loss of sympathetic nerves. Thus, due to the presence of redundant neural and hormonal systems, the adrenal medulla does not appear to play a critical role in cardiovascular or metabolic homeostasis. Nevertheless, as outlined in subsequent sections of this chapter, adrenal medullary dysfunction may contribute to several disorders or may be important in diseases such as diabetes that involve failure of other counterregulatory systems. B. Development and Morphology of the Adrenal Medulla 1. Development of the Adrenal Medulla While the steroid-producing adrenal cortex is of mesodermal origin, the catecholamine-producing adrenal medulla is derived from the neural crest. The adrenals are first noticeable during the sixth week of gestation as an accumulation of mesodermal cells. A mesonephronic capsule moves in on these “adrenal anlagen” at the 10 mm stage. At this point, sympathochromaffin cells from adjacent ganglionic masses invade the cortex to form the medulla. The venous sinuses develop along with radial capillaries of mesonephronic arterial origin. The rudimentary subcapsular zona glomerulosa cords of cells proliferate in between the vessels towards the medulla, becoming granular and eosinophilic and finally autolyzed. The involution of the gland occurs within the first 2 weeks after birth from the above-mentioned cells, which compose the fetal or provisional cortex. Once the glands reach 14 mm, a second stage of development occurs from mesenchymal cells of the mesothelium leading to the permanent cortex. The adrenal glands continue to grow postnatally, until they are fully differentiated around 12 years of age. Cells from the glomerular zone proliferate to form the fasciculata and reticular zone of the adult cortex. The adrenal medullary chromaffin cells, like the sympathetic neurons, derive from the neural crest and more specifically the sympathogonia. The medullary cells reach their final location in the center of the medulla after cell strands and fibers move ventrally and dorsally, where the chromaffin cells gradu-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ally develop over time. The adrenal medulla consists of three types of cells. The sympathetic cells (sympathogonia) are small cells with hyperchromatic nuclei and scanty cytoplasm. Pheochromoblasts are larger with larger nuclei, one or two prominent nucleoli, and abundant cytoplasm. Pheochromocytes have only one nucleolus. The pheochromocytes show the positive chromaffin reaction at the 50 mm stage or later. The sympathetic cells and the pheochromoblasts show a negative chromaffin response. While the pheochromocytes increase in number by division, the number of primitive cells gradually decreases. In humans substantial amounts of chromaffin tissue develop in extraadrenal locations, particularly as paraaortic tissue (17). Most of this regresses after birth so that in the adult only remnants exist and most chromaffin cells are confined to the adrenal medulla. In the fetus and at birth the medulla accounts for less than 1% of the total volume of the adrenal gland, but after birth undergoes rapid growth to account for about 9% of adrenal volume in the adult (18). Despite the underdeveloped nature of the fetal and neonatal adrenal medulla, increased release of epinephrine at birth is important for facilitating appropriate hemodynamic adjustments and stimulating production of surfactant by the lung (19). 2.

Morphology of the Adrenal Medulla

The adult human adrenals are flat, yellow-colored glands, located on the left and right side of the median plane, behind the peritoneum. They overlie the superior poles of the kidneys and are surrounded by loose connective tissue. Each adrenal measures about 50  30  10 mm. Individually the human adrenal glands are shaped differently, with a pyramidal right gland and a crescent-like left adrenal with a noticeable groove. Three adrenal parts can be distinguished—the head, body, and tail—with the head located medially. The medulla is predominantly located within the head and the body of the gland. Each adrenal gland consists of an outer part, the lipid-rich cortex with a thickness of 5 mm, and an inner grayish-colored central medulla consisting of chromaffin cells. The adrenal medulla is up to 2 mm thick and takes about one tenth of the entire weight of the gland. A connective tissue capsule that carries the blood vessels, nerves, and lymphatics surrounds the adrenal glands. Blood is supplied to adrenal medulla by direct arterial supply as well as from vessels draining centripetally from the cortex towards the medulla (20). The latter supply provides an important source of adrenocortical steroids for regulation of adrenal medullary function, but whether this reflects a true portal system is questionable (21). The adrenal medulla is composed of groups of rounded cells, called pheochromocytes, which are in close relationship with capillaries and venules. These cell groups are surrounded by a second group of cells, the sustentacular cells, which are not always seen in histological sections, but are easily demonstrated by immunostaining for S-100 protein. The medullary cells have poorly demarcated borders, a fine granular basophilic cytoplasm, and variably sized and Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

shaped nuclei, occasionally placed eccentrically. The nuclei have prominent eosinophilic nucleoli. An important characteristic feature of adrenal medullary cells is the presence of numerous catecholamine storage granules ranging in size from 100 to 300 nm in diameter. These granules turn brown when exposed to potassium bichromate solutions, ammoniacal silver nitrate, or osmium tetroxide due to the oxidation and polymerization of epinephrine and norepinephrine. This process is known as the “chromaffin reaction,” hence the terms chromaffin cells and chromaffin granules. At least two kinds of adrenal medullary chromaffin cells have been identified in most animal species based on differences in chromaffin granules (22). Norepinephrine-producing chromaffin cells possess dense-core granules eccentrically located in the vesicle, while cells storing epinephrine have less electrondense homogeneous granules (Fig. 1). The distinction in the human adrenal medulla is less clear (23), but this may reflect the extreme sensitivity of vesicle

Figure 1 Normal human adrenal chromaffin cells are immunostained with anti-chromogranin A, whereas cortical cells are stained with anti-cytochrome P450 17 antibodies. Note the irregular border of the medulla and cortex with intermingling of the two endocrine tissues.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

morphology from human chromaffin cells to any time-lag prior to fixation in glutaraldehyde (24). Evidence suggests that the two populations of norepinephrineor epinephrine-rich chomaffin cells may be controlled separately, enabling variable patterns of secretion of the two catecholamines according to the particular stimulus (25). This differential regulation may be regulated in part by differences in expression of receptors and second messenger systems (e.g., histamine receptors, glucagon receptors, calcium channels) that modulate exocytotic responses to neural input according to the presence of various autocrine, paracrine, and endocrine substances (26). C. Catecholamine Synthesis and Metabolism 1.

Catecholamine Synthesis

The rate-limiting step in catecholamine synthesis involves conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) by the enzyme tyrosine hydroxylase (27) (Fig. 2). Sources of catecholamines are therefore principally dependent on the presence of this enzyme, which is largely confined to dopaminergic and noradrenergic neurons of the central nervous system and to sympathetic nerves and chromaffin cells of the adrenal medulla and extraadrenal chromaffin tissue in the periphery. The DOPA formed by tyrosine hydroxylase is converted to dopamine by L-aromatic amino acid decarboxylase. Dopamine is then translocated into storage vesicles by the vesicular monoamine transporter and converted within vesicles to norepinephrine by dopamine -hydroxylase. The additional actions of phenylethanolamine N-methyltransferase (PNMT), an enzyme mainly found in chromaffin cells of the adrenal medulla, leads to conversion of norepinephrine to epinephrine. Since PNMT has a cytosolic location, synthesis of epinephrine depends on metabolism of norepinephrine that leaks into the cytoplasm from intravesicular sites of norepinephrine synthesis. 2.

Catecholamine Metabolism

Catecholamines are metabolized by a multiplicity of pathways catalyzed by an array of enzymes resulting in wide variety of metabolites (Fig. 3). This has created considerable confusion in the literature about how catecholamines are normally metabolized. As a result, the pathways of catecholamine metabolism described in modern text books and review articles are invariably incorrect and misleading. Most usually norepinephrine and epinephrine are erroneously reported to be deaminated to the acid metabolite, 3,4-dihydroxymandelic acid (DHMA), and from there, directly converted by catechol-O-methyltransferase (COMT) to vanillylmandelic acid (VMA). Deamination of catecholamines by MAO, however, requires one of two other types of enzymes—aldehyde dehydrogenases or aldehyde and aldose reCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Diagram illustrating the catecholamine biosynthetic pathway in an adrenal chromaffin cell. TH, Tyrosine hydroxylase; L-AADC, L-aromatic amino acid decarboxylase; DBH, dopamine -hydroxylase; PNMT, phenylethanolamine-N-methyltransferase.

ductases—to convert the reactive aldehyde intermediates into either acid or glycol deaminated metabolites (28,29). The aldehyde intermediate formed from dopamine is a good substrate for aldehyde dehydrogenase, but not aldehyde or aldose reductase. In contrast, the aldehyde intermediates formed from the -hydroxylated catecholamines, norepinephrine and epinephrine, are good substrates for aldehyde or aldose reductase, but poor substrates for aldehyde dehydrogenase. As a result norepinephrine and epinephrine are both deaminated to 3,4-dihydroxCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3 Pathways of metabolism of catecholamines. Enzymes responsible for each pathway are shown at the head of arrows. Solid arrows indicate the major pathways, whereas dotted arrows indicate pathways of negligible importance. Pathways of sulfate conjugation are not shown. DBH, Dopamine -hydroxylase; PNMT, phenolethanolamineN-methyltransferase; MAO, monoamine oxidase; COMT, catechol-O-methyltransferase; AR, aldose or aldehyde reductase; AD, aldehyde dehydrogenase; ADH, alcohol dehydrogenase; DOPET; 3,4-dihydroxyphenylethanol; DOPAC, 3,4-dihydroxyphenylacetic acid; MOPET, 3-methoxy-4-hydroxyphenyletanol; HVA, homovanillic acid; DHPG, 3,4-dihydroxyphenylglycol; DHMA, 3,4-dihydroxymandelic acid; MHPG, 3-methoxy-4-hydroxyphenylglycol; VMA, vanillylmandelic acid.

yphenylglycol (DHPG), the glycol metabolite. Deamination to the deaminated acid metabolite, DHMA, is not a favored pathway (30,31). DHPG is further Omethylated by COMT in nonneuronal tissues to 3-methoxy-4-hydroxyphenylglycol (MHPG), a metabolite also produced to a limited extent by deamination of normetanephrine and metanephrine (32). As a result of the above differences in substrate utilization, negligible VMA is formed by O-methylation of DHMA. Most VMA is formed by oxidation of MHPG catalyzed by alcohol dehydrogenase, an enzyme located largely in the liver (33,34). A small amount of VMA is also formed from oxidative deamination of normetanephrine and metanephrine. Thus, at least 90% of the VMA formed in the body is produced in the liver, mainly from local uptake and metabolism of circulating DHPG and MHPG (35). With the exception of VMA, all the catecholamines and their metabolites are metabolized to sulfate conjugates by a specific sulfotransferase isoenzyme (SULT1A3). In humans, a single amino acid substitution confers the enzyme with particularly high affinity for dopamine and the O-methylated metabolites of cateCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

cholamines, including normetanephrine, metanephrine, and methoxytyramine (36,37). The SULT1A3 isoenzyme is found in high concentrations in gastrointestinal tissues, which therefore represent a major source of sulfate-conjugated catecholamines and their metabolites (32,38). In humans, VMA and to a lesser extent the sulfate conjugates represent the main end products of norepinephrine and epinephrine metabolism. These metabolic end products are therefore eliminated mainly by urinary excretion, and as a result, their circulatory clearance is slow and plasma concentrations high relative to those of their precursors. 3.

Contribution of the Adrenal Medulla to Catecholamine Turnover

Compared to sympathetic nerves, the adrenal medulla makes a relatively minor contribution to the overall production and turnover of catecholamines. However, catecholamines are synthesized, metabolized, and released by adrenal medullary chromaffin cells differently than in sympathetic nerves (Table 1). Understanding these differences is important for interpretation of plasma and urinary catecholamines and their metabolites in clinical studies of the physiology and pathophysiology of the adrenal medulla. First and foremost, because PNMT is expressed mainly in adrenal chromaffin cells, over 90% of circulating epinephrine is derived from the adrenal medulla (39). This contrasts with circulating norepinephrine, over 90% of which is derived from sympathetic nerves, mostly those innervating mesenteric organs, kidneys, and skeletal muscle. Under resting conditions only about 7% of the norepinephrine and less than 2% of the dopamine released into the circulation are derived from the adrenal medulla. Table 1 Contribution of the Adrenals to Circulating Catecholamines and Metabolites

Catecholamines Epinephrine Norepinephrine Dopamine Metabolites Metanephrine Normetanephrine DHPG DOPAC

Adrenals (pmol/min)

Total body (pmol/min)

Adrenal contribution (%)

979 274 6

1075 3953

290a

91 7 2

449 91 665 300

494 392 13964

4120a

91 23 5 7

Values represent rates of release into the bloodstream derived from published data (39,190,191). a Estimates are for mesenteric organs only and therefore are underestimates of release into the blood stream from all tissues of the body (total body).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Another major difference between sympathetic nerves and adrenal chromaffin cells is that the former contain MAO, while the latter contain both MAO and COMT (Fig. 4). In sympathetic nerves, norepinephrine is metabolized by MAO to DHPG from two sources. At rest, most DHPG is derived from norepinephrine leaking from storage granules into the sympathetic axoplasm, with a smaller proportion derived from transmitter recaptured after release. In contrast, the additional presence of COMT—particularly the membrane-bound form of the enzyme—in adrenal medullary cells means that catecholamines leaking from chomaffin granules are additionally metabolized to the O-methylated metabolites normetanephrine and metanephrine (39–41). The above differences mean that in contrast to circulating DHPG, most of which is derived from sympathetic nerves, at least 90% of metanephrine and up to 40% of normetanephrine are formed from epinephrine and norepinephrine within the adrenals (39,40). This makes the adrenal medulla the single largest source of both normetanephrine and metanephrine in the body, exceeding the contribution of the liver. D. Neuropeptides, Secretory Proteins, and Growth Factors The adrenal medulla produces and secretes a wide variety of peptides, proteins, cytokines, and growth factors. These secretory products influence in both a paracrine and autocrine manner the development and differential function of the entire adrenal gland, including catecholamine-producing adrenal medullary chromaffin cells and adrenocortical steroid–producing cortical cells. 1.

Neuropeptides

Adrenal medullary chromaffin cells produce, store, and secrete a wide array of proteins and neuropeptides, many of which are stored together with the catecholamines in chromaffin vesicles. The first neuropeptides to be discovered in the adrenal medulla were the enkephalins (42). Met-enkephalin and Leu-enkephalin are by number the most prominent neuropeptides in chromaffin vesicles (43). Other neuropeptides that are costored with the adrenal medullary catecholamines include dynorphin, -endorphin, neuropeptide Y, substance P, neurokinin A, vasoactive intestinal peptide, calcitonin gene–related peptide, neurotensin, galanin, atrial natriuretic peptide, pituitary adenylate cyclase–activating peptide, somatostatin, adrenomedullin, proadrenomedullin 20 peptide, corticotropin-releasing hormone, corticotrophin, bombesin, vasopressin, vasotocin, and oxytocin (for review, see Ref. 44). Upon stimulation, these peptides are secreted together with the catecholamines. Many of the peptides are involved in a local, autocrine, or paracrine function in the differential regulation of adrenal medullary function (45,46). In

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 4 Model showing the contribution of the adrenal medulla in relation to other organs and tissues to production of circulating catecholamines and catecholamine metabolites. MAO, Monoamine oxidase; COMT, catechol-O-methyltransferase; SULT1A3, monoamine-preferring sulfotransferase; NE, norepinephrine; EPI, epinephrine; NMN, normetanephrine; MN, metanephrine; DHPG, 3,4-dihydroxyphenylglycol; DHMA, 3,4-dihydroxymandelic acid; MHPG, 3-methoxy-4-hydroxyphenylglycol; VMA, vanillylmandelic acid; NMN-SO4, normetanephrine sulfate; MN-SO4, metanephrine sulfate. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

addition, adrenal medullary secretory products influence adrenocortical steroidogenesis, exerting a predominantly stimulatory effect on steroidogenesis (44,47). 2.

Secretory Proteins

Chromaffin vesicles contain several proteins, including the vesicular monoamine transporters and dopamine–-hydroxylase, the enzyme responsible for conversion of dopamine to norepinephrine (see above). However, the major soluble proteins within chromaffin vesicles belong to the family of granins. This protein family consists of several secretory acidic glycoproteins, namely chromogranin A and B, and secretogranin II, with chromogranin A being the major representative in human adrenal medullary chromaffin vesicles. Multiple roles have been proposed for granins. Intracellularly, granins play a role in targeting peptide hormones and neurotransmitters to the vesicles of the regulated pathway in a pH- and calcium dependent manner (48). Extracellulary, proteolytic fragments of chromogranin A are involved the autocrine and paracrine regulation of hormone secretion, predominantly by inhibiting hormone secretion (for review see Ref. 49). Plasma chromogranin A levels are often increased in patients with pheochromocytoma, providing an alternative to catecholamines and catecholamine metabolites for detection of the tumor (50). Multiple populations of chromaffin cells exist within the medulla, which vary in their transmitter, peptide, and protein composition (for review see Ref. 51). The peptide and protein composition presumably depends on the concerted action of different neurotransmitters released from splanchnic nerves, which could act via various second messenger pathways as shown for acetylcholine and vasointestinal peptide (52), and on the intracellular interactions between different signaling pathways (52–54) and humoral or immune factors (54–58), perhaps of either intra- or extraadrenal origin. 3.

Growth Factors and Cytokines

Chromaffin cells produce and secrete several growth factors and cytokines. These include fibroblast growth factors, transforming growth factor  (TGF-) insulinlike growth factor (ILGF), interleukins 1 and 6 (IL-1, IL-6), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNT), and neurotropins (for review see Ref. 59). While some of these factors, such as pre-pro-TGF-, contain a signal peptide that makes it exportable to chromaffin granules and therefore released via the regulated pathway (59), others such as fibroblast growth factors are located in the nucleus or the cytoplasm (60). As in other tissues, the local production and release of these growth factors are involved in the development of the adrenal gland and maintenance of its function.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

E. Cellular Crosstalk of Chromaffin and Cortical Cells In the adult tetrapod adrenal gland, the steroid-producing cortical cells and the catecholamine-producing chromaffin cells are united in one gland. Among many nonmammalian species the combination of these two cell types varies. The chromaffin cells may be distributed in islets, as in amphibians and birds, or they can

Figure 5 Normal ultrastructure of rat chromaffin cells with characteristic secretory granules (magnification 8200).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

even be concentrated into one pole of the gland as in reptiles. In mammals, the conventional view is that the two different endocrine tissues are distinctly separated into an outer steroid-producing cortex and a central medulla. However, this is an oversimplification and the distribution of these cells is not be so clearly demarcated, but instead involves closer contact than often considered (Fig. 5). The presence of medullary cells in the zona glomerulosa was noted many years ago, but generally ignored. In most mammalian species, including humans, it is now accepted that adrenal chromaffin cells can be found in the adult adrenal cortex, either radiating through the cortex from the medulla or as islets or single cells. Occasionally such chromaffin cells may form larger cellular groups in the subcapsular region. Additionally, cortical cells can be found in the medulla, where they either form islets surrounded by chromaffin tissue or are in contact with the rest of the cortex. The adrenal medulla, in some parts, appears to be sprinkled with cortical cells (Fig. 5). This intimate intermingling of the two different cell types allows extensive contact zones for paracrine interaction. The control of epinephrine synthesis by adrenocortical steroids was first demonstrated by Wurtman and Axelrod in 1966 (61). In 1980 Carballeira and Fishman presented the hypothesis of an “adrenal functional unit” (62), but the interaction of the two adrenal stress systems was not studied in more detail until over 10 years later. The concept of a “neuro-adrenocortical axis” was introduced with findings that adrenocortical steroidogenesis could be controlled by the splanchnic nerves (63). Close intermingling and cellular contact zones between the chromaffin and cortical cell system in the human adrenal gave further support for crosstalk between the two systems (64). The adrenal medulla and cortex therefore exist in close functional interdependence. Secretory products of chromaffin cells regulate adrenocortical steroidogenesis, and steroids produced in the adrenal cortex influence the development and functioning of the adrenal medulla.

II. PATHOPHYSIOLOGY OF THE ADRENAL MEDULLA A. Age, Gender, and Obesity With advancing age the adrenal medulla increases in size, both in absolute mass and in proportion to the total adrenal mass (18). Increased age is also associated with a more irregular cortico-medullary border and increased intermingling of medullary and cortical cells. In contrast to the age-associated increase in adrenal medullary mass and the well-documented increases in sympathetic nerve firing and plasma norepinephrine concentrations, release of epinephrine by the adrenal medulla is unaffected or decreases with advancing age (65–68). Most studies examining epinephrine release have shown no age-related change in plasma epinephrine concentrations. Interpretation of these studies is, however, confounded by the anteCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

cubital venous site typically used for blood sampling and age-related changes in circulatory clearance of catecholamines. Due to highly variable local extraction, concentrations of epinephrine in forearm venous plasma samples vary widely between 20 to 80% those in arterial plasma. Sampling of arterial or central mixed venous blood is therefore superior for assessment of adrenal medullary epinephrine secretion (69). Moreover, decreased circulatory clearance of epinephrine with advancing age (66,70) results in an increase in plasma epinephrine concentrations that occurs independently of changes in epinephrine secretion by the adrenals. Using catecholamine radiotracer kinetic analyses and sampling from arterial blood to obviate the above confounding variables, Esler and colleagues showed that aging is associated with a decrease in adrenal epinephrine release that may not be apparent from measurements of plasma concentrations (66). Studies employing either measurements of urinary or plasma catecholamines have consistently indicated lower adrenal medullary release of epinephrine in females than males and no consistent difference in plasma levels of norepinephrine (40,71–74) (Fig. 6). The influence of gender on adrenal medullary epinephrine release is associated with a smaller size of the adrenal medulla in women than in men (75). Findings that plasma concentrations of metanephrine— a metabolite of epinephrine produced mainly in the adrenal medulla—are also lower in women than in men further indicate lower adrenal medullary stores of epinephrine in females than in males (40). Differences in body weight also influence the release of catecholamines from sympathetic nerves and the adrenal medulla in divergent directions. The sympathetic nervous system is activated by increased body mass index, a response that is implicated in the increase in blood pressure and other cardiovascular complications that accompany obesity (76). In contrast, increased body mass or adiposity is associated with decreased adrenal medullary secretion of epinephrine,

Figure 6 Plasma concentrations of epinephrine and metanephrine in males and females. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

and this may participate in the changes in fatty acid and energy metabolism that accompany and contribute to obesity (77–81). The above considerations of age, gender, and body weight illustrate how sympathetic and adrenal medullary systems respond heterogeneously to different influences. Importantly, the divergent influences of age, gender, and body weight on adrenal medullary and sympathoneural function are to some extent interdependent and likely contribute complexly with other hormonal systems to the pathology of disorders such as hypertension and diabetes. The age-associated activation of the sympathetic nervous system, for example, is at least in part related to age-dependent changes in body mass and differs according to gender (82,83). Similarly, age- and gender-associated differences in epinephrine release may also be related complexly to changes in body mass or differences in obesity and insulin resistance (80,81). Associated changes in adrenoceptor responsiveness to catecholamines add a further level of complexity to interactions of age, gender, and obesity in modulating the functional role of adrenal medulla and its contribution to disease pathology (70,84–87). B. Essential Hypertension Given the above associations of obesity and aging with heightened sympathetic outflow, but lowered adrenal medullary epinephrine release, it might be expected that essential hypertension, or at least obesity-induced hypertension, would be associated with increased norepinephrine and decreased epinephrine release. In line with the above considerations, increased sympathetic outflow is a well-established finding in essential hypertension (88,89). Additionally, at least one study has indicated that lower plasma concentrations of epinephrine in hypertensive than in normotensive individuals are dependent on differences in body mass index (74). Aside from this, however, most evidence indicates that essential hypertension is associated with increased adrenal medullary epinephrine release and not decreased release (40,90–94). Essential hypertension has a complex and poorly understood multifactorial etiology. In some patients, high blood pressure appears to have a clear hypernoradrenergic basis, whereas in others a contribution of the sympathetic nervous system is less clear (95,96). Similarly, any overall increase in plasma concentrations of epinephrine in hypertension likely reflects the presence of subgroups of patients with adrenal medullary activation. In these subgroups increased levels of circulating epinephrine may contribute to development of the disorder. Interest in the involvement of the adrenal medulla in the pathogenesis of essential hypertension stems from findings that epinephrine acts potently on presynaptic 2-adrenoceptors to stimulate release of norepinephrine from sympathetic nerve endings (97,98). As hypothesized from these findings of hypertension, stress-induced activation of the adrenal medulla leads to not only increased levels of circulating epinephrine, but also increased uptake and storage of epinephrine by Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

sympathetic nerves. The resulting increase in epinephrine coreleased with norepinephrine by sympathetic nerves leads to enhanced activation of presynaptic 2adrenoceptors, resulting in facilitation of transmitter release and enhanced vasoconstrictor responses to sympathetic nerve firing. Support for the epinephrine hypothesis of hypertension is provided by findings of co-release epinephrine by sympathetic nerves (99,100) and epinephrine infusion-induced increases in plasma norepinephrine and blood pressure that can be attenuated by -adrenoceptor blockade (93,101–103). As previously reviewed, the epinephrine hypothesis of hypertension remains unproved and has been a difficult hypothesis to test (104,105). If correct, it seems likely that expression of hypertension by this mechanism may be restricted to specific subgroups of individuals with altered adrenal medullary function or responsiveness to stress or sensitivity to activation of presynaptic adrenoceptors. C. Panic Disorder An abnormality in autonomic nervous function has long been hypothesized as a primary etiological factor in panic disorder. While panic attacks undoubtedly involve sympathoadrenal activation, whether such attacks reflect a primary underlying disturbance of autonomic function remains unclear. Tests of sympathoadrenal function, involving cardiovascular and catecholamine responses to change of posture, isometric exercise, and cold pressor testing, have failed to reveal any abnormalities in patients with panic disorder (106,107). Isolated findings of higher plasma concentrations of epinephrine and little difference in plasma norepinephrine suggest that panic disorder may be associated with selective activation of the adrenal medulla (108,109). For the most part, however, studies examining resting plasma concentrations or urinary excretion of catecholamines in patients with panic disorder have yielded inconsistent results (106,107,110–113). A more recent study utilizing direct sympathetic nerve recording and radiotracer-dilution methodology to estimate whole body and regional rates of catecholamine release into the bloodstream revealed no differences in resting sympathetic nerve activity (114). Baseline release of epinephrine was, however, increased in a subset of patients with panic disorder, but as a group this did not reach significance. Interestingly, however, resting release of epinephrine from the heart was substantially increased in patients with panic disorder. Since cardiac levels of epinephrine provide a temporally integrated index of adrenal release of epinephrine into the bloodstream (115), the elevated cardiac release of epinephrine in panic patients presumably reflects previous sympathoneural uptake from the circulation of epinephrine released during attacks. In patients experiencing a panic attack at the time of study, total body epinephrine release was found to increase dramatically, whereas there were small and variable changes in sympathetic outflow (114). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The above pattern of changes in sympathoneural and adrenal medullary activity during a panic attack resemble the pattern of predominantly adrenal medullary activation observed normally in response to psychological stress (116,117). Other findings of reduced -adrenoceptor responsiveness in panic patients (113,118) indicate that altered end-organ sensitivity to catecholamines is unlikely to contribute to susceptibility to panic, but may reflect receptor downregulation in response to panic-associated adrenal medullary activation. Rather than an underlying abnormality in autonomic or adrenal medullary function, the available data suggest that the core abnormality in panic disorder most likely involves inappropriate central nervous system processing of information from the environment. This conclusion is supported by findings that panic attacks can be easily precipitated in susceptible individuals by procedures otherwise relatively innocuous to normal individuals (119). Nevertheless, panic-induced adrenal medullary activation, resulting in a loading of sympathetic stores with epinephrine and associated effects on the cardiovascular system, may represent an important link in any excess mortality associated with panic disorder (120,121). D. Diabetes Due to the important actions of circulating epinephrine in blood glucose homeostasis, changes in function of the adrenal medulla in diabetes are critical to the progression of complications and associated disease pathology. Deficient glucagon counterregulatory responses to hypoglycemia, particularly in type I diabetes, are compensated for in the early stages of the disease by enhanced epinephrine responses (122,123). Impaired function of the adrenal medulla, and more specifically attenuated responsiveness to hypoglycemia, appears as an important complicating feature, generally occurring in the later stages of both type I and type II diabetes (124–127). In such patients the additional deficiency in epinephrine secretory responses can lead to frequent, severe, and prolonged hypoglycemia. Because of the potent physiological and psychological arousing effects of circulating epinephrine, impaired adrenal medullary function is believed to be a major contributing factor to hypoglycemia unawareness in diabetes (128). Impaired secretion of epinephrine in diabetes is well documented in diabetic autonomic neuropathy (127,129,130). Despite the association, there has been controversy over whether the neuropathy is the cause of impaired epinephrine secretion or reflects a separate aspect of the disease process (128,131). Intact epinephrine responses to exercise despite deficient responses to insulin hypoglycemia indicate that the defect is stimulus specific (125). Other mechanisms may be involved, including failure of central glucoreceptors to recognize hypoglycemia and activate the adrenal medulla. The presence of autoantibodies to adrenal medullary cells in some patients with type I diabetes suggests that autoimmune reactions may not be confined to

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

islet cells of the pancreas (132,133). An autopsy study confirmed the presence of fibrotic degenerative changes in adrenal medullae of patients with type 1 diabetes, further suggesting that the adrenal medulla may be another immunological target in this form of diabetes (134). Thus, rather than resulting from generalized neuropathy of the sympathoadrenal system, impaired adrenal medullary function in at least some type I diabetic patients may also reflect a similar pathological process responsible for damage to the pancreas. E. Autonomic Failure The most important debilitating clinical manifestation of autonomic failure syndromes, such as pure autonomic failure or multiple system atrophy, is orthostatic hypotension. In its most severe form, the patient is unable to remain in the upright posture for even a few minutes. The basis of the disorder involves failure of neurogenic vasoconstrictor responses secondary to defective sympathoneural release of norepinephrine. In pure autonomic failure, the lesion is postganglionic, involving degeneration of sympathetic nerves and lack of norepinephrine release, whereas in multiple system atrophy, sympathetic nerves are present but do not release norepinephrine appropriately due to a preganglionic lesion (135). There are also many other forms of autonomic failure involving sympathetic nerves, some of which have a genetic basis, some of which are caused by nutritional deficiencies, toxins, or drugs, and others that have an immunological or inflammatory basis. Because of the importance of sympathetically mediated vasoconstrictor responses to blood pressure homeostasis and because the adrenal medulla plays a negligible role in these responses, little attention has focused on the involvement of the adrenals in autonomic failure. However, similar to the pattern for norepinephrine, baseline plasma concentrations of epinephrine are normal in patients with multiple system atrophy and reduced in those with pure autonomic failure (136,137). This suggests that in pure autonomic failure the lesion not only involves loss of sympathetic nerve endings but also degeneration of the adrenal medulla, whereas in multiple system atrophy the adrenal medulla remains intact. However, in all forms of autonomic failure there can be impaired release of epinephrine in response to hypoglycemia (138–140). F. Disorders of Adrenocortical-Medullary Function 1.

Adrenocortical Insufficiency

In the primary adrenocortical insufficiency of Addison’s disease resulting from tuberculosis, the pathology involves destruction of the entire adrenal gland, including the medulla (141). Accordingly, this disorder includes impaired adrenal medullary secretion of epinephrine (142,143). In Addison’s disease, due to an auCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

toimmune adrenalitis of the adrenal cortex, the medulla is intact but plasma levels of epinephrine are decreased (144). This occurs despite glucocorticoid replacement, indicating that normal intraadrenal steroid levels are required for an adequate production of catecholamines in the human adrenal medulla (144). Epinephrine secretion is also impaired in secondary adrenocortical insufficiency due to deficiency of ACTH in hypopituitary children (145), further supporting the importance of a local source of steroids for adrenal medullary release of catecholamines. Secondary adrenocortical insufficiency may also result from exogenous glucocorticoid administration. The mechanism involves suppression of intraadrenal cortisol production through negative feedback of the hypothalamopituitary-adrenocortical axis. Thus, in rats chronic glucocorticoid treatment results in inhibition of adrenal medullary epinephrine secretion (146). Similarly, findings of low epinephrine levels in severe asthma patients treated with glucocorticoids may be explained by iatrogenic adrenocortical insufficiency (147). Similar impairment of adrenal medullary function might be expected in other patients on glucocorticoid treatment regimens, ranging from obstructive lung disease, inflammatory bowel disease, rheumatoid arthritis, to tumors. Since epinephrine plays a role in bronchodilatation and may have other critical physiological effects in the above diseases, it appears reasonable to consider possible confounding effects of impaired adrenal medullary function in patients exposed to glucocorticoid treatment. 2.

21-Hydroxylase Deficiency

21-Hydroxylase deficiency is characterized biochemically by reduced cortisol synthesis accompanied by a build-up and shunting of steroid precursors towards androgen production. The more severe cases also have aldosterone deficiency. These biochemical changes result in clinical symptoms of virilism with or without salt-wasting. In times of stress, patients may also experience episodes of dehydration, hypotension, and hypoglycemia. In a naturally occurring animal model of 21-hydroxylase deficiency, the above abnormalities in adrenal steroid synthesis were associated with impaired adrenal chromaffin cell function, including marked decreases in adrenal expression of PNMT and adrenal tissue levels of catecholamines (148). Similarly, patients with severe 21-hydroxylase deficiency had markedly decreased urinary excretion and plasma concentrations of epinephrine associated with incomplete formation of the adrenal medulla (149) (Fig. 7). These patients also had lowered plasma concentrations of metanephrine, consistent with the decrease in adrenal medullary stores of epinephrine found in the animal model of the disorder. The decreased plasma metanephrine levels correlated with the severity of the genetic defect and with the frequency of hospitalizations due to adrenal crises. The above findings raise the possibility that impaired adrenal medullary secretion of Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 7 Plasma concentrations of epinephrine and metanephrine in patients with simple virilizing (VZ) and more severe salt-wasting (SW) forms of 21-hydroxylase deficiency, compared to patients with bilateral adrenalectomy (AX) and age- and gender-matched control subjects (CT). (From Ref. 149.)

epinephrine may contribute to the cardiovascular instability and hypoglycemia observed in patients with the severe form of this disease. G. Pheochromocytoma Pheochromocytoma represents the most well-known disease of the adrenal medulla. A small proportion (10–15%) of these tumors also arise from extraadrenal chromaffin tissue (e.g., organs of Zuckerkandl). Pheochromocytomas are characterized by production of catecholamines, usually leading to hypertension and symptoms of catecholamine excess. The tumors are rare, occurring in less than 0.1% of patients with hypertension, with only about 2–5 cases detected annually per million population. Most pheochromocytomas are benign, and less than 15% are malignant. Thus, surgery provides effective treatment for most patients with pheochromocytoma, whereas left untreated the tumor can have catastrophic consequences for the patient. This and the high incidence of hypertension among the adult population means that pheochromocytomas are clinically important tumors that must be considered in large numbers of patients. However, due to the rarity of the tumor, pheochromocytoma remains an overlooked and underdiagnosed clinical entity. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

1. Structure and Morphology Pheochromocytomas are often encapsulated and on cross section are usually sharply circumscribed. Pheochromocytomas are highly vascular neoplasms, often appearing with hemorrhagic areas and, in large tumors, frequently showing necrotic or cystic degeneration. The microsopic appearance includes a mixture of alveolar (nesting or “zellballen”) patterns and trabecular arrangements of cells, identified as neurosecretory by their granulated catecholamine-containing vesicles. Pheochromocytoma cells can be identified immuno-histochemically by the presence of neuroendocrine tumor markers such as chromogranin A (Fig. 8). On the ultrastructural

Figure 8 Human pheochromocytoma stained with anti-chromogranin A illustrating characteristic appearance with tumor cells forming nests (Zellbullen) or cords separated by fibrovascular stroma and showing numerous small vascular channels.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

level, pheochromocytoma tumor cells resemble normal chromaffin cells. However, in contrast to the normal human adrenal medulla, pheochromocytomas usually contain more norepinephrine than epinephrine, but these proportions are highly variable and correlate well with different populations of secretory granules within tumor cells (150,151). A distinction between benign and malignant tumor cells is not possible on the basis of currently available histological or structural criteria. The distinction between benign and malignant pheochromocytomas is normally based on the presence of metastasis. 2.

Clinical Presentation

The presence of pheochromocytoma is usually indicated by clinical signs and symptoms that reflect the hemodynamic and metabolic actions of circulating catecholamines (Table 2). Hypertension is the most common sign and can be sustained or paroxysmal, but is usually labile. Symptoms include headache, palpitations, diaphoresis, palor, dyspnea, nausea, attacks of anxiety, and generalized weakness (152–154). Signs and symptoms that occur in paroxysms presumably reflect episodic catecholamine hypersecretion. Paroxysmal attacks may last from a few seconds to several hours, with intervals between attacks varying widely and as infrequent as once every few months.

Table 2 Clinical Symptoms and Signs of Sporadic and Familial Pheochromocytoma

Signs Hypertension sustained paraxysmal Symptoms Headache Diaphoresis Palpitations or tachycardia Anxiety or nervousness Nausea Tiredness or fatigue Dizziness Constipation

Sporadic

Familial

95 50 50

26 13 13

82 65 60 40 35 30 7 7

28 25 25 19 6 9 9 3

Numbers reflect percent of patients exhibiting signs and symptoms. Data in familial pheochromocytoma are from patients in whom the tumor was detected by periodic screening. Source: Adapted from Refs. 154, 167.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Although headache, palpitations, and sweating are nonspecific symptoms, they are frequently encountered in pheochromocytoma so that their associated presence with hypertension should arouse immediate suspicion of the tumor. Paroxysmal attacks can be precipitated by a diverse array of stimuli, including physical activity, administration of certain medications, and ingestion of foods, and during medical and diagnostic procedures (e.g., anesthesia, childbirth, endoscopy, radiographical contrast agents). Thus, paroxysmal attacks precipitated by the above or other conditions should also arouse a suspicion of pheochromocytoma. 3.

Hereditary Pheochromocytoma

Most pheochromocytomas are sporadic, but an important proportion, perhaps as much as 20%, occur in three known familial tumor syndromes: multiple endocrine neoplasia type 2 (MEN 2), von Hippel-Lindau (VHL) syndrome, and neurofibromatosis type 1. These disorders are inherited in an autosomal dominant fashion and are characterized by specific clinical manifestations. Patients with MEN 2a have a predisposition to medullary thyroid cancer and parathyroid disease, and those with the rarer form of MEN 2b are predisposed to additional cutaneous and mucosal neuromas. In VHL syndrome, family-specific mutations determine the varied clinical presentation of tumors, including retinal angiomas, central nervous system hemangioblastomas, pheochromocytomas, and tumors in the kidneys, pancreas, and epididyma (155). Pheochromocytomas in MEN 2 and VHL patients occur at an earlier age and present more frequently as bilateral or multicentric tumors than those in patients with sporadic pheochromocytoma (156). When pheochromocytomas are found by routine screening in familial settings, the patient is often normotensive and asymptomatic (157,158) (Table 2). Thus, clinicians should not rely on the presentation of the usual signs and symptoms before carrying out biochemical testing in such patients. Rather the high frequency of pheochromocytoma in MEN 2 and VHL patients makes it important to test for the tumor periodically as part of a routine screening and surveillance plan (156,159). Although neurofibromatosis type 1 is the most common familial cancer syndrome predisposing to pheochromocytoma, the risk of the tumor in this disorder is small, from 0.1 to 5.7% (160,161). Nevertheless, screening for pheochromocytoma in patients with von Recklinghausen’s disease and hypertension or before provocative procedures or pregnancy appears justified. 4.

Biochemical Phenotypes

Most pheochromocytomas secrete predominantly norepinephrine, many produce both norepinephrine and epinephrine, and more rarely others secrete predominantly epinephrine (162,163) (Fig. 9). Pheochromocytomas that secrete mainly dopamine have been described, but are extremely rare (164,165). These differ-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 9 Ratios of concentrations of epinephrine to norepinephrine (EPI/NE) in pheochromocytoma tumor tissue (upper panel) and plasma (middle panel) and of metanephrine to normetanephrine (MN/NMN) in plasma from patients with sporadic pheochromocytoma (sporadic) compared to familial pheochromocytoma due to von Hippel-Lindau syndrome (VHL) or multiple endocrine neoplasia type 2 (MEN 2). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ences in catecholamine secretion reflect differences in expression of catecholamine biosynthetic enzymes and can explain differences in presenting symptoms (166,167). Paroxysmal hypertension and symptoms such as palpitations, anxiety, dyspnea, and hyperglycemia are more common in patients with pheochromocytomas producing epinephrine than norepinephrine (163,168,169). Pheochromocytomas in patients with MEN 2 produce epinephrine and have an adrenergic phenotype, while those from VHL patients have a distinctive noradrenergic phenotype (167). Thus, differences in biochemical and clinical presentation of pheochromocytoma can reflect the underlying mutation. Presumably because expression of PNMT is dependent on high local concentrations of glucocorticoids, pheochromocytomas that produce comparatively large amounts of epinephrine typically have an adrenal location, whereas those in extraadrenal locations usually produce predominantly norepinephrine. Nevertheless, about a third of adrenal pheochromocytomas show a distinctly noradrenergic biochemical phenotype with very little epinephrine. Also, recurrent tumors with an adrenergic phenotype are occasionally found at extraadrenal locations. These exceptions suggest that rather than a continued proximity to adrenal steroids, the adrenergic or noradrenergic phenotype of a tumor may be dependent on development from specific noradrenergic or adrenergic subtypes of chromaffin cells. Perhaps because of the extraadrenal nature of metastases, malignant pheochromocytomas are typically noradrenergic. However, malignant pheochromocytomas are also often characterized by high tissue, plasma, and urinary levels of DOPA and dopamine (170–173) suggesting a biochemically dedifferentiated state. While elevations of plasma or urinary DOPA and dopamine are not in themselves particularly sensitive or specific markers of either pheochromocytoma or metastatic disease, when accompanied by elevations in plasma norepinephrine or other clinical evidence of pheochromocytoma, such elevations should arouse immediate suspicion of metastatic disease. 5.

Biochemical Diagnosis

Diagnosis of pheochromocytoma typically requires biochemical evidence of excessive catecholamine production by the tumor. This is usually achieved from measurements of catecholamines and catecholamine metabolites in urine or plasma (Table 3). With consideration of the potential dangers of a pheochromocytoma and the rarity of the tumor, the most important consideration in choice of initial biochemical test is the reliability of the test for exclusion of pheochromocytoma. Therefore, a suitably sensitive biochemical test remains the first choice in the initial work-up of the patient suspected to be harboring a pheochromocytoma. Measurements of plasma free metanephrines offer several advantages over other commonly available tests for diagnosis of pheochromocytoma (39–41). The larger contribution of the adrenal medulla than sympathetic nerves to circulating levels of free metanephrines compared to other analytes, including the parent Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Table 3 Sensitivity and Specificity of Biochemical Tests for Diagnosis of Pheochromocytoma Biochemical test Plasma metanephrines Plasma catecholamines Urinary catecholamines Urinary metanephrines Urinary vanillylmandelic acid

Sensitivity (%) (n  151)

Specificity (%) (n  349)

99 85 83 76 63

89 80 88 94 94

The sensitivities of tests of plasma metanephrines or plasma and urinary catecholamines were determined as the percentage of patients with pheochromocytoma with positive test results for either normetanephrine or metanephrine (i.e., for tests of plasma metanephrines) or with positive test results for either norepinephrine or epinephrine (i.e., for tests of plasma or urinary catecholamines). The specificities of tests of plasma metanephrines or plasma and urinary catecholamines were determined as the percentage of patients without pheochromocytoma with negative test results for both normetanephrine or metanephrine or with negative test results for both norepinephrine or epinephrine. The sensitivities and specificities of tests of urinary metanephrines reflect tests of urinary total metanephrines (i.e., the combined sum of free plus conjugated normetanephrine and metanephrine). Source: Ref. 178.

amines, means that pheochromocytomas produce proportionally larger increases above normal of plasma free metanephrines than of catecholamines or other metabolites. Moreover, the continuous production of metanephrines from catecholamines leaking from chromaffin granules of pheochromocytoma tumor cells makes measurements of metanephrines particularly useful for detection of tumors that secrete catecholamines intermittently or in low amounts. Because of the above factors, measurements of plasma free metanephrines offer a highly sensitive test with normal test results reliably excluding the presence of all but the smallest of familial pheochromocytomas (174–178). Where excluded, no other tests are necessary. However, unless of sufficient magnitude, elevations of plasma free metanephrines alone do not reliably prove or establish a pheochromocytoma. Similar to catecholamines, elevations in plasma free metanephrines and other metabolites may accompany a variety of other conditions or disease states. Because of this, most initial positive biochemical test results, particularly when elevations are marginal or mild, typically require follow-up tests, which may include clonidine suppression and glucagon stimulation (179,180). Use of the clonidine suppression test is particularly useful when there is suspicion that elevated plasma levels of norepinephrine are secondary to sympathetic activation rather than a pheochromocytoma. Lack of decrease of norepinephrine after clonidine or large increases in norepinephrine after glucagon provide strong evidence of a pheochromocytoma. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

6.

Tumor Localization

When attempting to locate a suspected pheochromocytoma, it is important to recognize the advantages and limitations of available imaging modalities and carry out these tests appropriately. A computed tomography (CT) or magnetic resonance imaging (MRI) scan of the entire abdomen is immediately appropriate when suspicion of a pheochromocytoma is strongly increased by the results of biochemical testing. CT has good sensitivity for detecting adrenal tumors (93–100%), particularly those larger than 1 cm, but is less sensitive for detecting extraadrenal tumors, especially those smaller than 2 cm. MRI has equal or lower sensitivity compared to CT for detecting adrenal tumors but is superior to CT for detecting extraadrenal tumors (181–183). Both CT and MRI, however, have inadequate specificity to positively identify a mass as a pheochromocytoma. A finding of an adrenal mass by CT or MRI, together with biochemical evidence and clinical signs and symptoms of a pheochromocytoma, might be all that is needed to justify surgery. However, just as failure to find an adrenal mass does not rule out a pheochromocytoma, the finding of an adrenal mass by CT or MRI does not necessarily indicate a pheochromocytoma. In both cases it may be appropriate to follow up with a different imaging modality, such as whole body MRI (for detection of extraadrenal tumors) or MIBG scintigraphy (for more specific detection of adrenal and extraadrenal tumors). Because of uptake by transporters localized to sympathetic neurons and chromaffin cells, MIBG scintigraphy has high specificity (95–100%) but is not highly sensitive (184–186). Sensitivity is increased using 123I-MIBG rather than 131 I-MIBG as the imaging agent. However, in the United States the former agent is not commercially available and is restricted to a few medical centers (187). Promising new methods for specific localization of pheochromocytoma include positron emission tomography (PET) coupled with agents such as 11C-hydroxyephidrine (188) or 18F-6-fluorodopamine (177), both substrates of the norepinephrine transporter expressed by chromaffin cells. In contrast, use of 18 F-fluorodeoxyglucose for locating a variety of tumors, including pheochromocytoma, depends on the high metabolic activity of such tumors (189). In rare cases where imaging studies are all negative, but where suspicion of a pheochromocytoma remains high, it may be appropriate to consider a vena caval sampling procedure to establish the source of the high circulating levels of catecholamines or free metanephrines.

REFERENCES 1.

Takamine J. The isolation of the active principle of the suprarenal gland. J Physiol 1901; 27:30P–39P. 2. Cannon WB. The Wisdom of the Body. New York: WW Norton, 1939.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

3.

4.

5. 6. 7.

8. 9.

10. 11. 12. 13.

14.

15.

16.

17. 18. 19. 20. 21.

von Euler US. A specific sympathomimetic ergone in adrenergic nerve fibres (sympathin) and its relations to adrenaline and nor-adrenaline. Acta Physiol Scand 1946; 12:73–96. Robertson DA, Johnson GA, Robertson RM, Nies AS, Shand DG, Oates JA. Comparative assessment of stimuli that release neuronal and adrenomedullary catecholamines in man. Circulation 1979; 59:637–643. Axelrod J, Kopin IJ. The uptake, storage, release and metabolism of noradrenaline in sympathetic nerves. Prog Brain Res 1969; 31:21–32. Ahlquist RP. A study of adrenotropic receptors. Am J Physiol 1948; 153:586–600. Hjemdahl P, Belfrage E, Daleskog M. Vascular and metabolic effects of circulating epinephrine and norepinephrine. Concentration-effect study in dogs. J Clin Invest 1979; 64:1221–1228. Warren JB, Dalton N. A comparison of the bronchodilator and vasopressor effects of exercise levels of adrenaline in man. Clin Sci 1983; 64:475–449. Freyschuss U, Hjemdahl P, Juhlin-Dannfelt A, Linde B. Cardiovascular and sympathoadrenal responses to mental stress: influence of beta-blockade. Am J Physiol 1988; 255:H1443–H1451. Macdonald IA, Bennett T, Fellows IW. Catecholamines and the control of metabolism in man. Clin Sci 1985; 68:613–669. Cryer PE. Adrenaline: a physiological metabolic regulatory hormone in humans? Int J Obes Relat Metab Disord 1993; 17(suppl 3):S43–46. Warren J. The adrenal medulla and the airway. Br J Dis Chest 1986; 80:1–6. Cryer PE, Tse TF, Clutter WE, Shah SD. Roles of glucagon and epinephrine in hypoglycemic and nonhypoglycemic glucose counterregulation in humans. Am J Physiol 1984; 247:E198–205. Hoelzer DR, Dalsky GP, Schwartz NS, Clutter WE, Shah SD, Holloszy JO, Cryer PE. Epinephrine is not critical to prevention of hypoglycemia during exercise in humans. Am J Physiol 1986; 251:E104–E110. Lenders JW, Peters JH, Pieters GF, Willemsen JJ, Thien T. Hemodynamic reactivity to sympathoadrenal stimulation in adrenalectomized women. J Clin Endocrinol Metab 1988; 67:139–143. Howlett K, Galbo H, Lorentsen J, Bergeron R, Zimmerman-Belsing T, Bulow J, Feldt-Rasmussen U, Kjaer M. Effect of adrenaline on glucose kinetics during exercise in adrenalectomised humans. J Physiol 1999; 519 Pt 3:911–921. Coupland RE. Post-natal fate of the abdominal para-aortic bodies in man. J Anat 1954; 88:455–464. Kreiner E. Weight and shape of the human adrenal medulla in various age groups. Virchows Arch A Pathol Anat Histol 1982; 397:7–15. Padbury JF. Functional maturation of the adrenal medulla and peripheral sympathetic nervous system. Baillieres Clin Endocrinol Metab 1989; 3:689–705. Coupland RE, Selby JE. The blood supply of the mammalian adrenal medulla: a comparative study. J Anat 1976; 122:539–551. Murakami T, Oukouchi H, Uno Y, Ohtsuka A, Taguchi T. Blood vascular beds of rat adrenal and accessory adrenal glands, with special reference to the corticomedullary portal system: a further scanning electron microscopic study of corrosion casts and tissue specimens. Arch Histol Cytol 1989; 52:461–476.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

22. 23. 24.

25.

26. 27. 28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

Hillarp NA, Hökfelt B. Evidence of adrenaline and noradrenaline in separate adrenal medullary cells. Acta Physiol Scand 1953; 30:55–68. Brown WJ, Barajas L, Latta H. The ultrastructure of the human adrenal medulla: with comparative studies of white rat. Anat Rec 1971; 169:173–183. Hansen JT, Notter MF, Okawara SH, Gash DM. Organization, fine structure, and viability of the human adrenal medulla: considerations for neural transplantation. Ann Neurol 1988; 24:599–609. Vollmer RR. Selective neural regulation of epinephrine and norepinephrine cells in the adrenal medulla—cardiovascular implications. Clin Exp Hypertens 1996; 18:731–751. Aunis D, Langley K. Physiological aspects of exocytosis in chromaffin cells of the adrenal medulla. Acta Physiol Scand 1999; 167:89–97. Nagatsu T, Levitt M, Udenfriend S. Tyrosine hydroxylase, the initial step in norepinephrine biosynthesis. J Biol Chem 1964; 239:2910–2917. Kawamura M, Eisenhofer G, Kopin IJ, Kador PF, Lee YS, Tsai JY, Fujisawa S, Lizak MJ, Sinz A, Sato S. Aldose reductase, a key enzyme in the oxidative deamination of norepinephrine in rats. Biochem Pharmacol 1999; 58:517–524. Eisenhofer G, Lamensdorf I, Kirk KL, Kawamura M, Sato S. Oxidative deamination of monoamines and biogenic aldehydes in neurodegeneration. In: Creveling CR, ed. Role of Quinone Species in Cellular Toxicity. Johnson City, TN: F.P. Graham, 2000:103–145. Eisenhofer G, Goldstein DS, Stull R, Ropchak TG, Keiser HR, Kopin IJ. Dihydroxyphenylglycol and dihydroxymandelic acid during intravenous infusions of noradrenaline. Clin Sci 1987; 73:123–125. Kawamura M, Kopin IJ, Kador PF, Sato S, Tjurmina O, Eisenhofer G. Effects of aldehyde/aldose reductase inhibition on neuronal metabolism of norepinephrine. J Auton Nerv Syst 1997; 66:145–148. Eisenhofer G, Pecorella W, Pacak K, Hooper D, Kopin IJ, Goldstein DS. The neuronal and extraneuronal origins of plasma 3-methoxy-4-hydroxyphenylglycol in rats. J Auton Nerv Syst 1994; 50:93–107. Blombery PA, Kopin IJ, Gordon EK, Markey SP, Ebert MH. Conversion of MHPG to vanillylmandelic acid. Implications for the importance of urinary MHPG. Arch Gen Psychiatry 1980; 37:1095–1098. Märdh G, Anggärd E. Norepinephrine metabolism in man using deuterium labelling: origin of 4-hydroxy-3-methoxymandelic acid. J Neurochem 1984; 42:43–46. Eisenhofer G, Aneman A, Hooper D, Rundqvist B, Friberg P. Mesenteric organ production, hepatic metabolism, and renal elimination of norepinephrine and its metabolites in humans. J Neurochem 1996; 66:1565–1573. Dajani R, Cleasby A, Neu M, Wonacott AJ, Jhoti H, Hood AM, Modi S, Hersey A, Taskinen J, Cooke RM, Manchee GR, Coughtrie MW. X-ray crystal structure of human dopamine sulfotransferase, SULT1A3. Molecular modeling and quantitative structure-activity relationship analysis demonstrate a molecular basis for sulfotransferase substrate specificity. J Biol Chem 1999; 274:37862–37868. Dajani R, Sharp S, Graham S, Bethell SS, Cooke RM, Jamieson DJ, Coughtrie MW. Kinetic properties of human dopamine sulfotransferase (SULT1A3) expressed in

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

38. 39.

40.

41.

42.

43.

44.

45. 46. 47.

48. 49. 50.

51. 52.

53.

prokaryotic and eukaryotic systems: comparison with the recombinant enzyme purified from Escherichia coli. Protein Expr Purif 1999; 16:11–18. Eisenhofer G, Coughtrie MW, Goldstein DS. Dopamine sulphate: an enigma resolved. Clin Exp Pharmacol Physiol 1999; 26:S41–S53. Eisenhofer G, Rundqvist B, Aneman A, Friberg P, Dakak N, Kopin IJ, Jacobs MC, Lenders JW. Regional release and removal of catecholamines and extraneuronal metabolism to metanephrines. J Clin Endocrinol Metab 1995; 80:3009–3017. Eisenhofer G, Friberg P, Pacak K, Goldstein DS, Murphy DL, Tsigos C, Quyyumi AA, Brunner HG, Lenders JW. Plasma metadrenalines: Do they provide useful information about sympatho-adrenal function and catecholamine metabolism? Clin Sci (Colch) 1995; 88:533–542. Eisenhofer G, Keiser H, Friberg P, Mezey E, Huynh T-T, Hiremagalur B, Ellingson T, Duddempudi S, Eijsbouts A, Lenders J. Plasma metanephrines are markers of pheochromocytoma produced by catechol-O-methyltransferase within tumors. J Clin Endocrinol Metab 1998; 83:2175–2185. Schultzberg M, Lundberg JM, Hokfelt T, Terenius L, Brandt J, Elde RP, Goldstein M. Enkephalin-like immunoreactivity in gland cells and nerve terminals of the adrenal medulla. Neuroscience 1978; 3:1169–1186. Winkler H, Apps DK, Fischer-Colbrie R. The molecular function of adrenal chromaffin granules: established facts and unresolved topics. Neuroscience 1986; 18:261–290. Ehrhart-Bornstein M, Hinson JP, Bornstein SR, Scherbaum WA, Vinson GP. Intraadrenal interactions in the regulation of adrenocortical steroidogenesis. Endocr Rev 1998; 19:101–143. Livett BG, Marley PD, Wan DC, Zhou XF. Peptide regulation of adrenal medullary function. J Neural Transm 1990; 29:S77–S89. Winkler H, Fischer-Colbrie R. Regulation of the biosynthesis of large dense-core vesicles in chromaffin cells and neurons. Cell Mol Neurobiol 1998; 18:193–209. Haidan A, Bornstein SR, Glasow A, Uhlmann K, Lubke C, Ehrhart-Bornstein M. Basal steroidogenic activity of adrenocortical cells is increased 10-fold by coculture with chromaffin cells. Endocrinology 1998; 139:772–780. Huttner WB, Natori S. Regulated secretion. Helper proteins for neuroendocrine secretion. Curr Biol 1995; 5:242–225. Hendy GN, Bevan S, Mattei MG, Mouland AJ. Chromogranin A. Clin Invest Med 1995; 18:47–65. Hsiao RJ, Neumann HP, Parmer RJ, Barbosa JA, O’Connor DT. Chromogranin A in familial pheochromocytoma: diagnostic screening value, prediction of tumor mass, and post-resection kinetics indicating two-compartment distribution. Am J Med 1990; 88:607–613. Langley K, Grant NJ. Molecular markers of sympathoadrenal cells. Cell Tissue Res 1999; 298:185–206. Tschernitz C, Laslop A, Eiter C, Kroesen S, Winkler H. Biosynthesis of large densecore vesicles in PC12 cells: effects of depolarization and second messengers on the mRNA levels of their constituents. Brain Res Mol Brain Res 1995; 31:131–140. Siegel RE, Eiden LE, Pruss RM. Multiple populations of neuropeptide-containing cells in cultures of the bovine adrenal medulla. Brain Res 1985; 349:267–270.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

54.

55.

56.

57.

58.

59. 60. 61. 62. 63.

64.

65. 66.

67.

68. 69. 70. 71.

Fischer-Colbrie R, Iacangelo A, Eiden LE. Neural and humoral factors separately regulate neuropeptide Y, enkephalin, and chromogranin A and B mRNA levels in rat adrenal medulla. Proc Natl Acad Sci 1988; 85:3240–3324. Pruss RM, Mezey E, Forman DS, Eiden LE, Hotchkiss AJ, DiMaggio DA, O’Donohue TL. Enkephalin and neuropeptide Y: two colocalized neuropeptides are independently regulated in primary cultures of bovine chromaffin cells. Neuropeptides 1986; 7:315–327. Eskay RL, Eiden LE. Interleukin-1 alpha and tumor necrosis factor-alpha differentially regulate enkephalin, vasoactive intestinal polypeptide, neurotensin, and substance P biosynthesis in chromaffin cells. Endocrinology 1992; 130:2252–2228. Beinfeld MC, Brick PL, Howlett AC, Holt IL, Pruss RM, Moskal JR, Eiden LE. The regulation of vasoactive intestinal peptide synthesis in neuroblastoma and chromaffin cells. Ann NY Acad Sci 1988; 527:68–76. Vaupel R, Jarry H, Schlomer HT, Wuttke W. Differential response of substance Pcontaining subtypes of adrenomedullary cells to different stressors. Endocrinology 1988; 123:2140–215. Unsicker K, Krieglstein K. Growth factors in chromaffin cells. Prog Neurobiol 1996; 48:307–324. Bieger SC, Henkel AW, Unsicker K. Localization of basic fibroblast growth factor in bovine adrenal chromaffin cells. J Neurochem 1995; 64:1521–1527. Wurtman RJ, Axelrod J. Control of enzymatic synthesis of adrenaline in the adrenal medulla by adrenal cortical steroids. J Biol Chem 1966; 241:2301–2305. Carballeira A, Fishman LM. The adrenal functional unit: a hypothesis. Perspect Biol Med 1980; 23:573–597. Bornstein SR, Ehrhart-Bornstein M, Scherbaum WA, Pfeiffer EF, Holst JJ. Effects of splanchnic nerve stimulation on the adrenal cortex may be mediated by chromaffin cells in a paracrine manner. Endocrinology 1990; 127:900–906. Bornstein SR, Gonzalez-Hernandez JA, Ehrhart-Bornstein M, Adler G, Scherbaum WA. Intimate contact of chromaffin and cortical cells within the human adrenal gland forms the cellular basis for important intraadrenal interactions. J Clin Endocrinol Metab 1994; 78:225–232. Linares OA, Halter JB. Sympathochromaffin system activity in the elderly. J Am Geriatr Soc 1987; 35:448–453. Esler M, Kaye D, Thompson J, Jennings G, Cox H, Turner A, Lambert G, Seals D. Effects of aging on epinephrine secretion and regional release of epinephrine from the human heart. J Clin Endocrinol Metab 1995; 80:435–442. McCarty R, Pacak K, Goldstein DS, Eisenhofer G. Regulation of peripheral catecholamine responses to acute stress in young adult and aged F-344 rats. Stress 1997; 2:113–122. Seals DR, Esler MD. Human ageing and the sympathoadrenal system. J Physiol 2001; 528:407–417. Best JD, Halter JB. Release and clearance rates of epinephrine in man: importance of arterial measurements. J Clin Endocrinol Metab 1982; 55:263–268. Marker JC, Clutter WE, Cryer PE. Reduced epinephrine clearance and glycemic sensitivity to epinephrine in older individuals. Am J Physiol 1998; 275:E770–E776. Davidson L, Vandongen R, Rouse IL, Beilin LJ, Tunney A. Sex-related differences

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

72.

73.

74.

75. 76.

77.

78. 79.

80. 81.

82.

83.

84.

85. 86.

87.

in resting and stimulated plasma noradrenaline and adrenaline. Clin Sci 1984; 67:347–352. Gerlo EA, Schoors DF, Dupont AG. Age- and sex-related differences for the urinary excretion of norepinephrine, epinephrine, and dopamine in adults. Clin Chem 1991; 37:875–878. Engstrom BE, Karlsson FA, Wide L. Gender differences in diurnal growth hormone and epinephrine values in young adults during ambulation. Clin Chem 1999; 45:1235–1239. Gonzalez-Trapaga JL, Nelesen RA, Dimsdale JE, Mills PJ, Kennedy B, Parmer RJ, Ziegler MG. Plasma epinephrine levels in hypertension and across gender and ethnicity. Life Sci 2001; 66:2383–2392. Swinyard CA. Growth of the human suprarenal glands. Anat Rec 1940; 87:141–150. Reaven GM, Lithell H, Landsberg L. Hypertension and associated metabolic abnormalities—the role of insulin resistance and the sympathoadrenal system. N Engl J Med 1996; 334:374–381. Leonetti DL, Bergstrom RW, Shuman WP, Wahl PW, Jenner DA, Harrison GA, Fujimoto WY. Urinary catecholamines, plasma insulin and environmental factors in relation to body fat distribution. Int J Obes 1991; 15:345–357. Landsberg L. Pathophysiology of obesity-related hypertension: role of insulin and the sympathetic nervous system. J Cardiovasc Pharmacol 1994; 23(suppl 1):S1–S8. Ward KD, Sparrow D, Landsberg L, Young JB, Vokonas PS, Weiss ST. The relationship of epinephrine excretion to serum lipid levels: the Normative Aging Study. Metabolism 1994; 43:509–513. Del Rio G. Adrenomedullary function and its regulation in obesity. Int J Obes Relat Metab Disord 2001; 24(suppl 2):S89–91. Lee ZS, Critchley JA, Tomlinson B, Young RP, Thomas GN, Cockram CS, Chan TY, Chan JC. Urinary epinephrine and norepinephrine interrelations with obesity, insulin, and the metabolic syndrome in Hong Kong Chinese. Metabolism 2001; 50:135–143. Jones PP, Davy KP, Alexander S, Seals DR. Age-related increase in muscle sympathetic nerve activity is associated with abdominal adiposity. Am J Physiol 1997; 272:E976–E980. Matsukawa T, Sugiyama Y, Watanabe T, Kobayashi F, Mano T. Gender difference in age-related changes in muscle sympathetic nerve activity in healthy subjects. Am J Physiol 1998; 275:R1600–R1604. Crampes F, Riviere D, Beauville M, Marceron M, Garrigues M. Lipolytic response of adipocytes to epinephrine in sedentary and exercise-trained subjects: sex-related differences. Eur J Appl Physiol Occup Physiol 1989; 59:249–255. Lonnqvist F, Nyberg B, Wahrenberg H, Arner P. Catecholamine-induced lipolysis in adipose tissue of the elderly. J Clin Invest 1990; 85:1614–1621. Jensen MD, Cryer PE, Johnson CM, Murray MJ. Effects of epinephrine on regional free fatty acid and energy metabolism in men and women. Am J Physiol 1996; 270:E259–E264. Borsheim E, Lonnroth P, Knardahl S, Jansson PA. No difference in the lipolytic response to beta-adrenoceptor stimulation in situ but a delayed increase in adipose tissue blood flow in moderately obese compared with lean men in the postexercise period. Metabolism 2001; 49:579–87.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

88. 89. 90.

91. 92.

93.

94.

95.

96.

97. 98. 99.

100.

101.

102.

103.

104.

Goldstein DS. Plasma catecholamines and essential hypertension. An analytical review. Hypertension 1983; 5:86–99. Esler M. The sympathetic system and hypertension. Am J Hypertens 2001; 13:99S–105S. Franco-Morselli R, Elghozi JL, Joly E, Di Giuilio S, Meyer P. Increased plasma adrenaline concentrations in benign essential hypertension. Br Med J 1977; 2:1251–124. Eliasson K, Hjemdahl P, Kahan T. Circulatory and sympathoadrenal responses to stress in borderline and established hypertension. J Hypertension 1983; 1:131–139. Cerasola G, Cottone S, D’Ignoto G, Grasso L, Contorno A, Carone MB, Fulantelli MA. Role of epinephrine in the development of essential hypertension. J Clin Hypertens 1987; 3:670–680. Nezu M, Miura Y, Adachi M, Adachi M, Kimura S, Toriyabe S, Ishizuka Y, Ohashi H, Sugawara T, Takahashi M. The effects of epinephrine on norepinephrine release in essential hypertension. Hypertension 1985; 7:187–195. Jacobs MC, Lenders JW, Willemsen JJ, Thien T. Adrenomedullary secretion of epinephrine is increased in mild essential hypertension. Hypertension 1997; 29:1303–1308. Goldstein DS, Levinson PD, Zimlichman R, Pitterman A, Stull R, Keiser HR. Clonidine suppression testing in essential hypertension. Ann Intern Med 1985; 102:42–49. Goldstein DS, Grossman E, Listwak S, Folio CJ. Sympathetic reactivity during a yohimbine challenge test in essential hypertension. Hypertension 1991; 18 (suppl III):III-40–III-48. Majewski H, Tung LH, Rand MJ. Hypertension through adrenaline activation of prejunctional beta-adrenoceptors. Clin Exp Pharmacol Physiol 1981; 8:463–468. Majewski H, Rand MJ. A possible role of epinephrine in the development of hypertension. Med Res Rev 1986; 6:467–486. Esler M, Eisenhofer G, Chin J, Jennings G, Meredith I, Cox H, Lambert G, Thompson J, Dart A. Is adrenaline released by sympathetic nerves in man? Clin Auton Res 1991; 1:103–108. Eisenhofer G, Smolich JJ, Esler MD. Disposition of endogenous adrenaline compared to noradrenaline released by cardiac sympathetic nerves in the anaesthetized dog. Naunyn Schmiedebergs Arch Pharmacol 1992; 345:160–171. Blankestijn PJ, Man in’t Veld AJ, Tulen J, van den Meiracker AH, Boomsma F, Moleman P, Ritsema van Eck HJ, Derkx FH, Mulder P, Lamberts SJ. Support for adrenaline-hypertension hypothesis: 18 hour pressor effect after 6 hours adrenaline infusion. Lancet 1988; 2:1386–1389. van den Meiracker AH, Man in ‘t Veld AJ, Boomsma F, Blankestijn PJ, Schalekamp MA. Epinephrine-induced enhancement of sympathetic activity in humans: inhibition by nonselective as well as beta 1-selective beta-adrenoceptor blockade. J Cardiovasc Pharmacol 1990; 15:610–617. Boudreau G, Peronnet F, De Champlain J, Nadeau R. Presynaptic effects of epinephrine on norepinephrine release from cardiac sympathetic nerves in dogs. Am J Physiol 1993; 265:H205–H211. Floras JS. Epinephrine and the genesis of hypertension. Hypertension 1992; 19:1–18.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

105. Goldstein DS, Golczynska A, Stuhlmuller J, Holmes C, Rea RF, Grossman E, Lenders J. A test of the “epinephrine hypothesis” in humans. Hypertension 1999; 33:36–43. 106. Stein MB, Asmundson GJ. Autonomic function in panic disorder: cardiorespiratory and plasma catecholamine responsivity to multiple challenges of the autonomic nervous system. Biol Psychiatry 1994; 36:548–558. 107. Roy-Byrne P, Cowley DS, Stein MB, Wingerson D, Veith R. Cardiovascular and catecholamine response to orthostasis in panic and obsessive-compulsive disorder and normal controls: effects of anxiety and novelty. Depress Anxiety 1997; 6:159–164. 108. Nesse RM, Cameron OG, Curtis GC, McCann DS, Huber-Smith MJ. Adrenergic function in patients with panic anxiety. Arch Gen Psychiatry 1984; 41:771–776. 109. Villacres EC, Hollifield M, Katon WJ, Wilkinson CW, Veith RC. Sympathetic nervous system activity in panic disorder. Psychiatry Res 1987; 21:313–321. 110. Nesse RM, Cameron OG, Buda AJ, McCann DS, Curtis GC, Huber-Smith MJ. Urinary catecholamines and mitral valve prolapse in panic-anxiety patients. Psychiatry Res 1985; 14:67–75. 111. Schneider P, Evans L, Ross-Lee L, Wiltshire B, Eadie M, Kenardy J, Hoey H. Plasma biogenic amine levels in agoraphobia with panic attacks. Pharmacopsychiatry 1987; 20:102–104. 112. Braune S, Albus M, Frohler M, Hohn T, Scheibe G. Psychophysiological and biochemical changes in patients with panic attacks in a defined situational arousal. Eur Arch Psychiatry Clin Neurosci 1994; 244:86–92. 113. Hoehn T, Braune S, Scheibe G, Albus M. Physiological, biochemical and subjective parameters in anxiety patients with panic disorder during stress exposure as compared with healthy controls. Eur Arch Psychiatry 1997; 247:264–274. 114. Wilkinson DJ, Thompson JM, Lambert GW, Jennings GL, Schwarz RG, Jefferys D, Turner AG, Esler MD. Sympathetic activity in patients with panic disorder at rest, under laboratory mental stress, and during panic attacks. Arch Gen Psychiatry 1998; 55:511–520. 115. Carlsson M, Carlsson A. Effects of mild stress on adrenal and heart catecholamines in male and female rats. J Neural Transm 1989; 77:217–226. 116. Dimsdale JE, Moss J. Plasma catecholamines in stress and exercise. JAMA 1980; 243:340–342. 117. Eisenhofer G, Lambie DG, Johnson RH. Beta-adrenoceptor responsiveness and plasma catecholamines as determinants of cardiovascular reactivity to mental stress. Clin Sci 1985; 69:483–492. 118. Maddock RJ, Carter CS, Magliozzi JR, Gietzen DW. Evidence that decreased function of lymphocyte beta adrenoreceptors reflects regulatory and adaptive processes in panic disorder with agoraphobia. Am J Psychiatry 1993; 150:1219–1225 119. Liebowitz MR, Gorman JM, Fyer AJ, Levitt M, Dillon D, Levy G, Appleby IL, Anderson S, Palij M, Davies SO. Lactate provocation of panic attacks. II. Biochemical and physiological findings. Arch Gen Psychiatry 1985; 42:709–719. 120. Coryell W, Coryell W, Noyes R, Clancy J. Panic disorder and mortality. Excess mortality in panic disorder. A comparison with primary unipolar depression. Arch Gen Psychiatry 1982; 39:701–703.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

121. 122.

123.

124.

125. 126.

127.

128. 129.

130.

131.

132.

133.

134. 135. 136.

Fleet R, Lavoie K, Beitman BD. Is panic disorder associated with coronary artery disease? A critical review of the literature. J Psychosom Res 2001; 48:347–356. Cryer PE, Gerich JE. Relevance of glucose counterregulatory systems to patients with diabetes: critical roles of glucagon and epinephrine. Diabetes Care 1983; 6:95–99. Shamoon H, Friedman S, Canton C, Zacharowicz L, Hu M, Rossetti L. Increased epinephrine and skeletal muscle responses to hypoglycemia in non-insulin-dependent diabetes mellitus. J Clin Invest 1994; 93:2562–2571. Bolli G, de Feo P, Compagnucci P, Cartechini MG, Angeletti G, Santeusanio F, Brunetti P, Gerich JE. Abnormal glucose counterregulation in insulin-dependent diabetes mellitus. Interaction of anti-insulin antibodies and impaired glucagon and epinephrine secretion. Diabetes 1983; 32:134–141. Hirsch BR, Shamoon H. Defective epinephrine and growth hormone responses in type I diabetes are stimulus specific. Diabetes 1987; 36:20–26. Kennedy FP, Bolli GB, Go VL, Cryer PE, Gerich JE. The significance of impaired pancreatic polypeptide and epinephrine responses to hypoglycemia in patients with insulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1987; 64:602–608. Kennedy FP, Go VL, Cryer PE, Bolli GB, Gerich JE. Subnormal pancreatic polypeptide and epinephrine responses to insulin-induced hypoglycemia identify patients with insulin-dependent diabetes mellitus predisposed to develop overt autonomic neuropathy. Ann Intern Med 1988; 108:54–58. Hoeldtke RD, Boden G. Epinephrine secretion, hypoglycemia unawareness, and diabetic autonomic neuropathy. Ann Intern Med 1994; 120:512–517. Hoeldtke RD, Boden G, Shuman CR, Owen OE. Reduced epinephrine secretion and hypoglycemia unawareness in diabetic autonomic neuropathy. Ann Intern Med 1982; 96:459–462. Horie H, Hanafusa T, Matsuyama T, Namba M, Nonaka K, Tarui S, Yamatodani A, Wada H. Decreased response of epinephrine and norepinephrine to insulin-induced hypoglycemia in diabetic autonomic neuropathy. Horm Metab Res 1984; 16:398–401. Ryder RE, Owens DR, Hayes TM, Ghatei MA, Bloom SR. Unawareness of hypoglycaemia and inadequate hypoglycaemic counterregulation: no causal relation with diabetic autonomic neuropathy. Br Med J 1990; 301:783–787. Scherbaum WA, Mogel H, Boehm BO, Hedderich U, Gluck M, Schernthaner G, Bottazzo GF, Pfeiffer EF. Autoantibodies to adrenal medullary and thyroid calcitonin cells in type I diabetes mellitus—a prospective study. J Autoimmun 1988; 1:219–230. Brown FM, Vinik AI, Ganda OP, Adri MN, Rabinowe SL. Different effects of duration on prevalence of anti-adrenal medullary and pancreatic islet cell antibodies in type I diabetes mellitus. Horm Metab Res 1989; 21:434–437. Brown FM, Smith AM, Longway S, Rabinowe SL. Adrenal medullitis in type I diabetes. J Clin Endocrinol Metab 1990; 71:1491–1495. Ziegler MG, Lake CR, Kopin IJ. The sympathetic-nervous-system defect in primary orthostatic hypotension. N Engl J Med 1977; 296:293–297. Polinsky RJ, Brown RT, Curras MT, Baser SM, Baucom CE, Hooper DR, Marini AM. Central and peripheral effects of arecoline in patients with autonomic failure. J Neurol Neurosurg Psychiatry 1991; 54:807–812.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

137.

138.

139.

140.

141. 142.

143. 144.

145.

146.

147. 148.

149.

150.

151. 152. 153.

Meredith IT, Esler MD, Cox HS, Lambert GW, Jennings GL, Eisenhofer G. Biochemical evidence of sympathetic denervation of the heart in pure autonomic failure. Clin Auton Res 1991; 1:187–194. Polinsky RJ, Kopin IJ, Ebert MH, Weise V. The adrenal medullary response to hypoglycemia in patients with orthostatic hypotension. J Clin Endocrinol Metab 1980; 51:1401–1406. Eisenhofer G, Lambie DG, Johnson RH, Tan E, Whiteside EA. Deficient catecholamine release as the basis of orthostatic hypotension in pernicious anaemia. J Neurol Neurosurg Psychiatry 1982; 45:1053–1055. Sasaki K, Matsuhashi A, Murabayashi S, Aoyagi K, Baba T, Matsunaga M, Takebe K. Hormonal response to insulin-induced hypoglycemia in patients with Shy-Drager syndrome. Metabolism 1983; 32:977–981. Guttman PH. Addison’s disease: A statistical analysis of five hundred and sixty-six cases and a study of the pathology. Arch Path 1930; 10:742–785. Luft R, von Euler US. Urinary excretion of cateholamines during insulin hypoglycemia in five patients with Addison’s disease and in one patient after bilateral adrenalectomy. Acta Endocrinol 1957; 26:96–100. Wegienka LC, Grasso SG, Forsham PH. Estimation of adrenomedullary reserve by infusion of 2-deoxy-D-glucose. J Clin Endocrinol Metab 1966; 26:37–45. Bornstein SR, Breidert M, Ehrhart-Bornstein M, Kloos B, Scherbaum WA. Plasma catecholamines in patients with Addison’s disease. Clin Endocrinol 1995; 42:215–218. Rudman D, Moffitt SD, Fernhoff PM, Blackston RD, Faraj BA. Epinephrine deficiency in hypocorticotropic hypopituitary children. J Clin Endocrinol Metab 1981; 53:722–728. Szemeredi K, Bagdy G, Stull R, Calogero AE, Kopin IJ, Goldstein DS. Sympathoadrenomedullary inhibition by chronic glucocorticoid treatment in conscious rats. Endocrinology 1988; 123:2585–2590. Mathe AA, Knapp PH. Decreased plasma free fatty acids and urinary epinephrine in bronchial asthma. N Engl J Med 1969; 281:234–238. Bornstein SR, Tajima T, Eisenhofer G, Haidan A, Aguilera G. Adrenomedullary function is severely impaired in 21-hydroxylase-deficient mice. FASEB J 1999; 13:1185–1194. Merke DP, Chrousos GP, Eisenhofer G, Weise M, Keil MF, Rogol AD, Van Wyk JJ, Bornstein SR. Adrenomedullary dysplasia and hypofunction in patients with classic 21-hydroxylase deficiency. N Engl J Med 2001; 343:1362–1368. Greenberg R, Rosenthal I, Falk GS. Electron microscopy of human tumors secreting catecholamines: correlation with biochemical data. J Neuropathol Exp Neurol 1969; 28:475–500. Lauper NT, Tyce GM, Sheps SG, Carney JA. Pheochromocytoma. Fine structural, biochemical and clinical observations. Am J Cardiol 1972; 30:197–204. Kvale WF, Roth GM, Manger WM, Priestley JT. Pheochromocytoma. Circulation 1956; 14:622–630. Gifford RW, Kvale WF, Maher FT, Roth GM, Priestley JT. Clinical features, diagnosis, and treatment of pheochromocytoma. A review of 76 cases. Mayo Clin Proc 1964; 39:281–302.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

154. 155.

156.

157.

158.

159.

160.

161. 162.

163.

164.

165.

166.

167.

Manger WM, Gifford RW. Clinical and Experimental Pheochromocytoma. Cambridge, MA: Blackwell Science, 1996. Choyke PL, Glenn GM, Walther MM, Patronas NJ, Linehan WM, Zbar B. von Hippel-Lindau disease: genetic, clinical, and imaging features. Radiology 1995; 194:629–642. Neumann HP, Berger DP, Sigmund G, Blum U, Schmidt D, Parmer RJ, Volk B, Kirste G. Pheochromocytomas, multiple endocrine neoplasia type 2, and von Hippel-Lindau disease. N Engl J Med 1993; 329:1531–1538. Walther MM, Reiter R, Keiser HR, Choyke PL, Venzon D, Hurley K, Gnarra JR, Reynolds JC, Glenn GM, Zbar B, Linehan WM. Clinical and genetic characterization of pheochromocytoma in von Hippel-Lindau families: comparison with sporadic pheochromocytoma gives insight into natural history of pheochromocytoma. J Urol 1999; 162:659–664. Pomares FJ, Canas R, Rodriguez JM, Hernandez AM, Parrilla P, Tebar FJ. Differences between sporadic and multiple endocrine neoplasia type 2A phaeochromocytoma. Clin Endocrinol 1998; 48:195–200. Calmettes C, Ponder BA, Fischer JA, Raue F. Early diagnosis of the multiple endocrine neoplasia type 2 syndrome: consensus statement. European Community Concerted Action: Medullary Thyroid Carcinoma. Eur J Clin Invest 1992; 22:755–760. Huson SM, Compston DA, Harper PS. A genetic study of von Recklinghausen neurofibromatosis in south east Wales. II. Guidelines for genetic counselling. J Med Genet 1989; 26:712–721. Walther MM, Herring J, Enquist E, Keiser HR, Linehan WM. von Recklinghausen’s disease and pheochromocytomas. J Urol 1999; 162:1582–1586. Kimura N, Miura Y, Nagatsu I, Nagura H. Catecholamine synthesizing enzymes in 70 cases of functioning and non-functioning phaeochromocytoma and extra-adrenal paraganglioma. Virchows Arch A Pathol Anat Histopathol 1992; 421:25–32. Ito Y, Fujimoto Y, Obara T. The role of epinephrine, norepinephrine, and dopamine in blood pressure disturbances in patients with pheochromocytoma. World J Surg 1992; 16:759–763. Proye C, Fossati P, Fontaine P, Lefebvre J, Decoulx M, Wemeau JL, Dewailly D, Rwamasirabo E, Cecat P. Dopamine-secreting pheochromocytoma: an unrecognized entity? Classification of pheochromocytomas according to their type of secretion. Surgery 1986; 100:1154–1162. Yasunari K, Kohno M, Minami M, Kano H, Ohhira M, Nakamura K, Yoshikawa J. A dopamine-secreting pheochromocytoma. J Cardiovasc Pharmacol 2001; 36(suppl 2):S75–S77. Feldman JM. Phenylethanolamine-N-methyltransferase activity determines the epinephrine concentration of pheochromocytomas. Res Commun Chem Pathol Pharmacol 1981; 34:389–398. Eisenhofer G, Walther MM, Huynh TT, Li ST, Bornstein SR, Vortmeyer A, Mannelli M, Goldstein DS, Linehan WM, Lenders JW, Pacak K. Pheochromocytomas in von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2 display distinct biochemical and clinical phenotypes. J Clin Endocrinol Metab 2001; 86:1999–2008.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

168. Page LB, Raker JW, Berberich FR. Pheochromocytoma with predominant epinephrine secretion. Am J Med 1969; 47:648–652. 169. Aronoff SL, Passamani E, Borowsky BA, Weiss AN, Roberts R, Cryer PE. Norepinephrine and epinephrine secretion from a clinically epinephrine- secreting pheochromocytoma. Am J Med 1980; 69:321–324. 170. Anton AH, Greer M, Sayre DF, Williams CM. Dihydroxyphenylalanine secretion in a malignant pheochromocytoma. Am J Med 1967; 42:469–475. 171. Goldstein DS, Stull R, Eisenhofer G, Sisson JC, Weder A, Averbuch SD, Keiser HR. Plasma 3,4-dihydroxyphenylalanine (dopa) and catecholamines in neuroblastoma or pheochromocytoma. Ann Intern Med 1986; 105:887–888. 172. McClean DR, Sinclair LM, Yandle TG, Nicholls MG. Malignant phaeochromocytoma with high circulating DOPA, and clonidine-suppressible noradrenaline. Blood Press 1995; 4:215–217. 173. John H, Ziegler WH, Hauri D, Jaeger P. Pheochromocytomas: can malignant potential be predicted? Urology 1999; 53:679–683. 174. Lenders JW, Keiser HR, Goldstein DS, Willemsen JJ, Friberg P, Jacobs MC, Kloppenborg PW, Thien T, Eisenhofer G. Plasma metanephrines in the diagnosis of pheochromocytoma. Ann Intern Med 1995; 123:101–109. 175. Eisenhofer G, Lenders JW, Linehan WM, Walther MM, Goldstein DS, Keiser HR. Plasma normetanephrine and metanephrine for detecting pheochromocytoma in von Hippel-Lindau disease and multiple endocrine neoplasia type 2. N Engl J Med 1999; 340:1872–1879. 176. Raber W, Raffesberg W, Bischof M, Scheuba C, Niederle B, Gasic S, Waldhausl W, Roden M. Diagnostic efficacy of unconjugated plasma metanephrines for the detection of pheochromocytoma. Arch Intern Med 2000; 160:2957–2963. 177. Pacak K, Linehan WM, Eisenhofer G, Walther MM, Goldstein DS. Recent Advances in Genetics, Diagnosis, Localization, and Treatment of Pheochromocytoma. Ann Intern Med 2001; 134:315–329. 178. Eisenhofer G. Biochemical diagnosis of pheochromocytoma. In: Pacak K, moderator. Recent advances in genetics, diagnosis, localization, and treatment of pheochromocytoma. Ann Intern Med 2001; 134:317–320. 179. Bravo EL, Tarazi RC, Fouad FM, Vidt DG, Gifford RW Jr. Clonidine-suppression test: a useful aid in the diagnosis of pheochromocytoma. N Engl J Med 1981; 305:623–626. 180. Grossman E, Goldstein DS, Hoffman A, Keiser HR. Glucagon and clonidine testing in the diagnosis of pheochromocytoma. Hypertension 1991; 17:733–741. 181. Manger WM, Gifford RWJr. Pheochromocytoma: current diagnosis and management. Clev Clin J Med 1993; 60:365–378. 182. Fink IJ, Reinig JW, Dwyer AJ, Doppman JL, Linehan WM, Keiser HR. MR imaging of pheochromocytomas. J Comput Assist Tomogr 1985; 9:454–458. 183. Schmedtje JF, Jr., Sax S, Pool JL, Goldfarb RA, Nelson EB. Localization of ectopic pheochromocytomas by magnetic resonance imaging. Am J Med 1987; 83:770–772. 184. Sisson JC, Frager MS, Valk TW, Gross MD, Swanson DP, Wieland DM, Tobes MC, Beierwaltes WH, Thompson NW. Scintigraphic localization of pheochromocytoma. N Engl J Med 1981; 305:12–17. 185. Shulkin BL, Shapiro B. Current concepts on the diagnostic use of MIBG in children. J Nucl Med 1998; 39:679–688.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

186.

187.

188.

189.

190.

191.

Shapiro B, Copp JE, Sisson JC, Eyre PL, Wallis J, Beierwaltes WH. Iodine-131 metaiodobenzylguanidine for the locating of suspected pheochromocytoma: experience in 400 cases. J Nucl Med 1985; 26:576–585. Eisenhofer G, Pacak K, Goldstein DS, Chen C, Shulkin B. 1231-MIBG scintigraphy of catecholamine systems: impediments to applications in clinical medicine. Eur J Nucl Med 2000; 27:611–612. Shulkin BL, Wieland DM, Schwaiger M, Thompson NW, Francis IR, Haka MS, Rosenspire KC, Shapiro B, Sisson JC, Kuhl DE. PET scanning with hydroxyephedrine: an approach to the localization of pheochromocytoma. J Nucl Med 1992; 33:1125–1131. Adams S, Baum R, Rink T, Schumm-Drager PM, Usadel KH, Hor G. Limited value of fluorine-18 fluorodeoxyglucose positron emission tomography for the imaging of neuroendocrine tumours. Eur J Nucl Med 1998; 25:79–83. Goldstein DS, Cannon RO, Quyyumi A, Chang P, Duncan M, Brush JE, Jr., Eisenhofer G. Regional extraction of circulating norepinephrine, DOPA, and dihydroxyphenylglycol in humans. J Auton Nerv Syst 1991; 34:17–35. Eisenhofer G, Aneman A, Friberg P, Hooper D, F~andriks L, Lonroth H, Hunyady B, Mezey E. Substantial production of dopamine in the human gastrointestinal tract. J Clin Endocrinol Metab 1998; 42:374–377.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

6 Autonomic Nervous System–Leptin Interactions Impact on Metabolic Rate and Body Weight Regulation Bulent Yildiz, Ma-Li Wong, and Julio Licinio David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A.

Metin Ozata Gulhane School of Medicine, Ankara, Turkey

Leptin is the peptide hormone product of the obese (ob) gene expressed predominantly in adipose tissue. Leptin has profound effects on the control of body weight, appetite, and energy balance. In studies of both obese and nonobese subjects, it has been shown that autonomic nervous system activity is also related to body weight regulation. The sympathetic nervous system is crucial in the regulation of lipolysis: sympathetic denervation leads to an increase in adipose tissue weight, while nerve stimulation results in fatty acid release and sympathetic blockade inhibits the mobilization of lipid. Recently, leptin has been shown to increase sympathetic nervous system activity. In fact, adipose tissues are innervated mostly by sympathetic fibers. Leptin gene expression and circulating leptin levels are affected by factors that either mimic or involve the sympathetic nervous system, such as catecholamines, fasting, cold, and exercise. Pulsatility and circadian variation in leptin could also be determined by changes in sympathetic nervous system activity. In the central nervous system, leptin seems to inhibit the pathways that stimulate food intake and promote weight gain, whereas it stimulates the pathways that promote anorexia and weight loss.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

It has been postulated that leptin and its multiple interactions with other neurochemical pathways in the hypothalamus may cause excess weight gain, increased sympathetic activity, and hypertension. Although leptin’s effects on energy balance have been extensively studied, its effects on sympathetic activity and cardiovascular function are not as well understood, and represent a fertile area for research.

I. INTRODUCTION Leptin is an adipocyte-derived hormone that is essential for normal body weight regulation (1). Leptin has potent effects on feeding behavior, thermogenesis and neuroendocrine responses (2,3). The landmark discovery of leptin established the fact that adipose tissue is not only an active regulator of body weight, but also an endocrine organ with feedback circuits possessing both afferent and efferent loops (4). While a number of neuroendocrine afferent signals contribute to body weight homeostasis, the sympathetic nervous system (SNS), which affects both energy expenditure and substrate utilization, is the major efferent pathway in the regulation of adipose tissue physiology and endocrine function. Studies of experimental starvation and overfeeding provide conclusive data that appetite, energy expenditure, and basal metabolic rate are under the control of the central and autonomic nervous systems. The autonomic nervous system (ANS) is divided into two systems, the SNS and the parasympathetic nervous system (PNS), which often oppose each other. The SNS evokes responses characteristic of the “fight-or-flight” response. These elements have been fully reviewed elsewhere in this volume. ANS activity is related to body weight regulation. In previous studies of both obese and never-obese subjects, it has been demonstrated that weight increase leads to increased sympathetic and decreased parasympathetic activity, whereas weight decrease leads to decreased sympathetic and increased parasympathetic activity (5). Recently, a possible impact of leptin on the ANS has been demonstrated. In fact, leptin has been shown to stimulate SNS activity in thermogenic and nonthermogenic organs in animal models (6,7). In the same experimental model, chronic leptin infusion has also been demonstrated to increase heart rate and arterial blood pressure (8). The hypothalamus regulates body fat and appetite through effects on endocrine function and the ANS. It is established that the hypothalamus integrates leptin signals into coordinated endocrine, behavioral and autonomic responses that maintain homeostasis. In this chapter we will review evidence that leptin– ANS interactions are a key element in the regulation of body weight and energy homeostasis.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

II. ADIPOSE TISSUE INNERVATION AND LEPTIN PRODUCTION Adipose tissues are innervated mostly, if not exclusively, by sympathetic fibers. These data have been obtained mainly from the Siberian hamster, which has clear innervation of both types of tissues, and have been confirmed in rats (9–11). It is recognized that brown adipose tissue (BAT) is much more innervated than white adipose tissue (WAT). In WAT, noradrenergic fibers are closely associated with the vasculature; however, there is evidence demonstrating the direct innervation of white fat cells (12). In BAT, both vessels and adipocytes are innervated (13). The neurotransmitter involved is noradrenaline, which binds to different noradrenergic-receptor subtypes depending on the fat pad and species (14). In adipose tissues, noradrenaline is the main lipolytic agent, while adrenaline has only a slight effect at physiological concentrations. Stimulation of sympathetic fibers results in lipolysis (14). Direct sympathetic control of lipolysis (15) suggests that leptin production is also under direct sympathetic control. The efferent sympathetic innervation probably influences the rate of fat mobilization because denervation increases fat pad mass and adipocyte number (16,17). Evidence for a functional sympathetic innervation of WAT has recently been confirmed in the Siberian hamster (17), which had increased wet weight relative to the contralateral pad in which innervation was still intact. This change was associated with an increase in the number of fat cells rather than an increase in their size. The simplest explanation for this change is that sympathetic activity directly decreases adipose tissue development, and probably leptin levels as well. The origin of part of the sympathetic innervation of WAT within the hypothalamus has been shown by retrograde tracer experiments involving the injection of pseudorabies virus (PRV) into inguinal and epididymal WAT pads in hamsters and rats (10). These experiments have demonstrated sympathetic connections through the intermediolateral cell group to the central autonomic nucleus of the spinal cord; in the brainstem, PRV-labeled cells were seen in a number of areas, including the solitary tract, which is known to be involved in the control of voluntary food intake and contains leptin receptors (OB-R) (18). In the forebrain, prominent PRV labeling was seen in regions of the paraventricular nucleus (PVN), while very little labeling was evident in the ventromedial hypothalamus, which has long been associated with sympathetic effects (19). Recently, the microinjection of leptin into this region has been shown to lead to an increase in circulating catecholamines (20). Stimulation of the PVN, which has sympathetic connections, is known to induce sympathetically mediated lipolysis (9). Sympathetic connections are also seen in the suprachiasmatic nucleus, which is known to be concerned with the initiation and regulation of circadian rhythms and may be related to the circadian variation in leptin levels. In rats, sympathetic nervous system activity in WAT increases with fasting and cold exposure (21–24) in conjunction with increased lipolysis in the tissue and decreased expression of lep-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

tin. Because fasting increases SNS activity in WAT in rodents, it is conceivable that the fasting-induced reduction in leptin levels in humans, known to be more rapid and profound than expected from the loss of body fat (25–28), might be mediated by increased SNS activity in the WAT.

III. SHORT-TERM REGULATION OF LEPTIN PRODUCTION AND SNS Although leptin gene expression and leptin levels are related to adiposity, leptin gene expression in WAT and circulating leptin are subject to short-term regulation. Factors known to increase leptin such as obesity and -methyl-para-tyrosine, or decrease it, such as noradrenaline, adrenaline, isoprenaline, 3-agonists, fasting, cold, and exercise, either mimic or involve the SNS. Leptin gene expression is subject to nutritional regulation, as fasting leads to a rapid and profound decrement in leptin gene expression, which is rapidly reversible (29) and therefore is not related to any change in body obesity. Leptin clearly responds acutely to a change in nutrient availability, although the role of the SNS in these responses was not apparent until similar profound falls in leptin gene expression seen on cold exposure and treatment with sympathomimetic amines were investigated (21,30). Again, the rapid fall of leptin levels after exposure to cold and reversal upon rewarming indicates its key role in nutritional regulation rather than reflecting changes in adiposity. Early studies indicated that exercise led to a fall in leptin production (31). However, results have varied and may represent changes in fat mass and/or sympathetic stimulation (32,33). As noted above, these major decreases in leptin gene expression or in leptin levels due to cold exposure or fasting can be reproduced by acute treatment with noradrenaline or, more potently, with the synthetic -agonist isoprenaline (21,34). Adrenaline also reduces leptin gene expression in human studies (35–37). It seems likely that the SNS is a key regulator of leptin production and that the administration of different sympathomimetic amines is able to mimic these effects. Both physiological perturbations are known to sympathetically stimulate WAT. Cold exposure leads to increased noradrenaline turnover in all tissues including WAT and increased sympathetic stimulation. The metabolic rate is raised, and fatty acids from adipose tissue are mobilized in order to maintain body temperature. Fasting, while in general decreasing sympathetic stimulation to most tissues (38), specifically causes sympathetic stimulation of WAT to provide fatty acids as an energy source (24). The lack of effect of cold exposure or fasting in rodent models of obesity is explained by the known down-regulation of -adrenoceptors in WAT (39–43) and decreased responsiveness to catecholamines in these animals (34,44).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Further indications of the major role of catecholamines in inhibiting leptin production has been obtained using mature adipocytes in vitro or using adipocytes in primary cell culture (45–47). The inhibition of leptin production by catecholamines in rodents seems to occur via 3-adrenoceptors as selective 3-agonists (BRL 35153A, CL31643, etc.) suppress leptin gene expression and decrease leptin levels (34,45,48–51). In humans, although there is some evidence that 3receptors are involved in fatty acid mobilization from WAT (52), the sympathetic regulation of WAT is likely to be mostly through 1/2-receptors (53). The use of different methods of adrenergic blockade can give further insight into the sympathetic regulation of leptin production. As mentioned above, the effects of fasting on leptin levels can be reversed by -adrenoceptor blockade. Changes in circulating leptin after fasting are blocked by high doses of propranolol, a predominant 1/2 antagonist (54). However, a combination of propranolol and the specific 3 antagonist SR 59230A is able to reverse the effects of cold exposure and increase the levels of leptin mRNA in warmly maintained control mice, suggesting that there is normally a tonic inhibition of leptin secretion from WAT by the sympathetic system (55). The suggestion of a tonic inhibition of the leptin system by the SNS is reinforced by the action of -methyl-para-tyrosine in mice. Alpha-methyl-para-tyrosine is an inhibitor of tyrosine hydroxylase, the rate-limiting step in catecholamine synthesis and has long been used in studies investigating noradrenaline turnover (38). Administration of -methylpara-tyrosine has been found to lead to an up to eight-fold increase in leptin levels within 8–10 hours in lean mice; -methyl-para-tyrosine more than reversed the fall in leptin levels and leptin mRNA in response to a 24-hour fast (56). In obese ob/ob mice, however, -methyl-para-tyrosine does not alter leptin gene expression, consistent with sympathetic down-regulation occurring in these animals. This result contrasts with a report done on humans that found that -methyl-paratyrosine does not alter plasma leptin levels (57), a difference that may reflect why a lower dose of the drug was, by necessity, administered in the human study. Administration of 6-hydroxydopamine, which chemically sympathectomises the peripheral sympathetic innervation without affecting brain adrenergic systems or the adrenal medulla, has been shown to raise leptin levels during fasting but to a lesser extent than -methyl-para-tyrosine (58). Leptin gene mRNA increased in BAT in this study, although not in WAT, suggesting that the change in leptin in response to 6-hydroxydopamine represents an increased output from BAT. It was concluded that -methyl-para-tyrosine increases leptin levels by inhibiting sympathetic activity but that the effects are complex and probably involve concomitant changes in insulin and glucose levels as well. However, 6-hydroxydopamine does not affect catecholamine secretion from the medulla, and, therefore, the increased effect of -methyl-para-tyrosine may indicate the involvement of adrenal medullary secretion in response to fasting.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

IV. CIRCADIAN VARIATION IN LEPTIN PRODUCTION AND SNS Leptin is a sleep hormone with high levels observed at night. In humans, leptin levels peak at night and fall to a nadir in the morning (59–62). Much of this diurnal pattern is entrained to meal patterns (60), although a circadian cycle of reduced amplitude continues in subjects undergoing continuous enteral feeding (61). This secretion pattern of leptin may be dependent on the changes in the activity of SNS associated with sleep. A few studies have demonstrated a lower SNS activity at night during sleep and a higher activity during the day in humans (63–65) and of changes in circulating noradrenaline and adrenaline concentrations (65–67). Thus, sleep episodes that are associated with lying down and arising are clearly associated with altered SNS activity. Activity of SNS is associated with suppression of circulating leptin concentrations in humans. In a male patient with pheochromocytoma, a very low leptin level and an absence of circadian variation were observed, and, after removal of the tumor, leptin levels increased and circadian variation became evident (68). However, other patients with pheochromocytoma showed little change in plasma leptin levels, possibly due to down-regulation of adrenergic receptors (69). Injection of a catecholamine, isoproterenol, directly inhibits secretion of leptin by human WAT fragment in culture (70). Stimulation of SNS activity in women by exposure to cold for 90 minutes is associated with a rapid decrease in plasma leptin concentrations (22). Thus, an inhibitory influence of SNS activity on leptin production during waking hours is a likely cause of the diurnal change in leptin concentration with a lesser inhibition during the nocturnal peak. In rodents, there is considerable evidence that the SNS can acutely suppress leptin levels (54,55,71). The existence of a regulatory feedback loop between leptin production by adipocytes and the brain that is mediated by the SNS has been suggested (71). There is a cold-induced suppression of leptin gene expression in the WAT of rodents, which is mediated primarily by the SNS (21,54,55). Inhibition of normal sympathetic tone (by inhibition of noradrenaline synthesis with methyl-para-tyrosine) increases leptin gene expression in WAT and increases circulating leptin concentrations (56). Circadian variation in leptin concentration in blood also occurs in rats and mice but, in keeping with their nocturnal habit, the peak is in the light period and the nadir in the nocturnal period when they are feeding (29,70,72). Thus, it seems that changes in SNS activity in adipose tissues could underlie changes in plasma leptin concentrations in human subjects. A plausible candidate for an inhibitor of leptin synthesis and secretion during pulses is noradrenaline, secreted from sympathetic nerve endings. Four lines of evidence support a role for noradrenaline in determining the pulsatility of leptin secretion: Pulsatility in SNS activity that corresponds to pulsatility in leptin concentration (in humans).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Rapid effects of catecholamines that change the plasma concentration of leptin in vivo (in humans) and inhibit leptin secretion by adipocytes (in rodents) Direct and indirect effects of leptin to stimulate SNS activity (demonstrated only for rodents and nonhuman primates). The presence of a sympathetic innervation in WAT (demonstrated only for rodents). A 24-hour study of plasma concentrations of noradrenaline and adrenaline with frequent sampling found 29 pulses of noradrenaline and 28 pulses of adrenaline (73), remarkably similar to the 32 detected for leptin (62). Pulsatility in SNS activity in humans is also suggested by pulsatile changes in systolic and diastolic blood pressures and in heart rate (64). Pulsatile inhibition of leptin secretion by noradrenaline could, therefore, conceivably occur. Thus, both the pulsatility and the circadian variation in leptin concentration could be determined by changes in SNS activity. Plasma leptin concentrations are rapidly and reversibly suppressed by the infusion of the -adrenergic agonist isoprenaline in humans (35). This would be consistent with the rapidity with which leptin levels increase during pulses, if these increases were due to release from inhibition by catecholamines. The stimulatory effects of leptin on SNS activity are known only from studies with rats and mice. Thus, leptin is able to increase SNS activity in both WAT and BAT, as ascertained from metabolic effects expected to be mediated by noradrenaline (6,74–81). Leptin has also been shown to increase the firing rate in sympathetic nerves of the hindlimb, adrenal, kidney, and BAT in rats (7,82–84). Moreover, intracerebroventricular administration of leptin into the brain in rhesus monkeys causes a rapid increase in circulating noradrenaline concentrations (85). Are the pulses of leptin and SNS activity pulses in humans reciprocally related? That is, does leptin increase SNS activity in humans as it does in rodents? It should be experimentally feasible to measure both leptin and noradrenaline in blood samples taken frequently during a 24-hour period, but this information is not yet available for humans. It can be speculated that during a pulse, an increase in plasma leptin concentration induces noradrenaline release via an action on the SNS, thus bringing about reciprocal suppression of leptin secretion. The question of whether there is indeed extensive sympathetic innervation of WAT in humans, as there is in rodents, remains to be elucidated. However, it is conceivable that a sympathetic nervous innervation of WAT in humans might be stimulated by leptin.

V. LEPTIN SIGNALING PATHWAYS IN CNS AND THE REGULATION OF ENERGY BALANCE Leptin is produced in WAT and crosses the brain–blood barrier through a saturable, unidirectional system to the hypothalamus. It then binds to cells expressCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ing the OB-R, thereby informing the CNS about the state of body fat. Leptin acts in the hypothalamus to regulate body weight and adipose tissue mass by reducing appetite and food intake and by increasing energy expenditure (thermogenesis) through sympathetic stimulation to BAT (86). Leptin receptors, single transmembrane proteins belonging to the cytokinereceptor superfamily, are expressed in several hypothalamic nuclei, including the arcuate nucleus, ventromedial hypothalamus, PVN, and dorsomedial hypothalamus (87). Leptin, acting principally on the arcuate nucleus, activates neuronal circuits that regulate body weight. This is supported by the decrease in food intake induced by local injection of leptin into this area (88) and the inability of central neural administration of leptin to affect food intake (89) after the arcuate nucleus has been destroyed. Other brain areas innervated by the arcuate nucleus neurons, such as the PVN and lateral hypothalamus, are considered as downstream neurons of second order in the pathway regulating neuronal activity by leptin (87). After activation of leptin receptors in the CNS, the signal is transduced by a series of integrated neuronal pathways that lead to changes in the nerve activity affecting different functions in the periphery. It has been proposed that two classes of neurons that are interconnected account for leptin sensitivity in the brain: those activated (catabolic pathway) and those inhibited (anabolic pathway) (90). Pathways that stimulate food intake and promote weight gain (e.g., neuropeptide Y, melanin-concentrating hormone, agouti-related protein, and orexins) appear to be inhibited by leptin and/or activated during fasting. In contrast, those pathways that promote anorexia and weight loss (e.g., melanocortins, cocaineand amphetamine-regulated transcript, thyrotropin-releasing hormone, and corticotropin-releasing hormone) are stimulated by leptin (91). Thus, a highly integrated and redundant system of neuronal pathways appears to mediate the CNS response to a change in leptin signaling. The importance of neurochemical pathways in the hypothalamus in modulating the chronic effects of leptin on sympathetic activity, thermogenesis, and arterial pressure is largely unexplored.

VI. PERIPHERAL LEPTIN SENSORS AND ANS Peripheral “leptin sensors” in rats have recently been identified. Gastric vagal afferents from the region of the stomach where leptin is expressed and where it is released in response to feeding (92) increase their firing rate in response to leptin, and the responsiveness of some of these afferents is enhanced by the presence of cholecystokinine (93). It is conceivable that a local, neurally mediated circuit between the stomach and brain provides leptin-mediated information about feeding that is different from the information provided by adipose tissue leptin.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Direct communication of WAT with the brain might occur via “leptin sensors” within the WAT itself. In rats, these sensors are capable of sensing leptin delivered by local injection and induce a reflex activation of efferent sympathetic nerves to WAT, BAT, adrenal medulla, pancreas, and liver that is mediated by impulses traveling to the brain via the afferent autonomic nerves from the tissue (94,95). Simultaneous suppression of firing rate in vagal pancreatic and hepatic nerves also occurs (95). These findings are preliminary and apply only to rats. However, if a similar leptin-responsive neural loop exists between human adipose tissue and the brain, it could potentially provide a basis for the similarity in pulsatility of leptin secretion and SNS activity. The existence of such a direct neural loop would obviate the need to explain how the pulsatile changes in leptin concentration in blood could be sensed by the brain and transduced into pulsatile changes in efferent SNS activity. They would be sensed locally in the adipose tissues where the leptin is released and transmitted via the afferent nerves. Such a link would provide a morphological basis for the proposed feedback regulatory loop between leptin production by adipose tissue and SNS activity in humans.

VII.

LEPTIN, ENERGY BALANCE, AND ANS

It has become evident that the SNS is pivotal in the regulation of lipolysis: sympathetic denervation leads to an increase in adipose tissue weight, while nerve stimulation results in fatty acid release and sympathetic or ganglionic blockade inhibits the mobilization of lipid (15). Changes in sympathetic activation in individual tissues have often been shown by alterations in the turnover of noradrenaline within the tissue. Noradrenaline turnover is increased in WAT in cold-exposed (23) and fasting rodents. Although fasting leads to a general decrease in noradrenaline turnover in many tissues (38), in WAT, there is increased turnover and, hence, elevated sympathetic activity (24) and raised fatty acid mobilization. Normally, circulating adrenaline levels (derived from the adrenal medulla) are less than the level of noradrenaline (derived principally from sympathetic overspill) and may not be high enough to be physiologically important in the stimulation of lipolysis in comparison with the direct sympathetic innervation. The 3-receptor is more likely to be stimulated by the high noradrenaline concentrations that are found in the synaptic cleft than by catecholamines derived from the circulation (48). However, circulating adrenaline levels may be raised at times of stress and may then contribute to the sympathetic response in adipose tissue. This may occur during fasting and cold exposure. Leptin decreases food intake when given peripherally to mice and rats (96–98). The ob/ob mice that have no leptin are particularly sensitive to the hormone. Intracerebroventricular leptin is more effective at lower doses than when given peripherally (98,99), as might be expected from a hormone thought to have

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

the hypothalamus as its primary site of action. Leptin is thought to increase energy expenditure (100,101). However, in mice at least, this seems to be caused by the prevention of sympathetically mediated reduction in expenditure that occurs in the light phase in thermoneutral conditions when the animals are not feeding (102). It has been postulated that the primary function of leptin is to prevent triglyceride overload by stimulating fatty acid metabolism (103,104). In isolated adipocytes, leptin-stimulated lipolysis is accompanied by up-regulation of mRNAs involved in fatty acid metabolism and, hence, only an increase in glycerol release into the medium was seen. In contrast, noradrenaline-stimulated lipolysis leads to increased glycerol and fatty acid release (105). In rodents, the metabolic effects of leptin involve the sympathetic stimulation of uncoupling protein (UCP) 1 activity in BAT (100) and are prevented by sympathetic denervation of BAT (77). Leptin increases noradrenaline turnover in BAT (6) and increases sympathetic nerve activity to the kidney, adrenals, and the hindlimb (7,106). Leptin (intracerebroventricularly or into the ventromedial hypothalamus) has been shown to increase circulating adrenaline and noradrenaline levels (20) and to increase glucose uptake in heart, BAT, and striated muscle (80) but not in WAT. This effect was blocked by preventing the release of noradrenaline from sympathetic nerves with guanethidine (ineffective centrally and on the adrenal medulla), but not by adrenal demedullation, indicating that sympathetic innervation rather than adrenaline from the adrenal medulla was responsible for these changes (107). Central leptin had a synergistic action with peripheral insulin sensitivity (108). Leptin has also been shown to increase glucose turnover and decrease hepatic glycogen content (78). Although leptin clearly has effects on energy expenditure in rodents, a recent comprehensive review has emphasized that this is not necessarily so for adult humans who have little or no BAT (33). Changes in energy expenditure have, however, been shown in species other than rodents and there are some indications that the control of intake and expenditure follow different pathways. For example, in rhesus monkeys, changes in energy expenditure occurred immediately after the introduction of intracerebroventricular leptin and were associated with increased circulating noradrenaline levels, but not adrenaline levels, indicating sympathetic stimulation of the adrenal medulla; changes in intake were rather more delayed and prolonged (85). However, peripheral leptin administration in primates resulting in changes in leptin concentration within the physiological range did not alter intake (85). Many species do not have discrete amounts of BAT (e.g., adult humans, adult sheep, pigs, many marsupials). Recently, two new mitochondrial uncoupling proteins, UCP 2 and UCP 3, have been discovered. UCP 2 is found in many tissues but prominently in WAT (109); UCP 3 is expressed predominantly in skeletal muscle but also in BAT (110). Leptin increases UCP 3 gene expression in skeletal muscle (111) and WAT, and UCP 2 expression in WAT (112,113). Al-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

though leptin alters UCP 2 and UCP 3 gene expression, the role of UCP 2 and UCP 3 in the sympathetic stimulation of energy metabolism in peripheral tissues is not completely established.

VIII. LEPTIN AND SNS: A LINK BETWEEN OBESITY AND HYPERTENSION? Recent observations suggest that leptin and its multiple interactions with other neurochemical pathways in the hypothalamus may account for the partial link between excess weight gain and increased sympathetic activity. Direct and indirect methods suggest that sympathetic activity is higher in obese than in lean subjects. For example, high caloric intake increases noradrenaline turnover in peripheral tissues and raises plasma noradrenaline concentration (114–116). High caloric intake also amplifies the rise in plasma noradrenaline associated with stimuli such as upright posture and isometric hand grip (114). Obese hypertensive subjects also have increased sympathetic activity in their muscles measured directly with microneurographic methods, compared with lean subjects (117,118). Moreover, weight loss markedly reduces sympathetic activity in obese subjects (119). Although there may be some variation in regional sympathetic activation, sympathetic activity is higher in several organs and tissues, including the kidneys, of obese subjects compared with lean ones (115,118). An interesting model of genetic obesity is the leptin-deficient ob/ob mouse (1). These mice are obese because of an inability to produce leptin and have reduced sympathetic activity compared with their lean counterparts (120). When fed a low-salt diet, ob/ob mice actually have lower blood pressures than their lean counterparts (120). Because increased leptin production may be an important mediator of sympathetic activation and hypertension in obesity, leptin deficiency may prevent the effect of increased adiposity in raising blood pressure in these mice. Some rodent models of obesity that have defective leptin receptors, such as Koletsky (fak/fak) obese spontaneously hypertensive rats, also have lower blood pressures than their lean controls when placed on a low-sodium diet (121). These observations suggest that important pathways normally linking obesity to hypertension, such as leptin, are absent in some of the genetic models of obesity. Hyperleptinemia is one of the possible links between obesity and sympathetic activation. Although leptin’s effects on energy balance have been extensively studied, its effects on sympathetic activity and cardiovascular function are not as well understood. Intravenous or intracerebroventricular infusions of leptin increase sympathetic activity in the kidneys, adrenals, and BAT (83,84). The short-term effect of leptin on sympathetic activity is dose-dependent and occurs in the absence of changes in plasma insulin or glucose (83). Also, the increase in sympathetic ac-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

tivity is slow in onset and may not be fully developed even after 2–3 hours of leptin administration (84). Despite an increase in sympathetic activity in several vascular beds, leptin administration often has little short-term effect on blood pressure (83,84), although small increases in arterial pressure have been observed in some studies when large doses of leptin are injected into the cerebral ventricles (122). The lack of an acute pressor effect (leptin) may be due to opposing depressor effects, such as stimulation of endothelial-derived nitric oxide (123), which offset the effects of increased sympathetic activity. Alternatively, the sympathetic stimulation caused by leptin may be too mild to cause marked peripheral vasoconstriction and transient increases in arterial pressure. However, even modest renal sympathetic stimulation could, over a period of several days, raise arterial pressure by increasing renal tubular sodium reabsorption. Although leptin has both pressor and depressor actions, and short-term leptin administration has little net effect on arterial pressure, long-term infusion of leptin raises blood pressure in rodents (8). Aizawa-Abe et al. (124) found that transgenic skinny mice in which leptin is secreted ectopically by the liver in large amounts also develop mild hypertension comparable to that produced by longterm leptin infusions (8). Agouti mice, which are obese and have high levels of circulating leptin, are also hypertensive despite antagonism of the hypothalamic melanocortin receptors by the high levels of circulating leptin. They are also hypertensive despite antagonism of the hypothalamic melanocortin receptors by the high levels of agouti protein (120,124). This suggests that leptin-induced hypertension may be at least partly independent of stimulation of the melanocortin system. The mechanisms by which increased circulating leptin chronically raise arterial pressure and heart rate are not entirely clear, but are consistent with activation of the SNS. It has been demonstrated that combined - and -adrenergic blockade completely abolished the usual increases in arterial pressure and heart rate during 14 days of leptin infusion (8). In fact, after - and -adrenergic blockade, long-term leptin infusion (8) reduced arterial pressure and heart rate, possibly due to decreased food intake and weight loss (8). Combined - and -adrenergic blockade, however, did not attenuate leptin-induced reductions in food intake or decreases in insulin and glucose levels. Also, administration of adrenergic or ganglionic blockers normalized blood pressure in transgenic skinny mice with leptin-induced hypertension (124). These observations indicate that increased adrenergic activity is essential for leptin-induced hypertension and tachycardia but does not have a major role in mediating the effects of leptin on insulin secretion or glucose homeostasis in nonobese rats. The finding that increasing plasma leptin raises arterial pressure in nonobese rats is consistent with the possibility that leptin may be an important link between obesity, sympathetic activity, and hypertension. However, if obesity is associated with resistance to the effects of leptin on the hypothalamus, and there-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

fore resistance to the effects of leptin on satiety and sympathetic activity, elevated leptin concentrations might cause minimal stimulation of sympathetic activity in obese subjects. The fact that most obese human subjects have high circulating leptin and continue to overeat has been interpreted as evidence for leptin resistance. In addition, some rodent models of obesity have a reduced responsiveness to anorexic effects of leptin (125). In some animal models, such as in the Zucker fatty rat, the leptin resistance is caused by an abnormality of the leptin receptor (126). However, in diet-induced obesity, there is little evidence for defective leptin receptor function. Whether diet-induced obesity attenuates the renal sympathetic responses to leptin is unknown. Nor have the long-term effects of leptin on blood pressure and heart rate been studied in obese subjects, compared with lean ones. Thus, a major issue that remains unresolved is whether there is resistance to the effects of leptin to renal sympathetic activity and whether leptin contributes to increased blood pressure in obese subjects. IX. CONCLUSIONS Leptin has a major role in the regulation of food intake and energy homeostasis. Multiple actions of leptin are relevant to feeding behavior, thermogenesis, and the control of body weight. Leptin also seems to have a role in cardiovascular regulation. Hyperleptinemia may be a partial link between excess weight gain, increased sympathetic activity, and hypertension. ANS activity impacts on metabolic rate and body weight regulation. The ANS is an important regulator of leptin production, and the effects of leptin on energy expenditure and metabolism in peripheral tissues seem to be mediated by changes in ANS activity. Therefore, the ANS is a key efferent mechanism in energy expenditure and metabolism. Further research is needed for a detailed understanding of the complex interactions between leptin and ANS activity and their functional roles in metabolic rate and the control of body weight. REFERENCES 1.

Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372(6505):425–432. 2. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998; 395(6704):763–770. 3. Woods SC, Seeley RJ, Porte D, Jr., Schwartz MW. Signals that regulate food intake and energy homeostasis. Science 1998; 280(5368):1378–1383.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

4. 5.

6. 7. 8. 9.

10.

11.

12. 13.

14. 15. 16.

17.

18. 19. 20.

21.

Mohamed-Ali V, Pinkney JH, Coppack SW. Adipose tissue as an endocrine and paracrine organ. Int J Obes Relat Metab Disord 1998; 22(12):1145–1158. Arone LJ, Mackintosh R, Rosenbaum M, Leibel RL, Hirsch J. Autonomic nervous system activity in weight gain and weight loss. Am J Physiol 1995; 269(1 Pt 2):R222–225. Collins S, Kuhn CM, Petro AE, Swick AG, Chrunyk BA, Surwit RS. Role of leptin in fat regulation. Nature 1996; 380(6576):677. Haynes WG, Morgan DA, Walsh SA, Mark AL, Sivitz WI. Receptor-mediated regional sympathetic nerve activation by leptin. J Clin Invest 1997; 100(2):270–278. Shek EW, Brands MW, Hall JE. Chronic leptin infusion increases arterial pressure. Hypertension 1998; 31(1 Pt 2):409–414. Bartness TJ, Bamshad M. Innervation of mammalian white adipose tissue: implications for the regulation of total body fat. Am J Physiol 1998; 275(5 Pt 2):R1399–1411. Bamshad M, Aoki VT, Adkison MG, Warren WS, Bartness TJ. Central nervous system origins of the sympathetic nervous system outflow to white adipose tissue. Am J Physiol 1998; 275(1 Pt 2):R291–299. Giordano A, Morroni M, Carle F, Gesuita R, Marchesi GF, Cinti S. Sensory nerves affect the recruitment and differentiation of rat periovarian brown adipocytes during cold acclimation. J Cell Sci 1998; 111(Pt 17):2587–2594. Youngstrom TG, Bartness TJ. Catecholaminergic innervation of white adipose tissue in Siberian hamsters. Am J Physiol 1995; 268(3 Pt 2):R744–751. Lever JD, Mukherjee S, Norman D, Symons D, Jung RT. Neuropeptide and noradrenaline distributions in rat interscapular brown fat and in its intact and obstructed nerves of supply. J Auton Nerv Syst 1988; 25(1):15–25. Lafontan M, Berlan M. Fat cell adrenergic receptors and the control of white and brown fat cell function. J Lipid Res 1993; 34(7):1057–1091. Rebuffe-Scrive M. Neuroregulation of adipose tissue: molecular and hormonal mechanisms. Int J Obes 1991; 15(suppl 2):83–86. Cousin B, Casteilla L, Lafontan M, Ambid L, Langin D, Berthault MF, et al. Local sympathetic denervation of white adipose tissue in rats induces preadipocyte proliferation without noticeable changes in metabolism. Endocrinology 1993; 133(5):2255–2262. Youngstrom TG, Bartness TJ. White adipose tissue sympathetic nervous system denervation increases fat pad mass and fat cell number. Am J Physiol 1998; 275(5 Pt 2):R1488–1493. Mercer JG, Moar KM, Hoggard N. Localization of leptin receptor (Ob-R) messenger ribonucleic acid in the rodent hindbrain. Endocrinology 1998; 139(1):29–34. Bray GA, Sclafani A, Novin D. Obesity-inducing hypothalamic knife cuts: effects on lipolysis and blood insulin levels. Am J Physiol 1982; 243(3):R445–449. Satoh N, Ogawa Y, Katsuura G, Numata Y, Tsuji T, Hayase M, et al. Sympathetic activation of leptin via the ventromedial hypothalamus: leptin-induced increase in catecholamine secretion. Diabetes 1999; 48(9):1787–1793. Trayhurn P, Duncan JS, Rayner DV. Acute cold-induced suppression of ob (obese) gene expression in white adipose tissue of mice: mediation by the sympathetic system. Biochem J 1995; 311(Pt 3):729–733.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

22. 23.

24.

25.

26. 27.

28.

29.

30.

31. 32.

33. 34.

35. 36.

37.

38.

Ricci MR, Fried SK, Mittleman KD. Acute cold exposure decreases plasma leptin in women. Metabolism 2000; 49(4):421–423. Garofalo MA, Kettelhut IC, Roselino JE, Migliorini RH. Effect of acute cold exposure on norepinephrine turnover rates in rat white adipose tissue. J Auton Nerv Syst 1996; 60(3):206–208. Migliorini RH, Garofalo MA, Kettelhut IC. Increased sympathetic activity in rat white adipose tissue during prolonged fasting. Am J Physiol 1997; 272(2 Pt 2):R656–661. Weigle DS, Duell PB, Connor WE, Steiner RA, Soules MR, Kuijper JL. Effect of fasting, refeeding, and dietary fat restriction on plasma leptin levels. J Clin Endocrinol Metab 1997; 82(2):561–565. Boden G, Chen X, Mozzoli M, Ryan I. Effect of fasting on serum leptin in normal human subjects. J Clin Endocrinol Metab 1996; 81(9):3419–3423. Kolaczynski JW, Considine RV, Ohannesian J, Marco C, Opentanova I, Nyce MR, et al. Responses of leptin to short-term fasting and refeeding in humans: a link with ketogenesis but not ketones themselves. Diabetes 1996; 45(11):1511–1515. Grinspoon SK, Askari H, Landt ML, Nathan DM, Schoenfeld DA, Hayden DL, et al. Effects of fasting and glucose infusion on basal and overnight leptin concentrations in normal-weight women. Am J Clin Nutr 1997; 66(6):1352–1356. Saladin R, De Vos P, Guerre-Millo M, Leturque A, Girard J, Staels B, et al. Transient increase in obese gene expression after food intake or insulin administration. Nature 1995; 377(6549):527–529. Bing C, Pickavance L, Wang Q, Frankish H, Trayhurn P, Williams G. Role of hypothalamic neuropeptide Y neurons in the defective thermogenic response to acute cold exposure in fatty Zucker rats. Neuroscience 1997; 80(1):277–284. Zheng D, Wooter MH, Zhou Q, Dohm GL. The effect of exercise on ob gene expression. Biochem Biophys Res Commun 1996; 225(3):747–750. Perusse L, Collier G, Gagnon J, Leon AS, Rao DC, Skinner JS, et al. Acute and chronic effects of exercise on leptin levels in humans. J Appl Physiol 1997; 83(1):5–10. Himms-Hagen J. Physiological roles of the leptin endocrine system: differences between mice and humans. Crit Rev Clin Lab Sci 1999; 36(6):575–655. Moinat M, Deng C, Muzzin P, Assimacopoulos-Jeannet F, Seydoux J, Dulloo AG, et al. Modulation of obese gene expression in rat brown and white adipose tissues. FEBS Lett 1995; 373(2):131–134. Pinkney JH, Coppack SW, Mohamed-Ali V. Effect of isoprenaline on plasma leptin and lipolysis in humans. Clin Endocrinol (Oxf) 1998; 48(4):407–411. Carulli L, Ferrari S, Bertolini M, Tagliafico E, Del Rio G. Regulation of ob gene expression: evidence for epinephrine-induced suppression in human obesity. J Clin Endocrinol Metab 1999; 84(9):3309–3312. Donahoo WT, Jensen DR, Yost TJ, Eckel RH. Isoproterenol and somatostatin decrease plasma leptin in humans: a novel mechanism regulating leptin secretion. J Clin Endocrinol Metab 1997; 82(12):4139–4143. Young JB, Landsberg L. Suppression of sympathetic nervous system during fasting. Science 1977; 196(4297):1473–1475.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

39.

40.

41.

42.

43. 44.

45.

46.

47. 48. 49.

50.

51. 52.

53.

Muzzin P, Revelli JP, Kuhne F, Gocayne JD, McCombie WR, Venter JC, et al. An adipose tissue-specific beta-adrenergic receptor. Molecular cloning and down-regulation in obesity. J Biol Chem 1991; 266(35):24053–24058. Collins S, Daniel KW, Rohlfs EM, Ramkumar V, Taylor IL, Gettys TW. Impaired expression and functional activity of the beta 3- and beta 1-adrenergic receptors in adipose tissue of congenitally obese (C57BL/6J ob/ob) mice. Mol Endocrinol 1994; 8(4):518–527. Collins S, Daniel KW, Petro AE, Surwit RS. Strain-specific response to beta 3adrenergic receptor agonist treatment of diet-induced obesity in mice. Endocrinology 1997; 138(1):405–413. Collins S, Daniel KW, Rohlfs EM. Depressed expression of adipocyte beta-adrenergic receptors is a common feature of congenital and diet-induced obesity in rodents. Int J Obes Relat Metab Disord 1999; 23(7):669–677. Breslow MJ, An Y, Berkowitz DE. Beta-3 adrenoceptor (beta-3AR) expression in leptin treated OB/OB mice. Life Sci 1997; 61(1):59–64. Hardie LJ, Rayner DV, Holmes S, Trayhurn P. Circulating leptin levels are modulated by fasting, cold exposure and insulin administration in lean but not Zucker (fa/fa) rats as measured by ELISA. Biochem Biophys Res Commun 1996; 223(3):660–665. Gettys TW, Harkness PJ, Watson PM. The beta 3-adrenergic receptor inhibits insulin-stimulated leptin secretion from isolated rat adipocytes. Endocrinology 1996; 137(9):4054–4057. Mitchell SE, Rees WD, Hardie LJ, Hoggard N, Tadayyon M, Arch JR, et al. ob gene expression and secretion of leptin following differentiation of rat preadipocytes to adipocytes in primary culture. Biochem Biophys Res Commun 1997; 230(2):360–364. Hardie LJ, Guilhot N, Trayhurn P. Regulation of leptin production in cultured mature white adipocytes. Horm Metab Res 1996; 28(12):685–689. Giacobino JP. Role of the beta3-adrenoceptor in the control of leptin expression. Horm Metab Res 1996; 28(12):633–637. Trayhurn P, Duncan JS, Rayner DV, Hardie LJ. Rapid inhibition of ob gene expression and circulating leptin levels in lean mice by the beta 3-adrenoceptor agonists BRL 35135A and ZD2079. Biochem Biophys Res Commun 1996; 228(2):605–610. Mantzoros CS, Qu D, Frederich RC, Susulic VS, Lowell BB, Maratos-Flier E, et al. Activation of beta(3) adrenergic receptors suppresses leptin expression and mediates a leptin-independent inhibition of food intake in mice. Diabetes 1996; 45(7):909–914. Collins S, Surwit RS. Pharmacologic manipulation of ob expression in a dietary model of obesity. J Biol Chem 1996; 271(16):9437–9440. Enocksson S, Shimizu M, Lonnqvist F, Nordenstrom J, Arner P. Demonstration of an in vivo functional beta 3-adrenoceptor in man. J Clin Invest 1995; 95(5):2239–2245. Sennitt MV, Kaumann AJ, Molenaar P, Beeley LJ, Young PW, Kelly J, et al. The contribution of classical (beta 1/2-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and right atrial appendage contraction by novel beta3-adrenoceptor agonists of differing selectivities. J Pharmacol Exp Ther 1998; 285(3):1084–1095.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

54.

55.

56.

57.

58.

59.

60. 61.

62.

63.

64.

65.

66.

67.

68. 69.

Trayhurn P, Duncan JS, Hoggard N, Rayner DV. Regulation of leptin production: a dominant role for the sympathetic nervous system? Proc Nutr Soc 1998; 57(3):413–419. Evans BA, Agar L, Summers RJ. The role of the sympathetic nervous system in the regulation of leptin synthesis in C57BL/6 mice. FEBS Lett 1999; 444(2–3):149–154. Rayner DV, Simon E, Duncan JS, Trayhurn P. Hyperleptinaemia in mice induced by administration of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine. FEBS Lett 1998; 429(3):395–398. Zimmermann RC, Krahn L, Rahmanie N, Sauer MV. Prolonged inhibition of presynaptic catecholamine synthesis does not alter leptin secretion in normal-weight men and women. Hum Reprod 1998; 13(4):822–825. Sivitz WI, Fink BD, Morgan DA, Fox JM, Donohoue PA, Haynes WG. Sympathetic inhibition, leptin, and uncoupling protein subtype expression in normal fasting rats. Am J Physiol 1999; 277(4 Pt 1):E668–677. Sinha MK, Ohannesian JP, Heiman ML, Kriauciunas A, Stephens TW, Magosin S, et al. Nocturnal rise of leptin in lean, obese, and non-insulin-dependent diabetes mellitus subjects. J Clin Invest 1996; 97(5):1344–1347. Schoeller DA, Cella LK, Sinha MK, Caro JF. Entrainment of the diurnal rhythm of plasma leptin to meal timing. J Clin Invest 1997; 100(7):1882–1887. Simon C, Gronfier C, Schlienger JL, Brandenberger G. Circadian and ultradian variations of leptin in normal man under continuous enteral nutrition: relationship to sleep and body temperature. J Clin Endocrinol Metab 1998; 83(6):1893–1899. Licinio J, Mantzoros C, Negrao AB, Cizza G, Wong ML, Bongiorno PB, et al. Human leptin levels are pulsatile and inversely related to pituitary–adrenal function. Nat Med 1997; 3(5):575–579. Furlan R, Guzzetti S, Crivellaro W, Dassi S, Tinelli M, Baselli G, et al. Continuous 24-hour assessment of the neural regulation of systemic arterial pressure and RR variabilities in ambulant subjects. Circulation 1990; 81(2):537–547. Degaute JP, van de Borne P, Linkowski P, Van Cauter E. Quantitative analysis of the 24-hour blood pressure and heart rate patterns in young men. Hypertension 1991; 18(2):199–210. Stene M, Panagiotis N, Tuck ML, Sowers JR, Mayes D, Berg G. Plasma norepinephrine levels are influenced by sodium intake, glucocorticoid administration, and circadian changes in normal man. J Clin Endocrinol Metab 1980; 51(6):1340–1345. Linsell CR, Lightman SL, Mullen PE, Brown MJ, Causon RC. Circadian rhythms of epinephrine and norepinephrine in man. J Clin Endocrinol Metab 1985; 60(6):1210–1215. Prinz PN, Halter J, Benedetti C, Raskind M. Circadian variation of plasma catecholamines in young and old men: relation to rapid eye movement and slow wave sleep. J Clin Endocrinol Metabl 1979; 49(2):300–304. Sakane N, Yoshida T, Mizutani T, Nakagawa Y. Serum leptin levels in a patient with pheochromocytoma. J Clin Endocrinol Metab 1998; 83(4):1400. Bottner A, Eisenhofer G, Torpy DJ, Ehrhart-Bornstein M, Keiser HR, Chrousos GP, et al. Lack of leptin suppression in response to hypersecretion of catecholamines in pheochromocytoma patients. Metabolism 1999; 48(5):543–545.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

70. 71. 72.

73.

74.

75.

76.

77. 78. 79.

80.

81.

82.

83. 84. 85.

86. 87.

Ricci MR, Fried SK. Isoproterenol decreases leptin expression in adipose tissue of obese humans. Obes Res 1999; 7(3):233–240. Trayhurn P, Hoggard N, Mercer JG, Rayner DV. Leptin: fundamental aspects. Int J Obes Relat Metab Disord 1999; 23(suppl 1):22–28. Ahima RS, Prabakaran D, Flier JS. Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding: implications for energy homeostasis and neuroendocrine function. J Clin Invest 1998; 101(5):1020–1027. Schofl C, Becker C, Prank K, von zur Muhlen A, Brabant G. Twenty-four-hour rhythms of plasma catecholamines and their relation to cardiovascular parameters in healthy young men. Eur J Endocrinol 1997; 137(6):675–683. Commins SP, Watson PM, Padgett MA, Dudley A, Argyropoulos G, Gettys TW. Induction of uncoupling protein expression in brown and white adipose tissue by leptin. Endocrinology 1999; 140(1):292–300. Cusin I, Zakrzewska KE, Boss O, Muzzin P, Giacobino JP, Ricquier D, et al. Chronic central leptin infusion enhances insulin-stimulated glucose metabolism and favors the expression of uncoupling proteins. Diabetes 1998; 47(7):1014–1019. Satoh N, Ogawa Y, Katsuura G, Numata Y, Masuzaki H, Yoshimasa Y, et al. Satiety effect and sympathetic activation of leptin are mediated by hypothalamic melanocortin system. Neurosci Lett 1998; 249(2–3):107–110. Scarpace PJ, Matheny M. Leptin induction of UCP1 gene expression is dependent on sympathetic innervation. Am J Physiol 1998; 275(2 Pt 1):E259–264. Kamohara S, Burcelin R, Halaas JL, Friedman JM, Charron MJ. Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 1997; 389(6649):374–377. Sarmiento U, Benson B, Kaufman S, Ross L, Qi M, Scully S, et al. Morphologic and molecular changes induced by recombinant human leptin in the white and brown adipose tissues of C57BL/6 mice. Lab Invest 1997; 77(3):243–256. Minokoshi Y, Haque MS, Shimazu T. Microinjection of leptin into the ventromedial hypothalamus increases glucose uptake in peripheral tissues in rats. Diabetes 1999; 48(2):287–291. Siegrist-Kaiser CA, Pauli V, Juge-Aubry CE, Boss O, Pernin A, Chin WW, et al. Direct effects of leptin on brown and white adipose tissue. J Clin Invest 1997; 100(11):2858–2864. Haynes WG, Morgan DA, Djalali A, Sivitz WI, Mark AL. Interactions between the melanocortin system and leptin in control of sympathetic nerve traffic. Hypertension 1999; 33(1 Pt 2):542–547. Haynes WG, Morgan DA, Walsh SA, Sivitz WI, Mark AL. Cardiovascular consequences of obesity: role of leptin. Clin Exp Pharmacol Physiol 1998; 25(1):65–69. Haynes WG, Sivitz WI, Morgan DA, Walsh SA, Mark AL. Sympathetic and cardiorenal actions of leptin. Hypertension 1997; 30(3 Pt 2):619–623. Tang-Christensen M, Havel PJ, Jacobs RR, Larsen PJ, Cameron JL. Central administration of leptin inhibits food intake and activates the sympathetic nervous system in rhesus macaques. J Clin Endocrinol Metab 1999; 84(2):711–717. Flier JS. Clinical review 94: What’s in a name? In search of leptin’s physiologic role. J Clin Endocrinol Metab 1998; 83(5):1407–1413. Schwartz MW, Woods SC, Porte D, Jr., Seeley RJ, Baskin DG. Central nervous system control of food intake. Nature 2000; 404(6778):661–671.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

88.

89.

90.

91.

92. 93.

94. 95.

96.

97.

98.

99.

100. 101.

102.

103.

104.

Satoh N, Ogawa Y, Katsuura G, Hayase M, Tsuji T, Imagawa K, et al. The arcuate nucleus as a primary site of satiety effect of leptin in rats. Neurosci Lett 1997; 224(3):149–152. Dawson R, Pelleymounter MA, Millard WJ, Liu S, Eppler B. Attenuation of leptinmediated effects by monosodium glutamate–induced arcuate nucleus damage. Am J Physiol 1997; 273(1 Pt 1):E202–206. Cowley MA, Smart JL, Rubinstein M, Cerdan MG, Diano S, Horvath TL, et al. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 2001; 411(6836):480–484. Schwartz MW, Baskin DG, Kaiyala KJ, Woods SC. Model for the regulation of energy balance and adiposity by the central nervous system. Am J Clin Nutr 1999; 69(4):584–596. Bado A, Levasseur S, Attoub S, Kermorgant S, Laigneau JP, Bortoluzzi MN, et al. The stomach is a source of leptin. Nature 1998; 394(6695):790–793. Wang YH, Tache Y, Sheibel AB, Go VL, Wei JY. Two types of leptin-responsive gastric vagal afferent terminals: an in vitro single-unit study in rats. Am J Physiol 1997; 273(2 Pt 2):R833–837. Niijima A. Afferent signals from leptin sensors in the white adipose tissue of the epididymis, and their reflex effect in the rat. J Auton Nerv Syst 1998; 73(1):19–25. Niijima A. Reflex effects from leptin sensors in the white adipose tissue of the epididymis to the efferent activity of the sympathetic and vagus nerve in the rat. Neurosci Lett 1999; 262(2):125–128. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995; 269(5223):540–543. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science 1995; 269(5223):543–546. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 1995; 269(5223):546–549. Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, et al. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 1995; 377(6549):530–532. Scarpace PJ, Matheny M, Pollock BH, Tumer N. Leptin increases uncoupling protein expression and energy expenditure. Am J Physiol 1997; 273(1 Pt 1):E226–230. Hwa JJ, Fawzi AB, Graziano MP, Ghibaudi L, Williams P, Van Heek M, et al. Leptin increases energy expenditure and selectively promotes fat metabolism in ob/ob mice. Am J Physiol 1997; 272(4 Pt 2):R1204–1209. Doring H, Schwarzer K, Nuesslein-Hildesheim B, Schmidt I. Leptin selectively increases energy expenditure of food-restricted lean mice. Int J Obes Relat Metab Disord 1998; 22(2):83–88. Shimabukuro M, Zhou YT, Levi M, Unger RH. Fatty acid–induced beta cell apoptosis: a link between obesity and diabetes. Proc Natl Acad Sci U S A 1998; 95(5):2498–2502. Unger RH, Zhou YT, Orci L. Regulation of fatty acid homeostasis in cells: novel role of leptin. Proc Natl Acad Sci U S A 1999; 96(5):2327–2332.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

105. 106.

107.

108.

109.

110.

111. 112.

113. 114. 115.

116. 117.

118. 119.

120.

121.

Wang MY, Lee Y, Unger RH. Novel form of lipolysis induced by leptin. J Biol Chem 1999; 274(25):17541–17544. Dunbar JC, Hu Y, Lu H. Intracerebroventricular leptin increases lumbar and renal sympathetic nerve activity and blood pressure in normal rats. Diabetes 1997; 46(12):2040–2043. Haque MS, Minokoshi Y, Hamai M, Iwai M, Horiuchi M, Shimazu T. Role of the sympathetic nervous system and insulin in enhancing glucose uptake in peripheral tissues after intrahypothalamic injection of leptin in rats. Diabetes 1999; 48(9):1706–1712. Shi ZQ, Nelson A, Whitcomb L, Wang J, Cohen AM. Intracerebroventricular administration of leptin markedly enhances insulin sensitivity and systemic glucose utilization in conscious rats. Metabolism 1998; 47(10):1274–1280. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, et al. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet 1997; 15(3):269–272. Boss O, Samec S, Paoloni-Giacobino A, Rossier C, Dulloo A, Seydoux J, et al. Uncoupling protein-3: a new member of the mitochondrial carrier family with tissuespecific expression. FEBS Lett 1997; 408(1):39–42. Liu Q, Bai C, Chen F, Wang R, MacDonald T, Gu M, et al. Uncoupling protein-3: a muscle-specific gene upregulated by leptin in ob/ob mice. Gene 1998; 207(1):1–7. Zhou YT, Shimabukuro M, Koyama K, Lee Y, Wang MY, Trieu F, et al. Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation. Proc Natl Acad Sci U S A 1997; 94(12):6386–6390. Scarpace PJ, Nicolson M, Matheny M. UCP2, UCP3 and leptin gene expression: modulation by food restriction and leptin. J Endocrinol 1998; 159(2):349–357. Landsberg L. Obesity, metabolism, and hypertension. Yale J Biol Med 1989; 62(5):511–519. Rumantir MS, Vaz M, Jennings GL, Collier G, Kaye DM, Seals DR, et al. Neural mechanisms in human obesity–related hypertension. J Hypertens 1999; 17(8):1125–1133. Masuo K, Mikami H, Ogihara T, Tuck ML. Weight gain–induced blood pressure elevation. Hypertension 2000; 35(5):1135–1140. Grassi G, Seravalle G, Cattaneo BM, Bolla GB, Lanfranchi A, Colombo M, et al. Sympathetic activation in obese normotensive subjects. Hypertension 1995; 25(4 Pt 1):560–563. Esler M. The sympathetic system and hypertension. Am J Hypertens 2000; 13(6 Pt 2):99S–105S. Grassi G, Seravalle G, Colombo M, Bolla G, Cattaneo BM, Cavagnini F, et al. Body weight reduction, sympathetic nerve traffic, and arterial baroreflex in obese normotensive humans. Circulation 1998; 97(20):2037–2042. Mark AL, Shaffer RA, Correia ML, Morgan DA, Sigmund CD, Haynes WG. Contrasting blood pressure effects of obesity in leptin-deficient ob/ob mice and agouti yellow obese mice. J Hypertens 1999; 17(12 Pt 2):1949–1953. Ernsberger P, Koletsky RJ, Friedman JE. Molecular pathology in the obese spontaneous hypertensive Koletsky rat: a model of syndrome X. Ann N Y Acad Sci 1999; 892:272–288.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

122. 123.

124.

125.

126.

Casto RM, VanNess JM, Overton JM. Effects of central leptin administration on blood pressure in normotensive rats. Neurosci Lett 1998; 246(1):29–32. Lembo G, Vecchione C, Fratta L, Marino G, Trimarco V, d’Amati G, et al. Leptin induces direct vasodilation through distinct endothelial mechanisms. Diabetes 2000; 49(2):293–297. Aizawa-Abe M, Ogawa Y, Masuzaki H, Ebihara K, Satoh N, Iwai H, et al. Pathophysiological role of leptin in obesity-related hypertension. J Clin Invest 2000; 105(9):1243–1252. El-Haschimi K, Pierroz DD, Hileman SM, Bjorbaek C, Flier JS. Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity. J Clin Invest 2000; 105(12):1827–1832. Ishizuka T, Ernsberger P, Liu S, Bedol D, Lehman TM, Koletsky RJ, et al. Phenotypic consequences of a nonsense mutation in the leptin receptor gene (fak) in obese spontaneously hypertensive Koletsky rats (SHROB). J Nutr 1998; 128(12): 2299–2306.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

7 The Autonomic Nervous System and Thermoregulation Quentin J. Pittman University of Calgary, Calgary, Alberta, Canada

The thermoregulatory system is a controlled system requiring sensors, a controller, and effectors. Sensation of temperature occurs in the skin, deep body tissues, and within the central nervous system (CNS) through specialized thermoreceptive fibers and neurons. Information about body temperature is integrated and compared to a reference temperature. This function is distributed in a number of CNS sites but is most highly concentrated in the hypothalamus. The hypothalamus utilizes endocrine, behavioral, and autonomic outputs to alter heat flow to the environment and to alter heat production. These functions are mediated by sympathetic fibers to a number of end organs, including vascular smooth muscle, sweat glands, and brown adipose tissue, and by outputs to both respiratory and skeletal muscles. There is a close relationship between the thermoregulatory system and the control of metabolic rate for purposes of body weight regulation. Under environmental extremes or in the case of pathology, hypothermia or hyperthermia may result. These conditions, which represent a breakdown of normal thermoregulatory control, are in contrast to fever, which is a regulated elevation of body temperature in response to cytokines and which is thought to be useful to the individual in fighting disease. An additional possible beneficial effect of altered body temperature is in the case of controlled hypothermia as a treatment for ischemic brain injury. I. INTRODUCTION Our body temperature is precisely regulated. The stability of this regulation is widely appreciated; even those individuals not scientifically trained are usually Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

aware that body temperature rests at approximately 37°C. Furthermore, deviations from this temperature have important diagnostic implications. As many schoolchildren quickly learn, the presence or absence of a fever can have a strong influence upon the likelihood of their parent or caregiver to agree to an absence from school. However, it is important to note that body temperature varies significantly at different loci in the body. Whereas internal body temperature is usually approximately 37°C, temperatures of more distally located structures are often much lower. Not only are their temperatures often lower, but distally located structures such as ears, arms, and legs display wide variations in temperature brought about by the actions of the autonomic nervous system. Thus, the concept has arisen that body temperature of the core is regulated at a relatively stable level while the periphery or shell is allowed to vary. It is important, however, to note that even within the body core, temperatures at all sites are not identical. For example, temperature around the colon can be over 1°C higher than that of central thoracic areas. Similarly, brain temperature is often higher than abdominal temperature. While temperature at any given site in the core may be precisely regulated, this does not mean that it is invariant. Body temperature follows a true circadian rhythm (1). In the active phase of the cycle, body temperature can be 0.5–1°C higher than in the in active phase. Thus, a rat, which is nocturnal, will have a higher body temperature during the dark phase then during the light phase of the cycle. Humans, who are generally active during the day, display a higher temperature during daytime hours. Over a longer time course, there is a systematic variation in body temperature in cycling females linked to the estrous cycle. Finally, body temperature is not regulated in isolation of other central control mechanisms, and nutritional status, physical exercise, and emotional states can influence body temperature. In order to achieve such precise control of body temperature, there must be a means by which the internal body temperature is monitored; this temperature must be compared to some arbitrary level, or setpoint, and an output signal generated to reduce any difference between the internal body temperature and the setpoint. The progression of scientific thought in our understanding of these mechanisms started with studies in the mid-1800s when Claude Bernard proposed that the constancy of internal body temperature or milieu interieur is achieved as a result of regulatory mechanisms. Subsequently, in the early decades of the twentieth century, Walter Cannon published an important work (2) in which he described the reflexive mechanisms used by animals to maintain their body temperature. This was followed by the recognition by Richter that, in addition to the autonomic mechanisms described by Cannon (and elaborated upon in this chapter), animals and humans also used “behavioral regulators” to maintain thermal homeostasis. With this brief background, the purpose of this chapter is to de-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

scribe the functional anatomy of the thermoregulatory system, examine its pathophysiological aspects, and finally show how the thermoregulatory system can be used for diagnostic and therapeutic purposes. Many attempts have been made to model the thermoregulatory system. Although these models can be based upon engineering, mathematical, or circuit principles, most have in common the idea of a control system based upon negative feedback loops. Such a system requires a series of sensors to provide information about body temperature to a controller that compares this temperature to a reference and then provides an appropriate correction to reduce the error signal between the sensed temperature and the reference. The major components of this system are outlined in Figure 1 and will be discussed in this section.

Figure 1 The major components of the thermoregulatory system.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

II. FUNCTIONAL ANATOMY A. Cutaneous Temperature Sensors While we are not generally aware of our internal body temperature, we can all appreciate our environmental temperature. This is important for our survival in a hostile environment in which temperatures may be considerably above or below those that are ideal for maintaining our 37°C body temperature. Environmental temperature is appreciated by monitoring of skin temperature with thermal sensors in the skin. In monitoring the temperature at the interface with our external environment, we can preempt our physiological thermoregulatory capabilities by employing behavioral means to minimize or maximize heat loss. Equally important, even in the absence of behavioral regulation, sensing environmental temperature allows the body to initiate appropriate compensatory mechanisms prior to significant fluctuation in internal body temperature. Stimulation of cutaneous thermal receptors leads to the perception of either warmth or cooling. At environmental temperatures only a few degrees below body temperature (32–34°C), there is no obvious thermal reception, whereas above or below this temperature warm or cold sensation results. A number of features determine both the threshold and the intensity of thermal sensation; these include the rate of temperature change, the area of skin that is affected, and the amount of temperature change. Cutaneous thermoreceptors are either warm receptors or cold receptors (3). Warm receptors, which exist as unmyelinated nerve endings of primary afferent C fibers, are activated at skin temperatures between 30 and 50°C. Cutaneous warm receptors share many properties of other receptors, having both static and dynamic discharge rates. As seen in Figure 2, a supra-threshold increase in temperature causes the free nerve endings to depolarize and action potential frequency is increased. It is thought that there are several populations of warm receptors having different thresholds for recruitment. Cold receptors, as would be expected, discharge at lower temperatures but do show activity between 10 and 40°C. These receptors, which appear to be supplied by either A or C fibers, generally show increased activity as skin temperature cools over the normal physiological range. At a comfortable environmental temperature, skin temperature is often around 30°C, and at this temperature both warm and cold thermoreceptors will show basal activity that will differentially increase in activity as skin temperature either warms or cools. Cutaneous thermal receptors are not distributed equally throughout the body surface. In primates, including humans, nonhairy skin on the face and hands contains many more thermal receptors then do other skin areas. Thus, the thermal sensitivity of the face can be two to three times more per unit area than that of the chest, abdomen, and thighs. While thermal sensitivities of various skin areas in other mammals have not been as intensively investigated, it is interesting to note Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Skin thermoreceptor properties. (A) In response to a step increase in skin temperature, a warm receptive fiber displays an increase in activity and a cold receptive fiber displays a reduction in activity. (B) Thermoreceptors display both dynamic and static properties in response to a step alteration in skin temperature. (From Ref. 3.)

that the scrotum of the rat is a tissue containing a rich supply of thermal receptors, which have been exploited for studies of thermal afferent pathways. Primary afferent fibers innervating thermoreceptors enter the dorsal horn of the spinal cord or the spinal nucleus of the trigeminal nerve in the case of the face. In the spinal cord, the afferent fibers synapse onto neurons in the marginal zone Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

and substantia gelatinosa of the dorsal horn, cross to the contralateral side of the cord, and ascend in the spinalthalamic tract to the thalamus. This information is then relayed to the sensory cortex. It has been suggested that these ascending axons also send collaterals to the brain stem reticular formation, which then relays information to the hypothalamus. In rats, there is good evidence that ascending axons carrying thermal information from the scrotum synapse in the nucleus raphe magnus, and this nucleus represents a critical link in the transfer of peripheral thermal information to higher CNS structures (4). In addition to the cutaneous thermal receptors described above, there are also deep body thermoreceptors that relay information to the brain from thoracic and abdominal structures. B. Central Control Mechanisms The hypothalamus has been described as the head ganglion of the autonomic nervous system (5), and this description is certainly appropriate in describing its involvement in thermoregulation. This capability comes about from its ability to sense its own temperature and to integrate this information with that from distant sites. In addition, the hypothalamus appears to have executive control of the temperature in that activation of neurons within specified sites in hypothalamus will lead to coordinated outputs to thermoregulatory effector organs leading to alterations in body temperature. The path to our understanding of hypothalamic function involved some of the premier neuroscientists of the previous century. Following reports from Isaac Ott in the United States and Charles Richter in France that damage to the base of the brain led to disruption in thermoregulation, much effort was directed at identifying the location of the “thermostat” in the brain. Sherrington, Bazett, Penfield, and others were able to report that decerebrate or spinally transected animals did not thermoregulate. The involvement of the hypothalamus was first experimentally shown when Ranson, Magoun, and others demonstrated that electrolytic lesions of the hypothalamus interfered with thermoregulation and that heating it activated heat loss mechanisms. While these early investigators proposed that the anterior hypothalamus was a heat-loss area and the posterior hypothalamus was a heat-production area, subsequent studies have refuted this compartmentation. Indeed, while the preeminent role of the hypothalamus in thermoregulation is undisputed, it has long been evident that other parts of the brain and spinal cord are also involved in the control of body temperature. As early as 1933, Keller demonstrated that animals could maintain rudimentary thermoregulatory function with transection below the hypothalamus, and Thauer showed that the spinal cord could support thermoregulatory functions. Thus, it is evident that there is a network of structures in the CNS which are involved in thermoregulation. It has been suggested that there is a hierarchical control, possibly with different structures subserving different functions. In particular, there is good evidence that thermoregulatory behavior may persist when the

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

hypothalamus is lesioned, suggesting that control may, under some conditions, rest outside of the hypothalamus. However, whatever the area involved, structures important in the control of thermoregulation have the ability to sense their own temperature by having a population of thermosensitive neurons. These neurons are most concentrated in the hypothalamus, and many studies have revealed important aspects of their function. C.

Hypothalamic Thermosensitive Neurons

With its location at the base of the brain and cradled within the circle of Willis, which distributes the blood from the carotid arteries to the brain, the hypothalamus is ideally situated to monitor central core temperature. Thermal changes of as little as 0.1°C in the hypothalamus cause significant thermoregulatory responses. The preoptic/anterior hypothalamic area (AHPOA) has the highest thermal sensitivity of all brain sites, and approximately 30% of neurons in this area are warm sensitive (in the rat) and about 10% are cold sensitive (6). Above a defined threshold, warm-sensitive neurons increase their firing rates in response to increases in temperature, whereas cold-sensitive neurons decrease firing rates as temperature increases. Available evidence indicates that warm-sensitive neurons appear to be inherently thermal sensitive whereas cold-sensitive neurons are synaptically driven by warm-sensitive neurons. The above physical basis of thermal sensitivity has been extensively investigated in hypothalamic warm-sensitive neurons. While increased action potential frequency in response to warming does not appear to be associated with membrane potential depolarization, it is associated with the rate of rise of a depolarizing prepotential that initiates action potential generation (Fig. 3). The rise time of the prepotential is controlled by a transient potassium current (IA), whose inactivation rate is temperature sensitive (7). It appears that in addition to sensing local brain temperature, hypothalamic thermoreceptors also receive afferent thermoregulatory information from the skin and other neural structures. Thus, they integrate thermal information from the environment, the body core, and the brain. In addition, hypothalamic neurons thought to be involved in thermoregulatory circuits are also responsive to nonthermal information. For example, some of these neurons receive synaptic inputs from the suprachaismastic nucleus where the body clock appears to be located, and it is likely that such inputs provide the basis for the circadian temperature rhythm. Other neurons are also responsive to a variety of metabolic and endocrine factors that may be important for integration of the thermoregulatory system with other autonomic functions. While the biophysical basis for the setpoint for body temperature is not yet known, a number of models employing neuronal circuits based in the hypothalamus have been proposed (8). It is uncertain as to how accurate these are. A par-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3 Intracellular recordings of warm sensitive neurons in tissue slices of the preoptic area of rats demonstrating salient features important in their thermosensitivity. Left: (A,B) action potential frequency increases as temperature is elevated and this is accompanied by inhibitory post-synaptic potential like activity (arrows); (C) when hyperpolarized (98 mV), there is no evidence of synaptic activity underlying the action potentials seen in A and B. Right: Cooling of an individual neuron (three different temperatures) reduces the rate of rise of the prespike potential. (From Ref. 7.)

ticularly interesting series of investigations carried out several years ago demonstrated that body temperature could be clamped at a specified level over a relatively long time by altering the ratio of sodium to calcium ions within the posterior hypothalamus. Thus, the ratio of sodium to calcium was proposed as the biophysical basis of the set point (9). Unfortunately, the synaptic and membrane mechanisms underlying this phenomenon are not yet known. As indicated above, the integrated and decision-making capabilities of the hypothalamus requires synaptic interactions. A wide variety of neurotransmitters have been shown to activate hypothalamic circuits involved in thermoregulation (10). The majority of hypothalamic synapses are either glutamatergic or GABAergic in nature, and it should come as no surprise that pharmacological interference with the receptors responding to these transmitters will profoundly alter body temperature. Similarly, monoamines such as noradrenaline, 5-hydroxytryptamine, and dopamine have all been implicated as neurotransmitters involved in thermoregulation. To add further complexity to the circuit diagram, the majority of the neuropeptides discovered to date are represented in at least some part of the hypothalamus, and virtually every one of these that has been injected into the hypothalamus will alter body temperature. While some of these, such as bombesin, Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

for example, appear to work by disabling the thermoregulatory control mechanisms to make the animal poikilothermic (11), others such as corticotrophin-releasing hormone (CRH) and leptin (12) exert coordinated actions within the hypothalamus to alter thermoregulatory outputs. D. Thermoregulatory Outputs The goal of the thermoregulatory effectors is to 1) maximize or minimize the heat exchange with the environment and 2) alter heat production. There are three outputs from the hypothalamic control areas to accomplish this—endocrine, behavioral and autonomic; all three outputs are recruited in a coordinated fashion. Over a longer time course, hormonal outputs alter cellular metabolism. An example would be release of thyrotropin-releasing hormone from hypothalamic tuberoinfundibular neurons to eventually cause increased levels of thyroxine (13). This cascade of hormonal events is thought to be activated in response to cold exposure in rodents and results in an increase in the basal metabolic rate due to the action of thyroxine. Behavioral thermoregulation provides a powerful means of controlling body temperature through its ability to alter heat exchange with the environment. It is, of course, very familiar to us when we engage in behavioral thermoregulation by putting on a coat before going out into the cool outdoors. In poikilotherms, body temperature can be maintained at a relatively stable level by moving between warm and cool environments. In the laboratory, rats can be trained to bar press for either hot or cold air to control their environment (14). There is some evidence that behavioral thermoregulation can occur in newborns prior to the appearance of coordinated autonomic outputs. Neural structures known to be involved in behavioral thermoregulation have not been defined in detail, but it is likely that cortical areas are involved. There is also evidence that areas such as the medulla and spinal cord can initiate behavioral thermoregulatory responses independent of the hypothalamus. For example, after lesions to the AHPOA in rats that disrupt autonomic thermoregulatory responses, operant responses for hot or cold are almost unaltered (15). E. Autonomic Thermoregulatory Control 1.

Regulation of Heat Exchange

Even at levels of basal metabolism, internal body temperature is usually greater than environmental temperature. Thus, there is a gradient of heat flow from the body core to the periphery. This heat exchange with the environment takes place at the skin and at sites within the respiratory tract. At these interfaces, heat exchange takes place using the physical principles of convection, conduction, radiation, and evaporation. Convection refers to the transfer of heat from the surface Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

of the body to the air in contact with it and the subsequent vertical air flow that results as the air is heated. Convective heat loss is increased as air flow over the body increases. Conduction refers to the transfer of heat from the body to a substance in physical contact with it. Conductive heat loss can include transfer of heat to air when there is water vapor in the air. This accounts for the increased heat loss (and the perception thereof) that occurs in a humid cold environment when compared to that in a dry environment at a similar temperature. Heat transfer occurs via radiation when heat flows from the body surface to a distant object in the environment, and this is facilitated by a greater differential in the temperature of the two objects. Of course, the converse can occur such as when we absorb heat by radiation, for example, when close to a hot fire or heating element. Finally, evaporative heat loss occurs because evaporation uses body heat to provide the energy required to change water from a liquid to a gaseous state. It is interesting that evaporation is the only means by which the body can cool itself when the environmental temperature is above body temperature. The body uses a number of means to enhance or limit physical heat exchange with the environment by altering convective, conductive, radiative, and evaporative heat loss. These include altering skin blood flow (vasomotion), increasing evaporative heat loss by sweating or increased respiratory rate, and increasing the insulative value of fur or feathers (piloerection) (Fig. 4).

Figure 4 The major pathways involved in the regulation of heat exchange between the body and the environment.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

2.

Vasomotor Control

Alteration of skin temperature represents an effective means of controlling heat loss from the body (16). Vasoconstrictor sympathetic fibers that utilize noradrenaline as a neurotransmitter innervate precapillary arterials. When activated, these fibers cause vasoconstriction, thereby reducing skin blood flow and preventing heat loss to the environment. In addition, in some parts of the body there are specialized shunts between arterials and venules that, when opened, greatly facilitate blood flow through these capillary beds. These arterio-venous anastomoses, as they are called, open in response to an elevation in body temperature and allow skin temperature to rise to levels close to that of the internal body core. There is some evidence that the vasodilatation is brought about by active vasodilator fibers whose transmitter is not known—suggestions include acetylcholine, vasoactive intestinal peptide, and bradykinin. Vasomotor control of the cutaneous circulation varies dramatically throughout the body. It is most effective in the fingertips, hand, ears, and toes and is much less active on the body trunk. Whereas skin blood flow can vary by a factor of 50–60 in the digits, this is reduced to about 7-fold at the trunk. Vasomotor control has been investigated extensively in laboratory animals. Just as is seen in humans and other primates, specialized parts of the body are particularly well adapted for vasomotor heat exchange. In the rabbit, the main structures important in vasomotor heat exchange are the ears, whose temperature can range from only slightly above 0°C at very low ambient temperatures to 35°C or more at warm ambient temperatures. In the rat, the tail functions as the major heat loss organ, and it has been suggested that up to 20% of the animal’s basal heat production can be lost through the tail. Both the rabbit ear and the rat tail contain arterio-venous anastomoses, as do human hands and fingers (among other sites). Skin blood flow is not the only circulatory organ involved in thermoregulation. There is also evidence that blood flow in the limbs (and tail) can be controlled to minimize or maximize heat exchange with the environment. In the cold, blood flow returning from the distal appendages appears to be directed to deep veins which lie in close apposition to arteries. By a countercurrent heat-exchange mechanism, blood leaving the body core is cooled as it loses heat to the returning venous blood in the nearby veins. This thus maintains heat within the body core and prevents loss of heat to the cool periphery. This is a highly advantageous arrangement for animals that must live in temperate and Arctic climates where they are faced with a large temperature differential between body temperature and ambient temperature. As a fortuitous side effect of the maintenance of the heat load within the body core, the resulting cool limb has less likelihood of melting ice and snow where the animal may be standing. The efferent pathways from the hypothalamus to peripheral vasoconstrictor fibers are probably similar to those utilized by other autonomic pathways. Nonetheless, a particularly elegant way of tracing such pathways is now available Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

with the use of the viral transneuronal labeling method. This technique utilizes the pseudorabies virus, which, when applied to a peripheral tissue, is taken up by the innervating nerve fibers and carried back along the axon to the CNS. The virus subsequently infects cells which are in synaptic contact with the initial neuron and so on up the neuraxis. With the use of this technique, a pathway within the brain has been elucidated that appears to be responsible for controlling vasomotor neurons innervating the rat tail artery (17). Synaptically connected neurons have been traced up to the anterior hypothalamic area with relay points in established autonomic nuclei throughout the pons, brain stem, and spinal cord. Interference with these descending pathways from the hypothalamus, for example, by a spinal cord lesion, results in profound loss of skin vasomotor control. This is illustrated in the human in a classic paper by Guttmann and colleagues (18) (Fig. 5). In addition to the descending control of vasomotor tone from the hypothalamus, there is also some evidence in humans for a spinal or lower brain stem reflex in which radiant heat applied to the trunk causes vasodilatation within a latency of a few seconds (19). The role or physiological significance of this is unknown. 3.

Evaporative Heat Loss

Evaporative heat loss is the only means of transferring heat to the environment at temperatures above body temperature. In humans, as environmental temperature approaches body temperature, evaporation accounts for over 90% of heat loss, whereas at an ambient temperature of 25°C it only accounts for 20–25% of heat loss. Evaporative heat loss through skin occurs in two forms in humans: insensible perspiration and sweat secretion. Insensible perspiration dissipates approximately 20% of the basal metabolic heat, but this can be increased dramatically (5to 6-fold) by sweating. Activity of sweat glands is controlled by the sympathetic nervous system but, in contrast to the noradrenergic postganglionic sympathetic nerves elsewhere in the body, those that innervate the eccrine sweat glands important in thermoregulatory control utilize acetylcholine as the postganglionic transmitter. Muscarinic receptors on the sweat glands mediate acetylcholine action. Sweat gland density varies considerably over the body surface with the face, palm, and sole of the foot having the greatest number of sweat glands. However, while density of sweat glands may be less on the trunk, sweat glands in this area are particularly effective in exuding large amounts of sweat. Lower mammals appear to rely less upon sweating for evaporative heat loss than do humans, although the pig appears to be notable exception. In some rodents, in lieu of sweating the animals spread saliva on their fur, and the subsequent evaporative heat loss presumably has an effect similar to that of sweating. An additional source of insensible evaporative heat loss occurs in the respiratory tract. In humans this does not appear to be highly regulated, although virtually all animals will increase their respiratory rates as body temperature is elevated. In some species, such as dogs, specialized respiratory mechanisms such as Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 5 Temperatures at various sites in (A) a normal man and (B) a patient with a C7 spinal cord transection in response to cold and warm ambient air. The normal individual maintains core (rectal) temperature upon cooling by vasoconstriction of the fingers and toes and, to a lesser degree, the trunk. The patient was unable to maintain core temperature in the cold because he was unable to induce vasoconstriction below the level of the lesion. (From Ref. 18.) Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

panting are employed to rapidly move air back and forth across the upper respiratory tract to increase evaporative (and convective) heat loss without associated changes in blood gases associated with respiration. 4.

Altering the Insulating Layer

While altering heat flow to the periphery through vasomotor control of blood flow to the skin provides an important means of regulating heat exchange, this is paralleled by other physiological and behavioral responses. In terms of physiological responses, the ability to create an insulating layer by trapping air in fur or feathers provides an efficient way of altering heat flow. By elevating fur or feathers, an animal can increase the thickness of its insulating layer. Piloerection, as it is called, is of course of limited utility in humans due to our loss of body hair. Nonetheless, the physiological response is maintained and is manifested as “goosebumps,” which occur due to the sympathetically induced (but cholinergically mediated) contraction of the arrectores pili muscles surrounding hair follicles. Of course, regulation of heat loss is achieved most effectively in humans by choosing clothing appropriate for the environment. As pointed out earlier, behavior represents a powerful means of thermoregulatory output. In a warm ambient temperature, animals will often assume a posture in which the limbs are positioned away from the body so as to maximize heat exchange. Conversely, in a cold environment, all appendages are placed close to the body to limit heat loss. Positions such as these can even be seen in newborn human babies. Such postures effectively alter the surface-to-mass ratio. In situations in which animals are in groups, surface-to-mass ratio can be enhanced by huddling. This is perhaps most dramatically illustrated in snakes that will overwinter in a large ball. F. Regulation of Heat Production All cells produce heat as a result of their normal metabolism. This metabolism contributes to the basal metabolic rate of the body, which in itself can be enhanced by activity, nutrient ingestion, and hormonal states. However, basal metabolic rate can be specifically enhanced, resulting in increased thermogenesis. Over longer time periods, a variety of body hormones can affect metabolism. An example alluded to earlier is the action of thyroxine to increase metabolic rate. More acutely, the body can also significantly alter metabolism via neuronal outputs to effector organs (Fig. 6). 1.

Nonshivering Thermogenesis

Two conditions under which thermogenesis can be specifically enhanced are dietinduced thermogenesis and nonshivering thermogenesis (NST). Diet-induced Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 6 The major pathways involved in the regulation of heat production in the body.

thermogenesis is an increase in metabolic rate associated with increased food intake, whereas NST occurs when the body specifically enhances heat production in response to either change in setpoint or to maintain body temperature in the face of cold challenge. The primary effector mechanism for NST is sympathetic activation of brown adipose tissue (BAT). BAT is found in virtually all mammalian neonates, Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

and it is often maintained into adulthood in rodents, particularly if they are coldacclimated. It is activated upon arousal from hibernation to warm animals from their hibernating temperature. BAT is found in the thoraco-lumbar areas, with major concentrations in the interscapular areas and in perirenal depots. Blood that is warmed in the interscapular BAT flows directly back to the heart, where it helps to maintain cardiac function in the cold. While human babies have abundant supplies of BAT in these areas, there it is some uncertainty as to whether adult humans maintain functional BAT. However, it is known that adult humans exhibit NST, making it possible that functional BAT does exist, but it is also quite likely that the NST occurs in muscle. The importance of BAT in thermogenesis is underscored by data from animals genetically deficient in BAT that demonstrate significantly impaired thermogenesis to a variety of stimuli. BAT is named because of its color, which differentiates it from white adipose tissue. The brown color comes both from the high degree of vascularization and from the large numbers of mitochondria in the fat cells. BAT is densely innervated by sympathetic nerves that activate -adrenergic receptors on the fat cells to degrade triglycerides to fatty acids via a cyclic AMP/protein kinase–mediated pathway. Fatty acids are substrates for mitochondrial oxidation. In BAT, oxidative phosphorylation is partially uncoupled (20). This means that the proton gradient generated by respiration is not coupled to the production of ATP from ADP, because the proton circuit is short-circuited, such that the energy stored in the proton gradient is released as heat rather than directed towards phosphorylation of ADP. This is facilitated in BAT mitochondria by the presence of an uncoupling protein (UCP). Sympathetic stimulation directly activates the UCP to facilitate heat production. The UCP found in BAT is called UCP1, and it is a member of several families of UCPs, numbered 1–4. It is likely that the other UCPs may support NST in tissue other than BAT. For example, considerable NST can take place in muscle, a site where some UCPs have been localized. The central nervous system sites responsible for activation of the sympathetic pathways to BAT have been investigated intensively utilizing lesion, electrical stimulation, and pharmacological stimulation (21). Among areas that have been implicated, those that have figured most prominently are the ventromedial hypothalamus, the paraventricular nucleus of the hypothalamus, and the medial preoptic area. These areas also figure prominently as structures that demonstrate functional connectivity with BAT as demonstrated by transneuronal viral tract tracing (22). They also appear to be activated during acute cold exposure, as revealed by Fos immunohistochemical studies in rats (23). While hypothalamic areas represent prominent sites for sympathetic activation of BAT, a variety of brain and brainstem structures undoubtedly are relays in the pathway from the hypothalamus to the preganglionic sympathetic neurons in the spinal cord. It appears that almost any neurotransmitter that has been implicated in thermogenesis is capable of activating NST (24). Likely candidates as transmitters in Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

thermogenic pathways involved in NST include GABA, glutamate, 5-HT, and a number of peptides, the best studied of which is CRH. 2.

Shivering Thermogenesis

Muscle tone contributes significantly to heat production, and this can be enhanced by muscle contractions. This is evident during exercise, when there is greatly increased heat production. In the face of a cold challenge, thermogenesis can be initiated voluntarily by activity, but this may be at the cost of increased convective heat loss. In fact, studies have shown that swimmers in cold water lose their body heat much more quickly than do subjects who float quietly. Shivering, which is a synchronous contraction of antagonist groups of skeletal muscles, provides a means to increase basal metabolism 3 to 5-fold without gross movement. It is entirely involuntary and is activated in response to cooling of the body core; the threshold for onset of shivering can be modified by skin temperature. Shivering usually only occurs when NST and heat conservation mechanisms are unable to maintain a normal body temperature. Shivering is strongly influenced by hypothalamic temperature, but spinal cord cooling is also effective (25). Despite being involuntary, shivering can be inhibited by conscious effort, suggesting an inhibitory cortical input. The posterior hypothalamus appears to play a prominent role in the control of shivering, but it receives descending inputs from septal areas and a variety of other hypothalamic nuclei, in addition to cortical inputs. In the rat, it appears that efferent signals from the hypothalamus descend through the medial forebrain bundle to rubrospinal or reticulospinal tracts, which then activate the ventral horn motor neurons that innervate skeletal muscle (26). Thus, thermoregulation, usually considered to be under the control of the autonomic nervous system, in this case recruits skeletal muscles, which are usually considered to be under voluntary control, for thermoregulatory purposes.

III. THERMOREGULATION AND PATHOPHYSIOLOGY As has been discussed above, the regulation of body temperature depends upon the ability to sense both core and skin temperatures, integrate this information, and compare it to a reference, setpoint temperature and initiate appropriate behavioral and physiological responses to maintain a stable body temperature. Pathological states can alter thermoregulation at any part of this control system. Peripheral neuropathies can alter both thermal sensation as well as motor outputs important in vasomotor control, shivering, and NST (27). Similarly, lesions in many areas of the neuraxis can interfere with thermoregulation. Most obvious is a spinal cord injury, in which patients are unable to vasoconstrict or shiver below the level of the lesion. However, vascular accidents or tumors within the central nervous system, Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

in particular those of the brain stem or of the hypothalamus, may be associated with impaired thermoregulation. For example, pituitary tumors often invade the hypothalamus, resulting in a constellation of autonomic and endocrine disorders, among which are thermoregulatory problems. In addition, there are a number of genetic disorders associated with altered thermoregulation; usually, these are also associated with alterations in body weight. As a result of studies of a number of genetically altered animal models, it is now recognized that there is an intimate relationship between the thermoregulatory system and that involved in the control of metabolism and body weight (28). Two critical components of this relationship are leptin and NST. A. Thermoregulation and Obesity In normal animals, it has long been recognized that increased food intake is associated with increased metabolism, a phenomenon known as diet-induced thermogenesis (DIT). This is a sympathetically driven response, involving BAT and possibly skeletal muscle thermogenesis, which effectively elevates body temperature in order to reduce weight gain (29,30). Conversely, animals (31) and humans (32) that are starved have been known for many years to regulate their body temperature at a lower level. The hypothermia associated with starvation does not appear to result from a detriment in thermogenic capability but rather appears to be a regulated reduction in metabolic rate brought about by a true reduction in setpoint (33). The discovery of leptin, a hormone that is secreted mainly by fat cells and thought to signal fat stores to the hypothalamus, has provided us with new insights about the relationships between metabolism and obesity and the role of DIT in this relationship. Critical evidence supporting this relationship comes from studies of mice that lack either the gene responsible for the production of leptin (ob/ob) or its receptor (db/db). Such mice become obese because they do not recruit BAT thermogenesis to reduce body weight. The Zucker rat has also long been known to have difficulty in defending its body temperature in the cold, and we know now that this rat, which is also susceptible to obesity, has a deficit in leptin signaling (34). There have now been a number of human families who suffer from obesity and thermoregulatory problems whose problems have been traced to leptin-signaling deficits. It is perhaps not surprising that there is a close relationship between thermoregulation and regulation of body weight. Both functions are controlled by a network of hypothalamic neurons that are synaptically connected; of particular importance are the ventral medial, dorsal medial, and paraventricular nuclei, which the reader will recognize have been implicated in both heat loss and thermogenesis pathways. Furthermore, it appears that many of the same neurotransmitters are involved in the regulation of both functions. For example, in addition

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

to its role as the peripheral signal of fat stores, leptin is also found in the brain in hypothalamic structures. CNS injection of leptin or of a number of other central neuropeptides including orexin, NPY, and MSH has been shown to simultaneously affect energy balance and body temperature (35). B. Hypothermia Even in the face of normal thermoregulation, there can be a failure of thermoregulation due to externally imposed environmental conditions which overwhelm the thermoregulatory system. When body temperature falls below 35°C in the human, a condition known as hypothermia is said to exist. In contrast to the regulated hypothermia that may occur during starvation, hypothermia that occurs in the absence of the change in setpoint is considered pathological. That is, even though setpoint temperature remains at or about 37°C, inability to increase heat production or to reduce heat loss in the face of the cold environment results in a progressive fall in core temperature (Fig. 7). It may have a number of etiologies (36). While moderate hypothermia is not considered dangerous, it can be very unpleasant. Furthermore, as temperature falls to below 32–33°C, there is susceptibility to cardiac arrhythmia (37). Equally dangerous perhaps is the fact that as the brain cools, its function becomes impaired and our most important thermoregulatory output, namely behavior, is altered. Hypothermic individuals may become confused and disoriented and may make inappropriate choices that further increase their risk of hypothermia. As hypothermia becomes more profound, there is sig-

Figure 7 Relationship between the setpoint and the core temperature under conditions of normothermia, hypothermia, hyperthermia, and fever.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

nificant alteration in thermoregulatory function. For example, shivering disappears, metabolic rate declines, and cardiac output falls. Blood gases and acid-base regulation are altered and renal function is reduced. These are serious problems that can lead to a fatal outcome. However, susceptibility to hypothermia and its consequences varies greatly. Hypothermia occurs more readily in individuals in which there is a large surface-to-mass ratio such as is seen in babies. Elderly individuals also appear to be more susceptible to hypothermia, possibly due to reduced thermogenic capacity. Recent studies indicate this may be associated with a reduction of UCP levels in skeletal muscle. Drugs that cause peripheral vasodilatation or that interfere with thermogenesis increase susceptibility to hypothermia. Examples of drugs that may be associated with hypothermia include alcohol, barbiturates, and tranquilizers such as chlorpromazine. Neonatal animals and young children appear to be particularly resistant to the deleterious effects of hypothermia. In northern climates, newspapers occasionally report miraculous recoveries of babies who have fallen into cold water or who have been exposed to adverse environmental temperatures without proper clothing. Indeed, babies with no apparent signs of life, i.e., with no heartbeat, respiration, or brain electrical activity and with body temperatures as low as 15–16°C, have been revived to live apparently normal lives. As a result, medical students are taught that there is no such thing as a cold, dead person; only after body temperature is raised to near normal levels without return of function can such a conclusion be reached. C. Hyperthermia When body temperature is elevated above normal, in the absence of a change in setpoint, the condition known as hyperthermia exists (Fig. 7). Hyperthermia can be very serious, leading to fatal heat stroke as body temperature rises above 40°C (38). Heat stroke is often associated with tropical climes and is an annual problem during the pilgrimage to Mecca carried out by Moslems during the Hajj. However, it can also be a problem in temperature climates. A recent heat wave in Chicago was estimated to have caused several hundred fatalities due to heat stroke. Heat stroke can be brought about by exercise in very hot environments, particularly if this is associated with dehydration and salt depletion brought about by excessive sweating. There is some evidence that heavy exercise is associated with development of a low-grade fever brought about by translocation of bacteria from the gut to the abdomen due to ischemia of the vasculature of the gut (38). The associated rise in body temperature may predispose such individuals to hyperthermia and subsequent heat stroke. Symptoms of heat stroke include an elevation of body temperature, generally over 41.1°C, lack of sweating, associated with a hot dry skin, and disordered cognition, ataxia, and coma. Susceptibility to heat stroke can be reduced by preCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

vious acclimatization and or acclimation, possibly due to reduced thresholds for engagement of heat loss mechanisms. Because of the severity of hyperthermia and heat stroke, it is important that body temperature be reduced as quickly as possible. This usually takes the form of external cooling, although in some settings peritoneal dialysis with cold fluids and assisted respiration with cold air have also been used.

IV. DIAGNOSTIC AND CLINICAL IMPLICATIONS A. Fever In contrast to hyperthermia, where body temperature rises above setpoint levels, fever represents the condition in which the body setpoint is elevated and body temperature is increased to the new setpoint level (Fig. 7). When setpoint is elevated during the initial phases of the fever, an error signal is generated between the setpoint and the body temperature, and heat production and conservation mechanisms are activated until the body temperature is elevated to the new setpoint level; this is called the pyretic phase (Fig. 8). The febrile temperature is maintained until such time as the fever breaks and the setpoint returns to normal. Again, an error signal is generated and the elevated body temperature is then reduced through engagement of heat loss mechanisms and a reduction in heat production. This is called the antipyretic phase, and it can also be initiated by exogenous antipyretics such as aspirin. During a febrile illness, the setpoint can fluctuate regularly, leading to cycles of rather bizarre behaviour wherein individuals are alternately cold and shivering and, a short time later, overheated and sweating.

Figure 8 The two phases of fever, pyresis and antipyresis, and the change in setpoint and core temperature that accompanies them.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Fever is a phylogenetically ancient phenomenon and is one of the most important indicators of disease. At normal febrile temperatures, fevers are not considered dangerous and they do not lead to hyperthermia and heat stroke. While in very young children fevers can be associated with febrile convulsions, these are seldom seen in older individuals. Indeed, in addition to being diagnostic of illness, fever is now considered to be, at worst, benign and most likely helpful in fighting disease. This is because it is also an important participant, along with other hostdefense responses, in the neuro-immune response to infection. Animals prevented from developing fever have greater morbidity and mortality than those permitted to develop fever. Because fever appears to be a hallmark of infection throughout the phylogenetic tree and has been evolutionarily conserved, the concept has arisen that fever has survival value (39). When microorganisms (gram-negative infections are best understood, but viral infections also cause fever) invade our bodies, they expose our immune system to large lipopolysaccharide (LPS) molecules, often called exogenous pyrogens or endotoxins. LPS binds to a soluble, circulating LPS-binding protein, and this complex binds to the CD14 and toll-like receptors found on certain monocytes and macrophages. These in turn synthesize and release a variety of endogenous proteins; those thought to be most important in fever are interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF). During fever, a number of humoral changes take place, collectively called the acute phase response. In addition, a number of behavioral changes take place, which are collectively called sickness behaviors (40). Here we will discuss the regulated rise in body temperature characteristic of fever. There is good evidence that fever involves the CNS (41); some of the possible players in the CNS are illustrated in Figure 9. The mechanism by which peripherally generated cytokines or other peptides activate the brain has been intensively investigated. Evidence exists for several possible avenues, depending upon the route and dose of administration of cytokines (reviewed in Refs. 42, 43); these include direct transport across the blood-brain barrier, entry at circumventricular organs, local stimulation of perivascular and meningeal cells, and activation of peripheral nerves. Whatever the route of administration, it appears that most cytokines activate an inducible cyclooxygenase (COX 2), most likely in glia and in endothelial cells, to cause intracerebral synthesis of prostaglandins, largely of the E series (PGE) (reviewed in Refs. 43, 44). Peripheral immune stimuli activate many autonomic and endocrine nuclei, as revealed by Fos expression (45–47), but it is difficult to distinguish which pathways are involved in the fever response and which are involved in the many other autonomic responses (cardovascular, gastrointestinal, etc.) associated with immune activation, especially at the high doses often employed in these studies. Prostaglandins are known to act in several sites to activate central sympathetic pathways (reviewed in Ref. 44), but the most sensitive of these for the purposes of fever generation appears to be a small nucleus in the ventral medial preoptic area (VMPOA) (43). Among other

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 9 Some of the neural pathways and likely agents involved in the febrile response.

projections of this nucleus, that to the paraventricular nucleus (PVN) and nearby perifornical area appear to be particularly important sites for activation of heat conservation and thermogenesis to cause fever. In addition to the prostaglandin link, intense (i.e., high-dose) peripheral immune activation causes synthesis within the brain of a variety of cytokines and certain transcription factors (48). While application of IL-1 or TNF directly to the brain by intracerebroventricular injection will cause a fever, and receptors of such cytokines are present in the brain, the involvement of this brain cytokine system in the responses to peripheral immune stimuli is not well understood. Nonetheless, for some models of fever, particularly those with long latencies, injection of an IL-1 receptor antagonist (IL-1ra) into the brain will inhibit fever due to peripheral inflammation (49). It has now been reported in many labs that fever size and duration can change dramatically between males and females and also in association with the reproductive cycle. Fevers subside, either naturally by inactivation of causative organisms or due to active pharmacological intervention (i.e., aspirin inhibits PGE synthesis). There is now good evidence that defervescence and fever suppression is a controlled process involving release and action of the neuropeptides arginine vasopressin (AVP) and alpha-melanocyte–stimulating hormone (MSH). While the

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

evidence for MSH is not as extensive as that for AVP, its injection reduces fever and an antagonist elevates fever (50,51). Salient facts supporting a role for AVP as an antipyretic are summarized as follows: 1.

AVP, introduced into the brain (ventral septal area (VSA) or amygdala), reduces febrile, but not normal body temperature via an action at V1 type receptors. 2. Activation of endogenous AVP pathways causes antipyresis. 3. Interference with AVP release or action results in elevated, prolonged fevers. 4. During defervescence, increased quantities of AVP are released into the brain. The exact locus of AVP action within the brain is not known, nor is the mechanism by which it acts to reduce fever. A source of the AVP involved in brain antipyresis is a group of AVP-immunoreactive cell bodies located in the bed nucleus of the stria terminalis (BST); these are different from the AVP-containing neurons found in the magnocellular PVN and supraoptic nuclei (SON). The latter cell bodies project to the pituitary, where they release AVP into the circulation to regulate renal and cardiovascular function. Nonetheless, AVP in this pathway is also released during fever, but its role in the host defense response is not known. Central AVP may also be important in causing a condition called endogenous antipyresis. This is a state in which the normal febrile response to a pyrogen is reduced; it can be seen in certain neonates, in some types of hypertension, in acute hypotension, and in parturient animals (52). In addition to central antipyretics, a variety of peripheral hormones and some cytokines are also effective antipyretics. Some cytokines such as II-4 and an II-1 receptor antagonist are synthesized along with the pyrogenic cytokines and appear to antagonize their action. Glucocorticoids act both peripherally and centrally to lower fever (53). Of course, the best-known antipyretics are those that we take to suppress fever and inflammation, the nonsteroidal anti-inflammatories such as aspirin (acetylsalicylic acid), acetaminophen, ibuprofen, etc.

V. THERAPEUTICS Manipulation of body temperature has a long history as a means to treat disease; for example, prior to the emergence of antibiotic therapy, fever therapy was used to treat syphilis. More recently, elevated body temperature has sometimes been used to treat some types of cancer (54). The most promising manipulation of body temperature, however, appears to be the use of hypothermia in brain ischemia (stroke) and neurotrauma. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The effectiveness of hypothermia in reducing ischemic cell death was recognized nearly 50 years ago in the classic experiments of Rosomoff, Bigelow, and DeBakey (55–57). Its use in cardiac surgery has been extensive (reviewed in Ref. 58). Nearly 15 years ago Busto and colleagues (59) recognized that mild hypothermia during ischemia was neuroprotective, and many studies since that time have shown that hypothermia can reduce ischemic damage both in vivo in global and focal stroke models (reviewed in Ref. 60) and in vitro (61). Conversely, in both patient and in animal studies the appearance of hyperthermia during ischemia or fever following a stroke is almost invariably associated with a worsened outcome (62). Furthermore, the neuroprotective properties of some “neuroprotective” pharmacological agents can be attributed to their hypothermic action. Hypothermia initiated after reperfusion is also effective, and its effectiveness is enhanced with longer cooling of up to 48 hours (63). There are many possible explanations and mechanisms as to why hypothermia is so effective, and it is suggested that it is the constellation of effects that is important (reviewed in Ref. 64). However, the fact that it is effective postischemia would appear to make it a practical treatment for patients appearing at hospital after a stroke. Unfortunately, results for published and in progress neurotrauma/stroke studies have been equivocal (65). Although rodent studies have been encouraging, there remain highly discouraging reports from higher mammalian studies, and even recent small-scale stroke trials in humans report significant morbidity and mortality. VI. SUMMARY An understanding of autonomic control of thermoregulation is critical to an understanding of many other physiological, behavioral, and pharmacological processes. Interference with thermoregulation will affect such diverse events as learning in a maze and the rate of metabolism of a drug. It shares many of the same neural structures as do other autonomic functions. While only passing reference was made to other autonomic functions in this chapter, it is important for the reader to refer to other chapters in this text that deal in more detail with these related functions. REFERENCES 1.

Refinetti R, Menaker M. The circadian rhythm of body temperature. Physiol Behav 1992; 51(3):613–637. 2. Cannon WB. The Wisdom of the Body. New York: Norton, 1932. 3. Hensel HH. Thermoreception and Temperature Regulation. London: Academic Press, 1981. 4. Hellon RF, Taylor DC. An analysis of a thermal afferent pathway in the rat. J Physiol 1982; 326:319–328.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

5.

6. 7.

8. 9. 10.

11. 12.

13.

14. 15. 16. 17.

18. 19. 20. 21. 22. 23.

Sharkey KA, Pittman QJ. The autonomic nervous system: peripheral and central integrative aspects. In: Gregor R, Windhorst U, eds. Comprehensive Human Physiology—From Cellular Mechanisms to Integration. New York: Springer-Verlag, 1996:335–353. Boulant JA. Hypothalamic neurons. Mechanisms of sensitivity to temperature. Ann NY Acad Sci 1998; 856:108–115. Curras MC, Kelso SR, Boulant JA. Intracellular analysis of inherent and synaptic activity in hypothalamic thermosensitive neurones in the rat. J Physiol 1991; 440:257–271. Bligh J. The central neurology of mammalian thermoregulation. Neuroscience 1979; 4(9):1213–1216. Myers RD, Veale WL. Body temperature: possible ionic mechanism in the hypothalamus controlling the set point. Science 1970; 170(953):95–97. Pittman QJ, Thornhill JA. Neuropeptide mechanisms affecting temperature control. In: Ganten D, Pfaff D, eds. Current Topics in Neuroendocrinology. Vol. 10. Behavioral Aspects of Neuroendocrinology. New York: Springer-Verlag, 1990: 223–241. Tache Y, Pittman QJ, Brown M. Bombesin induced poikilothermy in rats. Brain Res 1980; 188:525–530. Luheshi GN, Gardner JD, Rushforth DA, Loudon AS, Rothwell NJ. Leptin actions on food intake and body temperature are mediated by IL-1. Proc Natl Acad Sci USA 1999; 96(12):7047–7052. Arancibia S, Rage F, Astier H, Tapia-Arancibia L. Neuroendocrine and autonomous mechanisms underlying thermoregulation in cold environment. Neuroendocrinology 1996; 64:257–267. Gordon CJ. 24-hour control of body temperature in rats. I. Integration of behavioral and autonomic effectors. Am J Physiol 1994; 267(1 Pt 2):R71–R77. Satinoff E, Rutstein J. Behavioral thermoregulation in rats with anterior hypothalamic lesions. J Comp Physiol Psychol 1970; 71(1):77–82. Rowell LB. Cardiovascular aspects of human thermoregulation. Circ Res 1983; 52(4):367–379. Smith JE, Jansen AS, Gilbey MP, Loewy AD. CNS cell groups projecting to sympathetic outflow of tail artery: neural circuits involved in heat loss in the rat. Brain Res 1998; 786(1–2):153–164. Guttmann L, Snell ES, Cranston WI. Thermoregulation in spinal man. J Physiol (Lond) 1958; 142:406–419. Kerslake DM, Cooper KE. Vasodilatation in the hand in response to heating the skin elsewhere. Clin Sci 1950; 9:31–47. Ricquier D, Bouillaud F. Mitochondrial uncoupling proteins: from mitochondria to the regulation of energy balance. J Physiol 2000; 529 Pt 1:3–10. Rothwell NJ. Central control of brown adipose tissue. Proc Nutr Soc 1989; 48(2):197–206. Bamshad M, Song CK, Bartness TJ. CNS origins of the sympathetic nervous system outflow to brown adipose tissue. Am J Physiol 1999; 276(6 Pt 2):R1569–R1578. Baffi JS, Palkovits M. Fine topography of brain areas activated by cold stress. A fos immunohistochemical study in rats. Neuroendocrinology 2000; 72(2):102–113.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

24. Rothwell NJ. CNS regulation of thermogenesis. Crit Rev Neurobiol 1994; 8(1–2):1–10. 25. Mercer JB. The shivering response in animals and man. Arctic Med Res 1991; 50(suppl 6):18–22. 26. Nagashima K, Nakai S, Tanaka M, Kanosue K. Neuronal circuitries involved in thermoregulation. Auton Neurosci 2000; 85(1–3):18–25. 27. Spallone V, Menzinger G. Autonomic neuropathy: clinical and instrumental findings. Clin Neurosci 1997; 4(6):346–358. 28. Lowell BB, Spiegelman BM. Towards a molecular understanding of adaptive thermogenesis. Nature 2000; 404(6778):652–660. 29. Lowell BB. Adaptive thermogenesis: turning on the heat. Curr Biol 1998; 8(15):R517–R520. 30. Rothwell NJ, Stock MJ. A role for brown adipose tissue in diet-induced thermogenesis. Obes Res 1997; 5(6):650–656. 31. Hamilton CL. Effect of food deprivation on thermal behavior of the rat. Proc Soc Exp Biol Med 1959; 100:354–356. 32. Grande F, Anderson JT, Keys A. Changes of basal metabolic rate in man in semistarvation and refeeding. J Appl Physiol 1958; 12:230–238. 33. Sakurada S, Shido O, Sugimoto N, Hiratsuka Y, Yoda T, Kanosue K. Autonomic and behavioural thermoregulation in starved rats. J Physiol 2000; 526(Pt 2):417–424. 34. Schalling M, Johansen J, Nordfors L, Lonnqvist F. Genes involved in animal models of obesity and anorexia. J Intern Med 1999; 245(6):613–619. 35. Forbes S, Bui S, Robinson BR, Hochgeschwender U, Brennan MB. Integrated control of appetite and fat metabolism by the leptin-proopiomelanocortin pathway. Proc Natl Acad Sci USA 2001; 98(7):4233–4237. 36. Harchelroad F. Acute thermoregulatory disorders. Clin Geriatr Med 1993; 9(3):621–639. 37. Paton BC. Accidental hypothermia. Pharmac Ther 1983; 22:331–377. 38. Hales JR. Hyperthermia and heat illness. Pathophysiological implications for avoidance and treatment. Ann NY Acad Sci 1997; 813:534–544. 39. Kluger MJ, Kozak W, Conn CA, Leon LR, Soszynski D. Role of fever in disease. Ann NY Acad Sci 1998; 856:224–233. 40. Dantzer R, Bluthe RM, Laye S, Bret-Dibat JL, Parnet P, Kelley KW. Cytokines and sickness behavior. Ann NY Acad Sci 1998; 840:586–590. 41. Cooper KE. Fever and Antipyresis—The Role of the Nervous System. Cambridge: Cambridge University Press, 1995. 42. Blatteis CM, Sehic E. Fever: How many circulating pyrogens signal the brain? News Physiol Sci 1997; 12:1–9. 43. Elmquist JK, Scammell TE, Saper CB. Mechanisms of CNS response to systemic immune challenge: the febrile response. Trends Neurosci 1997; 20:565–570. 44. Blatteis CM, Sehic E. Prostaglandin E2: A putative fever mediator. In: Mackowiak PA, ed. Fever: Basic Mechanisms and Management. Philadelphia: Lippincott-Raven Publishers, 1997:117–145. 45. Rivest S, Laflamme N. Neuronal activity and neuropeptide gene transcription in the brains of immune-challenged rats. J Neuroendocrinol 1995; 7:501–525.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

46.

47.

48. 49.

50.

51.

52. 53.

54. 55.

56.

57.

58. 59.

60.

61.

Wan W, Wetmore L, Sorensen CM, Greenberg AH, Nance DM. Neural and biochemical mediators of endotoxin and stress-induced c-fos expression in the rat brain. Brain Res Bull 1994; 34(1):7–14. Elmquist JK, Scammell TE, Jacobson CD, Saper CB. Distribution of Fos-like immunoreactivity in the rat brain following intravenous lipopolysaccharide administration. J Comp Neurol 1996; 371:85–103. Hopkins SJ, Rothwell NJ. Cytokines and the nervous system I: Expression and recognition. Trends Neurosci 1995; 18:83–88. Luheshi G, Hopkins SJ, Lefeuvre RA, Dascombe MJ, Ghiara P, Rothwell NJ. Importance of brain IL-1 type II receptors in fever and thermogenesis in the rat. Am J Physiol 1993; 265:E585–E591. Huang QH, Entwistle ML, Alvaro JD, Duman RS, Hruby VJ, Tatro JB. Antipyretic role of endogenous melanocortins mediated by central melanocortin receptors during endotoxin-induced fever. J Neurosci 1997; 17(9):3343–3351. Rajora N, Boccoli G, Burns D, Sharma S, Catania AP, Lipton JM. alpha-MSH modulates local and circulating tumor necrosis factor-alpha in experimental brain inflammation. J Neurosci 1997; 17(6):2181–2186. Pittman QJ, Wilkinson MF. Central arginine vasopressin and endogenous antipyresis. Can J Physiol Pharmacol 1992; 70:786–790. Coelho MM, Luheshi G, Hopkins SJ, Pelá IR, Rothwell NJ. Multiple mechanisms mediate antipyretic action of glucocorticoids. Am J Physiol Reg Integr Comp Physiol 1995; 269:R527–R535. Falk MH, Issels RD. Hyperthermia in oncology. Int J Hyperthermia 2001; 17(1):1–18. Rosomoff HL. Hypothermia and cerebral vascular lesions. I. Experimental interruption of the middle cerebral artery during hypothermia. J Neurosurg 1956; 13:244–255. Pontius RG, Bloodwell RD, Cooley DA, DeBakey ME. The use of hypothermia in the prevention of brain damage following temporary arrest of cerebral circulation: experimental observations. Surg Forum 1954; 5:224–228. Bigelow WG, Lindsay WK, Greenwood WF. Hypothermia: Possible role in cardiac surgery: investigation of factors governing survival in dogs at low body temperature. Ann Surg 1950; 132:849–866. Tisherman SA, Rodriguez A, Safar P. Therapeutic hypothermia in traumatology. Surg Clin North Am 1999; 79(6):1269–1289. Busto R, Dietrich WD, Globus MY, Valdes I, Scheinberg P, Ginsberg MD. Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury. J Cereb Blood Flow Metab 1987; 7(6):729–738. Corbett D, Thornhill J. Temperature modulation (hypothermic and hyperthermic conditions) and its influence on histological and behavioral outcomes following cerebral ischemia. Brain Pathol 2000; 10(1):145–152. Bruno VM, Goldberg MP, Dugan LL, Giffard RG, Choi DW. Neuroprotective effect of hypothermia in cortical cultures exposed to oxygen-glucose deprivation or excitatory amino acids. J Neurochem 1994; 63(4):1398–1406.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

62.

Thornhill J, Asselin J. Increased neural damage to global hemispheric hypoxic ischemia (GHHI) in febrile but not nonfebrile lipopolysaccharide Escherichia coli injected rats. Can J Physiol Pharmacol 1998; 76(10–11):1008–1016. 63. Corbett D, Hamilton M, Colbourne F. Persistent neuroprotection with prolonged postischemic hypothermia in adult rats subjected to transient middle cerebral artery occlusion. Exp Neurol 2000; 163(1):200–206. 64. Colbourne F, Sutherland G, Corbett D. Postischemic hypothermia. A critical appraisal with implications for clinical treatment. Mol Neurobiol 1997; 14(3):171–201. 65. Correia M, Silva M, Veloso M. Cooling therapy for acute stroke. Cochrane Database Syst Rev 2000; (2):CD001247.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

8 Phylogenesis of the Autonomic Nervous System from Fish to Mammals C. Liana Bolis University of Milan, Milan, Italy, and AIREN, Geneva, Switzerland

Andrea Zenone Dalla Valle and Margherita Piccolella University of Milan, Milan, Italy

I. INTRODUCTION When comparative studies were first made on mammals and the other animals, the researchers paid attention principally to differences in anatomy. It was only later, when technology improved, that they started to study comparative physiology and biochemistry. Galen (1) made the earliest recorded reference to the visceral nervous system in the second century. He gave the first account of the paravertebral sympathetic chains, but he made the mistake of describing the sympathetic and vagal trunks as one structure originating within the cranium. This gave rise to an error, which persisted for 1500 years. Galen was the first to note that the denervated heart maintained its beat. Following these observations of Galen, little progress was made through the ensuing 14 centuries until the time of Vesalius (1543) (2), who depicted a combined vagosympathetic trunk arising from the brain stem. Stephanos (1545) (3) and later Eustachius (1563) (4) were the first to distinguish the two separate nerves. In the seventieth century Willis (1664) published a remarkably clear account of the ganglionated chains and their connections with the intercostal nerves (5). He described the cardiac branches and stated that the great mesenteric plexus sent its nerve fibers like rays in all directions; hence, it came to be called the solar plexus. He considered that its function was to place the heart and viscera in con-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

nection with the brain so that they could act in harmony. The modern nomenclature of the cranial nerves originated with Willis. In addition, he gave an accurate description of the vagus or “wandering nerve” with a true understanding of its apparent union with the cervical sympathetic in some of the lower mammals and its separate course in man. He even noted the branch given off to the aortic arch “so it may react to changes in the pulse.” According to Sheehan (6), who describes the conception of the autonomic nervous system at the end of the seventeenth century, the “intercostal” (sympathetic) and “wandering” (vagus) nerves, though clearly separated anatomically, remained physiologically one system, possessing a double function. When one considers that this concept was based almost entirely on anatomical observations, it constitutes a most remarkable hypothesis. In 1732 the Danish anatomist Winslow (7) gave the name “sympathetic” to nerves that he demonstrated by dissection to lie outside the main cerebrospinal pathways. Neubauer (1772) published an illustration of the vagus and sympathetic nerves in the neck and thorax that ranks as one of the best anatomical plates produced to date (8). As in the other fields of medical science, anatomical knowledge developed far ahead of physiological knowledge. In 1669 Lower (9) published the earliest observations on stimulation of the vagus. Further experiments by Ens (1745) (10) and by the Webers (1846) (11) a century later finally established the role of the vagus in inhibition of the heart. The discovery that the sympathetic trunks originate below the cranium and not from the brain stem, as described by Galen and all subsequent anatomists, was worked out by Du Petit in 1727 (12). He was likewise the first to observe the pupillary paralysis that follows cervical sympathectomy, thus antedating Bernard and Horner by over 100 years. The first appreciation of involuntary movements and visceral sensation developed out of the experiments of Whytt (1751) (13). He was the first to gain an insight into such fundamental concepts as the tone of skeletal muscle, the reflex responses of the pupils to light, and the fact that “the distension of hollow muscles has a remarkable influence towards exciting them into action.” The ultimate expression of this theory had been reached by Willis, who wrote that “sympathy” was due to communications of the nerve tubes, which issued from the cerebellum and the brain stem, more especially those belonging to the “eighth pair” and the “intercostal nerve.” Whytt (1765) revised this traditional view by stating that “sympathy” presupposed feeling and must therefore be dependent on nerves (14). Although earlier workers had recognized that the viscera were not under voluntary control of the nervous system, they had not observed the structural differences between skeletal muscle and the muscular coats of the hollow viscera. This discovery was made by Muller (1840) (15), who did not recognize that arteries possess a true muscular coat. The histological description of the muscular

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

layer was given by Von Kolliker (1849) (16), and its innervation by a periarterial sympathetic plexus was described by Henle (1868) (17). Langley’s final establishment of the two great divisions of the involuntary nerves depended on Hirschmann’s discovery in 1863 that moderate doses of nicotine prevent pupillary dilatation when the cervical sympathetic trunk is stimulated. In fact, the term “autonomic nervous system” was proposed by Langley in 1898 to describe “the sympathetic system and the allied nervous system of the cranial and sacral nerves, and for the local nervous system of the gut” (18). Early work on the functional anatomy of the autonomic nervous system included studies on tetrapods other than mammals, e.g., amphibians and birds. Young studied both teleost and elasmobranch fish, describing the morphology of the autonomic innervation of the iris (19–21) and viscera (19,22,23). The comparative approach makes it possible to understand how animals can adjust various physiological parameters in response to changing environmental or internal cues. The evolutionary changes governed by natural selection have covered 400 million years of vertebrate evolution. For example, all vertebrates have an alimentary canal of some sort, but the control systems are not identical in all the animal groups. This study has been very important for understanding pathways that are fundamental in the human being. The mammalian autonomic nervous system is divided into a sympathetic, a parasympathetic, and an enteric portion. In the original definitions, the sympathetic pathways ran from thoracic and lumbar segments of the spinal cord, while the parasympathetic pathways ran in the cranial nerves and from the sacral segments. This anatomical division was upheld by the perceived functional differences, where the terms sympathetic and adrenergic have become almost synonymous in the literature dealing with mammals. The discovery of a vast number of nonadrenergic, noncholinergic neurotransmitters, notably peptides, clearly challenges the idea of “sympathetic  adrenergic.” Similarly, a functional subdivision based on transmitter content of the neurons has been used for several types of nerves.

II. FISH In fish, the cranial autonomic pathways are probably restricted to the oculomotor and vagus nerves, although autonomic fibers in the facial and glossopharyngeal nerves in elasmobranchs and teleosts have been postulated by some workers (24). There are no lachrymal or salivary glands in fish, and the main targets for the cranial autonomic pathways within the head are the eye and the gills. The paired sympathetic chains are very well developed in teleosts. In contrast to the situation in other vertebrates, these chains continue into the head, bearing ganglia in contact with the cranial nerves.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

In the elasmobranchs, the paravertebral ganglia are not always longitudinally connected to form proper sympathetic chains of the teleost or tetrapod type. The autonomic nervous system of cyclostomes is fragmentary. In the African and South American lungfish, cranial autonomic pathways are restricted to the vagus, but in the Australian lungfish there may be additional pathways to the eyes running in the oculomotor nerve (24). The transmitter substances found in the fish groups are similar to those found in the tetrapods, with the notable exception of the neuropeptides, where the amino acid sequences differ (25). Thus, both acetylcholine and the catecholamines (adrenaline and noradrenaline) occur in autonomic nerves of fish. Adrenaline is the dominant catecholamine in adrenergically innervated organs of teleost and amphibians, while noradrenaline dominates in the other groups. Contrary to popular belief, noradrenaline is not the sole catecholamine in mammalian adrenergic neurons (26). In fact, adrenaline release from mammalian adrenergic nerves can be quite substantial, and this is also true for birds. Serotonin is now recognized as an important transmitter substance in the peripheral system of both mammals and nonmammalian vertebrates. Serotonin participates together with the autonomic sympathetic and parasympathetic fibers in the control of gastrointestinal and vascular functions. A. Cyclostomes The spinal autonomic pathway in cyclostomes is sparse and not organized as in mammals. This is due to the ill-defined nature of tissues that could be considered to be components of these pathways (24). Cyclostomes do not possess sympathetic ganglia like those seen in mammals, and it is most probable that they do not have autonomic ganglion cells closely associated with the roots of their spinal nerves. In lampreys, small collections of cells, presumed to be postganglionic neurons, are clustered around the cloaca and distal portions of the hindgut and ureters. Ultrastructural studies have demonstrated that the neurons do receive synaptic inputs (27), which presumably originate from the more caudal spinal nerves (28). Although there is no obvious widespread system of spinal autonomic neurons, there are defined aggregations of chromaffin cells throughout the body of lamprey (29). The largest collection of chromaffin cells is located in the walls of the anterior part of the posterior cardinal veins. Smaller irregular collections of chromaffin cells occur in the walls of the segmental veins, especially near the dorsal root ganglia. Some of the chromaffin cells are multipolar and are innervated by fibers running from the dorsal rather than ventral roots. Although these fibers have been considered to be autonomic, there is no functional evidence that this is really the case. The spinal autonomic outflow in hagfish (Myxine) is difficult to interpret. Cells considered to be autonomic neurons have been described along the dorsal aorta (24,30). In addition, there is a subcutaneous nervous plexus, formed by

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

terminations of the spinal nerves, which is particularly dense around the slime glands; these cells have been interpreted as being peripheral autonomic neurons lying in spinal pathways, which presumably provide motor innervation to smooth muscle surrounding the glands and the subcutaneous venous sinuses (31). B. Elasmobranchs Sympathetic ganglia and their corresponding spinal pathway are clearly present in elasmobranchs. They still possess some of the characteristics of the cyclostome spinal autonomic system, the most notable being more or less segmentally arranged aggregations of chromaffin tissue. Sympathetic ganglia are absent from cranial and caudal regions (32). Adjacent ganglia usually are interconnected by a loose network of fine nerve trunks, but there is no well-demarcated sympathetic chain as seen in mammals, and some ganglia may lack direct connections with their neighbors. On the other hand, there may be connections between ganglia on each side of the animal. The more cranial ganglia lie on the dorsal walls of the posterior cardinal sinuses at levels corresponding to the nearby segmental arteries. The more caudal ganglia are located more ventrally; however, in Heterodontus (33), the most cranial sympathetic is significantly larger then the others, since it is the origin of the largest splanchnic nerve, following the celiac artery to the stomach. In the region of the branchial plexus of sharks there is a postbranchial plexus (34), containing smaller pregastric ganglia (35), especially at regions where the plexus is joined by visceral rami of the vagus nerve (34). Nevertheless, there are no prevertebral ganglia associated specifically with the celiac and mesenteric arteries of elasmobranchs. Although there are some ganglion cells scattered around the urinogenital tract, there are no well-defined pelvic ganglia. Postganglionic fibers do not travel with the spinal nerves; rather, they follow segmental blood vessels directly in the splanchnic nerves to the gastrointestinal tract and perhaps other viscera (36). The most characteristic feature of the elasmobranch sympathetic ganglia is their close association with prominent collections of chromaffin tissue. The largest collections of chromaffin tissue are aggregated around the gastric ganglia, and the combined gastric ganglion-chromaffin cell mass is known as the “axillary body.” In Heterodontus the chromaffin tissue extends along the interganglionic connectives, thereby forming a continuous cord on the dorsal surface of the posterior cardinal vein from the axillary body to the cranial end of the kidney (33). The chromaffin tissue releases catecholamines into the circulation in a way analogous with the adrenal medulla of mammals. The most likely targets of these circulating catecholamines are the heart, which also contains chromaffin tissue but which generally lacks a direct sympathetic innervation, and perhaps the branchial vasculature (36).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

C. Teleosts The sympathetic ganglia of teleost fish are generally arranged into well-organized paravertebral sympathetic chains, with adjacent ganglia being connected by a well-defined sympathetic trunk. In most teleost species, there are usually five pairs of cranial sympathetic ganglia associated with the cranial nerves (34). The first two ganglia are associated with the trigeminal-facial sensory ganglion complex. One ganglion is primarily associated with the trigeminal component, while the other is associated with the facial nerve component. The third ganglion lies ventral to the sensory ganglion of the glossopharyngeal nerve, while the fourth and fifth ganglia lie at either end of the vagal sensory ganglion complex. In Menidia, a relatively primitive teleost, there is a more rostral small group of ganglion cells lying on the cranial margin of the trigeminal ganglion near the roots of the ciliary nerves and probably connecting with the palatine plexus (37). In some teleost species, the cranial sympathetic ganglia fuse to varying degrees. The great majority of the neurons in the cranial sympathetic ganglia synthesize catecholamines (30). Postganglionic sympathetic fibers run out with their corresponding cranial nerves to supply blood vessels, pigment cells, and perhaps glands in the cranial and branchial region (34). In the trunk, the more cranial parts of the spinal sympathetic chain lie on either side of the vertebral column. However, in Uranoscopus, Platycephalus, and Serranus they fuse in the region of the kidneys where the posterior cardinal veins also fuse and run as a single sympathetic trunk, ventral to the vertebral column, toward the end of the abdominal cavity (19,34,38). As the cranial sympathetic ganglia, the postganglionic neurons in the spinal sympathetic ganglia directly enter their corresponding spinal nerve via communicating rami to innervate blood vessels, pigment cells, and perhaps cutaneous glands. Nevertheless, preganglionic fibers project for considerable distances in both directions along the chain, as well as from one side to the other via the commissures (19,38). Although there are ganglia associated with the first two spinal segments and the branchial plexus, their preganglionic inputs probably leave the spinal cord at the third and fourth spinal segments (19,38). In Platycephalus, preganglionic outflow from the third and fourth spinal segments runs cranial, while the outflow from the fifth and sixth segments runs caudal along the sympathetic chain (38). Many teleost species possess some form of a celiac ganglion that is absent from the primitive chondrostean fish, as is the case in elasmobranchs (38). In Gadus, the celiac ganglion seems to be comprised of the fused second and third spinal sympathetic ganglia on the right side (30), while in Uranoscopus and the stomachless flatfish, Rhombosolea and Ammotretis, the celiac ganglion is connected by short splanchnic nerves to the first and second spinal sympathetic ganglia of the right side only (19,40).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Most of the postganglionic neurons in the sympathetic chains, celiac ganglion, or scattered along the splanchnic nerves contain catecholamines (30). However, there is pharmacological evidence that the pigment cells in the skin of catfish (Parasilus) are innervated by cholinergic sympathetic neurons, presumably arising from the chain ganglia (30). While there are some chromaffin cells in the sympathetic ganglia, the main collection of chromaffin tissue surrounds the posterior cardinal veins at the cranial ends of the kidneys, forming the so-called “head kidney.” In Gadus, there is a small sympathetic ganglion associated with this tissue. Despite this, the chromaffin tissue is innervated by spinal preganglionic neurons (30,41). There is a similar distribution of chromaffin tissue in ganoid fish, with the major aggregations being around the posterior cardinal (39,42,43). D. Dipnoans As in teleosts, the sympathetic ganglia of lungfish are arranged into well-defined paravertebral sympathetic chains. However, like tetrapods, there are no cranial sympathetic ganglia. Furthermore, there may not be any connections between the sympathetic trunk and the vagus or any of the other cranial nerves. An unusual feature of the dipnoan sympathetic chain is that it tends to form loops around the segmental arteries, with a small ganglion lying on both the cranial and caudal ends of the loop. The ganglia are so small that they may not be visible macroscopically (44,45). Lungfish possess segmentally arranged aggregations of chromaffin tissue lying around the origins of the segmental arteries, in addition to accumulations in the walls of the posterior cardinal veins and atrium (45,46). This is reminiscent of the distribution of chromaffin tissue in elasmobranchs and cyclostomes (30).

III. AMPHIBIANS There are marked differences in the organization of the spinal autonomic pathways between the three major groups of existant amphibians, ranging from the primitive urodeles through the familiar anurans to the highly specialized legless amphibians. Of the amphibian groups, the most primitive arrangement of the sympathetic ganglia is found in the urodeles. In some species the more cranial sympathetic trunk on each side is doubled, with a component lying on either side of each lateral aorta. The largest sympathetic ganglia occur in the region of the branchial plexus, with a relatively large ganglion lying cranial and caudal to the origin of the subclavian artery. These ganglia are interconnected with each other and other smaller ganglia to form a subclavian plexus.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

In contrast with the urodeles, the sympathetic chains of anuran amphibians are well organized, with prominent segmentally arranged ganglia. Anurans have a greatly reduced number of vertebrae and spinal segments, and there are usually only 10 pairs of sympathetic ganglia, corresponding to the number of spinal nerves (47). There are no cranial sympathetic ganglia, although the sympathetic trunk extends into the head as far as the ciliary nerves (48). The two most cranial pairs of ganglia tend to be larger than the rest (49). The sympathetic chain in caecilians (Caecilia) is much reduced compared with other amphibians and with any other tetrapods. This reduction of the sympathetic system may be a consequence of their limbless condition with a concomitant reduction in the necessity for specific regional control of the circulation, for example.

IV. REPTILES In general, the spinal sympathetic pathways of reptiles and birds are well organized, with clearly defined paravertebral chains. Some reptiles and birds share features not seen in other vertebrates. The paravertebral sympathetic chains of the lizard may contain 15–25 pairs of ganglia, each one corresponding to a spinal nerve. The most cranial ganglion is at the level of the branchial plexus and is considerably larger than the rest (50,51). Virtually all of the postganglionic neurons in the sympathetic chain of lizards synthesize catecholamines. The great majority of them are likely to innervate the cardiovascular system, although most of the viscera do have a sympathetic innervation (52,53). Furthermore, in chameleons there is a well-documented sympathetic innervation of the cutaneous chromatophores (54). The arrangement of the sympathetic ganglia in chelonians seems to be similar to that in lizard (55). There is a particularly large ganglion at the level of the branchial plexus, which gives rise to cardiac and pulmonary nerves. The cervical sympathetic trunk may bear two to three small ganglia; these ganglia also may contribute to the cardiac nerves, as well as to the innervation of cranial structures (56,57). The spinal sympathetic ganglia of crocodilians have a number of peculiarities in their arrangement (55). The most cranial of the thoracic ganglia lies at the level of the branchial plexus, and, as in the case of the lizard, it is much larger than the other paravertebral ganglia. In addition to feeding into the branchial plexus, the ganglion gives rise to a prominent cardiac nerve. Consequently, it has been named the “cardiac ganglion” (56), although it clearly corresponds to the stellate ganglion of mammals.

V. BIRDS The sympathetic chains of birds extend from upper cervical to sacral levels. The ganglia are well formed and are segmentally arranged with short communicating rami to their corresponding spinal nerves. As in crocodiles, ganglia are present in Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

the cervical sympathetic trunk, which runs through the transverse foramina of the cervical vertebrae. There is convincing functional and histological evidence that preganglionic outflow to the sympathetic chains has a much more restricted distribution, extending only from the last cervical segment to the fourth lumbar segment (57,58). The major targets of these neurons are blood vessels and the pennamotor muscles, which may be innervated by two functionally distinct classes of neurons: one raising the feathers and one lowering them (58). In contrast with reptiles, there are well-developed prevertebral ganglia in birds (47,59). It seems clear that the increase in control complexity of the peripheral autonomic nervous system across vertebrate phylogeny is parallel to that seen in the central nervous system. Presumably, it is related most closely to the requirements for increasingly efficient control of the cardiovascular system, gut, and other internal organs in response to the high energy demands imposed by lifestyles progressively more independent of environmental constraints (60–62).

VI. MAMMALS For many years, conventional wisdom has stated that there are two main ganglia associated with the autonomic outflows of the facial nerve and one main ganglion in the glossopharyngeal nerve autonomic pathway. Thus, the sphenopalatine ganglion and the submandibular ganglion lie on branches of the facial nerve, and the otic ganglion lies on a branch of the glossopharyngeal nerve. However, scattered all along the various ramifications of both the facial and glossopharyngeal nerves are many small aggregations of autonomic nerve cell bodies forming microganglia. Furthermore, there are many interconnections between the facial and glossopharyngeal nerves, especially in the region of the tympanic and cavernous plexuses (63,64), and it is not always obvious from anatomical considerations alone within which pathway a particular ganglion or microganglion lies. All the neurons in the ganglia lying on facial and glossopharyngeal nerve pathways are thought to be cholinergic, although this has only been demonstrated directly for some of the ganglia in rat (65,66). The sphenopalatine ganglion lies on the medial side of the orbit and receives preganglionic fibers from the facial nerve via the nerve of the pterygoid canal and the greater superficial petrosal nerve. Although the ganglion has close anatomical associations with the maxillary division of the trigeminal nerve, no preganglionic nerves take that route. In many species (67) there is at least one small ganglion on the Vidian nerve, proximal to the main ganglion. Other small ganglia occur in fine nerves running from the sphenopalatine ganglion towards the eye as part of a retro-orbital plexus. The sphenopalatine ganglion contains predominantly secretomotor and vasodilator neurons supplying the nasal mucosa, the lacrimal gland, and the uvea. It is also likely to provide vasodilator innervation to the muscles of the jaws and face. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The submandibular ganglion lies in close association with the lingual nerve. Although it receives most of its preganglionic input from the facial nerve via the chorda tympani, there is also likely to be some input from the glossopharyngeal nerve via connections with the chorda tympani in the tympanic plexus (68). In addition to supplying secretomotor and vasodilator innervation to the submandibular salivary gland, the ganglion also innervates the sublingual gland (69). The otic ganglion lies just outside the base of the skull close to the mandibular division of the trigeminal nerve. Some species, such as goats and horses, actually possess a paired otic ganglion, one on either side of the mandibular nerve, and interlinked by commissural fibers. In cats there seems to be a complex of three or more microganglia rather than a single otic ganglion (70). The otic ganglion receives most of its preganglionic input from the glossopharyngeal nerve via the lesser petrosal nerve. In addition to the main autonomic ganglia of the facial and glossopharyngeal nerve pathways, other aggregations of ganglion cells are found along various ramifications of these nerves. Although there seem to be some species differences, relatively constant collections of microganglia occur in the tympanic plexus, in the cavernal plexus, and along the lingual nerve within the tongue (67,71). These ganglion cells are surprisingly abundant, but little is known about the function of most of these ganglia; they probably cause vasodilatation of local blood vessels: they are nonadrenergic and commonly contain vasoactive intestinal polypeptide (VIP) (66,67). Furthermore, retrograde tracing studies have shown that a prominent microganglion within the cavernous plexus of rats innervates the cerebral circulation (66). The intralingual neurons receive preganglionic inputs from both the facial nerve (72) and the glossopharyngeal nerve (73,74). At least some of the neurons in the cavernous plexus are likely to lie on facial nerve pathways, but an additional preganglionic contribution from the glossopharyngeal nerve cannot be ruled out (75). The autonomic pathways in the vagus nerve of mammals are complex. The vagus provides autonomic innervation to the foregut as well as to the esophagus, stomach, duodenum, airways, thyroid gland, and pancreas, in addition to the heart. There is also some anatomical evidence for a vagal innervation of more distal structures such as the proximal colon and kidney. Microganglia are scattered along the course of the vagus nerve (76) and also occur in plexuses associated with the target tissue, such as the heart, airways, thyroid gland, and pancreas (75). In the gastrointestinal tract, the vagus provides preganglionic input to some enteric neurons within the myenteric plexus (77). Any particular target tissue may be innervated by more than one vagal autonomic pathway, each of which may have opposing effects. A long series of physiological and morphological experiments has demonstrated that each of the major microganglia located in or near the heart has a separate role in regulating the rate and force of the heart beat (78). Indeed the left vagus has functions different from those

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

of the right vagus (78). Immunohistochemical observations in guinea pigs suggest that some of these cardiac ganglia may be distinguished by their neuropeptide content (79). In addition to the principal ganglion cells, the cardiac ganglia of most mammalian species also contain small collections of chromaffin cells (80). The functional neuroanatomy of the spinal autonomic pathways is best known in mammals, especially guinea pigs, cats, and humans. Although sympathetic preganglion outflows are restricted to thoraco-lumbar levels of the spinal cord, sympathetic ganglia themselves extend from upper cervical to sacral levels. The sympathetic ganglia can be separated into two main groups on the basis of their location and their peripheral projections and functions. They are usually called the “paravertebral” and the “prevertebral” ganglia. In addition to the principal ganglion cells, most sympathetic and pelvic ganglia contain small aggregations of chromaffin cells. However, the largest collection of chromaffin tissue in adult mammals occurs in the adrenal medulla. The paravertebral sympathetic ganglia lie beside the vertebral column, forming two largely independent sympathetic chains. From thoracic to sacral levels, the paravertebral ganglia are more or less segmentally arranged. Adjacent paravertebral ganglia are connected with each other by interganglionic connectives, thereby forming the characteristic chain of ganglia. The largest ganglion is the stellate ganglion. It is located at the lower cervical–upper thoracic level of the chain and connects with the brachial plexus and cervical sympathetic trunk as well as giving rise to the cardiac sympathetic nerves. Cervical ganglia are also present. In marsupials and eutherians, the most prominent is the superior cervical ganglion. It has a close anatomical relationship with the bifurcation of the common carotid artery and with the vagus nerve and provides sympathetic innervation to cranial tissues. However, in monotremes the main cervical ganglion is found more caudally, around the level of the sixth cervical spinal nerve (81). Preganglionic fibers reach the postganglionic neurons within the ganglia via the ventral spinal roots and communicating rami. The axons of postganglionic neurons may leave the ganglia directly, or they may project along the chain for a number of segments before they leave. In either case, postganglionic fibers may then enter the spinal nerves via communicating rami to be distributed to the rest of the body, or they may project to the viscera by well-defined nerve trunks, such as the carotid nerves in the head, the cardiac nerves in the thorax, or the splanchnic nerves in the abdomen. Small collections of ganglion cells may lie in the rami communicating with the spinal nerves and along the carotid, cardiac, and splanchnic nerves (47,75,82). The prevertebral sympathetic ganglia are associated with the main ventral branches of the aorta. Thus, there is a celiac ganglion at the origin of the celiac artery, a superior mesenteric ganglion at the origin of the superior mesenteric artery, and an inferior mesenteric ganglion at the origin of the inferior mesenteric artery and a renal ganglion at the origin of each renal artery.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

There are usually thought to be no prevertebral ganglia in the thorax. However, in most mammalian species examined, there is a prominent mediastinal ganglion, which lies near the arch of the aorta (83) and which is regarded as a thoracic prevertebral ganglion. It is closely associated with the cardiac nerves running from the stellate ganglion, and it also may have connections with the vagus nerve. Most of the postganglionic neurons in the prevertebral ganglia are noradrenergic, and they mainly innervate the abdominal and pelvic viscera. They receive multiple preganglionic inputs from thoraco-lumbar preganglionic neurons via the splanchnic nerves that run through the mesentery from the sympathetic chain. However, some cells in the inferior mesenteric ganglion have preganglionic input from the sacral spinal levels via the pelvic nerves. Such neurons would be functionally “parasympathetic” according to conventional criteria based on Langley’s classification. Furthermore, many of the neurons also receive peripheral synaptic inputs from enteric neurons with cell bodies in the myenteric plexus of the intestine (77,84,85). Thus some of the prevertebral neurons, such as those controlling secretion from the small intestine, are integral components of peripheral reflex arcs (77). As the preaortic plexus extends caudally from the inferior mesenteric ganglion, it is often known as the hypogastric plexus, which in turn runs into the pelvic plexus. The major ganglion in this region is known as the hypogastric or anterior pelvic ganglion in males and the paracervical ganglion in females. In both sexes, these ganglia are associated with a series of smaller ganglia and contain noradrenergic and nonadrenergic neurons. Functional studies have shown that some neurons in these ganglia receive preganglionic inputs from lumbar levels of the spinal cord and, therefore would be considered to lie within “sympathetic” pathways. However, other neurons in the same ganglia have preganglionic inputs from sacral levels of the spinal cord and would therefore be regarded as lying in “parasympathetic” pathways. Furthermore, some individual neurons probably have inputs from both levels of the spinal cord (86), so that they cannot be classified unambiguously as lying in either a sympathetic or a parasympathetic pathway. Within the more caudal parts of the pelvic plexus, most of the neurons are nonadrenergic and most of their preganglionic inputs arise from sacral levels via the pelvic nerves. In most species the caudal pelvic neurons are aggregated into small ganglia near and within the walls of the distal colon and rectum, the urinary bladder and ureters, and, in males, at the base of the penis. When the various neuronal populations within the different ganglia on the spinal autonomic pathways of mammals are compared using functional, anatomical, and neurochemical criteria, it is evident that the prevertebral and pelvic ganglia form a graded continuum. Thus using Langley’s criteria, the celiac-superior mesenteric ganglion complex can be seen as being mainly “sympathetic,” in that most of the postganglionic neurons are noradrenergic and the preganglionic inputs

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

arise almost entirely from thoraco-lumbar levels. Conversely, the caudal pelvic ganglia can be considered mainly “parasympathetic,” in that most of the postganglionic neurons are nonnoradrenergic, and preganglionic inputs arise predominantly from the sacral spinal cord. However, the inferior mesenteric ganglia and the anterior pelvic ganglia contain mixtures of neurons lying in both the sympathetic and parasympathetic pathways, with the inferior mesenteric ganglion containing relatively more “sympathetic” neurons and the anterior pelvic ganglion containing relatively more “parasympathetic” neurons. Consequently, the ganglia on the spinal autonomic pathways should be designated simply as “paravertebral,” “prevertebral,” or “pelvic” ganglia (87) according to their locations, without any implications as to the origin of preganglionic inputs, neurochemistry, or functions of their constituent neurons.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11.

12.

13. 14.

Galen C. De usu partium corporis humani (translation by Nicolao Rigio Calabro), 1854. Vesalius A. De fabrica humani corporis. Basel: J. Oporini, 1543. Stephanos C. De dissectione partium corporis humani. Paris: S. Collins, 1545. Eustachius B. Opuscola Anatomica. Venice: V. Luchinus, 1563. Willis T. Cerebri anatome; cui accessit nervorum descriptio et usus. London: J. Flesher, 1664. Sheehan D. Discovery of the autonomic nervous system. Arch Neural Psychiatry 1936; 35:1081–1115. Winslow JB. Exposition anatomique de la structure du corps humain. Paris: G. Desprez, 1732. Neubauer JE. Descriptio anatomica nervorum cardiacorum. Sectio prima de nervo intercostali cervicali, dextri imprimis lateralis. Frankfurti et Lipsiae, in off. Fleisheriana, 1772; 230 (5,6,257). Lower R. Tractatus de corde, item de mota, et colore sanguinis et chyli in eum transita. London: 1669. Ens A. De causa, vices cordis alternas producente. Utrecht: J. Broedelet, 1745. Weber E, Weber EH. Experiences qui prouvent que les nerfs vagues, stimules par l’appareil de rotation galvano-magnetique, peuvent retarder et meme arreter le mouvement du coeur. Arch Gén Méd 1846; 12:12–13. Petit du FP. Memoire dans lequel il est demontre que les nerfs intecostaux fournissent des rameaux qui portent des esprits dans les yeux. Hist Acad Roy Sci 1727; I–19 (7,82). Whytt R. An Essay on the Vital and Other Involuntary Motions of Animals. Edinburgh: Hamilton, Balfour and Neill, 1751. Whytt R. Observations on the Nature, Causes and Cure of Those Disorders Which Have Been Commonly Called Nervous, Hypochondriac or Hysteric; to Which Are Prefixed Some Remarks on the Sympathy of the Nerves. 2nd ed. Edinburgh: T. Becket, 1765.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

27. 28. 29. 30. 31.

32. 33.

34. 35. 36. 37.

Muller J. Elements of Physiology. 2 Vols. 2nd ed. London: Taylor and Walton, 1928. Kolliker von A. Zur Lehre von der Contractilitat menschlicher Blut- und Lymphgefasse. Z Wiss Zool 1849; 1:257–260. Henle FG. Handbuch der systematischen Anatomie des Mensches. Vol. 3 Brunswick: F. Vieweg u. Sohn, 1868. Langley J.N. On the union of the cranial autonomic (visceral) fibres with the nerve cells of the superior cervical ganglion. J Physiol (London) 1898; 208:240–270. Young JZ. On the autonomic nervous system of the teleostean fish, Uranoscopus scaber. QJ Microsc Sci 1931; 74:491–535. Young JZ. Comparative studies on the physiology of the iris. I. Selachians. Proc Roy Soc Ser B. Biol Sci 1933; 112:228–241. Young JZ. Comparative studies on the physiology of the iris. II. Uranoscopus and Lophius. Proc Roy Soc Ser B Biol Sci 1933; 112:242–249. Young JZ. The autonomic nervous system of selachians. Quart J Micr Sci 1933; 75:571–624. Young JZ. The innervation and reactions to drugs of the viscera of teleostean fish. Proc Roy Soc 1936; 120:303–318. Nicol J. Autonomic nervous system in lower chordates. Biol Rev 1952; 27:1–49. Holmgren S, Jensen J. In: Comparative Physiology and Evolution of the Autonomic Nervous System. Chur, Switzerland: Harwood Academic, 1994:69–995. Euler von US. A specific sympathomimetic ergone in adrenergic nerve fibres (symphathin) and its relation to adrenaline and noradrenaline. Acta Physiol Scand 1946; 12:73–97. Nakao T, Ishizawa A. An electron microscopic study of autonomic nerve cells in the cloacal region of the lamprey, Lampetra japonica. J Neurocytol 1982; 11:517–532. Johnels AG, Ostlund E. Anatomical and physiological studies on the enteron of Lampetra fluviatilis (L.). Acat Zool (Stockholm) 1958; 39:9–12. Gaskell WK. The distribution and physiologic action of the suprarenal medullary tissue in Petromizon fluviatilis. J Physiol 1912; 44:59–67. Nilsson S. Autonomic Nerve Function in Vertebrates. Berlin: Springer-Verlag, 1983. Bone Q. Some observation upon the peripheral nervous system. In: Bell DJ, Freeman BM, eds. Physiology and Biochemistry of the Domestic Fowl. Vol 2. London: Academic Press, 1963:641–673. Shore TW. On the minute anatomy of the vagus nerve in Selachians with remarks on the segmental value of the cranial nerves. J Anat Physiol 1889; 23:428–451. Gannon BJ, Campbell GD, Satchell GH. Monoamine storage in relation to cardiac regulation in the Port Jackson shark, Heterodontus portusjacksoni. Z Zellforsch Mikroskop Anat 1972; 131:437–450. Chevrel R. Sur l’anatomie du systeme nerveux grand sympathique des elasmobranches et des poissons osseux. Arch Zool Exp Gen 1889; 2 series (suppl. 5):1–194. Nicol J. The autonomic nervous system of the chimaeroid fish, Hydrolagus colliei. QJ Microscop Sci 1950; 91:379–399. Nilsson S, Holmgren S. The autonomic nervous system. In: Shuttleworth TJ, ed. Physiology of Elasmobranch Fishes. Berlin: Springer-Verlag, 1988:143–169. Herrick CJ. The cranial and first spinal nerves of Medinia: a contribution upon the nerve components of bony fishes. J Comp Neurol 1899; 9:153–455.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

38.

39. 40.

41. 42. 43.

44. 45. 46.

47. 48. 49. 50. 51. 52.

53.

54. 55.

56. 57.

Soust M. Studies on the sympathetic nervous system of the flathead (Platycephalus bassensis). B. Sc. (Hons) thesis, University of Melbourne, Department of Zoology, 1981. Chevrel R. Recherches anatomiques sur le systeme nerveux grand sympathique de l’esturgeon (Acipenser sturio.) Arch Zool Exp Gen 1894; 3 series (2):401–444. Grove DJ, Campbell G. The role of extrinsic and intrinsic nerves in the coordination of gut motility in the stomachless flatfish, Rhombosolea tapirina and Ammotretis rostrata, Guenther. Comp Biochem Physiol 1979; 63C:143–159. Nilsson S. Adrenergic control system in fish. Marin Biol Lett 1984; 5:127–146. Nilsson S. On the adrenergic system of ganoid fish: the Florida spotted gar, Lepisosteus platyrhincus (Holostei). Acta Physiol Scand 1981; 111:447–454. Balashov NV, Fange R, Govyrin VA, Leont’eva GR, Nilsson S, Prozorovskaya MP. On the adrenergic system of ganoid fish: the beluga, Huso huso (Chondrostei). Acta Physiol Scand 1981; 111:435–440. Jenkin PM. Note on the sympathetic nervous system of Lepidosiren paradoxa. Proc R Soc Edimburgh 1928; 48:55–69. Holmes W. The adrenal homologues in the lungfish Protopterus. Proc R Soc Lond 1950; series B (137):549–565. Abrahamsson T, Holmgren S, Nilsson S, Petersson K. On the chromaffin system of the African lungfish, Protopterus aethiopicus. Acta Physiol Scand 1979; 107:135–139. Pick J. The Autonomic Nervous System: Morphological, Comparative, Clinical and Surgical Aspects. Philadelphia, J.B. Lippincott, 1970. Morris JL. The autonomic innervation of the iris of the toad (Bufo Marinus). B.Sc. (Hons) thesis, University of Melbourne, Dep. of Zoology, 1975. Huber GC. A contribution on the minute anatomy of the sympathetic ganglia of the different classes of vertebrates. J Morphol 1900; 16:27–86. Hirt A. Der Grenzstrang des Sympathicus bei einigen Saurien. Z Anat Entwicklungsgesch 1921; 62:536–551. Adams WE. Observation on the lacertilian sympathetic system. J Anat 1942; 77:6–11. McLean JR, Burnstock, G. Innervation of the urinary bladder of the sleepy lizard (Trachysaurus rugosus). I. Fluorescent histochemical localization of catecholamines. Comp Biochem Physiol 1967; 20:667–673. McLean JR, Burnstock G. Innervation of the lungs of the sleepy lizard (Trachysaurus rugosus). I. Fluorescent histochemistry of catecholamines. Comp Biochem Physiol 1967; 22:809–813. Sand A. The comparative physiology of colour response in reptiles and fishes. Biol Rev 1935; 10:361–382. Hirt A. Die vergleichende Anatomie des sympathischen Nervensystem. In: Handbuch der vergleichende Anatomie der Wirbeltiere, Band 2, Teil 1. Berlins Bolk, 1934:685–776. Gaskell WK, Gadow H. On the anatomy of the cardiac nerves in certain cold-blooded vertebrates. J Physiol (London) 1884; 5:362–372. Terni T. Il simpatico cervicale degli Amnioti. Ricerche di morfologia comparata. Z Anat Entwicklungsgesch 1931; 96:289–426.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

58. 59. 60. 61. 62. 63. 64. 65.

66.

67.

68. 69. 70. 71.

72. 73. 74. 75. 76. 77. 78.

Langley JN. On the sympathetics of birds and the muscles that move the feathers. J Physiol (London) 1904; 30:221–252. Bennett T. The peripheral and autonomic nervous system. In: Farmer DS, King JR, eds. Avian Biology, Vol. IV, New York: Academic Press, 1974:1–79. Bray AA. The evolution of the terrestrial vertebrates: environmental and physiological considerations. Phil. Trans R Soc Lon 1985; ser. B (309):289–322. Carroll RL. Vertebrate Paleontology and Evolution. New York: W.H. Freeman, 1988. Little C. The Terrestrial Invasion: An Ecophysiological Approach to the Origin of Land Animals. Cambridge: Cambridge University Press, 1990. Rosen S. The tympanic plexus. Arch Otolaryngol 1950; 52:15–18. Suzuki N, Hardebo JE. The pathway of parasympathetic nerve fibres to cerebral vessels from the otic ganglion in the rat. J Auton Nerv Syst 1991; 36:39–46. Leblanc G, Trimmer BA, Landis SC. Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: co-existence with vasoactive intestinal peptide and choline acetyltransferase. Proc Natl Acad Sci USA 1987; 84:3511–3515. Suzuki N, Hardebo JE, Owman C. Origin and pathways of choline acetyltransferasepositive parasympathetic nerve fibres to cerebral vessels in rat. J Cereb Blood Flow Metab 1990; 10:399–408. Gibbins IL, Brayden JE, Bevan JA. Perivascular nerves with immunoreactivity to vasoactive intestinal polypeptide in cephalic arteries of the cat: distribution, possible origins and functional implications. Neuroscience 1984; 13:1327–1346. Laage-Hellman JE, Stromblad BC. Secretion from human submaxillary gland after section of the chorda tympani. J Appl Physiol 1960; 15:295–297. Kuntz A. The Autonomic Nervous System. 3rd ed. Philadelphia: Lea and Febiger, 1945. Ohkubo M. Macroscopic study of the otic ganglion in domestic animals. Acta Anat Nippon 1979; 54:322–333. Ichikawa H, Nishikawa S, Wakisaka S, Matsuo S, Takano Y, Akai M. Co-existence of vasoactive intestinal polypeptide and substance P-like immunoreactivities in the tongue of the guinea pig. Neurosci Lett 1988; 89:283–285. Hellekant G. Circulation in the tongue. Wenner-Gren Centre International Symposia Series 1972; 20:127–136. Fitzgerald M, Alexander R. The intramuscular ganglia of the cat’s tongue. J Anat 1969; 105:27–46. Gomez H. The innervation of the lingual salivary glands. Anat Rec 1961; 139:69–76. Gibbins IL. Peripheral autonomic nervous system. In: Paxinos G, ed. The Human Nervous System, New York: Academic Press, 1990: 23–34. Botar J, Afra D, Moritz P, Schiffman H, Scholz M. Die Nervenzellen und Ganglien des N. vagus. Acta Anat 1950; 10:284–314. Furness JB, Costa M. The Enteric Nervous System, Edinburgh: Churchill Livingstone, 1987. Randall WC. Selective autonomic innervation of the heart. In: Randall WC, ed. Nervous Control of Cardiovascular Function. New York: Oxford University Press, 1984:40–67.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

79. 80.

81. 82. 83. 84.

85.

86. 87.

Gibbins IL, Morris JL. Colocalization of transmitters in cardiac nerves. Proc Austr Physiol Pharmacol Soc 1991; 22:64–72. Jacobowitz D. Histochemical studies of the relationship of chromaffin cells and adrenergic nerve fibres to the cardiac ganglia of several species. J Pharmacol Exp Ther 1967; 158:227–240. Van den Broek AJP. Untersuchungen über den Bau des sympathischen Nervensystems der Saugtiere. I. Der Halssympathicus. Morph Jhrb 1908; 37:202–289. Gabella G. Structure of the Autonomic Nervous System. London: Chapman and Hall, 1976. Teitelbaum HA, Uhlenhuth E. The mediastinal ganglion and its relation to the innervation of the viscera. Anat Rec 1932; 52:241–251. Masuko S, Chiba T. Projection pathways, co-existence of peptides and synaptic organization of nerve fibres in the inferior mesenteric ganglion of the guinea-pig. Cell Tissue Res 1988; 253:507–516. Webber RH, Heym C. Immunohistochemistry of biogenic polypeptides in nerve cells and fibres of the guinea-pig inferior mesenteric ganglion after perturbations. Histochemistry 1988; 88:287–297. Szurszewski JH. Physiology of mammalian prevertebral ganglia. Ann Rev Physiol 1981; 43:53–68. Kuntz A. The evolution of the sympathetic nervous system in vertebrates. J Comp Neurol 1911; 21:215–236.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

9 Clinical Studies of the Sympathetic Nervous System Murray Esler and Magdalena S. Rumantir Baker Medical Research Institute and Monash University, Melbourne, Victoria, Australia

The application of clinical methods for measuring regional sympathetic nervous activity has led to a quantum leap in the understanding of human sympathetic nervous physiology and of sympathetic pathophysiology in diseases, most notably of the cardiovascular system. Conditions characterized by acute and chronic sympathetic activation and by acute and chronic sympathetic inhibition and failure have been identified. The introduction of therapies directed at appropriately augmenting or inhibiting these disturbances of sympathetic nervous function has followed. The widespread and successful use of -adrenergic blockers in patients with cardiac failure, based as it is on recognition that there is excessive stimulation of the heart through activation of the cardiac sympathetic outflow, is a telling demonstration of the potential clinical utility of research on clinical cardiovascular neuroscience. I. INTRODUCTION After many years as a “Cinderella” of internal medicine, the sympathetic nervous system has moved towards center stage, particularly in the field of cardiovascular medicine. Where previously the clinical application of research on the sympathetic nervous system and catecholamines was confined largely to the diagnosis of syndromes of autonomic nervous failure and phaeochromocytoma, more recently the impact has been wide-ranging. Sympathetic nervous system activation in the pathogenesis of human heart failure, ventricular arrhythmias, and essential hypertension has been widely studied and the therapeutic value of sympathetic nervous inhibition amply demonstrated. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

A. Cardiac Failure More than 25 years ago, the nearly universal view was that the heart was anatomically and functionally sympathetically denervated in human heart failure (1). In a technically innovative series of studies involving the measurement of noradrenaline isotope dilution, the sympathetic nerves of the failing heart were demonstrated to be intact and in reality firing at a very high level (2–5). This research provided the theoretical evidence justifying the use of -adrenergic blocking drugs in heart failure. In recently completed multicenter trials, -adrenergic blocking drugs were shown to lower mortality rates in heart failure by 35–65% (6,7).

B. Cardiac Arrhythmias There has recently been a major revolution in the medical management of cardiac arrhythmias. The drugs previously widely used for treatment of ventricular tachyarrhythmias, introduced into clinical practice on the basis of effects on isolated cardiac myocyte membrane electrolyte fluxes and membrane potential, were found in the Cardiac Arrhythmia Suppression Trial (CAST) study to substantially increase the risk of sudden death (8). This has led to greater recognition of both the importance of autonomic nervous mechanisms in arrhythmia development and the therapeutic value of drugs with antiadrenergic activity, such as -adrenergic blockers and amiodarone (9). These developments have also provided support for psychosomatic concepts of arrhythmia development, where life stresses, depressive illness, and panic disorder (10–12) are seen as triggering ventricular arrhythmias by neural mechanisms.

C. Essential Hypertension In primary human hypertension, analysis of regional sympathetic nervous system function has demonstrated activation of the sympathetic nervous outflows to the heart, the kidneys, and skeletal muscle vasculature, particularly in younger patients (13,14). This sympathetic activation no doubt contributes to blood pressure elevation in human hypertension, but in addition it seems to have adverse metabolic and other consequences beyond this (15). Obesity-related hypertension is a common variant of essential hypertension in which disturbed sympathetic nervous function similarly appears to be important both in the development of the high blood pressure and in contributing to the risk of cardiovascular complications. These developments provide the backdrop to this chapter, in which contemporary methods for investigating the human sympathetic nervous system will be described. The normal physiology of the sympathetic nervous system and the clinical pathophysiology of human disease delineated by these methods will be reCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

viewed, leading to a consideration of the implications in diagnosis and the existing and potential applications in therapy.

II. METHODOLOGY FOR STUDYING HUMAN REGIONAL SYMPATHETIC NERVOUS FUNCTION Until recently, knowledge of sympathetic nervous pathophysiology in human disease has been rather sketchy, largely because of the rather rudimentary nature of the tests of sympathetic nervous system function available to investigate clinical medicine. Among such tests, measurement of the plasma concentration of the sympathetic neurotransmitter noradrenaline has occupied center stage, although other neurochemical measures of aspects of sympathetic neuronal function, such as that provided by the arterial plasma concentration of dopa, which provides information concerning overall synthesis rates of noradrenaline, and the plasma concentration of dihydroxyphenylglycol (DHPG), which is indicative of intraneuronal metabolism of noradrenaline, have more recently been usefully employed (17). A. Limitations of Plasma Noradrenaline Concentration Measurements in the Study of Human Sympathetic Nervous Function The potential value of noradrenaline release rate measurements as an index of nerve firing was seen early, with demonstration that the washout of noradrenaline from an organ was proportional to the rate of electrical stimulation of its sympathetic nerves (18). The development of isotope-derivative methods for determining the concentration of catecholamines in plasma was quickly followed by their enthusiastic application to clinical research. But plasma noradrenaline measurements, although providing a useful guide to sympathetic nervous system function, do have substantial limitations (18). One of these is the dependence of plasma noradrenaline concentrations on rates of removal of the neurotransmitter from plasma, not just sympathetic tone and noradrenaline release. An example is provided by the demonstration that a time-honored clinical test for detecting sympathetic nervous failure in the setting of postural hypotension, measurement of the plasma noradrenaline response to upright posture, is invalid due to the confounding influence of a large, posture-dependent fall in noradrenaline plasma clearance in the autonomic insufficiency patients. This elevates their plasma noradrenaline concentration during the postural test (19). A second limitation of plasma noradrenaline measurements, and perhaps the principal one, is that global indices of sympathetic nervous function provide no information on regional sympathetic nervous function. This limitation applies also Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

to other related static plasma concentration measurements, such as those of DOPA and DHPG. Sympathetic nervous system responses typically show regional differentiation, with the sympathetic outflow to some organs being activated while those to other regions may be unchanged or inhibited, so that methods providing information about regional sympathetic nervous activity are more instructive (18). B. Clinical Methods for Assessing Regional Sympathetic Nervous System Function Clinical measure of sympathetic nerve firing and noradrenaline release, radioscanning methodologies visualizing the sympathetic innervation of an organ, and more indirect hemodynamic and pharmacological indices of neural control of the circulation have provided a basis for the development of clinical tests of regional sympathetic nervous function (Fig. 1). These tests are complementary rather than competing methodologies, measuring various aspects of sympathetic nervous function, but they do differ in their validity and utility. 1. Clinical Microneurography This technique provides a method for studying nerve firing rates in subcutaneous sympathetic nerves distributed to skin and skeletal muscle. Fine tungsten electrodes are inserted through the skin, with positioning of the electrode tip in sympathetic fibers of, most commonly, the common peroneal or median nerves. Multifiber recordings of “bursts” of nerve activity (13,14), synchronous with the heart beat, or single fiber tracings (20), are generated. 2. Noradrenaline Spillover Rate Measurements Sympathetic neurotransmitter release can be studied clinically using radiotracerderived measurements of the appearance rate of noradrenaline in plasma from individual organs (21) or for the body as a whole (22). With microneurographic methods for studying sympathetic nerve firing rates, the nerves to skeletal muscle and skin only can be studied. An important limitation in clinical research is the inaccessibility to testing of the sympathetic nerves to internal organs. Noradrenaline spillover measurements are more helpful in this regard. The relationship that holds in general between the sympathetic nerve firing rate of an organ and the rate of spillover of noradrenaline into its venous effluent provides the experimental justification for using measures of regional noradrenaline release as a clinical index of sympathetic nervous tone in individual organs (18). During constant rate infusion of radiolabeled noradrenaline, the regional rate of spillover of noradrenaline (NA) to plasma can be determined by isotope dilution (2–5,18): Regional NA spillover  [(CVCA)  CAE]PF Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 1 Available methods for measuring sympathetic nervous system activity in humans. Nerve firing rates can be measured subcutaneously in postganglionic sympathetic fibers distributed in skin and the skeletal muscle vasculature using clinical microneurography. Measurement of the overflow of noradrenaline to plasma from individual organs provides a means of quantifying regional sympathetic activity in the limbs and viscera. Cardiac sympathetic nerve scans can be used to visualize the anatomy of the innervation of the heart. Heart rate spectral analysis has been proposed as a means of quantifying cardiac sympathetic tone, but lacks specificity for this purpose.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

where CV and CA are the plasma concentration of noradrenaline in regional venous and arterial plasma, E is the fractional extraction of tritiated noradrenaline, and PF is the organ plasma flow. It should be emphasized that noradrenaline spillover is measured, and from this noradrenaline release (and sympathetic activity) is inferred. Released noradrenaline is subject to several possible fates: reuptake into sympathetic nerves, Omethylation after uptake into extraneuronal cells, or diffusion into plasma. The rate of overflow of noradrenaline is determined not only by the rate of noradrenaline release (and hence sympathetic nerve firing and nerve density) but also by the activity of the competing disposition mechanisms of uptake, metabolism, and diffusional flow to the circulation, the latter being influenced by factors such as regional blood flow and the exchange conductivity of the capillary and postcapillary venular bed. 3. Radio-scanning Methodologies Imaging methodologies of several types, utilizing both positron emission tomography and single photon emission computed tomography scanning, can be used to demonstrate the anatomy of sympathetic innervation of an organ (13,23). The scanning agents most widely used are [123I]meta-iodobenzylguanidine (MIBG), 6-[18F]fluorodopamine, and [11C]hydroxyephedrine (13,23). Sympathetic denervation can be readily demonstrated with imaging techniques in patients with pure autonomic failure in whom postganglionic sympathetic nerve fibers are absent and after the sympathetic nerve section and degeneration that accompanies cardiac transplantation. Use of these agents to estimate sympathetic “activity” (nerve firing and noradrenaline turnover) is more problematic. MIBG and [11C]hydroxyephedrine, unlike noradrenaline, are not stored in the neurotransmitter vesicles and are not subject to electrically coupled vesicular release (13).* [18F]fluorodopamine is more satisfactory in this regard, as the tracer is converted to [18F]fluoronoradrenaline after it as taken up into sympathetic nerves. This probe has been successfully applied, for example, in studying the various syndromes causing postural hypotension and has allowed differentiation of the diverse forms of underlying autonomic failure (23). 4. Heart Rate Spectral Analysis Spontaneous, superimposed circulatory rhythms partly under sympathetic nervous control can be investigated using power spectral analysis. With this technique, mathematical partitioning is used to identify individual, superimposed

*Some of the views on MIBG are not unanimous; see Chapter 12.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

rhythms producing cyclical variation in heart rate (24). Underlying independent high-frequency (~0.3 Hz) and low-frequency (~0.1 Hz) rhythmic influences on heart rate can be recognized. The autonomic nervous system provides the principal effector mechanism for this heart rate variability. The low-frequency variability derives in part from the influence of the cardiac sympathetic nerves (24). Lowfrequency variability in heart rate does not, of course, strictly provide a measure of the activity of the cardiac sympathetic nerves (13,25,26), despite widespread and often uncritical interpretation of the variability measurement in this way, even its semi-official endorsement in the report of the Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology (27). Low-frequency heart rate variability is determined by the combined influences of the intrinsic central nervous oscillation in sympathetic outflow, the gain of the relevant circulatory reflex loops, and the postsynaptic chronotropic responsiveness of the heart. 5. Pharmacological Autonomic Blockade Pharmacological blockade of the autonomic nervous system can be used to provide, by subtraction, semi-quantitative information concerning the prevailing level of sympathetic cardiovascular tone (28,29). The method developed initially was to utilize the fall in arterial pressure after ganglionic blockade to gauge the overall “neurogenic component” in blood pressure maintenance. A similar approach is to study the change in regional vascular resistance after -adrenergic blockade (29) and the change in heart rate and cardiac output after -adrenergic blockade of the heart (28). Measured changes are determined by neural traffic, but also by additional factors, including cardiovascular hypertrophy and the efficiency of effector organ transmembrane signal transduction.

III. NORMAL PHYSIOLOGY OF THE HUMAN SYMPATHETIC NERVOUS SYSTEM A. Regionalization of Sympathetic Responses It is now recognized as a truism that sympathetic nervous responses are regionally patterned or differentiated, rather than globally affecting all sympathetic outflows identically (13,18). This is evident in a variety of diseases (see Sec. IV) and also characterizes the normal physiology of the sympathetic nervous system. Examples are provided by the preferential activation of the cardiac sympathetic outflow with mental stress, cigarette smoking, and healthy aging, preferential activation of the renal sympathetic outflow with restriction of dietary sodium intake, and preferential renal sympathetic inhibition with aerobic exercise training (13,18). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

In experiments testing sympathetic nervous responses to laboratory mental stress, difficult mental arithmetic produces pronounced and disproportionate activation of the sympathetic nerves of the heart, indicated by approximately a trebling of cardiac noradrenaline overflow to plasma (18). The increase in noradrenaline spillover for the body as a whole, indicative of the overall sympathetic response, is less, typically only 50–80%. Muscle sympathetic nerve firing increases even less, actually falling in some cases (13). For the human renal sympathetic nerves also it has been possible to demonstrate differentiated neural responses. One example concerns the sympathetic nervous response to dietary sodium restriction. Experiments in laboratory animals have indicated the importance of the renal sympathetic nerves, directly innervating the renal tubules, in sodium homeostasis (30). The sympathetic fibers exert a sodium-conserving influence. Dietary sodium restriction in healthy young men has been shown to produce a selective activation of the renal sympathetic outflow, sparing the sympathetic nerves of the heart (31). This presumably represents a homeostatic, antinatriuretic response. A second instance of selective modification of renal sympathetic nerve firing is seen with aerobic exercise training, in this case reduction in renal noradrenaline spillover in the absence of any effect on cardiac noradrenaline spillover, indicating that, perhaps surprisingly, the cardiac sympathetic nerves do not participate in exercise training sympathoinhibition (32). B. Discordance in Sympathetic Nervous and Adrenal Medullary Responses An analogous phenomenon is mismatching of adrenal medullary and sympathetic nervous system responses. Increased secretion of adrenaline accompanies sympathetic inhibition in some or all sympathetic outflows with fasting, cigarette smoking, tricyclic antidepressant administration, and vasovagal syncope (13). Conversely, the sympathetic activation of healthy aging occurs in the presence of lowered adrenaline secretion rates (33). Insulin hypoglycemia massively increases adrenaline secretion, but accompanied by a much lower level of sympathetic activation, involving some but not all sympathetic outflows. C. Mechanisms of Regional Patterning of Sympathetic Nervous Responses The neural basis of the regional differentiation of human sympathetic nervous system responses, such as described above, remains incompletely understood, but considerable progress has been made in experiments performed in laboratory animals (34). These show that it is the norm rather than the exception for sympathetic nervous responses to be regionally patterned, rather than global, one of the Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

very few examples of the latter being the sympathetic activation seen during a near maximum exercise response. An underlying topographical organization within postganglionic sympathetic fibers exists, contributing to this functional differentiation of sympathetic responses. In humans one obvious example of this is the anatomical organization of sympathetic fibers evident during clinical microneurography studies, where skeletal muscle vasoconstrictor sympathetic fibers are anatomically separated from skin vasoconstrictors, typically running in motor and sensory nerve bundles, respectively. Within the central nervous system, a complex, neuranatomical organization of functional sympathetic responses is evident (34). Two of many examples are the topographic arrangement of ventral medullary neurons that differentially control sympathetic vasoconstrictor tone to various end organs (35), and the presence of discrete hypothalamic nuclei, which exert a relatively selective excitatory effect on the hepatic circulation but have no influence on the systemic circulation (36). Clearly there is scope for considerable specificity and sensitivity in sympathetic nervous responses. The concept that the typical sympathetic response is a global “fright, fight, and flight” reaction is an anachronism (34). D. Noradrenergic Control of Human Sympathetic Outflow Central nervous system noradrenergic mechanisms are of importance in the regulation of the sympathetic nervous system. Noradrenergic projections from the brain stem to forebrain centers are typically sympathoexcitatory and pressor (37,38), while the bulbar noradrenergic nuclei mediating baroreceptor blood pressure responses are depressor (39). Electrophysiological and anatomical studies carried out in animals provide evidence of connections linking the pressor nuclei of the hypothalamus and the amygdala with the sympathetic preganglionic neurons in the thoracolumbar cord, either directly or more commonly via neuronal groups of the rostral ventrolateral medulla (37,38). Given the importance of central noradrenergic neurotransmission in influencing sympathetic nervous outflow from the brain, the relation of human CNS noradrenergic turnover to sympathetic nervous system activity has been investigated with sampling from high in the internal jugular vein, analogous to the measurement of norepinephrine spillover from peripheral organs. Noradrenaline is released into the cerebrovascular circulation from brain neurons rather than cerebrovascular sympathetic nerves (88–90). Intravenous infusion of the ganglionic blocker trimethaphan in healthy men at a rate sufficient to produce a reduction of approximately 20 mmHg in supine systolic blood pressure increased MHPG spillover from the brain fivefold (Fig. 2). This presumably represents a compensatory response in pressor, sympathoexcitatory forebrain noradrenergic neurons (37,38) in the face of an interruption in sympathetic neural traffic and fall in arterial pressure. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Controlled hypotension produced by ganglionic blockade with trimethaphan was associated, as expected, with reduction in noradrenaline (NA) spillover to plasma from sympathetic nerves. Brain noradrenaline turnover, measured with internal jugular venous sampling to quantify overflow of noradrenaline and the lipophilic metabolites MHPG and DHPG, increased in sympathoexcitatory brain neurons in an adaptive but futile (given the ganglionic blockade) reflex response to the hypotension.*p  0.05.

Differentiating the pattern of cerebral drainage into the internal jugular veins allows selective measurement of subcortical norepinephrine turnover. Advantage is taken of the fact that the human cerebral venous drainage is asymmetrical to study the regional origins of monoamine spillover into the cerebrovascular circulation. Using a technesium-99 cerebral venous sinus scan to delineate the Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

pattern of venous drainage in individual subjects, subcortical and cortical neurotransmitter turnover can be distinguished (40,42). The more common pattern is for the right internal jugular vein to have the superior sagittal sinus as its major tributary and the cerebral cortex as its predominant field of drainage; here, subcortical venous drainage is into the left internal jugular vein. Sometimes the sinus drainage pattern is reversed, with cortical venous drainage being into the left internal jugular vein. In a minority of cases the drainage pattern is nonlateralizing, with ready admixture of blood occurring at the confluence of the sagittal and straight sinuses. Several studies provide evidence of a functional link between human forebrain noradrenaline turnover and peripheral sympathetic activity. In young men, muscle sympathetic nerve activity at rest measure by microneurography and subcortical noradrenaline turnover are directly related. With the sympathetic activation accompanying healthy human aging, the same relationship applies (43). Clinical studies in hypertension and heart failure patients (see Sec. IV) provide additional evidence of a link between human cerebral neuronal noradrenergic activity and sympathetic nervous outflow. E. Sympathetic Nervous Cotransmission Constituents of sympathetic vesicles, including adrenaline, chromogranins, neuropeptide Y, dopamine--hydroxylase, and adenosine, are coreleased with noradrenaline from sympathetic nerves and overflow into plasma. 1. Adrenaline In healthy young men, no extraadrenal release of adrenaline is evident at rest, but with the very high rates of sympathetic nerve firing occurring during near maximal aerobic exercise, overflow of adrenaline from the sympathetic nerves of the heart into the coronary sinus is detectable (44). In a variety of clinical contexts (see Sec. IV) adrenaline is continuously coreleased from the heart, where it possibly acts presynaptically to augment sympathoneuronal noradrenaline release and perhaps contribute to the development of hypertension and ventricular arrhythmias. 2. Neuropeptide Y Most of the neuropeptide Y in human plasma derives from the venous drainage of the gut and enters the system circulation via the hepatic vein (45). As for sympathetic adrenaline cotransmission, with the high rate of sympathetic nerve firing accompanying exercise, release of neuropeptide Y from the heart into coronary sinus plasma is evident (45). In cardiac failure, neuropeptide Y is continuously released from the heart to plasma in measurable quantities (see Sec. IV)(46). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

IV. CLINICAL SYMPATHETIC NERVOUS SYSTEM PATHOPHYSIOLOGY A. Acute Sympathetic Nervous Inhibition This is seen in purest form in the simple faint. The commonly used term “vasovagal syncope,” is something of a misnomer in that although increased vagal tone in the heart contributes to heart rate slowing, the acute hypotension that characterises the condition is primarily due to sudden, reversible, almost total sympathetic nerve inhibition. During the faint, near-absent sympathetic tone has been documented, with microneurography, in the sympathetic outflow to skeletal muscle vasculature, and in the heart and kidneys, where noradrenaline spillover falls to zero (13,18). Fainting is most commonly triggered by a homeostatic challenge to blood pressure control, such as prolonged standing, or by an emotional reaction, of which that to venepuncture is perhaps most typical. The central nervous mechanisms integrating the fainting response in humans remain unknown. B. Syndromes of Chronic Sympathetic Nervous Failure Hypotensive syndromes exist in which, in contrast to vasovagal syncope, the causative sympathetic nervous underactivity is chronic and unremitting. The pathophysiological pattern of sympathetic failure encompasses pure autonomic failure, characterized by peripheral sympathetic nerve degeneration, Shy-Drager syndrome, in which there is degeneration of the CNS centers involved in integrated sympathetic circulatory control, and dopamine--hydroxylase deficiency, in which there is a genetic synthetic defect in noradrenaline and adrenaline synthesis. The clinical presentation typically is with disabling postural hypotension. 1.

Pure Autonomic Failure

This disorder is characterized by widespread degeneration of postganglionic sympathetic fibers. Near total sympathetic denervation of the heart is the norm (23,47). The cause remains uncertain, although the absence of the neurotrophin nerve growth factor in the venous drainage of the heart and in arterial plasma (Fig. 3)(48) suggests that defective neurotrophic support of sympathetic nerves might be the pathogenic mechanism. 2.

Dopamine--Hydroxylase Deficiency

In this rare syndrome of sympathetic failure, sufferers present with postural hypotension and a variety of allied symptoms attributable to the underlying defect, total lack of capacity to synthesize noradrenaline and adrenaline (49,50). The key to diagnosis is the high concentration of dopamine and its metabolites in plasma and urine, resulting from the synthesis block at the point of hydroxylation of Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3 Healthy subjects (black bars) had measurable amounts of the neurotrophin nerve growth factor (NGF) in arterial plasma and demonstrable release of NGF from the heart into the coronary sinus (CS). In patients with pure autonomic failure and sympathetic nerve degeneration (grey bars), NGF was barely detectable in arterial plasma and not released by the heart, suggesting that in them the loss of sympathetic innervation is due to lack of neurotrophic support by NGF.*p  0.05.

dopamine to noradrenaline (49,50) Sympathetic nerve firing rates are high at rest, to increase further with upright posture, but there is electrochemical dissociation in the sympathetic neuron as the vesicles contain no noradrenaline, and postural hypotension occurs (51). Very effective treatment is available in the form of oral administration of the noradrenaline precursor L-dihydroxyphenylserine (LDOPS), which is decarboxylated to noradrenaline directly, in a process bypassing the dopamine -hydroxylation step. C. Acute Sympathetic Nervous Activation Short-term physiological sympathetic nervous system activation occurs as one response element in the integrated changes accompanying upright posture, exercise, eating, cold exposure, and the affective states of anger and anxiety (13,18). In acute mental stress reactions, preferential sympathetic activation occurs in the cardiac sympathetic outflow, accompanied by increased adrenal medullary secretion of adrenaline (13). Nerve firing rates in postganglionic sympathetic fibers directed to the skeletal muscle vasculature are little changed or, in fact, sometimes fall. Cardiac output increases under the influence of cardiac sympathetic activation, vagal withdrawal, and elevated arterial plasma concentration of adrenaline. The elevation of blood pressure coupled with decreased regional vascular resistance from the effects of adrenaline and reduced sympathetic activation increase skeletal muscle blood flow. These changes, in their totality, are adaptive, Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

subserving the vigilance and physical exercise requirements of the fight or flight reaction. Much recent attention has been given to a potential deleterious aspect of the mental stress response, as a trigger for clinical presentation of coronary heart disease in the form of myocardial infarction, ventricular arrhythmias, and sudden death (11,52,53). Epidemiological evidence unequivocally demonstrates such a phenomenon with the severe acute mental stress accompanying natural disasters such as earthquakes and also during panic attacks in panic disorder sufferers (52). In panic attacks sympathetic nervous activation is clearly evident (Fig. 4)(12). The mediating mechanisms of the adverse cardiac event are coronary artery spasm, adrenaline-induced hypokalemia, fissuring and rupturing of atherosclerotic plaques in the coronary artery wall leading to thrombosis, and the generation of ventricular arrhythmias through the action in concert of sympathetic activation and myocardial ischemia. D. Diseases with Chronic Sympathetic Nervous Activation A number of diseases are characterized by chronic sympathetic nervous activation. The causal mechanisms and regional pattern of the sympathetic stimulation differ (13,18), but in each case the sympathetic nervous overactivity is thought to directly contribute to the pathogenesis of the disorder and in some is specifically targeted with antiadrenergic drugs. 1.

Heart Failure

The demonstration that the level of sympathetic nervous drive to the failing heart in patients with severe heart failure is a major determinant of prognosis (5) and that mortality in heart failure is reduced by -adrenergic blockade (6,7) indicates the potential clinical relevance of cardiovascular neuroscience research. The cardiac sympathetic nerves are preferentially stimulated in severe heart failure, with the application of isotope dilution methods for measuring cardiac norepinephrine release to plasma, indicating that in untreated patients cardiac norepinephrine spillover is increased as much as 50-fold, similar to levels of release seen in the healthy heart during near maximal exercise (2). This preferential activation of the cardiac sympathetic outflow contributes to arrhythmia development and to progressive deterioration of the myocardium and has been linked to mortality in both mild and severe cardiac failure. Although the central nervous system mechanisms involved in the sympathetic nervous activation at present remain uncertain, increased intracardiac diastolic pressure seems to be one peripheral reflex stimulus (46) and increased forebrain noradrenaline turnover an important central mechanism (41).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 4 Muscle sympathetic nerve activity in a patient with panic disorder at rest (top panel), during a spontaneous panic attack in the laboratory (middle panel), and after the attack had settled (bottom panel). During an attack there was a large increase in the size of multiunit firing bursts.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Production of the sympathetic nerve trophin nerve growth factor by the cardiac myocytes of the failing heart is reduced in experimental and human heart failure (54). Consequent failure of neurotrophic support is the probable basis for the well-documented sympathetic nerve depletion present. Additional neurophysiological abnormalities present in the failing human heart include release of the sympathetic cotransmitters epinephrine and neuropeptide Y at high levels more typical of their release during exercise in healthy subjects (46) and the possible presynaptic augmentation of norepinephrine release from the cardiac sympathetic nerves by the regionally released epinephrine (55). 2.

Essential Hypertension

Measurement of nerve firing in postganglionic sympathetic efferents directed to the skeletal muscle vasculature with microneurography and regional rates of noradrenaline spillover utilizing isotope dilution techniques demonstrates activation of the sympathetic nerves of the heart, kidneys, and skeletal muscle vasculature. The central nervous mechanism operating is uncertain, but is thought to involve sympathoexcitatory noradrenergic projections from the brainstem to the forebrain (see Sec. III)(40). The increase in sympathetic activity is thought to both initiate and sustain the blood pressure elevation and additionally to contribute to adverse cardiovascular events (13,15,18). The high renal sympathetic tone contributes to hypertension development by stimulating renin secretion and through promoting renal tubular reabsorption of sodium. Sympathetic overactivity seems to particularly influence systolic pressure by increasing the rate of left ventricular ejection, by reducing aortic compliance through increasing neural arterial tone, and via arteriolar vasoconstriction, by promoting rebound of the reflected arterial wave from the periphery. Sympathetically mediated vasoconstriction in skeletal muscle vascular beds, in reducing delivery of glucose to muscle, is a basis for insulin resistance and hyperinsulinemia (56). Cardiac sympathetic stimulation contributes to the development of left ventricular hypertrophy and, most likely, to the genesis of ventricular arrhythmias and sudden death. 3.

Obesity and Obesity-Related Hypertension

As obesity prevalence soars in industrialized countries and progressively increases in the third world with the appearance of altered patterns of nutrition and a reduction in work-related energy expenditure, obesity-related hypertension has become a truly global health issue. Despite the scale of the problem, the mechanisms of the blood pressure elevation accompanying overweight are poorly understood. On the issue of possible sympathetic nervous pathophysiology in obesity there have been two enduring hypotheses. The first hypothesis is that sympathetic

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

nervous system underactivity is present in human obesity, as it commonly is in animal models (57), and provides a metabolic basis for the obesity. Sympathetic underactivity, in not providing the normal calorie-wasting thermogenic response to overeating, could potentially lead to positive energy balance and weight gain. The second hypothesis, attributable to Landsberg (58), is that in obesity sympathetic nervous activation occurs with chronic overeating, where it facilitates energy balance and weight stabilization but at the cost of adverse consequences attributable to chronic stimulation of the sympathetic nervous system, in particular elevation in blood pressure. These are not mutually exclusive hypotheses, as obesity is a heterogeneous disorder. The sympathetic nervous underactivity hypothesis of human obesity development now appears to be disproven. Selective activation of the sympathetic nerves to the kidneys and skeletal muscle vasculature is present in normotensive human obesity, although admittedly accompanied by reduced rates of noradrenaline spillover from the heart (13,16). Since only approximately 3% of whole body energy production derives from the heart, reduced cardiac sympathetic activity, however, could make only a trifling contribution to overall energy balance. What might be the basis for activation of the renal sympathetic nerves? Hyperinsulinemia and hyperleptinemia accompanying obesity are candidates, but as yet the evidence for both is inconclusive. The reduction in cardiac sympathetic activity in the normotensive obese also defies ready explanation. It is possible that cardiac sympathetic nervous activity is reflexly depressed in response to circulatory overloading brought on by enhanced renal sympathetic nervous activity and sodium retention. Cardiopulmonary blood volume, stroke volume, and cardiac output are increased substantially in the obese. In patients with obesity-related hypertension, there is a an elevation of renal noradrenaline spillover similar in degree to that present in the normotensive obese, but without suppression of cardiac sympathetic outflow, as in them cardiac noradrenaline spillover is more than double that of normotensive obese and 25% higher than in healthy volunteers (16). Accordingly, the increase in sympathetic outflow to the kidneys appears to be a necessary but apparently not a sufficient cause for the development of clinical hypertension, commonly being present also in overweight people with blood pressure in the normotensive range. High renal sympathetic tone in the latter, of course, may well have contributed to the level of their pressure, although not in sufficient degree to cause clinical hypertension. 4.

Depressive Illness

Sympathetic nervous activity is chronically increased in depressive illness (18). The underlying CNS mechanisms are unclear, as is the regional pattern of sympathetic activation. Because patients with depressive illness are at increased risk of myocardial infarction and sudden death (10), it has been surmised that the sym-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

pathetic outflow to the heart is chronically stimulated, but this is yet to be adequately tested. The typical absence of hypertension in depressive illness suggests that the renal sympathetic nerves are spared and that the sympathetic activation is not generalized. 5.

Hepatic Cirrhosis

Cirrhosis is characterized hemodynamically by systemic and pulmonary vasodilatation of uncertain origin, with compensatory reflex sympathetic nervous activation producing an elevated cardiac output. The sympathetic outflow to the heart, kidneys, skeletal muscle vasculature, and hepatomesenteric circulation is activated (59). The renal sympathetic nervous stimulation contributes to sodium retention and edema, while the high cardiac sympathetic tone provides a mechanism for the sudden cardiac death to which patients with cirrhosis are predisposed. There is a neurogenic increase in postsinusoidal vascular resistance in the liver, underlying the portal hypertension commonly present. The latter has been targeted with antiadrenergic drugs to protect against the catastrophic gastrointestinal bleeding that portal hypertension can cause (see Sec. VI). E. Adrenaline Cotransmission in Sympathetic Nerves Adrenaline, the principal hormone of the adrenal medulla, is present also in low concentrations in extraadrenal tissues, largely contained within sympathetic nerves. Adrenaline in extraadrenal tissues appears to have been largely derived from hormone circulating in plasma, although synthesis in situ has also been documented. Adrenaline within sympathetic nerves may be released with noradrenaline as a cotransmitter, facilitating the release of the major transmitter through stimulation of presynaptic -adrenoceptors on sympathetic nerves and increasing the amount of noradrenaline released per nerve impulse. In healthy young men no regional extraadrenal adrenaline release can be demonstrated at rest. In contrast, in elderly men, adrenaline release from the heart is evident at rest (33). Several diseases, notably essential hypertension, cardiac failure, and panic disorder, are characterized by co-release of adrenaline with noradrenaline from the heart (12,55,60). Whether this adrenaline is derived from local synthesis or accumulated in the heart by neuronal uptake from plasma is uncertain. Both mechanisms may be operative. These three disorders and healthy aging are characterized by chronic or recurrent acute sympathetic activation, which does seem to have the capacity to induce the adrenaline-synthesising enzyme phenlyethanolamine methyltransferase (PNMT) in extraadrenal tissues. To this point, whether pathophysiological neuronal release of adrenaline contributes materially to disease development or to complications in heart failure, hypertension, or panic disorder is unknown. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

V. IMPLICATIONS FOR DIAGNOSIS A. Discrimination of Neurogenic Essential Hypertension from Pheochromocytoma and Syndromes of Sympathetic Activation When associated with elevated blood pressure, the clinical features in some other disorders may suggest a possible diagnosis of neurally mediated essential hypertension. If blood pressure appears to be highly variable, or if heart rate is high, neural mechanisms are often thought to be operating. Heightened blood pressure variability, however, is not a phenotypic marker of neurally mediated essential hypertension (Table 1). 1.

Panic Disorder

The symptomatology and blood pressure peaks of panic attacks can cause confusion. Sympathetic nervous activity, adrenaline secretion, heart rate, and blood pressure do increase during panic attacks, but patients with panic disorder typically have both normal blood pressure and normal sympathetic nervous activity between attacks (12). 2.

White Coat Hypertension

Another form of anxiety, the situational anxiety of “white coat hypertension,” may also masquerade as neural essential hypertension, especially if heart rates are high in the clinic. Such people showing an alerting response to medical examination, with blood pressure elevation, need to be differentiated from neural essential hypertension, in which the hypertension on 24-hour ambulatory blood pressure monitoring is sustained. 3.

Pheochromocytoma

Persistent tachycardia in patients with catecholamine-secreting tumors can also cause diagnostic uncertainty in the present context. The need to identify patients

Table 1 Typical Features of the Syndrome of Neural Essential Hypertension Age  50 years Hypertension typically mild Increased spillover of noradrenaline from the heart and kidneys High rates of muscle sympathetic nerve firing Adrenal medullary secretion of adrenaline normal High heart rate  increased cardiac output Elevated renal renin release and plasma renin activity

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

with possible pheochromocytoma and confirm the diagnosis by measurement of urinary excretion of catecholamines and their metabolites is self-evident. It is unusual for the level of sympathetic activation in neural essential hypertension to be such that urinary noradrenaline values are so elevated as to create diagnostic difficulty. 4.

“Labile Hypertension”

Sometimes the term labile hypertension is equated with neurogenic hypertension. Labile hypertension, however, does not exist. It is a misnomer typically applied to patients with borderline blood pressure elevations (61). In those with borderline hypertension, blood pressure recorded in the clinic, while showing the usual degree of variability, creates an illusion of greater fluctuation or variability than normal by oscillating around the cut-off point for the diagnosis of established hypertension. While the earlier suggestion was that spontaneous variability of arterial pressure was greater in borderline hypertension, 24-hour ambulant blood pressure monitoring in borderline hypertension has disclosed unremarkable pressure traces, with blood pressure fluctuation no greater than in healthy subjects (61).

B. Syndromes of Autonomic Failure Chronic disabling postural hypotension from sympathetic failure results primarily from central nervous system disorders impairing the reflex control of the circulation (Shy-Drager syndrome), idiopathic degeneration of sympathetic nerves (pure autonomic failure), Parkinson’s disease, and diabetic autonomic neuropathy. Differentiating between the first three on clinical grounds is often difficult but is important in terms of both prognostication and treatment. The presence of parkinsonian symptoms in Shy-Drager syndrome, in particular, can cause diagnostic confusion. Recently described differences in pathophysiology uncovered by noradrenaline isotope dilution methodology and myocardial sympathetic nerve scanning provide the key to successful diagnosis (23,47). Patients with pure autonomic failure have very low rates of whole body noradrenaline spillover at rest and near total sympathetic denervation in the heart demonstrable with cardiac noradrenaline spillover measurements and myocardial scanning (23,47). In contrast, in Shy-Drager syndrome resting noradrenaline release rates are normal and there is no sympathetic denervation of the heart (47). Unequivocal differentiation of Parkinson’s disease with autonomic failure from Shy-Drager syndrome is now possible; unexpectedly, in Parkinson’s disease with autonomic failure, the heart has been found to be extensively sympathetically denervated (47).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

C. Recurrent Vasovagal Syncope Fainting from prolonged standing or with emotional stimuli is associated in the public mind with adolescence and young adulthood. Incapacitating recurrent presyncope and fainting, however, can occur in middle and old age from disturbances in reflex postural circulatory control. The characteristics of the faint appear to be similar or identical to those found in vasovagal syncope in the young: bradycardia and hypotension associated with adrenaline secretion, sympathetic withdrawal, and increased vagal tone (13). Elaborate, highly stylized protocols of tilt table testing have been advocated to ascertain whether recurrent fainting in individual patients is due to so-called neurocardiogenic syncope. These often involve prolonged head-up tilting at steep angles, with additional pharmacological provocation if the orthostatic challenge is well tolerated at first, until a positive result (presyncope or syncope) is obtained. Such testing, as expected, is notoriously unreliable, with an unacceptably high level of false-positive results. VI. IMPLICATIONS FOR THERAPY The emergence of a better understanding of sympathetic nervous system pathophysiology has led to clinical management strategies involving targeted antiadrenergic therapies in disorders with harmful sympathetic nervous overactivity and, conversely, measures to augment sympathetic function in syndromes of autonomic failure. A. Heart Failure The cardiac sympathetic nerves are preferentially stimulated in severe heart failure. Cardiac norepinephrine spillover is increased as much as 50-fold, similar to levels of release seen in the healthy heart during near maximal exercise (2). This preferential activation of the cardiac sympathetic outflow contributes to arrhythmia development and to progressive deterioration of the myocardium and has been linked to mortality in both mild and severe cardiac failure. The level of sympathetic nervous drive to the failing heart in patients with severe heart failure is, in fact, the major determinant of prognosis (5). These findings provided the theoretical justification for the highly successful introduction of -adrenergic blockade as a heart failure therapy. Mortality in heart failure is reduced 30–60% by -adrenergic blockade (6,7). Following on this demonstrable benefit of -adrenergic blockade in heart failure, additional antiadrenergic measures including central suppression of sympathetic outflow with imidazoline-binding agents such as clonidine, blocking of norepinephrine synthesis by dopamine--hydroxylase inhibition, and antagonism of neuropeptide Y are now under investigation.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

B. Essential Hypertension and Obesity-Related Hypertension Measurement of nerve firing in postganglionic sympathetic efferents directed to the skeletal muscle vasculature with microneurography and regional rates of noradrenaline spillover utilizing isotope dilution techniques demonstrates that activation of the sympathetic nerves of the heart, kidneys, and skeletal muscle vasculature is common in patients with essential hypertension. Preferential activation of the renal sympathetic outflow is characteristic of obesity-related hypertension. The high renal sympathetic tone contributes to hypertension development by stimulating renin secretion and through promoting renal tubular reabsorption of sodium (15,18). Sympathetically mediated vasoconstriction in skeletal muscle vascular beds, in reducing delivery of glucose to muscle, is a basis for insulin resistance and hyperinsulinemia (56). Cardiac sympathetic stimulation contributes to the development of left ventricular hypertrophy and, most likely, to the genesis of ventricular arrhythmias and sudden death (15). Given that sympathetic activation in essential hypertension seems to contribute both to blood pressure elevation and to other adverse metabolic and cardiovascular effects—although it should be said that this issue has not been definitively investigated—it would be logical to specifically recommend therapies inhibiting the sympathetic nervous system in patients with essential hypertension. The current attention given by many pharmaceutical companies to research on pharmacogenetic guidance of antihypertensive therapy illustrates the attractiveness of this concept of “tailored” antihypertensive therapy. In obesity-related hypertension, the two nonpharmacological measures most commonly applied, dietary calorie restriction and an exercise program, are well known to suppress sympathetic nervous system activity. Negative energy balance from calorie restriction lowers sympathetic tone and blood pressure in humans (13,18). Aerobic exercise training preferentially reduces renal sympathetic activity (32) and would seem to be indicated as first-line therapy in obesity-related hypertension because of this very specific and potentially beneficial neurophysiological effect and, perhaps more obviously, from the empirically demonstrated effects of exercise in promoting negative energy balance, reducing body weight, and lowering blood pressure. Attempts at reduction in body weight, although pivotal in the treatment of obesity-related hypertension, more often than not fail, so that antihypertensive drug therapy is needed. What are the preferred drugs? Whether obesity-related hypertension has a specific sensitivity to antiadrenergic drugs, in fact, has not been adequately investigated. C. Hepatic Cirrhosis and Portal Hypertension Cirrhosis is characterized hemodynamically by systemic and pulmonary vasodilatation, with compensatory reflex sympathetic nervous stimulation. The sympathetic outflow to the hepatomesenteric circulation is activated, with a neurogenic Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

increase in postsinusoidal vascular resistance in the liver causing elevation in portal venous pressure (59). The portal hypertension has been targeted with antiadrenergic drugs, to protect against the catastrophic gastrointestinal bleeding it can cause. -Adrenergic blocking drugs reduce portal venous pressure via reduction in blood vein blood flow, while imidazoline receptor–binding drugs, which inhibit central sympathetic outflow, have the theoretical advantage over -blockers of reducing postsinusoidal vascular resistance and portal pressure while maintaining portal venous blood flow (59).

D. Psychosomatic Heart Disease Patients with panic disorder and depressive illness are at increased risk of sudden death and myocardial infarction. Sympathetic nervous activity is chronically increased in depressive illness (13,18), although whether the cardiac sympathetic outflow is involved is not known. In patients with panic disorder, during panic attacks acute sympathetic activation occurs. This sympathetic nervous stimulation involves the heart, leading uncommonly to coronary artery spasm (53). Compared to the treatment of another coronary heart disease risk factor, hypercholesterolemia, in which synthesis inhibition with statin drugs is so helpful, modification of psychosomatic heart disease coronary risk is largely unexplored territory. Primary therapy of the psychiatric disorder, selective use of antiadrenergic drugs, and the use of calcium channel blocking drugs to guard against coronary artery spasm (53) may possibly prove to be of value.

E. Postural Hypotension from Autonomic Failure Hypotensive syndromes exist in which the causative sympathetic nervous underactivity is chronic and unremitting. The pathophysiological pattern of sympathetic failure encompasses pure autonomic failure, characterized by peripheral sympathetic nerve degeneration, Shy-Drager syndrome, in which there is degeneration of the CNS centers involved in reflex sympathetic circulatory control, and parkinsonism with autonomic failure, in which there is widespread loss of postganglionic sympathetic fibers (23). All are helped by the sodium-retaining synthetic mineralocorticoid fludrocortisone, but beyond this treatment responses differ. The indirect sympathomimetic ephedrine, which acts by releasing noradrenaline from sympathetic nerves, is beneficial in Shy-Drager syndrome but, as expected, not in pure autonomic failure, where sympathetic innervation is absent. The partial adrenergic agonist dihydroergotamine, which is a relatively selective venoconstrictor, works best in the presence of adrenoceptor denervation hypersensitivity in patients with pure autonomic failure. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ACKNOWLEDGMENT Funded by an Institute Grant to the Baker Medical Research Institute from the National Health and Medical Research Council of Australia and by a Monash University Postgraduate Publications Award.

REFERENCES 1.

2.

3.

4.

5.

6.

7. 8.

9. 10. 11.

12.

13.

Chidsey CA, Braunwald E, Morrow AG, Mason DT. Myocardial norepinephrine concentration in man: Effects of reserpine and of congestive heart failure. N Engl J Med 1963; 269:653–658. Hasking G, Esler M, Jennings G, Burton D, Johns J, Korner P. Norepinephrine spillover to plasma in congestive heart failure: evidence of increased overall and cardiorenal sympathetic nervous activity. Circulation 1986; 73:615–621. Rundqvist B, Elam M, Bergmann-Sverrirsdottir Y, Eisenhofer G, Friberg P. Increased cardiac adrenergic drive precedes generalized sympathetic activation in human heart failure. Circulation 1997; 95:169–175. Eisenhofer G, Friberg P, Rundqvist B, Quyyumi AA, Lambert G, Kaye DM, Kopin IJ, Goldstein DS, Esler MD. Cardiac sympathetic nerve function in congestive heart failure. Circulation 1996; 93:1667–1676. Kaye DM, Lefkovits J, Jennings GL, Bergin P, Broughton A, Esler MD. Adverse consequences of high sympathetic nervous activity in the failing human heart. J Am Coll Cardiol 1995; 26:1257–1263. Packer M, Bristow MR, Colucci WS, Fowler MB, Gilbert EM, Shusterman NH. The effect of carvedilol on morbidity and mortality in patients with chronic heart failure. N Engl J Med 1996; 334:1349–1355. CIBIS II Investigators and Committees. The cardiac insufficiency bisoprolol study II (CIBIS II). Lancet 1999; 353:9–13. The CAST Investigators. Preliminary Report: effect of encainide and flecainide on mortality in a randomized trial of arrhythmia suppression after myocardial infarction. N Engl J Med 1989; 321:406–412. Kaye DM, Dart AM, Jennings GL, Esler MD. Antiadrenergic effect of amiodarone therapy in human heart failure. J Am Coll Cardiol 1999; 33:1553–1559. Musselman DL, Evans DL, Nemeroff CB. The relationship of depression to cardiovascular disease. Arch Gen Psychiatry 1998; 55:580–592. Kawachi I, Colditz GA, Ascherio A, Rimm EB, Giovannucci E, Stampfer MJ, Willett WC. Prospective study of phobic anxiety and risk of coronary heart disease in men. Circulation 1994; 89:1992–1997. Wilkinson DJC, Thompson JM, Lambert GW, Jennings GL, Schwarz RG, Jefferys D, Turner AG, Esler MD. Sympathetic activity in patients with panic disorder at rest, under laboratory mental stress and during panic attacks. Arch Gen Psych 1998; 55:511–520. Grassi M, Esler M. How to assess sympathetic activity in humans. J Hypertens 1999; 17:719–734.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

14. 15. 16.

17.

18.

19.

20. 21.

22.

23. 24.

25.

26. 27.

28. 29.

30. 31.

Sundlof G, Wallin BG. The variability of muscle nerve sympathetic activity in resting recumbent man. J Physiol (London) 1977; 272:383–397. Esler M. High blood presure management: Potential benefit of I1 agents. J Hypertens 1998; 16 (suppl 3):S19–S24. Rumantir MS, Vaz M, Jennings GL, Collier G, Kaye DM, Seals DR, Wiesner GH, Brunner-La Rocca HP, Esler MD. Neural mechanisms in human obesity-related hypertension. J Hypertens 1999; 17:1125–1133. Goldstein DS, Eisenhofer G, Stull R, Folio CJ, Keiser HR, Kopin IJ. Plasma dihydroxyphenylglycol and the intraneuronal disposition of norepinephrine in humans. J Clin Invest 1988; 81:213–220. Esler M, Jennings G, Lambert G, Meredith I, Horne M, Eisenhofer G. Overflow of catecholamine neurotransmitters to the circulation: Source, fate and functions. Physiol Rev 1990; 70:963–985. Meredith IT, Eisenhofer G, Lambert GW, Jennings GL, Esler MD. Plasma noradrenaline responses to head-up tilt are misleading in autonomic failure. Hypertension 1992; 19:628–633. Macefield V, Wallin BG, Vallbo AB. The discharge behaviour of single vasoconstrictor motoneurones in human muscle nerves. J Physiol (Lond) 1994; 481:799–809. Esler M, Jennings G, Korner P, Blombery P, Sacharias N, Leonard P. Measurement of total and organ-specific norepinephrine kinetics in humans. Am J Physiol 1984; 247:E21–E28. Esler M, Jackman G, Bobik A, Kelleher D, Jennings G, Leonard P, Skews H, Korner P. Determination of norepinephrine apparent release rate and clearance in humans. Life Sci 1979; 25:1461–1470. Goldstein DS, Holmes C, Cannon RO III, Eisenhofer G, Kopin IJ. Sympathetic cardioneuropathy in dysautonomias. N Engl J Med 1997; 336:696–702. Akselrod S, Gordon D, Madwed JB, Snidman NC, Shannon DC, Cohen RJ. Hemodynamic regulation: investigation by spectral analysis. Am J Physiol 1985; 249:H867–H875. Kingwell BA, Thompson JM, Kaye DM, Mc Pherson GA, Jennings GL, Esler MD. Heart rate spectral analysis, cardiac norepinephrine spillover and muscle sympathetic nerve activity during human sympathetic nervous activation and failure. Circulation 1994; 90:234–240. Eckberg DL. Spectral balance: a critical appraisal. Circulation 1997; 96:3224–3232. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Heart rate variability. Circulation 1996; 93:1043–1065. Julius S, Pascual A, London R. Role of parasympathetic inhibition in the hyperkinetic type of borderline hypertension. Circulation 1971; 44:413–418. Egan B, Panis R, Hinderliter A, Schork N, Julius S. Mechanism of increased alphaadrenergic vasoconstriction in human essential hypertension. J Clin Invest 1987; 80:182–186. DiBona G. Neurogenic regulation of renal tubular sodium reabsorption. Am J Physiol 1977; 233:F73–F81. Friberg P, Meredith I, Jennings G, Lambert G, Fazio V, Esler M. Evidence of increased renal noradrenaline spillover rate during sodium restriction in man. Hypertension 1990; 16:121–130.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

32.

33.

34. 35.

36. 37.

38. 39. 40.

41.

42.

43. 44.

45. 46.

47.

Meredith IT, Friberg P, Jennings GL, Dewar EM, Fazio VA, Lambert GW, Esler MD. Regular exercise lowers renal but not cardiac sympathetic activity in man. Hypertension 1991; 18:575–582. Esler MD, Kaye DM, Thompson JM, Jennings GL, Cox HS, Turner AG, Lambert GW, Seals DR. Effects of aging on epinephrine secretion, and on regional release of epinephrine from the human heart. J Clin Endocrinol Metab 1995; 80:435–442. Janig W, McLachlan EM. Specialized functional pathways are the building blocks of the autonomic nervous system. J Autonom Nerv Syst 1992; 41:3–14. Dean C, Seagard JL, Hopp FA, Kampine JP. Differential control of sympathetic activity to kidney and skeletal muscle by ventral medullary neurons. J Autonom Nerv Syst 1992; 37:1–10. Tysbenko VA, Yanchuk PI. Central nervous control of hepatic circulation. J Autonom Nerv Syst 1991; 33:255–266. Van Huysse JW, Bealer SL. Central nervous system norepinephrine release, hypotension and hyperosmolality in conscious rats. Am J Physiol 1991; 260:R1071–1076. Huangfu D, Koshiya N, Guyenet P. A5 noradrenergic unit activity and sympathetic nerve discharge in rats. Am J Physiol 1991; 261:R393–R402. Chalmers, J., Pilowsky, P. Brainstem and bulbospinal neurotransmitter system in the control of blood pressure. J Hypertens 1991; 9:675–694. Ferrier C, Jennings GL, Eisenhofer G, Lambert G, Cox HS, Kalff V, Kelly M, Esler MD. Evidence for increased noradrenaline release from subcortical brain regions in essential hypertension. J Hypertens 1993; 11:1217–1227. Lambert GW, Kaye DM, Lefkovits J, Jennings GL, Turner AG, Cox HS, Esler MD. Central nervous system monoamine neurotransmitter turnover and its association with sympathetic nervous activity in treated heart failure patients. Circulation 1995; 92:1813–1818. Lambert GW, Kaye DM, Thompson JM, Turner AG, Cox HS, Vaz M. Jennings GL, Wallin BG, Esler MD. Internal jugular venous spillover of noradrenaline and metabolites and their association with sympathetic nervous activity. Acta Physiol Scand 1998; 163:155–163. Seals DR, Esler MD. Topical review: human ageing and the sympathoadrenal system. J Physiol (London) 2000; 528:407–417. Esler M, Eisenhofer G, Chin J, Jennings G, Meredith I, Cox H, Lambert G, Thompson J, Dart A. Is adrenaline released by sympathetic nerves in man? Clin Autonom Res 1991; 1:103–108. Morris MJ, Cox HS, Lambert GW, Kaye DM, Jennings GL, Meredith IT, Esler MD. Region-specific plasma NPY Hypertension 1997; 29:137–143. Kaye DM, Lambert GW, Lefkovits J, Morris M, Jennings GL, Esler MD. Neurochemical evidence of cardiac sympathetic activation and increased central nervous system norepinephrine turnover in severe congestive heart failure. J Am Coll Cardiol 1994; 23:570–578. Meredith IT, Esler MD, Cox HS, Lambert GW, Jennings GL, Eisenhofer G. Biochemical evidence of sympathetic denervation of the heart in pure autonomic failure. Clin Autonom Res 1991; 1:187–194.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

48. 49.

50.

51.

52. 53.

54.

55.

56.

57.

58.

59.

60.

61.

Vaddadi G. Focus on nerve growth factor, pure autonomic failure and cardiac failure. Bachelor of medical science thesis, University of Melbourne, 1999. Man in’t Veld AJ, Boomsma F, Moleman P, Schalekamp MADH. Congenital dopamine--hydroxylase deficiency: a novel orthostatic syndrome. Lancet 1987; 1:183–187. Robertson D, Goldberg MR, Hollister AS, Onrot J, Wiley V, Thompson JG, Robertson RM. Isolated failure of autonomic noradrenergic neurotransmission: Evidence for impaired beta-hydroxylation of dopamine. N Engl J Med 1986; 314:1494–1497. Thompson JM, O’Callaghan CJ, Kingwell BA, Lambert GW, Lennings GL, Esler MD. Total norepinephrine spillover, muscle sympathetic nerve activity and heart rate spectral analysis in a patient with dopamine-beta-hydroxylase deficiency. J Aut Nerv System 1995; 55:198–206. Leor J, Poole WK, Kloner RA. Sudden cardiac death triggered by an earthquake. N Engl J Med 1996; 334:413–419. Mansour VM, Wilkinson DJC, Jennings GL, Schwarz RG, Thompson JM, Esler MD. Panic disorder: coronary spasm as a basis for coronary risk? Med J Austr 1998; 168:390–392. Kaye DM, Vaddadi G, Grushkin SL, Du X-J, Esler MD. Reduced myocardial nerve growth factor expression in human and experimental heart failure. Circ Res 2000; 86:e80–e84. Kaye DM, Cox H, Lambert G, Jennings GL, Turner A, Esler MD. Regional epinephrine kinetics in severe heart failure: Evidence for extra-adrenal, non-neural release. Am J Physiol 21995; 69:H182–H188. Bray GA, York DA, Fisler JS. Experimental obesity: a homeostatic failure due to defective nutrient stimulation of the sympathetic nervous system. Vitam Horm 1989; 45:1–125. Julius S, Gundrandsson T, Jamerson K, Andersson O. The interconnection between sympathetics, microcirculation and insulin resistance in hypertension. Blood Pressure 1992; 1:9–19. Landsberg L: Diet, obesity and hypertension: an hypothesis involving insulin, the sympathetic nervous system, and adaptive thermogenesis. Q J Med 1986; 236:1081–1090. Esler M, Dudley F, Jennings G, Debinski H, Lambert G, Jones P, Crotty B, Colman J, Willett I. Increased sympathetic nervous system activity, and effects of its pharmacological inhibition, in alcoholic cirrhosis. Ann Intern Med 1992; 116:446–455. Rumantir MS, Jennings GL, Lambert GW, Kaye DM, Seals DR, Esler MD. The “adrenaline hypothesis” of hypertension revisited: evidence for adrenaline release from the heart of patients with essential hypertension. J Hypertens 2000; 18:717–723. Esler M. Hyperadrenergic and “labile” hypertension. In: John Swales, ed. Textbook of Hypertension. London: Blackwell, 1994:741–749.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

10 Autonomic Regulation and Dysregulation of the Heart Insights from Spectral Analysis of Cardiovascular Oscillations Massimo Pagani and Daniela Lucini University of Milan and L. Sacco Hospital, Milan, Italy

The finger pointing to the moon is not the moon Old Taoist saying

In a traditional scenario, people seek medical help most often because of disturbing symptoms, while doctors engage in treatments aiming at curing patients: the underlying model is that of disease causing organic damage within individuals. In the emerging new approach, driven by aging populations, improved medical technology, and awareness of limited resources, medicine provides the means to optimize the dynamic process between health and illness, mostly focusing on subjective outcomes, compressing disease and fostering prevention. The change of paradigm is from a doctor-centered to a patient-centered medical practice, maintaining and measuring the quality of the process. The aim thus might shift from a sole interest in hard endpoints, easily measurable from organic changes according to population standards, to incorporate patient’s preferences and individual performance, based also on individual functional measures (1), which are obviously more difficult to assess, particularly in real-life conditions outside of hospitals. Consequently, it easy to foresee the need for new diagnostic and therapeutic methods, inclusive of a new alliance between doctors and patients, likely to lead, in short, to a new epistemology, built on the increasing societal pervasiveness of information technology and the foreseeable birth of virtual medicine. A part of this process might comprise a redefinition of the relationship between organ damage (objective disease) and symptoms (subjective illness). As an intermediate step, we Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

should consider the possibility that (measurable) functional equivalents might accompany, or even antedate, the occurrence of (subjective) disturbances in absence of any (objective) organ damage. Conversely, it is well recognized that hidden damage may be present before the occurrence of any symptom. In our clinic, we address the tenuous, often ill-defined, boundary between organ disease (based on standardized population evidence) and subjective perceptions (based on often deceptive individual self-description) in the context of a psychosomatic approach. The general model is that functional equivalents, such as those provided by markers of autonomic activity, usually accompany the occurrence of symptoms. This approach may be useful in the application of autonomic physiology to cardiovascular medicine, particularly considering the major role of cardiovascular diseases in the western world and the emergence of individualized preventive strategies. Readers are referred to the many excellent reviews (2–11) that have been published in recent years on the topic of autonomic cardiac innervation; in this chapter we will specifically focus on theoretical and practical aspects of spectral analysis of heart period and arterial pressure variability as means to infer normal and abnormal regulation, as we routinely perform in our clinical work.

I. INFORMATION, THE AUTONOMIC NERVOUS SYSTEM, AND CARDIOVASCULAR OSCILLATIONS Information theory dictates that, in the realm of all possible varieties, information and noise cannot be identified per se, but only within a specified goal-oriented system. Accordingly, information is generated out of noise by coding or structures. In the context of the various levels of functional complexity of the nervous system, the autonomic nervous system (ANS) can be viewed as one of its many subsystems (12). Its aim is to handle information in order to guarantee the dynamic adaptation of the organism to the flow of time-dependent stimuli and tasks arising from the environment, be it external or internal. Applying a cybernetic approach to the relationship between noise and information, both negative and positive feedback (13) modalities appear in theory crucial to permit not only various quantities of a given state, but also several states in the variety of possible functions. The experimental observation that both negative and positive feedback circuits (Fig. 1) (14) are operative in the functional design of the autonomic cardiovascular control complies with this model. Informationally, the simplest difference observable in a system is the dyad one/zero (active/inactive), corresponding in the nervous system to the modality excitation/inhibition. At the organism level this corresponds to complex operational programs or different states such as activity and rest, wakefulness and sleep. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 1 Opposing feedback mechanisms that, in addition to central integration, regulate the state of sympathovagal balance. (From Ref. 14.)

Behaviorally, intact humans and animals can be considered as open systems, separated from the environment by boundaries that exchange a continuum of matter, energy, and information. It can easily be shown that the flow of information, both between the whole organism or its subsystems, such as the ANS, and its external, or internal, environment is characterized not only by quantitative, but by qualitative differences as well. It follows that energy and information cannot be equated, but should be conceived in their goal-oriented relationship. In the specific case of the ANS, the energy (amount of bits, or nerve impulses per unit time) cannot be simply equated to the information they carry, which pertains also, if not mostly, to the attendant changes in the controlled function.

II. ANS, NEURAL CODES, AND BEYOND To extract information from neural signals pertaining to ANS activity, it is therefore necessary to consider not only specific functional aspects, i.e., the set of relevant goals, such as changes in heart rate, arterial pressure and flow, myocardial contractility, and so on, but also a matrix of structures, such as vagal and sympathetic circuits with their reciprocal interactions (within the ANS and with respiratory neuronal pools, with humoral control systems, and with the postsynaptic transduction leading to target function) and attending neural codes. Usually simple neural structures carry information employing, in spite of its intrinsic lesser efficiency, a frequency rather than amplitude modulation. This latter modality, in fact, guarantees greater protection from noise and errors, which is an important feature to assure functional stability. Thus, accuracy of information transmission might be more important than mere energetic or mathematical effiCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ciency, in line with the extreme redundancy of neural organization. Complex neural structures, conversely, might handle information also in schemes rather different from the simple number of action potentials per unit time (i.e., intensity code, level of tonic activity), so easily translated into some average chemical equivalent (e.g., messenger spillover to plasma). In fact, neural structures possess a rich repertoire of elementary algorithms (15), supporting highly structured temporal codes based on correlation and synchronization. In cardiovascular-respiratory organization this might more easily translate into a code based on different patterns, associated with the state (active/inactive) of various oscillatory circuits, rather than simple changes in absolute values of derived parameters of individual oscillators, examined independently from one another. These considerations might help better focus various attempts to infer autonomic cardiovascular control from computer analysis of spontaneous cardiovascular beat-by-beat variabilities. In keeping with the elegant synergetic description made by Haken (16), statements about complex auto-organizing systems should consider all available information (and thus not to disregard any of it, even if apparently trivial) and rely on educated best guesses (based on probability levels, rather than on an impossible absolute certainty). Consequently, to extract the information hidden in cardiovascular variability signals, it is convenient to explicitly address the underlying structures (neural circuits, target organ transduction properties, system’s functional aims) and codes (how information is stored and transferred in neural circuits). Regarding this latter, it should be realized that up to now the available literature on ANS (12) has given larger credit to the intensity code (neural activity, tone, or the equivalent plasma level of messengers) than to other more complex codes (such as rhythm or pattern) (Table 1). Accordingly, it should be no surprise if studies suggesting the use of second (or higher)-order codes find it difficult to gain acceptance from more conservative readers. Notably, time integration of spike activity provides some information on how ANS drives the target performance over time (e.g., increases in sympathetic firing rate are usually followed by tachycardia). Conversely, changes in pattern do not provide direct information on target control, rather they can be used to infer more subtle changes in cardiovascular regulation. For example, the reduced baroreflex gain observed during exercise (17) indicates that for any given change in pressure (e.g., a rise) there is a smaller attending directionally similar variation (i.e., a lengthening) in RR interval; the reduced baroreflex sensitivity (BRS) following a myocardial infarction is a negative prognostic indicator. We might thus conclude that the type of information carried by these two modalities is likely to be different. In the following section we will assume that transduction properties at the target level and the gain of the signaling molecular chain can be treated (for simplicity) as an invariant. Specific exceptions, such as genetically determined adrenergic overexpression (18) or disease-mediated up- or downregulation, will be explicitly indicated. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Table 1 Percentage of Papers Containing Terms Related to Neural or Autonomic Activity and to Different Code Modalities (Medline database, 1966 to 2000) Key terms

Pattern

Information

Correlation

Rhythm

Oscillation

Synchronization

Code

Neural activity Autonomic activity Sympathetic activity Parasympathetic activity MSNA Heart rate variability

13.067 10.979 4.174 3.463 2.632 5.528

9.760 4.451 2.505 3.463 1.316 5.692

4.853 4.748 5.318 7.792 10.526 9.541

2.293 3.858 2.690 6.494 0.439 5.897

2.133 0.890 0.247 0.216 0.439 0.942

1.333 0.000 0.340 0.000 0.439 0.123

1.120 0.000 0.000 0.000 0.000 0.041

MSNA, muscle sympathetic nerve activity. Note the small and decreasing values of the frequency of terms referring to various aspects of complex coding modalities. Source: Ref. 12. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

III. ESSENTIALS OF CARDIAC INNERVATION Within hemodynamic performance, cardiac function is regulated in a beat-to-beat fashion by the following components: cardiac vagal and sympathetic nerves (both comprising afferent and efferent fibers) the receptor transduction chain and the local humoral milieu, conversely vascular neural regulation entails a simpler neural organization, involving only (afferent and efferent) sympathetic nerve fibers (endothelial-derived constrictor and dilator substances add a fine balance to peripheral vasomotion control). Given the tight anatomical and functional integration of cardiac, pulmonary, and vascular structures, all aimed at the common goal of delivering at the periphery appropriate quantities of oxygen and nutrients and of carrying away metabolic waste products and carbon dioxide, it is rather artificial, as in this chapter, to consider cardiac innervation separately from the rest of the cardiovascular-respiratory system. Overall, both the vagus and the sympathetic nerves are mixed in nature, as they are comprised of both sensory and motor fibers (11). As is customary with peripheral nervous structures, the different functional effects depend on the specific circuit and chemical messenger involved. Vagal and sympathetic afferent fibers feed the centers, with coded sensory signals about both mechanical and chemical events occurring within the myocardium and in the coronary beds: thus information on both normal and abnormal events is carried, at various levels, to the central nervous structures with the same neural sensory channels. Characteristics of the sensory experience associated to any given condition, such as the occurrence of pain during myocardial ischemia, depend on the central processing of the information. The relationship between a given stimulus and derived sensation is not, however, a simple one, because it is subject to sensory amplification and distortion, which involves a complex chain of events inclusive of dynamic alterations of the receptor chain, and may be affected by prior physiological or psychological experiences. Personal, social, and cultural aspects conjoin to generate a unique mix giving rise to cardiac sensations, many of which linger below the level of awareness and are colored by the emotionally charged notion that cardiac diseases are the most frequent and often unexpected killers. This complexity is confirmed by clinical experience indicating that not only may a stimulus be present and the sensation absent, as shown by the observation that a majority of ischemic attacks may be without pain, but that the sensation might be there and the stimulus absent, as demonstrated by patients with implanted defibrillators who while asleep may “feel” a phantom defibrillator discharge without any such discharge having occurred (19). These considerations might also help in the clinical judgment of other cardiac symptoms, such as dyspnea in congestive heart failure or palpitations, which are so frequently divorced from objective evidence of cardiac disease.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Regarding cardiac pain, sympathetic afferent fibers (20) represent the major sensory channel conveying neural information to the centers, although some contribution from vagal afferents could also occur. Malliani et al. proposed a spatiotemporal theory (21), which, integrating elements of the intensity and of the specificity theories, makes it possible to interpret available evidence on the “elusive” link between myocardial ischemia and pain. The intensity theory dictates that cardiac pain derives from excessive stimulation of normal cardiac sensory fibers: pain would be decoded from the abnormally great number of action potentials reaching the centers. The specificity hypothesis entails the existence of cardiac nociceptor fibers, usually silent, but recruited by noxious events: pain would be decoded from recruitment of a specific group of sensory fibers, which would act as part of an alarm system. Experimental evidence of such recruitment is, however, lacking; conversely, the spatio-temporal theory accounts for available experimental findings. In anesthetized animals cardiac sympathetic afferent fibers, under physiological hemodynamic conditions, are always active, although with a low firing rate; their activity increases drastically with stimuli, such as intense ischemia (Fig. 2), which are clinically associated with pain. In addition, various chemicals my modulate and facilitate afferent discharge (22–24). In conscious animals, after full recovery from experimental surgery, even extremely intense and diffuse excitatory chemical stimuli (such as produced by large amounts of intracoronary bradykinin) do not provoke behavioral signs of pain (25), but localized mechanical stretch of the coronary bed may do so, particularly when injury to tissue, as related to recent surgery, is also present (21). The sensory code for cardiac pain would therefore reside in the elevated firing of a restricted population of sensory fibers, excited on top of a diffusely increased sensory activity, or augmented sensibility thereof. Interpretation of cardiac-related sensations should also incorporate the observation of convergence from somatic sensory fibers and complex central processing at different hierarchical levels (26): for instance, in humans myocardial ischemia is felt as painful if afferent information activates both thalamic and cortical structures (27), while no pain is elicited if only the thalamic level is activated by the chain of neural changes initiated by the ischemia-related sensory input, suggesting some crucial role for cortical integration in the genesis of ischemic pain. Similar considerations apply to other cardiac-related sensations, such as the dyspnea of acute cardiogenic pulmonary edema, which is closely related to the activation of the complex sensory supply of the left side of the heart and of the lungs by way of increased filling pressures. Pharmacological reduction of excessive -adrenergic effects, possibly reflecting preexisting microvascular genetic or disease-mediated functional alterations, is finally capable of significantly improving overall myocardial performance (28), with potential implications for the treatment of chronic myocardial ischemia.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Activity of an afferent sympathetic nonmyelinated nerve fiber with a left ventricular sensory field. Tracings represent, from top to bottom, systemic arterial pressure, coronary perfusion pressure, nerve impulse activity (cathode-ray oscilloscope recordings). (a) Interruption of the left main coronary artery perfusion; (b) intercoronary administration beginning at the arrow, of bradykinin, 5 ng/kg; (c) intracoronary administration of bradykinin, 10 ng/kg; (d) intracoronary administration of bradykinin, 30 ng/kg; (e) electrical stimulation of the left inferior cardiac nerve activating the afferent fiber; the biphasic first deflection is the artifact of the fiber: The approximate length of the nerve was 8 cm. The conduction velocity calculated for this fiber was 0.45 m/s. (f) Mechanical probing, marked by a bar, of an area of the external surface of the left ventricle. (From Ref. 20.)

Information on a possible physiological role for the rich cardiac innervation is usually gauged from experimental studies of cardiogenic reflexes, considering the stimulus-response properties of target functions: either efferent neural activity (vagal or sympathetic) or hemodynamic variables. In this latter case, it is also important to exclude that observed hemodynamic changes (e.g., reductions in arterial pressure) are not directly caused by the stimuli, such as the immediate cardiac depression produced by acute myocardial ischemia. Neural ablation and stimulation (frequently mimicked by pharmacological treatment) or the new elegant ap-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

proach provided by the use of genetically modified animals, with programmed neural manipulations, can demonstrate the hypothetical involvement of specific neural pathways. Although in a sense obvious, it is not always recognized that only studies conducted on conscious preparations best reflect the physiology of real-life conditions (29), inclusive of psychological aspects. In general, in the interpretation of experimental data in the field of cardiac neurophysiology it may be best to avoid extrapolations and excessive simplifications (16). Selecting and summarizing in the context of this report, (overall) (11) cardiogenic reflexes produce negative feedback, as they produce bradycardia and hypotension when the neural circuits involve primarily a vagal activation [e.g., Bezold-Jarisch effect (30)]; conversely, positive feedback, indicated by tachycardia and hypertension, is produced by a prevailing sympathetic activation [e.g., Bainbridge reflex (31)]. The selective assessment of individual vagal and sympathetic contribution to cardio-cardiac reflexes requires complex electrophysiological techniques or selective neuro-surgical manipulations to be unraveled. Figure 3 provides a representative example of the ischemia-induced reflex vagal predominance and sympathetic inhibition in the neurally intact anesthetized acute cat preparation, transformed after vagotomy in sympathetic activation (32). The influence of time and recovery from surgery-induced tissue damage on the directional sign of cardiogenic reflexes is shown in a conscious dog (25) in Figure 4. A Bezold-Jarisch negative feedback cardio-cardiac inhibitory reflex is produced by intracoronary bradykinin soon after surgery, at a time when tissue damage is still present, but the same stimulus produces the usual, positive feedback, excitatory cardio-cardiac sympathetic activation when complete recovery has occurred, weeks later, notably in absence of any behavioral sign of pain. It is important to note that crucial information on cardiovascular regulation depends also on several other inputs, particularly reflexogenic areas from the arterial side coding mechanical (systemic arterial, i.e, high-pressure, or pulmonary, i.e., low-pressure, baroreceptors) and chemical (chemoreceptor) information that is continuously integrated in the central structures. Arterial baroreflexes, initiating bradycardia and hypotension, typically result in negative feedback. Chemoreflexes, conversely, are more complex, as they provoke divergent effects of bradycardia and hypertension, together with augmented respiratory activity (capable of initiating a subsequent chain of events, leading to additional changes in coronary resistance). In congestive heart failure, the potential role of activation of cardiopulmonary sympathetic afferents by the abnormally elevated local pressures, and subsequent engagement of spinal reflexes, may be suggested by experiments in chronic cats with a spinal section at C7 and muscarinic blockade (33). An infusion of saline, mimicking the increase in filling cardiac and pulmonary pressures, and activating a cardio-cardiac sympathetic excitatory reflex, reproduced the clinical findings of tachycardia and hypertension frequently found in acute pulmonary edema. Severing the neural loop abolished sympathetic excitatory responses. This

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3 Effects of global ischemia in a cat with chronic sinoaortic denervation, before and after sectioning the vagi. The tracings represent, from top to bottom: AP, arterial pressure; MAP, mean arterial pressure; LVP, left ventricular pressure; LVdP/dt, left ventricular dP/dt; HR, heart rate; Symp, cardiac efferent sympathetic nerve activity: Bars indicate the periods of the left main coronary artery occlusions (From Ref. 32.)

finding suggests a physiopathological rationale for the clinical efficacy of drugs, such as opiods, the beneficial effects of which during acute pulmonary edema outweigh the simple reduction of associated anxiety, and might also relate to their capacity to centrally reduce sympathetic responsiveness. Thus, simplified overall models might be useful to include neural control principles in cardiovascular practice. Malliani and coworkers proposed a model of continuous interaction of positive and negative feedback circuits (13) (Fig. 1). Accordingly, the continuous flow of information reaching the centers from the sensory innervated cardiovascular periphery is schematically divided according to their capacity to initiate negative (mostly through baroreceptors and vagal afferents) and positive (through sympathetic afferents) feedback reflexes, which are under the regulatory control of supraspinal centers. Behavioral programs, central integration, and sensory information from the periphery all contribute to the dyCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 4 Contrasting effects of intracoronary bradykinin (100 ng/kg) in a conscious dog when examined early (left panel) and late (right panel) after surgery. Note that while a depressor response was obtained on the third postoperative day, it reverted to a pressor response at a time of complete recovery from surgery (20 th postoperative day). (From Ref. 32.)

namic regulation of beat by beat cardiovascular performance, accommodating peripheral demands and individual goals. Obviously in this model pathophysiology does not have any finalistic nature (11).

IV. RECORDING OF ANS ACTIVITY IN HUMANS Available techniques to study autonomic nerve activity in humans do not allow any direct measure of vagal efferent activity. Some investigators, however, suggest using heart rate variability (HRV) or baroreflex gain (34) as a surrogate measure of vagal control of the sinoatrial (SA) node, in spite of the fact that these two parameters have a complex, integrated nature and derive from the interplay of several different neural circuits. For instance, animal studies clearly showed that both Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

transient (35) and long-term changes (36) in sympathetic afferent activity have a profound inhibitory action on baroreflex gain. Clinical studies show that this latter is in turn tightly linked to simple measures of HRV, such as RR 2 (37). In spite of these issues, clinical use of both HRV and baroreflex gain is proving extremely promising to gauge cardiovascular prognosis. Regarding sympathetic nerve activity, several clinical studies have indicated the extremely interesting potentials of two of the most widely employed techniques: norepinephrine spillover to plasma and electroneurographic recordings of muscle sympathetic nerve activity (MSNA) (38). To these, neural imaging techniques are adding still largely unexplored potential to the investigative armamentarium. Importantly, however, none of these methods appears suitable for regular clinical use in large numbers of patients or in real-life conditions outside of the clinical laboratory. Moreover, it should be recalled that the various techniques available to assess average (frequently indicated as tonic) global or regional sympathetic activity (such as MSNA and norepinephrine spillover) should be considered complementary and used in combination and coupled with the additional evaluation of end-organ cardiac and vascular adrenergic response. Power spectral analysis of cardiovascular variabilities (39) may in addition furnish a tool providing information on autonomic cardiovascular regulation (i.e., modalities of autonomic codes), according to the model (and constraints) of the sympatho-vagal balance, functionally related to central oscillatory structures coding various levels of arousal (excitation/quiet), which are thereby (indirectly) linked to levels of activity.

V. APPLICATIONS OF CARDIOVASCULAR OSCILLATIONS: INFERRING NORMAL AND ABNORMAL AUTONOMIC FUNCTION FROM HRV When, following the provocative suggestions of Akselrod et al. (40), we approached the study of dynamic cardiovascular autonomic regulation employing spectral analysis of cardiovascular beat-by-beat variability, our goal was to infer useful information (12,39) on neural regulation of the cardiovascular system, particularly in real life and dynamic conditions, where commonly used techniques are not applicable to the analysis of transition across different functional states. Accordingly, heuristics and practical relevance suggested switching from studies of reflex cardiovascular control, which in a clinical setting are limited to standardized laboratory conditions, to the analysis of cardiovascular variability, which can more easily be applied to the continuous changes of real-life conditions. To allow widespread use of the approach, the choice of the initial parameter to study fell on the simple ECG, a physiological variable that could be easily obtained in ambulant conditions by way of Holter recordings or radiotelemetry. The addition of ar-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

terial pressure measurements (also available with a noninvasive device) allowed the simultaneous assessment of markers of efferent sympathetic vascular modulation and of baroreflex gain. The major caveats with ambulatory recordings derive from the frequently unrecognized effects of biological and experimental noise, such as that associated with slow breathing (41) and physical activity (42), on cardiovascular rhythms and hence on the greater indetermination affecting derived inferences.

VI. HRV IN RESTING AND STIMULATED PHYSIOLOGICAL CONDITIONS Under normal resting conditions, RR variability spectra contain three major components (14,39,43) (see Fig. 5): one centered at 0 Hz [usually called direct current (DC) or very low frequency (VLF), in the range 0.00–0.03 Hz], a low-frequency (LF range 0.03–0.14 Hz, centered at 0.1 Hz), and a high-frequency, respiratory, component (HF range 0.15–0.35 Hz, centered at 0.25 Hz). Spectral powers are presented both in absolute (i.e., msec2) and normalized units (n.u.). Normalized units are computed by dividing the power of every LF or HF component by total power (i.e., variance) after having subtracted from it the VLF (⬇noise) component and multiplying the ratio by 100. The presence of several small noise components, in the low- or high-frequency region of the spectrum, dictates that the sum of LF and HF is nearly always less than 100. The relative powers of the LF and HF components are similar in young subjects at rest, so that the LF/HF ratio is approximately equal to one. With aging there is a decline in variance, particularly evident in the early decades; such a decline levels off in old age (39). Conversely, the spectral powers, in n.u., remain largely stable with aging [with the notable exception of old age, when stand-induced changes are particularly reduced (44)]. Conditions of enhanced sympathetic drive, such as tilt (39) (Fig. 5) [or simply standing up (45)] (Fig. 6), moderate mental (Fig. 6) or physical exercise (Fig. 7) (46), or the arousal of waking up in the morning (47) are all associated with a shift in spectral profile towards the low-frequency region. This corresponds to an increase in the normalized power of LF (as well as LF/HF) and a decrease in HF. When using FFT algorithms and forcing a simple division of the spectral power in two predetermined bands of interest (48), the increased computational noise might render the model of sympatho-vagal balance and subsequent inferences on autonomic cardiac regulation inapplicable (12). In view of the strong nonlinear modulation by respiration of cardiovascular, and notably R-R, variability (41), it is crucial to factor in the analysis of HRV the influence of respiration. Respiration at about 0.1 Hz determines a drastic augmentation of the LF component (entrainment), while paced breathing at higher frequencies leads to a shift of spectral power towards the HF region [and a reduction of tilt induced changes (39)]: Sim-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 5 Spectral analysis of HRV in a young subject at rest and during 90° tilt. The RR interval time series (i.e., tachograms) are illustrated in the top panels. The middle panels contain the autospectra (PSD  power spectral density), which indicate the presence, in addition to VLF (very low frequency, centered at 0 Hz), of two major components (LF  low frequency; HF  high frequency). During tilt, the LF component becomes largely predominant. In this example the total variance is markedly reduced during tilt and consequently LF and HF powers are both decreased when expressed in absolute units. The use of normalized units (nu) clearly indicates the altered relation between LF and HF during tilt as represented by the pie chart, which shows the relative distribution together with the absolute power of the two components represented by the area.

ilar enhancements of the HF component and reduction of LF are produced by increases in respiratory volume (49); however, the more complex recording instrumentation is a likely reason for the limited number of studies formally addressing this issue. When respiration is markedly altered, any information that would be inCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 6 Example of autoregressive spectral analysis of simultaneous tachogram, systogram, and respirogram of a control subject. Notice the increase in the LF component, of R-R interval, and of systolic arterial pressure variabilities during both STAND and MA (mental arithmetic). Furthermore, during this latter condition, the respiratory autospectrum becomes flat and broadened as a consequence of the altered respiration pattern present during talking. (From Ref. 45.)

ferred from R-R variability on the basis of a code implying the most frequent physiological respiratory pattern in the linear region ~0.15–0.35 Hz may be questionable. As a practical corollary, in all instances of spectral analysis of R-R variability, it is strongly recommended always to obtain at least are independent assessment of respiratory rate. The addition of spectral analysis of arterial pressure variability furnishes an indirect means to infer sympathetic regulation of vascular oscillations (50), which are more directly explored by MSNA. As shown in Figure 8, MSNA variability contains HF and LF components. Infusions of vasoactive substances in order to shift the autonomic balance towards a state of sympathetic (with i.v. nitroprusside) or vagal (with i.v. phenylephrine) predominance induce, respectively, a LF or HF prevalence. (See Ref. 51 for further details.) Results indicated a compelling relationship between changes in autonomic drive and cardiovascular oscillations. The balance between LF and HF components of cardiovascular variability tracks closely the changes in directly measured peripheral sympathetic activity. During stress consistent with parasympathetic activation, the increased HF component and decreased LF component in not only R-R interval but also MSNA suggests an Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 7 Example of the effects of R-R interval variability and on respiration of stepwise bicycle exercise, performed in the recumbent position, at moderate intensity (approximately 30% of maximum theoretical effort for age and sex). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 8 Power spectra of MSNA, R-R interval, SAP variabilities, and respiration (Resp) in a single subject during infusions of saline (control), nitroprusside, and phenylephrine. During sympathetic activation induced by nitroprusside (left), the LF component of neural and cardiovascular variability signals predominates relative to the HF component. Conversely, during sympathetic inhibition and vagal activation induced by phenylephrine, there is an increase of the HF component relative to the LF component. AU indicates arbitrary units. (From Ref. 51.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

intimate interaction between parasympathetic and sympathetic neural oscillatory structures. The high coherence (Fig. 9) between HF and LF fluctuations in MSNA and R-R interval and the persistence of this synchrony of rhythms across a range of arterial pressure perturbations provide functional evidence to support the concept of common central mechanisms governing sympathetic and parasympathetic rhythmic activity. Overall, the study (51) supported the concept of using changes in the LF component of systolic arterial pressure (SAP) variability as a marker of

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

changes in sympathetic efferent nerve activity to peripheral vasculature. The simultaneous analysis of R-R interval and systolic arterial pressure variability, by way of the index , provide in addition, a means to quantify the gain of the negative feedback arterial pressure–heart period baroreflex (52), providing results similar to those obtained with the phenylephrine slope, but without requiring artificial hemodynamic stimuli. An assessment of the positive feedback loop can be approximated by nonbaroreflex slopes (53), but the specific underlying model is far from resolved (54). The inclusion of respiration in the computation makes it possible to separate the gains of the arterial from the cardiopulmonary circuits (see Ref. 55 for details).

VII.

THE DYNAMIC MODEL: REST AND STAND PARADIGM VS. 24HOUR RECORDINGS

Neural signals regulating cardiovascular targets, as customary for neural activity in general, are characterized by a large variability that, accordingly, must be accounted for in the algorithm employed for their analysis and interpretation. The wide extremes of cardiovascular performance, ranging typically from a R-R interval of about 800 msec at rest, associated to a variance of about 2000–3000 msec2, to a R-R interval of slightly more than 300 msec and variance of 300 msec2, respectively, during maximal exercise (46), suggested that a description of dynamics of neural regulation should incorporate in the study protocols conditions of activation. However, experimental paradigms considering several levels of arousal, although very informative, and at times essential for specific focused questions, such as the progressive autonomic effects of increasing gravitational load (gradual tilt) or exercise (gradual treadmill or bicycle load), are impractical for routine clinical use, either because of their complexity or because of the excessive length of experimental time required, which may make patient stability impossible. The ancillary issue of a gradual mental load (56), although of importance in specialized settings, will not be dealt with in this chapter. In our laboratory we routinely employ a rest-stand (or the largely similar rest-tilt) paradigm that minimizes the time required to gather experimental data, while simultane-

Figure 9 Average coherence values (K2) between (from top to bottom) LF components of R-R interval and MSNA, HF components of R-R interval and MSNA, HF components of respiration (Resp) and MSNA, and LF components of SAP and MSNA. The dotted lines indicate the threshold value (0.5) above which there is a significant correlation between oscillations in different variables. Correlation should not be taken as a proof of causality, but only of exchange of information between variables. C indicates control; Phenyl-epi, phenylephrine. (From Ref. 51.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ously furnishing results relating to at least two different states of activation of the sympatho-vagal balance (12). During stand, at 1G (i.e., at sea level), the reflex activation of sympathetic efferent activity, and simultaneous vagal withdrawal, are the result of a complex interplay of changes in the input signals to the cardiovascular neuronal pools, which could be schematically comprised of arterial and cardiopulmonary baroreceptors, and afferents from the antigravitational muscles. Other sensory inputs, such as the chemoreceptors, are usually stable and thus disregarded in usual conditions. Therefore, the change from rest to stand can furnish a standardized method to approximate the trajectory (16) followed by autonomic regulation passing from quite to activation. The paradigm furnished by the dynamics of 24-hour recordings of the ECG in ambulant subjects would in addition permit one to explore changes in the state of the sympatho-vagal balance induced by real-life occurrences, such as the sleep-wake cycle or moderate physical and mental activity (47). It must be mentioned, however, that 24-hour recordings do not usually address explicitly the noise related to subjects’ movements that might influence the VLF components (42). The same principle applies to experiments employing pharmacological (e.g., vasoactive drugs) or physiological stimuli (e.g., lower body negative pressure, Valsalva maneuver) to alter the state of autonomic balance.

VIII. ENHANCED SYMPATHETIC ACTIVITY: PHYSICAL AND MENTAL EXERCISE Spectral profiles of the R-R interval, systolic arterial pressure, and respiration variability in a control subject examined at rest and mental arithmetic during active stand (14,39) are shown in Figure 6 (43). It is apparent that the (relative) power of the LF component becomes predominant in both the tachogram and systogram spectra, signaling the stand-induced sympathetic excitation and vagal withdrawal. The quantitative changes in spectral characteristics are in the case of the tachogram best followed, using stochastic, rather than deterministic, algorithms and normalized, rather than absolute, units. The use of normalization (and the LF/HF ratio), accounting for changes in absolute power across individuals and conditions, furnishes a formal means to reduce the uncertainty in assessing the distribution of spectral power when variance, or the VLF component, undergoes divergent changes with the autonomic balance (see Fig. 5), as frequently observed in experimental conditions (e.g., physical and mental exercise, sleep, -adrenergic or muscarinic blockade). The more recent introduction of time-variant algorithms, improving the time resolution of the methodology, furnished a technique adequate to follow rapid changes in autonomic balance and capable of linking, for example, some cases of orthostatic intolerance to autonomic dysregulation (57) that would not be

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

otherwise recognizable. The further refinement of batch- and time-variant algorithms suggests that other indicators, such as the instantaneous center frequency (58), or the baricenter, might be added to the investigative armamentarium. These physically defined measures (Hz) could help reconcile some of the still ongoing debate (59,60) on the validity and meaning of the mathematical manipulations of spectral measures of HRV employed to enhance the signal-to-noise ratio. Figure 7 depicts the changes in spectral profile produced in a normotensive subject by physical exercise on both R-R interval and arterial pressure variability (46). It is apparent that during exercise the spectral power shifts towards the LF region in both signals. However, the drastic reduction in R-R variance renders the shift in power observable only with normalized units; in addition, with greater loads the increased biological and methodological noise might render interpretation difficult. Accordingly this approach might be better suited to paradigms of exercise limited to lower intensities or to selected muscle masses. Conversely, in conscious dogs (46,61) the changes from a rest condition of largely prevailing vagal modulations, as signaled by a predominant HF component, to a predominant LF is more apparent. During exercise the  index signals as well a clear reduction of baroreflex gain (17,46). With higher levels of exercise the spectra of R-R interval variance are characterized by an extremely reduced absolute power and the sole presence of a respiratory component that is mechanically (not neurally) (62) driven: as a corollary, in this and other extreme conditions (e.g., severe congestive heart failure) the sympatho-vagal model may be inapplicable (12). Because of the engagement of respiration, as occurs with talking, the assessment of the autonomic effects of mental exercise must factor in this variable. In addition, influences of the context and the possible reinforcing effect of human confrontation and the subject’s system of beliefs should be considered. Among the most frequently laboratory paradigms employed, mental arithmetic (or public speaking) entails talking and human confrontation (56); reaction time interval [e.g., the interactive K test (KT), or computer games] is performed silently and without human interaction; simple reading may be performed aloud (i.e., talking) or silently and is likely to be related to a very limited “mental load” (63). The study of real-life conditions or of situational stress requires specific protocols and instrumentation, e.g., ambulatory or telemetric recordings (45). In humans both acute experimental (i.e., mental arithmetic) (46) and possibly long-term real-life stressors (64) increase heart rate and arterial pressure, reduce baroreflex gain, and lead to a shift of the spectral balance towards the LF component, both in RR and systolic arterial pressure variability (Fig. 6). The possibility of an entrainment of LF by slow respiration must always be considered (63). Similar changes are observed also in animals (65) or humans (64) exposed to situational stress. Under these conditions the LF prevalence produced by stress is independent of respiration and reflects central arousal. Marked hemodynamic changes, such as hypertension, tachycardia, as well as increased coronary blood flow, with attendant en-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

hanced propensity to arrhythmias (66) and ischemia (67), are observed in conscious dogs exposed to aversive environments or to rage and confrontation. The superiority of natural, as opposed to artificial stimuli in the assessment of behavioral effects on cardiovascular regulation was shown some 20 years ago by Baccelli et al. (68) with elegant experiments on conscious cats. The use of noninvasive approaches permits one to explore more easily, in humans as well, the effects of behavioral interventions on autonomic regulation. For example, relaxation training, while maintaining a normal autonomic profile at rest, induces a complex array of effects, such as a reduction of sympathetic responsiveness to both physical and mental stimuli, similar to that obtained with adrenergic blockade (69), and a more regular breathing pattern (70). Spectral analysis of cardiovascular variability and telemetric recordings are also useful in assessing the effects of extreme environmental conditions, such as those related to the stress of space environment. Permanence in microgravity (71), among other effects, impairs cardiovascular regulation, reducing baroreflex gain and limiting vascular sympathetic responsiveness (72), resulting, on return to 1G, in transient orthostatic intolerance. The addition of the assessment of subjective appraisal of emotional components with standardized techniques might further refine the information obtainable about the interaction between the subject and his or her environment.

IX. ENHANCED VAGAL ACTIVITY: THE VASO-VAGAL REACTION A majority of studies (43) considers that an increase in absolute values of R-R variance (or its time domain equivalent standard deviation (SD)), an elevated baroreflex gain, or a shift in spectral power towards the high-frequency region (corresponding to a higher HF component, either in absolute or normalized units) signals an augmented vagal activity, or modulation. Obviously, the absence of a technique capable of directly assessing in humans vagal efferent drive calls for caution in interpreting experimental findings, although results from animal investigations largely support the sympatho-vagal model. Also in the case of vagal drive, average activity is not equal to oscillatory modulation: accordingly it should be of no surprise if the HF component of R-R variability increases in the absence of an increase in average efferent vagal activity and clear bradycardia, as elegantly shown by acute experiments in dogs (73). The reverse may also occur—an increase in sympathetic efferent modulation in the absence of increased heart rate. Recordings of efferent vagal and sympathetic activity in cats or rats also demonstrated the presence of two oscillatory components in both sympathetic and vagal efferent nerve activity (74), displaying changes in oscillatory power congruent with the model of sympatho-vagal balance, but only weakly related to their respective average drive.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

In humans, mechanical or electrical stimulation of vagal afferents (75), as well as metronome driven (39) or expanded respiration (49), increases the normalized power of HF component of RR variability and simultaneously reduces LF. In addition, the response to excitatory stimuli is limited, as shown by the reduced increase in LF with tilt performed during paced breathing (39) and by the reduced chemoreflex mediated rise of LF during hyperventilation performed in conditions of controlled hypoxia (49). These frequently unrecognized changes in responsiveness of spectral indices of autonomic modulation in conditions of altered respiratory physiology, if not explicitly factored in the studies of cardiovascular variability, might flaw the interpretation of results, as in the study by Pomeranz et al. (76), which only considered paced breathing. Normalized units (or LF/HF ratio) (39) and simultaneous independent assessment of neural oscillatory properties with, e.g., MSNA recordings (77) are of particular importance in addressing the effects of muscarinic blockade in humans (Fig. 10). Small doses of atropine centrally enhance vagal drive and do not affect SA responsiveness: accordingly a clear increase in HF is observable, both in R-R and in MSNA variability. Conversely, large doses of atropine nearly abolish R-R variability. Using sensitive techniques, a marked prevalence of the LF component can be observed in the R-R interval variability spectra, along with a marked reduction of the absolute power of all components; while MSNA variability unveils the maintained presence of both LF and HF oscillations in efferent sympathetic traffic, indicating that pharmacological blockade of muscarinic effects at the SA node dissociates neural from target oscillations. Conditions of quiet, as during sleep (47), are associated to enhanced signs of vagal modulation of the SA node [bradycardia, elevated R-R variance, a shift towards HF in spectral profile, and an increase in baroreflex gain (52)]. Relaxation training seems to more simply reduce signs of sympathetic responsiveness to excitatory stimuli (69). Athletic training leads, instead, to a complex rearrangement of cardiac autonomic drive: during holidays, away from training routines, bradycardia and a prevailing HF must be contrasted, during the active competitive season, with bradycardia and an elevated LF, reflecting the aftereffects of the intense daily training routines (78). A vagal predominance, as suggested by enhanced HF (and reduced LF or LF/HF) may be induced by abnormal stimulation of the gastrointestinal sensory supply and has been observed in conditions of gastric fullness or in patients with a prior duodenal ulcer (79) (although in this latter case a central or peripheral change in the gain of involved circuits could not be excluded).

X. HRV IN PATHOLOGICAL CONDITIONS In view of the growing importance of subjective components in modern medicine, the hypothesis that heart rate variability might provide measures of functional

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 10 Spectral oscillations of R-R interval, muscle sympathetic nerve activity (MSNA), systolic arterial pressure (SAP), and respiration (Resp.) in a single individual during control measurements and after low-dose and high-dose atropine. After high-dose atropine, R-R interval oscillations are affected by effects of peripheral muscarinic blockade. Nevertheless, a relative increase in the high-frequency component of MSNA is evident. Because of blockade of vagal effects on the sinoatrial node by high-dose atropine and a decrease in R-R interval variance, the residual oscillatory component of the R-R interval is limited predominantly to low frequency, as seen in the 15-fold magnified inset. (From Ref. 77.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

equivalents of subjective symptoms, and of the interaction with the environment, might be of practical importance. In this context it is relevant to recall that spectral parameters can be obtained through simple transtelephonic approaches (80) from either the doctor’s office or patients’ homes. Accordingly, it might be envisaged that in several pathological conditions in which autonomic dysregulation might play a part, HRV analysis could become a convenient method to add to standard diagnostic routines. In the following sections we will focus on specific aspects of selected conditions in which an assessment of the autonomic nervous systems plays a role as either a diagnostic or therapeutic component of individual treatment. A. Essential Hypertension Overall arterial hypertension is characterized, particularly in its early phases, by enhanced sympathetic and reduced vagal drives at rest. This corresponds to a shift of spectral profiles (81) to a prevailing LF (Fig. 11) (and higher LF/HF) of R-R variability, reduced baroreflex gain (52), and enhanced LFSAP (82). However, the marked spread of data renders the use of sole rest values questionable, since they are affected by a large individual variance. Hence, as mentioned earlier (12), a dynamic protocol, as obtained with the addition of a stand (or tilt) stimulus, appears more robust to assess the autonomic dysregulation of arterial hypertension. In fact, in essential hypertension the response to the stand (or tilt)-induced increase in markers of sympathetic modulation appears blunted (83), similar to that observed in the 24-hour variability profile extracted from Holter recordings (84). Spectral analysis of R-R and arterial pressure variability may also be useful to detect changes in autonomic regulation of the SA node produced by specific pharmacological or behavioral treatments. For instance, -adrenergic blockers reduce arterial pressure, improve baroreflex gain, and increase the HF component of R-R variability (37); conversely, equipotent hypotensive doses of short-acting dihydropiridines may produce an increase in sympathetic drive and enhancement of LF responses of arterial oscillations (85) (which are an indirect marker of vascular sympathetic modulation). Such an increase in sympathetic vascular responsiveness does not occur with longer-acting dihydropiridines, which might accordingly be better suited in the treatment of patients with coexisting cardiovascular diseases. On the other hand, aerobic training might improve baroreflex gain, with potential beneficial effects on cardiovascular risk profile (52). Over time, the occurrence of target organ changes, such as left ventricular hypertrophy or congestive heart failure, might induce more clear alterations in resting values of spectral indices of SA modulation, as discussed below. The possibility of autonomic alterations in secondary forms of hypertension, as in the case of cyclosporine-associated hypertension, has also been examined with spectral analysis of cardiovascular variability. In a study from our group (86), we observed that autonomic regulation was largely maintained in human

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 11 Effects of chronic -adrenergic blockade (atenolol 100 mg once daily for 2 weeks) or R-R variability in a hypertensive subject. Note the predominance of low frequency (LF) both at rest and during tilt before treatment and its reduction during treatment. (From Ref. 83.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

transplant recipients in whom immunotherapy had induced a secondary rise in arterial pressure. B. Myocardial Ischemia After myocardial infarction, reduced measures of heart rate variability and of baroreflex gain provide strong prognostic indicators of future events (87). Reduced HRV is also an important predictor of cardiac events in the general population (88). For instance, patients with standard deviation of normal R-R intervals (SDNN) less than 50 msec (87) at 2 weeks post–anterior myocardial infarction (post-AMI) have a 5.3-fold increase in mortality rate. Reduced HRV correlates to transient ischemic events (89), left ventricular dilatation (90), atrial fibrillation (91), or tachyarrhythmias (92); accordingly, this parameter might be capable of improving risk stratification in patients with coronary artery disease. Here, we will not address alterations in nonlinear properties of R-R variability (11) that might follow acute myocardial infarction (MI). Acute myocardial ischemia produces sympathetic excitatory reflexes, which are paralleled by an increase in the LF component of R-R and SAP variability, independent of baroreflex engagement (Fig. 12) (61). A marked LF shift in sympathovagal balance during acute transient myocardial infarction in man has been shown by studies in patients undergoing coronary angioplasty (93). Soon after myocardial infarction (94) the spectral profile at rest shifts towards LF prevalence, and reduced responses to standing up are also observed, suggestive of an increased sympathetic and reduced vagal modulation—changes that slowly disappear over about 6 months. After MI there is also a reduced baroreflex gain that may be improved, together with R-R variance, by rehabilitation training (95), beyond what is observed as a consequence of the natural recovery. Large population studies have clearly indicated the importance of altered autonomic regulation as a determinant of prognosis, but the extent to which cardiac autonomic alterations play a causative role or are just markers of hidden disturbances remains unclear. It is conceivable that spectral analysis of HRV might serve as a means to titrate autonomic changes induced by ischemic heart disease and its treatment, particularly in relation to psychosocial factors (96). C. Congestive Heart Failure Among the hemodynamic hallmarks of congestive heart failure there is a rise in filling pressures of the heart (and lungs), representing, from a neural control point of view, an altered input responsible for progressive autonomic dysregulation, characterized by gradually increased sympathetic activity and reduced baroreflex gain. An important role for excessive stimulation of sympathetic afferents in this context has been shown by acute experiments on cats (94), in which a spinal sec-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 12 Effects of a transient, nonhypotensive, coronary artery occlusion (c.a.o. between arrows, B) on R-R and systolic arterial pressure (SAP) variabilities power spectral density (PSD) in a conscious dog. Note that during occlusion there is a reduction in variance from control (A) (see also the tachogram in B), together with appearance of a large LF component in both R-R and SAP power spectrum, as a reflection of increase in sympathetic efferent activity. LVP: Left ventricular pressure; dP/dt: rate of change of left ventricular pressure. (From Ref. 61.)

tion at C7 and muscarinic blockade had limited cardiac control to sympatho-sympathetic spinal circuits. In these animals acute saline infusions producing a marked increase in atrial pressure initiated a reflex sympathetic tachycardia [Bainbridge effect (31)]. In conscious dogs (97), experimental congestive failure, as produced by fast atrial pacing, leads to a shift of the spectral profile towards LF prevalence, largely dependent upon an intact left ventricular sensory supply (98), with an additional baroreflex modulation. In patients, progressive levels of chronic heart failure (CHF) (99), as indicated by New York Heart Association (NYHA) class, are characterized by a gradual reduction in R-R variance and a LF shift in spectral profile. In the extremes of CHF, however, R-R variance becomes drastically reduced, and only a HF component can be observed in both R-R interval and MSNA variability, while average MSNA activity is markedly increased (Fig. 13) (100). It follows that, like with exercise, in conditions of extreme stimulation the sympatho-vagal model should not be applied to simply infer autonomic regulation from HRV (12). Of potential interest is the ancillary observation of a worse prognosis in CHF patients who lack a LF variability component (100). Reversal of the hemodynamic disturbance with Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

either a left ventricular assist device (101) or a cardiac transplantation restores the LF oscillations (102). However, in cardiac transplant recipients average MSNA remains elevated as compared to healthy controls, again pointing to the likely different information (12) carried by average nerve activity and oscillatory modulations. Spectral analysis of HRV has also been employed to assess the beneficial effects of pharmacological or rehabilitation treatments in patients with CHF, con-

Figure 13 From top to bottom the following are represented: Electrocardiogram (ECG), sympathetic efferent activity from the peroneal nerve (neurogram and MSNA), respiration, and autospectra (PSD) of related tachogram (RRI), MSNA, and respiration. The left panels are from a control subject, the right panel from a patient with severe CHF. Notice in this latter case the extremely elevated sympathetic activity (83 beats/min vs. 28 bursts/min) and the lack of a LF component in both RRI and MSNA autospectra. (From Ref. 100.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

sidering, for instance, digitalis (103), angiotensin-converting enzyme (ACE) inhibitors (104,105), or aerobic training (106). Limited information is available on genetics of HRV (107) or the influence of inflammatory cytokines. D. Arrhythmias and Sudden Death Increased sympathetic activity and reduced vagal (108) drive have long been recognized as independent risk factors for arrhythmias and sudden death, both in clinical and experimental conditions. In this context the pro-arrhythmic effect of acute or chronic psychological stress (96), possibly through the attending increase in sympathetic drive (and humoral changes), must also be considered in the clinical management of patients. Accordingly, the possibility to monitor the state of the sympathovagal balance with HRV analysis (47) and the widespread use of Holter monitoring might be of practical value once large-scale trials will have established accepted standards. In general, all types of arrhythmias have been associated with altered neural modulation of the heart, and conversely restoration of a normal neural regulation has been sought as a goal in medical treatment of arrhythmias. However, it is still unclear whether simple restoration of a normal oscillatory pattern might suffice, or if causal therapy should be always preferably instituted. For instance, scopolamine (109,110) administration might increase the HF and reduce the LF power in patients, thus restoring a balanced spectral profile, but this might be per se insufficient to regain a normal risk level. In fact, arrhythmic risk (111) is determined by a series of factors, among which left ventricular function (as assessed, e.g., by LV ejection fraction) or baroreflex gain (usually assessed by phenylephrine slopes) may influence spectral profile of HRV.

XI. CARDIOVASCULAR OSCILLATIONS TO TAILOR INDIVIDUAL TREATMENT Cardiovascular risk profile depends on an array of interacting factors, many of which can be linked to altered autonomic regulation, such as sympathetic overactivity and vagal inhibition. We will here address the hypothesis that individual assessment of HRV might be useful to promote and monitor lifestyle changes as a means to facilitate focused integrated preventive strategies (Table 2). Awareness and personal commitment to change on the part of the patient are the key components of successful therapies, combining drugs with, at times aggressive, physiological and psychological interventions (112). In our outpatient clinic the study of cardiovascular variability is integrated with usual clinical routines and may be of value in assessing the results of treatment so that patients may verify the efficacy of therapies on individual surrogate endpoints. Accordingly, smoking, sedentary lifestyle, obesity, and excessive stress are all

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Table 2 Schematic Summary of Modifiable Cardiovascular Risk Factors and Possibly Related Autonomic Abnormalities Risk factor

Autonomic profile

Smoking

Sympathetic activation

Hypertension

Sympathetic activation

Diabetes

Autonomic neuropathy

Obesity

Sympathetic activation

Sedentary lifestyle Stressa

⬃normal Sympathetic activation

Hypercholesterolemia

Endothelial dysfunction

Spectral profile at rest Reduced R-R 2; LFRR predominance; reduced baroreflex gain ⬃R-R 2; LFRR predominance; reduced baroreflex gain Reduced R-R 2; initially maintained spectral distribution; reduced baroreflex gain Progressive LFRR predominance ⬃normal ⬃R-R 2; LFRR predominance; reduced baroreflex gain —

Spectral changes with stand Reduced increase in LFRR

Reduced increase in LFRR

Reduced increase in LFRR

Progressively blunted increase in LFRR ⬃normal Reduced increase in LFRR

—

a

Some patients with depression or hostility might be included in this category.

treated as components of a single complex maladaptive lifestyle. Patients are invited to focus on progressive, stepwise changes, according to their personal preferences (113) and beliefs, but also targeting a more balanced autonomic profile. We recognize that there is still a long way to go before this hypothesis becomes fully documented. Our preliminary results with prevention on selected aspects, such as smoking cessation (114) (Fig. 14) or cardiovascular rehabilitation (115), justify an optimistic outlook in including spectral analysis of cardiovascular variables for the individual assessment of autonomic regulation in patients. The recent possibility to perform HRV analysis with a transtelephonic technique (80), in addition, is widening the horizon of this approach to unprecedented possibil-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 14 Example of the spectral analysis of R-R interval variability (top) and of respiration (bottom), in the same subject, during the smoking period (left), the nicotine patch treatment (middle), and the placebo patch treatment (right). (From Ref. 114.)

ities that are undergoing active testing. Among the areas of application, this approach is also being tested as an integral component of a strategy aiming at the maintenance of optimal stress responses in extreme conditions, such as with elite athletes (116) and astronauts (72). Time and new data from large prospective studies will show if tailored preventive strategies can profit from the addition of autonomic testing with HRV analysis, aiming to address both organic and integrated aspects in individual patients.

ACKNOWLEDGMENTS We gratefully acknowledge the secretarial help of Giovanna Macciò. Part of this work was supported by ASI, ESA, IRCEA, and COFIN 2000–2001.

REFERENCES 1. Clancy CM, Eisenberg JM. Outcomes research: measuring the end results of health care. Science 1998; 282:245–246. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

2. 3. 4. 5.

6. 7. 8. 9.

10. 11. 12. 13.

14. 15. 16.

17. 18.

19. 20. 21.

Longhurst JC. Cardiac receptors: their function in health and disease. Prog Cardiovasc Dis 1984; 27:201–222. Thames MD. Contribution of cardiopulmonary baroreceptors to the control of the kidney. Fed Proc 2001; 37:1209–1213. Thoren PN, Donald DE, Sheperd JT. Role of the heart and lung receptors with nonmedullated vagal afferents in circulatory control. Circ Res 1976; 38:2–9. Secher NH, Jacobsen J, Friedman DB, Maurer G. Bradycardia during reversible hypovolaemic shock: associated neural reflex mechanisms and clinical implications. Clin Exp Pharmacol Physiol 1992; 19:733–743. Rutecki P. Anatomical, physiological, and theoretical basis for the antiepileptic effect of vagus nerve stimulation. Epilepsia 2001; 31:S1–S6. James TN, Rossi L, Hageman GR. On the pathogenesis of angina pectoris and its silence. Trans Am Clin Climatol Assoc 1988; 100:81–99. Imaizumi T, Takeshita A. Influence of ANP on sympathetic nerve activity and chronotropic regulation of the heart. J Cardiovasc Electrophysiol 1993; 4:719–729. Wooley G, Staszewska-Wolley J. A role for cyclic GMP in the initiation of cardiac pressor reflexes by bradykinin and capsaicin. Pol J Pharmacol Pharm 1990; 42:249–257. Ludbrook J. Cardiovascular reflexes from cardiac sensory receptors. Austr NZ J 1990; 20:597–606. Malliani A. Principles of Cardiovascular Neural Regulation in Health and Disease. Dordrecht: Kluwer Academic Publishers, 2000. Pagani M, Malliani A. Interpreting spontaneous oscillations of muscle sympathetic nerve activity and of heart rate variability. J Hypertens 2000; 18:1709–1719. Malliani A, Pagani M, Lombardi F. Positive feedback reflexes. In: Zanchetti A, Tarazi RC, eds. Handbook of Hypertension. Amsterdam: Elsevier Science Publishing Co., 1986:69–81. Malliani A, Pagani M, Lombardi F, Cerutti S. Cardiovascular neural regulation explored in the frequency domain. Circulation 1991; 84:482–492. Gerstner W, Kreiter AK, Markram H, Herz AVM. Neural codes: firing rates and beyond. Proc Natl Acad Sci USA 1997; 94:12740–12741. Haken H. Synergetics—An Introduction: Nonequilibrium Phase Transitions and Self-Organization in Physics, Chemistry and Biology. 3rd ed. New York: SpringerVerlag, 1983. Lucini D, Trabucchi V., Malliani A, Pagani M. Analysis of initial autonomic adjustments to moderate exercise in humans. J Hypertension 1995; 13:1660–1663. Uechi M, Asai K, Osaka M, Smith A, Sato N, Wagner TE, Ishikawa Y, Hayakawa H, Vatner DE, Shannon RP, Homcy CI, Vatner SF. Depressed heart rate variability and arterial baroreflex in conscious transgenic mice with overexpression of cardiac Gsalpha. Circ Res 1998; 82:416–423. Kowey PR, Marinchak RA, Rials SJ. Things that go bang in the night. N Engl J Med 1992; 327:1884. Malliani A. Cardiovascular sympathetic afferent fibers. Rev Physiol Biochem Pharmachol 1982; 94:11–74. Malliani A, Pagani M, Lombardi F. Visceral versus somatic mechanisms. In: Wall PD and Melzack, eds. Textbook of Pain. Edinburgh: Churchill Livingstone Edinburgh, 1984:100–109.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

22. 23.

24. 25.

26. 27. 28.

29. 30. 31. 32.

33.

34. 35.

36.

37.

38. 39.

Huang HS, Stahl GL, Longhurst JC. Cardiac-cardiovascular reflexes induced by hydrogen peroxide in cats. Am J Physiol 1995; 268:H2114–H2124. Gnecchi-Ruscone T, Montano N, Contini M, Guazzi M, Lombardi F, Malliani A. Adenosine excites cardiac sympathetic afferent fibers and potentiates their response to coronary artery occlusion. J Autonom Nerv Syst 1995; 53:175–184. Ustinova EE, Schultz HD. Activation of cardiac vagal afferents by oxygen-derived free radicals in rats. Circ Res 1994; 74:895–903. Pagani M, Pizzinelli P, Furlan R, Guzzetti S, Rimoldi O, Sandrone G, Malliani A. Analysis of the pressor sympathetic reflex produced by intracoronary injections of bradykinin in conscious dogs. Circ Res 1985; 56:175–183. Critchley HD, Mathias CJ, Dolan RJ. Neuroanatomical basis for first-and secondorder representations of bodily states. Nature Neuroscience 2001; 4:207–212. Rosen S, Paulesu E, Frith C., Frackowiak R.S.J., Davies GJ, Jones T, Camici PG. Central nervous pathways mediating angina pectoris. Lancet 1994; 344:147–150. Heusch G, Baumgart D, Camici PG, Chilian WM, Gregorini L, Hess O, Indolfi C, Rimoldi O. Alpha-adrenergic coronary vasoconstriction and myocardial ischemia in humans. Circulation 2000; 101:689–694. Vatner DE, Braunwald E. Cardiovascular control mechanisms in the conscious state. N Engl J Med 1975; 293:970–976. Zucker IH. Left ventricular receptors:physiological controllers or pathological curiosities? Basic Res Cardiol 1986; 81:539–557. Boettcher DH, Zimpfer M, Vatner DE. Phylogenesis of the Bainbridge reflex. Am J Physiol 2001; 242:R244–R246. Lombardi F, Casalone C, Della Bella P, Malfatto G, Pagani M, Malliani A. Global versus regional myocardial ischaemia: differences in cardiovascular and sympathetic responses in cats. Cardiovas Res 1984; 18:14–23. Bishop VS, Lombardi F, Malliani A, Pagani M, Recordati G. Reflex sympathetic tachycardia during intravenous infusions in chronic spinal cats. Am J Physiol 1976; 230:25–29. Barron HV, Lesh MD. Autonomic nervous system and sudden cardiac death. J Am Coll Cardiol 1996; 27:1053–1060. Pagani M, Pizzinelli P, Bergamaschi M, Malliani A. A positive feedback sympathetic pressor reflex during stretch of the thoracic aorta in conscious dogs. Circ Res 1982; 50:125–132. Gnecchi Ruscone T, Lombardi F, Malfatto G, Malliani G. Attenuation of baroreceptive mechanisms by cardiovascular sympathetic afferent fibers. Am J Physiol 1987; 253:H787–H791. Lucini D, Pagani M, Mela GS, Malliani A. Sympathetic restraint of baroreflex control of heart period in normotensive and hypertensive subjects. Sci 1994; 86:547–556. Grassi G, Esler M. How to assess sympathetic activity in humans. J Hypertension 1999; 17:719–734. Pagani M, Lombardi F, Guzzetti S, Rimoldi O, Furlan R, Pizzinelli P, Sandrone G, Malfatto G, Dell’Orto S, Piccaluga E, Turiel M, Baselli G, Cerutti S, Malliani A. Power spectral analysis of heart rate and arterial pressure variabilities as a marker of sympathovagal interaction in man and conscious dog. Circ Res 1986; 58:178–193.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

40.

41.

42.

43.

44.

45. 46.

47.

48.

49.

50. 51.

52.

53.

54.

Akselrod S, Gordon D, Ubel FA, Shannon DC, Cohen RJ. Power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control. Science 1981; 213:220–222. Brown TE, Beightol LA, Koh J, Eckberg DL. Important influence of respiration on human R-R interval power spectra is largely ignored. J Appl Physiol 1993; 75:2310–2317. Bernardi L, Valle F, Coco M, Calciati A, Sleight P. Physical activity influences heart rate variability and very-low-frequency components in Holter electrocardiograms. Cardiovasc Res 1996; 32(2):234–237. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Heart rate variability. Standards of measurement, physiological interpretation, and clinical use. Circulation 1996; 93:1043–1065. Lucini D, Bertoni L, Pitto G, Frassetto G, Pagani M, Malliani A. Reduced response with ageing to sympathoexcitatory and sympathoinhibitory stimuli in humans. J Hypertension 1993; 11:S170–S171. Pagani M, Lucini D, De Bernardi F, Pozzi A, Pizzinelli P, Signorini MG, Cerutti S, Malliani A. Mental stress. J Ambul Monitor 1992; 5:235–244. Pagani M, Lucini D, Rimoldi O, Furlan R, Piazza S, Biancardi L. Effects of physical and mental exercise on heart rate variability. In: Malik M, Camm AJ, Heart Rate Variability. New York: Futura Publishing Company Inc., 1995:245–266. Furlan R, Guzzetti S, Crivellaro W, Dassi S, Tinelli M, Baselli G, Cerutti S, Lombardi F, Pagani M, Malliani A. Continuous 24-hour assessment of the neural regulation of systemic arterial pressure and RR variabilities in ambulant subjects. Circulation 1990; 81:537–547. Malliani A, Pagani M, Lombardi F. Importance of appropriate spectral methodology to assess heart rate variability in the frequency domain. Hypertension 1994; 24:140–142. van de Borne P, Montano N, Narkiewicz K, Degaute JP, Malliani A, Pagani M, Somers VK. Importance of ventilation in modulating interaction between sympathetic drive and cardiovascular variability. Am J Physiol (Heart Circ Physiol) 2001; 280:H722–H729. Pagani M, Rimoldi O, Malliani A. Low-frequency components of cardiovascular variabilities as markers of sympathetic modulation. TiPS 1992; 13:50–54. Pagani M, Montano N, Porta A, Malliani A, Abboud FM, Birkett CL, Somers VK. Relationship between spectral components of cardiovascular variabilities and direct measures of muscle sympathetic nerve activity in humans. Circulation 1997; 95:1441–1448. Pagani M, Somers VK, Furlan R, Dell’Orto S, Conway J, Baselli G, Cerutti S, Sleight P, Malliani A. Changes in autonomic regulation induced by physical training in mild hypertension. Hypertension 1988; 12:600–610. Legramante JM, Raimondi G, Massaro M, Peruzzi G, Iellamo F. Investigating feed forward neural regulation of circulation from analysis of spontaneous arterial pressure and heart rate fluctuations. Circulation 1999; 99:1760–1766. Baselli G, Cerutti C, Civardi S, Malliani A, Pagani M. Cardiovascular variability signals: towards the identification of a closed-loop model of the neural control mechanisms. IEEE 1988; 35:1033–1046.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

55.

56.

57.

58.

59. 60. 61.

62.

63.

64.

65.

66.

67. 68.

69.

Lucini D, Porta A, Milani O, Baselli G, Pagani M. Assessment of arterial and cardiopulmonary baroreflex gains from simultaneous recordings of spontaneous cardiovascular and respiratory variability. J Hypertension 2000; 18:1–6. Pagani M, Mazzuero G, Ferrari A, Liberati D, Cerutti S, Vaitl D, Tavazzi L, Malliani A. Sympathovagal interaction during mental stress. A study using spectral analysis of heart rate variability in healthy control subjects and patients with prior myocardial infarction. Circulation 1991; 83:II-43–II-51. Furlan R, Piazza S, Dell’Orto S, Barbic F, Bianchi A, Mainardi LT, Cerutti S, Pagani M, Malliani A. Cardiac autonomic patterns preceding occasional vasovagal reactions in healthy humans. Circulation 1998; 98:1756–1761. Jasson S, Medigue C, Maison-Blanche P, Montano N, Meyer L, Verneiren C, Mansier P, Coumel P, Malliani A. Instant power spectrum analysis of heart rate variability during orthostatic tilt using a time-/frequency-domain method. Circulation 1997; 96:3521–3526. Eckberg DL. Sympathovagal balance. A critical appraisal. Circulation 1997; 96:3224–3232. Malliani A, Pagani M, Montano N, Mela GS. Sympathovagal balance: a reappraisal. Circulation 1998; 98:2640–2643. Rimoldi O, Pierini S, Ferrari A, Cerutti S, Pagani M, Malliani A. Analysis of shortterm oscillations of RR and arterial pressure in conscious dogs. Am J Physiol 1990; 258:H-967–H-976. Bernardi L, Salvucci F, Suardi R, Soldà PL, Calciati A, Perlini S, Falcone C, Ricciardi L. Evidence for an intrinsic mechanism regulating heart rate variability in the transplanted and the intact heart during submaximal dynamic exercise? Cardiovasc Res 1990; 24:969–981. Bernardi L, Wdowczyck-Szulc J, Valenti C, Castoldi S, Passino C, Spadacini G, Sleight P. Effects of controlled breathing, mental activity and mental stress with or without verbalization on heart rate variability. JACC 2000; 35:1462–1469. Lucini D, Pagani M, Norbiato G, Clerici M. Altered cardiovascular regulation in healthy subjects exposed to mild chronic stress. Psychom Med 2001; 63:177–178. Pagani M, Rimoldi O, Pizzinelli P, Furlan R, Crivellaro W, Liberati D, Cerutti S, Malliani A. Assessment of the neural control of the circulation during psychological stress. J Autonom Nerv Syst 1991; 35:33–42. DeSilva RA, Verrier RL, Lown B. The effects of psychological stress and vagal stimulation with morphine on vulnerability to ventricular fibrillation (VF) in conscious dogs. Am Heart J 2001; 95:197–203. Verrier RL, Hagestad EL, Lown B. Delayed myocardial ischemia induced by anger. Circulation 1987; 75:249–254. Baccelli G, Albertini R, Del Bo A, Mancia G, Zanchetti A. Role of sinoaortic reflexes in hemodynamic patterns of natural defense behaviors in the cat. Am J Physiol 1981; 240:H421–H429. Lucini D, Covacci G, Milani R, Mela GS, Malliani A, Pagani M. A Controlled study of the effects of mental relaxation on autonomic excitatory responses in healthy subjects. Psychosom Med 1997; 59:541–552.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

70.

71. 72.

73.

74. 75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

Signorini MG, Saliani V, Lucini D, Pagani M, Cerutti S. Parameters from deterministic chaos in the processing of respiration signals during mental stress. J Ambul Monit 1992; 5:139–152. Blomqvist GC, Raven PB, White R. International. Med Sci Sports Exercise 1996; 28(suppl):S1–S112. Pagani M, Iellamo F, Lucini D, Cerchiello M, Castrucci F, Pizzinelli P, Porta A, Malliani A. Selective impairment of exitatory pressor responses after prolonged simulated microgravity in humans. In: Autonom Neurosci Basic Clin 2001; 91:85–95. Hedman AE, Tahvanainen KUO, Hartikainen JEK, Hakumäki MOK. Effect of sympathetic modulation and sympatho-vagal interaction on heart rate variability in anaesthetized dogs. Acta Physiol Scand 1995; 155:205–214. Montano N. Evidence for central organization of cardiovascular rhythms. Physiologist 2000; 43:279. Kamath MV, Hollerbach S, Bajwa A, Fallen EL, Upton ARM, Tougas G. Neurocardiac and cerebral responses evoked by esophageal vago-afferent stimulation in humans: effect of varying intensities. Cardiovasc Res 1998; 40:591–599. Pomeranz B, Macaulay JB, Caudill MA, Kutz I, Adam D, Gordon D, Kilborn KM, Barger AC, Shannon DC, Choen RJ, Benson H. Assessment of automonic function in humans by heart rate spectral analysis. Am J Physiol 1985; 248:H151–H153. Montano N, Cogliati C, Porta A, Pagani M, Malliani A, Narkyewicz C, Abboud FM, Birkett CL, Somers VK. Central parasympathetic effects of atropine modulate spectral oscillations of sympathetic nerve activity. Circulation 1998; 98:1394–1399. Furlan R, Piazza S, Dell’Orto S, Gentile E, Cerutti S, Pagani M, Malliani A. Early and late effects of exercise and athletic training on neural mechanisms controlling heart rate. Cardiovasc Res 1993; 27:482–488. Lucini D, Cerchiello M, Basilisco G, Cainelli M, Bianchi P.A., Fiorelli G, Malliani A, Pagani M. Autonomic control of heart period in duodenal ulcer patients. Insight from spectral analysis of heart rate variability. Autonom Neurosci Basic Clin 2000; 84:122–129. Lucini D, Cerchiello M, Porta A, Pagani M. Assessing autonomic disturbances of hypertensive patients in the office of general practitioners: a transtelephonic approach to spectral analysis of HRV. XI Europ Congr Hypertension, Milan, 2001. Malliani A, Pagani M, Lombardi F, Furlan R, Guzzetti S, Cerutti S. Spectral analysis to assess increased sympathetic tone in arterial hypertension. Hypertension 1991; 17:III-36–III-42. M. Pagani and D. Lucini. Autonomic dysregulation in essential hypertension: Insights from heart rate and arterial pressure variability. Autonom Neurosci Basic Clin 2001; 90:76–82. Guzzetti S, Piccaluga E, Casati R, Cerutti S, Lombardi F, Pagani M, Malliani A. Sympathetic predominance in essential hypertension: a study employing spectral analysis of heart rate variability. J Hypertens 1988; 6:711–717. Guzzetti S, Dassi S, Pecis M, Casati R, Masu AM, Longoni P, Tinelli M, Cerutti S, Pagani M, Malliani A. Altered pattern of circadian neural control of heart period in mild hypertension. J Hypertens 1991; 9:831–838.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

Lucini D, Mela GS, Malliani A, Pagani M. Evidence of increased sympathetic vasomotor drive with shorter acting dihydropyridine calcium channel antagonist in human hypertension: a study using spectral analysis ot RR interval and systolic arterial pressure variability. J Cardiovasc Pharmacol 1997; 29:676–683. Lucini D, Milani R, Ventura HO, Mehra MR, Messerli FH, Pagani M. Study of the arterial and autonomic effects of cyclosporine in humans. Hypertension 2000; 35:1258–1263. Kleiger RE, Miller JP, Bigger JT Jr, Moss AJ. Decreased heart rate variability and its association with increased mortality after acute myocardial infarction. Am J Cardiol 1987; 59:256–262. Tsuji H, Venditti FJ, Manders ES, Evans JC, Larson MG, Feldman CL, Levy D. Reduced heart rate variability and mortality risk in an elderly cohort. The Framingham Study. Circulation 1994; 90:878–883. Lanza GA, Pedrotti L, Rebuzzi AG, Pasceri V, Quaranta G, Maseri A. Usefulness of the addition of heart rate variability to Holter monitoring in predicting in-hospital cardiac events in patients with unstable angina pectoris. Am J Cardiol 1997; 80:263–267. Dambrink JH, Tuininga YS, van Gilst WH, Peels KH, Lie KI, Kingma JH. Association between reduced heart rate variability and left ventricular dilatation in patients with a first anterior myocardial infarction. CATS Investigators. Captopril and Thrombolysis Study. Br Heart J 1994; 72:514–520. van den Berg E, Haaksma J, Brouwer J, Tieleman RG, Mulder G, Crijns HJ. Heart rate variability in patients with atrial fibrillation is related to vagal tone. Circulation 1997; 96:1209–1216. Valkama JO, Huikuri HV, Koistinen MJ, Yli-Mayry S, Airaksinen KEJ, Myerburg RJ. Relation between heart rate variability and spontaneous and induced ventricular arrhythmias in patients with coronary artery disease. J Am Coll Cardiol 1995; 25:437–443. Airaksinen KEJ, Ikäheimo MJ, Linnaluoto MK, Tahvanainen KUO, Huikuri HV. Gender difference in autonomic and hemodynamic reactions to abrupt coronary occlusion—analysis of risk factors from 8207 procedures. J Am Coll Cardiol 1998; 31:301–306. Lombardi F, Sandrone G, Pernpruner S, Sala R, Garimoldi M, Cerutti S, Baselli G, Pagani M, Malliani A. Heart rate variability as an index of sympathovagal interaction after acute myocardial infarction. Am J Cardiol 1987; 60:1239–1245. Iellamo F, Legramante JM, Massaro M, Raimondi G, Galante A. Effects of a residential exercise training on baroreflex sensitivity and heart rate variability in patients with coronary artery disease. A randomized controlled study. Circulation 2000; 102:2588–2592. Rozanski A, Blumenthal JA. Impact of psychological factors on the pathogenesis of cardiovascular disease and implications for therapy. Circulation 1999; 99:2192–2217. Binkley PF, Nunziata E, Haas GJ, Nelson SD, Cody RJ. Parasympathetic withdrawal is an integral component of autonomic imbalance in congestive heart failure: demonstration in human subjects and verification in a paced canine model of ventricular failure. J Am Coll Cardiol 1991; 18:464–72.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107. 108. 109.

110.

111.

112.

Rimoldi O, Sato N, Uechi M, Shen YT, Pagani M. Role of cardiac and baroreceptor innervation on changing sympatho-vagal balance at baseline and during the development of cardiac heart failure. Circulation 1996; 94:C1911. Guzzetti S, Cogliati C, Turiel M, Crema C, Lombardi F, Malliani A. Sympathetic predominance followed by functional denervation in the progression of chronic heart failure. Eur Heart J 1995; 16:1100–1107. van de Borne P, Montano N, Pagani M, Oren RM, Somers VK. Absence of low-frequency variability of sympathetic nerve activity in severe heart failure. Circulation 1997; 95:1449–1454. Cooley RL, Montano N, Cogliati C, van de Borne P, Richenbacher W, Oren RM, Somers VK. Evidence for a central origin of the low-frequency oscillation in RR-interval variability. Circulation 1998; 98:556–561. van de Borne P, Montano N, Narkiewicz K, Degaute JP, Oren RM, Pagani M, Somers VK. Sympathetic rhythmicity in cardiac transplant recipients. Circulation 1999; 99:1606–1610. Krum H, Bigger JT Jr, Goldsmith RL, Packer M. Effect of long-term digoxin therapy on autonomic function in patients with chronic heart failure (see comments). J Am Coll Cardiol 1995; 25:289–294. Kamen PW, Krum H, Tonkin AM. Low-dose but not high-dose captopril increases parasympathetic activity in patients with heart failure. J Cardiovasc Pharmacol 1997; 30:7–11. Guedon-Moreau L, Pinaud A, Logier R, Caron J, Lekieffre J, Dupuis B, Libersa CH. Effect of ramipril of heart rate variability in digitalis-treated patients with chronic heart failure. Cardiovasc Drugs Therapy 1997; 11:531–536. Lin JL, Chan HL, Du CC, Lai CW, Lin KT, Wu CP, Tseng YZ, Lien WP. Long-term beta-blocker therapy improves autonomic nervous regulation in advanced congestive heart failure: a longitudinal heart rate variability study. Am Heart J 1999; 137:658–665. Singh JP, Larson MG, O’Donnell CJ, Tsuji H, Levy D. Heritability of heart rate variability: the Framingham Heart Study. Circulation 1999; 99:2251–2254. Lown B. Sudden cardiac death: The major challange confronting contemporary cardiology. Am J Cardiol 1979; 43:313–328. Venkatesh G, Fallen EL, Kamath MV, Connolly S, Yusuf S. Double blind placebo controlled trial of short term transdermal scopolamine on heart rate variability in patients with chronic heart failure. Heart 1996; 76:137–143. Hull SS, Vanoli E, Adamson PB, De Ferrari GM, Foreman RD, Schwartz PJ. Do increases in markers of vagal activity imply protection from sudden death? The case of scopolamine. Circulation 1995; 91:2516–2519. La Rovere MT, Bigger JT, Marcus FI, Mortara A, Schwartz PJ. Baroreflex sensitivity and heart-rate variability in prediction of total cardiac mortality after myocardial infarction: ATRAMI (Autonomic Tone and Reflexes After Myocardial Infarction). Lancet 1998; 351(9101):478–484. Ornish D, Brown SE, Scherwitz LW, Billings JH, Armstrong WT, Ports TA, McLanahan SM, Kirkeeide RL, Brand RJ, Gould KL. Can lifestyle changes reverse coronary heart disease? Lancet 1990; 336:129–133.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

113.

Kassirer JP. Incorporating patients’ preferences into medical decisions. N Engl J Med 1994; 330:1895–1896. 114. Lucini D, Bertocchi F, Malliani A, Pagani M. Autonomic effects of nicotine patch administration in habitual cigarette smokers: a double-blind, placebo-controlled study using spectral analysis of RR interval and systolic arterial pressure variabilities. J Cardiovasc Pharmacol 1997; 31:714–720. 115. Lucini D, Milani RV, Costantino G, Levie CJ, Porta A, Pagani M. Effects of cardiac rehabilitation and exercise training on autonomic regulation in patients with coronary artery disease. Am Heart J 2002, in press. 116. Iellamo F, Legramante I, Pigozzi F, Spataro A, Norbiato G, Lucini D, Pagani M. Conversion from vagal to sympathetic predominance with strenuous training in high performance world class athletes. Circulation 2002; 105:2719–2724.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

11 Cardiovascular Autonomic Dysregulation Vilho V. Myllylä, Juha T. Korpelainen, Tarja H. Haapaniemi, Uolevi Tolonen, Timo H. Mäkikallio, Kyösti A. Sotaniemi, and Heikki V. Huikuri University of Oulu, Oulu, Finland

Mikko P. Tulppo Merikoski Rehabilitation and Research Centre, Oulu, Finland

The autonomic nervous system (ANS) is known to have an important role in the maintenance of homeostasis in the body by adjusting the functions of various organs to changing circumstances of endogenous and exogenous origin. Therefore, the reactions of ANS are coupled to almost all physiological and pathological conditions. Additionally, there are many diseases that may primarily or secondarily alter or modify ANS functions. Dysregulation of ANS is seen as secondary to diseases of the peripheral nervous system (PNS) and to those of the central nervous system (CNS) as well as a primary, selective disease affecting only ANS. In patients with diabetes the manifestation of autonomic neuropathy was first shown to worsen the prognosis and even the life expectancy of the patients. Excessive mortality was explained by increased frequency of cardiac deaths. In coronary heart disease diminished heart rate variability, especially a loss of circadian oscillation, has been associated with an increased risk of cardiac arrhythmia and sudden death. Similar findings have been reported in stroke patients. Cardiovascular dysregulation can lead to serious complications such as chronic degenerative diseases of the CNS. Increasing knowledge about autonomic dysfunction in patients with multiple system atrophy (MSA) has yielded new tools for the diagnosis of the disorder, especially for differentiating between MSA and Parkinson’s disease. The same studies have provided us with new data on the role of ANS in the integration between cortical and visceral inputs essential for establishing and maintaining homeostasis in the body. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The wide individual and interindividual variations of the autonomic responses make it necessary to develop objective methods for verification of the dysfunction. In this area great advances have been made recently. The physiology and pathology of heart rate variation is best known. At the moment the role of nonlinear methods in assessing the cardiovascular dysregulation seems to be emphasized.

I. CLINICAL SIGNIFICANCE OF STUDYING CARDIOVASCULAR REGULATION The most important physiological role of the ANS is to rapidly adjust the functions of various organs to the changing circumstances of endogenous or environmental origin, i.e., it is essential for the maintenance of homeostasis of the body. Therefore, reactions of ANS are linked to almost all physiological and pathological conditions. Furthermore, there are many diseases that may primarily or secondarily affect ANS and a great number of drugs that are known to modify ANS functions. However, our knowledge of the significance of ANS disturbances is still inadequate and contradictory, which may be related to the limited number and reliability of the methods used for the detection and assessment of ANS disorders. Dysregulation of ANS is seen as secondary to diseases of the PNS and CNS as well as a primary, selective disease involving only ANS, often called progressive autonomic failure (PAF) (1). We also know that autonomic failure may be encountered after toxic exposures, especially after excessive use of alcohol. According to Bannister (1) PAF can be seen as an independent disease or in combination with degenerative CNS diseases, such as Parkinson’s disease (PD) and MSA. During recent years our understanding of ANS dysfunction in cardiovascular disease has greatly increased. It is well known that the manifestation of autonomic neuropathy worsens the prognosis and even the life expectancy of diabetic patients (2). Excessive mortality has been explained by increased frequency of cardiac sudden death linked with autonomic neuropathy (3,4). There are some reported cases with total denervation of the heart in diabetics (5), which may lead to circumstances where the heart does not react at all to external stimuli. Silent myocardial infarcts seem to be more prevalent among diabetics than in the normal population (6). In coronary heart disease diminished heart rate (HR) variability, especially a loss of circadian oscillation, has been found to be associated with an increased risk of cardiac arrhythmia and sudden death (7–9). It is probable that suppressed vagal activity during the night is the underlying cause of unfavorable consequences due to unopposed sympathetic activity and imbalance between the sympathetic and parasympathetic regulatory systems (7–9). On the other hand, it is interesting that acute cerebrovascular diseases may cause prognostically

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

unfavorable suppression of HR variability similar to that observed in coronary heart disease (10–15). Autonomic disturbances can also lead to serious complications of CNS diseases, e.g., syringomyelia. A majority of PD patients do suffer from vegetative symptoms (16,17), and the disturbances can be objectively verified by commonly used bedside methods. However, the exact clinical significance of these disturbances still remains to be shown. Because the role of ANS in the regulation of biological functions seems to be essential for maintenance of life, it is important that scientific research continue providing us new data on the role of ANS in integration between cortical and visceral inputs essential for establishing and maintaining homeostasis in the body. The wide individual and interindividual variations of autonomic responses make it important that objective recording methods be further developed. Until now, the physiology and pathology of HR variability is best known. It seems probable that the role of nonlinear methods applying long-term registrations will be further emphasized in the near future. The function of the heart can be approached as a target of autonomic innervation, which as we know can be disturbed or modified by many neurological and other diseases. Analogously, the function of ANS can be seen to be dependent on the condition and diseases of other organs, especially of the heart. We feel that the interactions between ANS and other systems deserve further research, including interactions with the endocrinological and immunological systems.

II. METHODS FOR ASSESSING CARDIOVASCULAR AUTONOMIC DYSFUNCTION Measurement of standard cardiovascular reflexes has become the golden standard in the clinical testing of autonomic functions (18). This involves continuous HR, blood pressure (BP), and breathing monitoring under standardized conditions, e.g., rest, deep breathing, upright positioning, and isometric work, to define circulatory responses. Analysis of tonic HR variability from ambulatory ECG recordings has now become an important method for assessment of autonomic regulation (19). Using microneurographic recording technique, muscle and skin sympathetic activity can be directly measured in humans (20). This method provides direct information about sympathetic impulse traffic to skin and muscle and is well suited for the study of sympathetic physiology and pathophysiology but is not applicable for routine diagnostic work. The respiratory related variability of HR, known as respiratory sinus arrhythmia, is generated by autonomic brain stem reflexes and is primarily mediated by vagal innervation of the heart (21), but it is also influenced by the changes in BP (22). More subtle changes in beat-to-beat fluctuation and BP due to sympa-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

thetic excitation, sympathovagal balance, and arterial BP oscillations also occur, and they can be obtained as very-low-frequency (VLF) and low-frequency (LF) components in the spectral analysis of HR and BP variability (23). A. Cardiovascular Autonomic Reflexes Physiological variability of HR and BP is considerable: respiratory sinus arrhythmia is higher at rest than during activity, but HR and BP are low at rest and high during activity. Moreover, HR and BP variability is also different in different sleep stages, respiratory variability being high during sleep stages 3 and 4 and low in rapid eye movement (REM) sleep, but slow variability being high during REM sleep and low during sleep stages 3 and 4 (24). Increasing sympathetic drive, especially in children, may lower respiratory HR variability in deep breathing tests. Therefore, a careful standardization of cardiovascular test performance is important. The test should be performed under standardized environmental conditions: ideally in a warm and silent room in a comfortable bed. The registration time in the morning or in the afternoon does not affect the results (25). The test should be performed after a normal night’s sleep, without drinking coffee or ingesting food for several hours (3 hours in our laboratory) before the test. Alcohol is not permitted for 12–14 hours before testing. Drugs affecting ANS are discontinued before testing: anticholinergics, 9--fludrocortisone, diuretics, and sympathomimetic and parasympathomimetic agents, as well as - and -antagonists. Nowadays the signals are recorded by computer. In addition to ECG, the breathing signal using nasal thermistore and continuous blood pressure monitoring with a plethysmographic instrument such as a Finapress (Ohmeda, Englewood, CO) should be used. During both the recording and the off-line analysis, the raw signals could be visualized to ensure the optimal test performances (26). Before the beginning of the tests, a patient’s maximum contracting power (handgrip) of the dominant or healthy hand is performed with a dynamometer for the isometric work test. Furthermore, the maximum blowing power is tested by the Valsalva maneuver. At the end of 30 minutes, resting BP is measured. Thereafter, the subtests are performed in the following order. The interval between the subtests must be standardized: in our laboratory the next test is not begun until HR and BP have returned to the resting level. In the normal breathing test the consecutive R-R intervals for 5–10 minutes are recorded. Both time domain and frequency domain parameters for HRV are calculated (27). SD is the most used time domain indicator of HR variability. Frequency domain analysis can be performed either using fast Fourier transform or autoregressive model techniques. The VLF peak of 0.04 Hz, the LF peak of 0.04–0.15 Hz, as well as the high-frequency (HF) peak of 0.15–0.4 Hz corresponding to respiratory frequency can be found. Manual selection of the frequency bands, especially if the spectrum of respiration is used to define the HF band, gives the most relevant data. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

In the deep breathing test a paced respiration of 6 breaths per minute, deeply but not maximally (low CO2 reduces sinus arrhythmia), is performed to obtain the maximal respiration arrhythmia (Fig. 1). The ratio of the median (or highest or mean) of the longest (expiration) to the shortest (inspiration) R-R interval is calculated as the test measure. Continuous BP monitoring is suggested because in deep breathing the arterial BP changes has a significant influence on the magnitude of HR variation (22). In the Valsalva maneuver, the patient maintains at 40 mmHg or at least 50% of the individual maximum expiratory pressure for 15 seconds a small leak in the blowing tube, ensuring that the epiglottis is not closed. The Valsalva ratio (the longest R-R interval after blowing to the shortest one during blowing or immediately after it) is used as the test indicator (Fig. 1). The sympathetically mediated tachycardia ratio (pulse increase during blowing) may be of importance especially in people over 65 year of age (25). In early adrenergic failure, BP changes occur

Figure 1 Normal pulse variation of (A) a healthy subject of 55 years of age and (B) a patient 51 years of age with autonomic nervous system deficit. 1  Normal breathing; 2  deep breathing; 3  Valsalva manouver. Pulse rate on y-axis (upper rectangles) and time (seconds) on x-axis. Respiration signal (1–3) in the lower rectangles. (From Tolonen U. et al., unpublished data.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

without HR changes. Continuous BP monitoring during the Valsalva maneuver is therefore suggested. Upright positioning may be performed either actively or by using the tilt table test. The pulse reaction is followed during active standing, but also during passive tilting using quick (2 s) and steep tilting up to 80°–90° (28,29). The ratio of the longest R-R interval around beat 30 to the shortest one around beat 15 is calculated as the “30:15 ratio.” Orthostatic hypotension, defined as a BP decrease of

20 mmHg, can be diagnosed after standing or head-up tilting at an angle of at least 60°. In the tilt table test, the duration of standing must be 3–10 minutes (30). The BP response is quantified as the largest drop or the lowest increase in systolic and diastolic pressures. In the spectral analysis of HRV with autoregressive model, disappearance of slow oscillations may precede clinical sings of syncope (31). In the isometric work test the patient grips a dynamometer at 30% of the maximum voluntary power for 5 minutes. The largest increase in systolic and diastolic BP is used as the test measure. Time from 4 to 5 minutes is the most valuable in differentiating the patients from the controls. HR values are age and pulse rate dependent, but BP values are not (32). Increasing body mass index decreases HR variability as well (33). In the isometric work test the values of males and females must be analyzed separately. In the spectral analysis of HR, the LF and HF bands may overlap and decomposition analysis is useful (34). B. Analysis Methods of Heart Rate Variability from Long-Term ECG Recordings Heart rate variability can be easily analyzed from 24-hour electrocardiographic recordings (35). This 24-hour heart rate variability has high intraindividual and intersubject reproducibility (35). There are numerous indexes to describe R-R interval fluctuation during a 24-hour period (27). An ideal method would be an index that could be easily computed with a simple and widely available analysis method. Currently there is no consensus about the best available index of HR variability recorded from a 24-hour period despite the efforts of the Task Force of the North American Society of Pacing and Electrophysiology and the European Society of Cardiology to unify and standardize the methodology (27). Noisy data, artifacts, trends, and ectopic beats are among the most important problems in heart rate variability analysis. Reviews of these problems are described in detail elsewhere (36,37). I.

Time Domain Indices of Heart Rate Variability

Traditionally, heart rate fluctuation has been assessed by calculating indices based on statistical operations on R-R intervals (means and variance). The most widely Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

used time domain parameters are average heart rate and standard deviation of all normal-to-normal R-R intervals over a specific time period (27). The latter is probably also the best-known heart rate variability index. The standard deviation of all R-R intervals over an entire recording (usually over 24 hours) (SDNN) and the standard deviation of the means for all 5-minute R-R intervals over the entire recording (SDANN) are simple time domain methods. These variables are considered to reflect both parasympathetic and sympathetic influences on heart. The other time domain approach is based on an analysis of the differences between adjacent R-R intervals. These include, e.g., the square root of the mean squared differences of successive R-R intervals (RMSSD), the standard deviation of successive difference of R-R intervals (SDSD), and the percentage of differences between adjacent normal R-R intervals of 50 ms (pNN50). These are considered to be reliable indices of cardiac parasympathetic activity independent of long-term trends and to reflect mainly the respiratory component of HR variability (38–40). All the time domain measure indices may be affected by artifacts and outliers, and these measures therefore require data from which artifacts and ectopic beats have been carefully eliminated. 2.

Geometrical Indices of Heart Rate Variability

Geometrical methods present R-R intervals in geometric patterns. The triangular index is a measure where the length of R-R intervals serves as the x-axis of the plot and the number of each R-R interval length serves as the y-axis. Triangular index approximates the R-R interval distribution and correlates highly with the standard deviation of all R-R intervals, but it is highly insensitive to artifacts and ectopic beats, because they are left outside the triangle. This reduces the need for preprocessing of the recorded data (41). The two-dimensional Poincaré plots method (also called return maps) is another geometrical method that provides a beat-to-beat visual (42) and quantitative analysis of R-R intervals (43,44). This quantitative method of analysis is based on the notion of different temporal effects of changes in the vagal and sympathetic modulation of heart rate on the subsequent R-R intervals without a requirement for a stationary quality of the data (43). The shape of the plot can be used to classify the signal into one of several classes (42,45,46), and the irregular shapes quantified from Poincaré plots may then be classified as nonlinear. In quantitative analysis, short-term R-R interval variability and long-term R-R interval variability of the plot can be separately quantified (Fig. 2). 3.

Spectral Indices of Heart Rate Variability

The power spectrum of R-R intervals reflects the amplitude of heart rate fluctuations at different oscillation frequencies (Fig. 2) providing useful frequency-specific information from heart rate behavior (47). Methods based on fast Fourier Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Representative examples of R-R interval tachogram (upper panel), power spectra (middle panel, left side), two-dimensional vector analyses of Poincaré plot (middle panel, right side), detrended fluctuation analysis (DFA) (lower panel, left side), and a scaling slope of long-term fluctuations of heart rate variability. R-R interval tachogram is derived from 5-minute recording at night and spectrum, Poincaré plot and DFA analysis are derived from 1-hour portion at night in healthy subject. The long-term scaling slope is derived from entire 24-hour recording. Abbreviations: HR, heart rate; VLF, very-low-frequency spectral component; LF, low-frequency spectral component; HF, high-frequency spectral component; SD1, short-term beat-to-beat R-R interval variability from Poincaré plot; SD2, long-term R-R interval variability from Poincaré plot; 1, short-term fractal scaling exponent (from 4 to 11 beats) derived by DFA method; 2, intermediate-term fractal scaling exponent ( 11 beats) derived by DFA method; , long-term scaling slope for entire 24-hour recording. (From Refs. 54–55.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

transformation and autoregressive analysis are most commonly used to transform signals into the frequency domain. Practically speaking, both yield similar results. Investigators usually divide the power spectrum into different spectral bands and calculate the powers in these bands. The boundaries of these bands are defined differently by different authors. The spectrum is usually divided into three or four different bands. The boundaries of the most commonly used frequency bands are as follows: ultra low frequency, 0.0033 Hz; very low frequency, 0.0033–0.04 Hz; low frequency, 0.04–0.15 Hz; high frequency, 0.15–0.4 Hz. The boundaries that should be used in physiological studies have been recommended by the European Society of Cardiology and the North American Society of Pacing and Electrophysiology (27). These recommendations are based on a suggested, but only partly proven, physiological background of heart rate variability. 4.

Nonlinear Indices of Heart Rate Variability

Analysis methods derived from nonlinear system theory have opened up a new approach to studying and understanding the characteristics of heart rate behavior. These analysis methods differ from the traditional measures of heart rate variability because they are not designed to assess the magnitude of variability, but rather quality, scaling, and correlation properties of the signal. Numerous algorithms have been developed to describe nonlinear fluctuation of heart rate data. Some of these methods have already provided valuable information on abnormalities in heart rate behavior (48–52). The detrended fluctuation analysis technique is one such a measure (Fig. 2). It quantifies the presence or absence of fractal correlation properties of R-R intervals and has been validated for time series data (48,53–55). It was developed to characterize fluctuations in scales of multiple lengths. The self-similarity occurring over a large range of time scales can be defined for a selected time scale with this method. The details of this method have been described by Peng et al. (48). Normal healthy subjects have shown scaling exponent values near 1, indicating fractal-like heart rate behavior, and altered fractal-like behavior has been reported in patients with cardiovascular diseases and with advancing age (53–58). Another commonly used nonlinear measure is approximate entropy, which was developed for time series to classify complex systems (49). It measures the regularity and complexity of time series data by quantifying the likelihood that runs of patterns that are close remain close on next incremental comparisons. This measure is widely used in various heart rate variability analyses (49,58–61). The obvious advantage of this method is its ability to discern changing complexity from a relatively small amount of data, which makes it applicable to a variety of contexts. Although sophisticated nonlinear techniques seem to be helpful for analyzing complex mechanisms involved in cardiovascular regulation, scaling analysis techniques based on the power spectral density of the signal have been proposed Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

as a valid alternative for the study of the complex fluctuations of heart rate time series data (Fig. 2) (50,62). A plot of spectral power and frequency on bilogarithmic scale and the slope of this relation has been found to be altered among patients with cardiovascular disorders (55,62). Taken together, increasing evidence from multiple studies supports the utility of nonlinear heart rate variability analysis methods in various settings.

III. CARDIOVASCULAR DYSFUNCTION IN NEUROLOGICAL DISEASES A. Stroke and Traumatic Brain Injury Brain lesions caused by cerebrovascular accidents and traumatic brain injury may entail a wide spectrum of autonomic failure including cardiovascular, gastrointestinal, urogenital, sudomotor, and thermoregulatory disorders (63,64). Focal damage may affect the autonomic regulatory centers or pathways selectively, while large lesions cause more complicated and extensive involvement related to increased intracranial pressure. Despite differences in etiology, precipitating factors, and concomitant diseases between stroke and trauma, the associated autonomic complications are discussed together in the context of cardiovascular consequences as the localization of an acute cerebral lesion is more essential than its primary pathoanatomic nature. It is also common to these conditions that their relationship with cardiovascular autonomic failure may not be only consequential but also causal and cumulative, both the cerebral and cardiac dysfunction being potentially capable of impairing each other further. The most common cardiovascular autonomic complications associated with cerebral lesions are electrocardiographic changes, arrhythmias, ischemic myocardial damage, congestive heart failure, sudden cardiac death, and blood pressure changes (63–68). They are known to be prevalent particularly in the acute phase of cerebral damage, which the majority of the available information addresses, but long-lasting autonomic failure can also occur (12). Cardiovascular autonomic failure may compromise and even ruin an otherwise favorable outcome of a patient with stroke or cerebral trauma, and because many of its manifestations are potentially preventable and treatable, it deserves routine consideration. 1.

Electrocardiographic Changes and Myocardial Damage

Among the most common ECG changes observed in association with acute cerebral lesions are prolonged QT intervals, inverted or flat T waves, depressed or elevated ST segments, U waves, prominent P wave, and high-amplitude QRS complex. These abnormalities have been reported in up to 40% of patients with ischemic stroke and up to 70–90% of patients with hemorrhagic stroke Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

(65–67,69–71). These changes may of course also reflect the presence of concomitant heart disease but are known to be due to disturbed cardiac autonomic regulation (72). In contrast to their immediate presence in the case of primary ischemic myocardial damage, ECG changes related to cerebral lesions often appear later, within a few days, and may be detected for up to 1–2 weeks (65). The ECG changes may cause differential diagnostic problems, and the possibility of coincident myocardial infarction must be kept in mind because of a strong correlation between acute ischemic stroke and myocardial disease (67). Furthermore, changes such as prolongation of the QT interval may lead to life-threatening ventricular arrhythmias. The ECG changes in cerebral damage may evolve without any evidence of true myocardial pathology, but there is also evidence of subendocardial hemorrhages, myofibrillar degeneration, and other histopathological abnormalities in cases with a primary cerebrovascular accident and without coronary heart disease (73). In a study on 54 patients with ECG changes, hypothalamic damage was observed in 49 cases, of which 42 had signs of necrotic myocardial injury (74). Patients who had received propranolol did not develop myocardial necrosis in spite of verified hypothalamic involvement. In addition to ECG and histological changes, elevated plasma creatine kinase enzyme levels reflecting myocardial damage have also long been known to be associated with cerebrovascular accidents and traumas, and they have been found prognostically unfavorable (66,75–77). Like ECG changes, the biochemical markers of myocardial ischemia due to brain lesions tend to occur later than in primary cardiac disease and revert to normal in 1–2 weeks (76). ECG changes may present with or without plasma creatine kinase concentration increase. 2.

Cardiac Arrhythmias and Blood Pressure Changes

Another manifestation of disturbed centrally mediated cardiovascular autonomic control is the occurrence of cardiac arrhythmias in patients with acute stroke or traumatic brain injury in the absence of myocardial damage or prior incidents (65,66,71,77,78). Among a large variety of arrhythmias reported, the most common are atrial fibrillation, supraventricular tachycardia, ectopic ventricular contractions, ventricular fibrillation, and multifocal ventricular tachycardia. Arrhythmias are well known to be more common in hemorrhagic than in ischemic brain accidents. Both in ischemic stroke and in traumatic cerebral injury, it is important to consider its function not only as a consequence of the brain damage but also as its potential cause. Cerebral lesions may also lead to failure in blood pressure regulation. Arterial hypertension is a much more frequent finding than hypotension in stroke, but in both instances precipitating factors other than the potential presence of autonomic failure should be kept in mind (79). Beginning with the acute phase after ischemic and particularly after hemorrhagic stroke, both diastolic and systolic Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

pressures tend to be elevated for a period of several days before returning to basal values (80,81). Paroxysmal neurogenic hypertension associated with increased sympathetic activity has been associated with both hemispheral and brain stem lesions (82–87) and particularly with insular cortex lesions (83) in the former and nucleus tractus solitarius (86) lesions in the latter region. Diurnal fluctuation of blood pressure is known to be reduced shortly after a cortical and subcortical lesion (82,84,85,88). Neurogenic hypotension after severe head trauma is a relatively rare but serious complication with high mortality (89). Orthostatic hypotension is considered an uncommon problem in stroke patients (90). The treatment of arrhythmias and blood pressure abnormalities is largely symptomatic and follows the general therapeutic regimens applied to these disorders. Considering the obviously increased sympathetic overdrive behind many of the manifestations mentioned above, -blocking agents often are the first choice in these conditions. Optimal values of blood pressure in cerebrovascular accidents have remained an issue of argumentation, but there is a general agreement that only markedly elevated blood pressure should be treated in case of ischemic stroke (79). There are no controlled studies comparing different treatment strategies or preventive interventions relating to various aspects of cardiovascular autonomic dysfunction in stroke or traumatic brain injury. 3.

Neurogenic Pulmonary Edema

Neurogenic pulmonary edema is a relatively rare but life-threatening complication, which may cause important differential diagnostic problems in cardiovascular evaluation in stroke and brain injuries. A well-known etiological factor is increased intracranial pressure, and mortality is high (91). Recent reports show that medullary brain stem structures are important anatomical sites of origin for this condition (92,93), and experimental studies suggest a role for neuropeptide Y in its pathophysiology (94). 4.

Heart Rate Variability

The application of power spectral analysis in the investigation of heart rate and its regulation has opened up more sophisticated tools to study the cardiovascular autonomic control than the conventional methods discussed above. The beat-to-beat variation of heart rate represents continuous and physiological fluctuation of sympathetic and parasympathetic control activity, and lack of this normal heart rate variation indicates failure of the central cardiovascular autonomic regulation. In general, the low-frequency power is considered to reflect mainly the sympathetic activity, while the high-frequency components are measures of vagal tone (see sec. I). Heart rate analysis in acute cerebral lesions has been studied mainly in cerebrovascular accidents, the common main finding being that heart rate variability

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

is suppressed or lost. This is considered to reflect uncoupling of the central regulation mechanisms and the cardiovascular system, which ranges from complete in the terminal phases of large destructive lesions [e.g., brain death (95,96)] to partial in less severe accidents (10–15,64,96–98). Cardiovascular reflex tests used to investigate respiratory sinus arrhythmia in a group of 27 patients with a variety of acute cerebrovascular accidents or traumatic brain injury showed that heart rate oscillation was markedly reduced in 20 cases about 2 days after the event, and in only 4 patients was the finding normal (10). Follow-up showed that the diminished heart rate variability was present in the acute phase of the accident, thought to reflect reduced supratentorial excitatory influence on vagal cardioinhibitory activity. However, in case secondary brain stem compression developed, the variability was greatly increased, which finding was more complicated to explain in this heterogeneous group. Twenty-four patients with various acute cerebrovascular accidents were compared with healthy controls and patients with transient ischemic attacks in another study (13) using heart rate variability during deep breathing, tilting, and isometric work. Compared with left-sided stroke, the controls’ right-sided lesions were associated with a reduced heart rate variability during breathing attributable to reduced parasympathetic influence on the heart, while no effect of the lesion lateralization was found in the other tests. A further study (12) with a more homogeneous patient cohort, 40 consecutive patients of whom 25 had a hemispheral and 15 had a brain stem infarct, and with a wider battery of cardiovascular reflex tests and a healthy control group, revealed suppression of heart rate responses to parasympathetically mediated stimuli. During the first few days following onset of the event, heart rate responses were significantly impaired according to normal breathing, the Valsalva maneuver, tilting, and isometric work. One month later most of the responses were still diminished, and even 6 months after the acute phase significant impairment could still be verified in normal breathing test. The findings represent long-lasting parasympathetic hypofunction associated with cerebral infarction, which may be contributing to shifting the cardiovascular autonomic balance towards sympathetic overactivity (12). The use of time domain and frequency domain measures of heart rate variability has further contributed to the present knowledge of the complexity of cardiovascular autonomic control failure in acute cerebral lesions. Heart rate power spectral analysis in a group of 37 pediatric patients with traumatic, ischemic, anoxic, and hemorrhagic brain lesions showed associations between low-frequency heart rate power and severity of neurological dysfunction and outcome (95). Maximum values for low-frequency components and the minimum value for high-frequency components predicted likelihood for favorable outcome. The lowfrequency components were particularly decreased in brain-dead patients. In a study (11) comparing 20 patients with right-hemisphere and 20 patients with left-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

hemisphere ischemic stroke, the low-frequency components were reduced in either lateralization of the lesion but significantly more so when the right hemisphere was involved. Most of the available power spectral heart rate studies in cerebral accidents have been devoted to investigating the acute phase of these diseases, with longterm follow-up reports being few. Thirty-one consecutive hemispheral infarct patients were included into a 6-month follow-up study (15). All the measured components of heart rate variability comprising standard deviation of R-R intervals, total power, low-frequency power, and high-frequency power were significantly reduced when compared with those of healthy controls, and this difference persisted through the follow-up period. The impairment of heart rate variability correlated with the severity of the neurological deficits and disability. In patients with large infarcts causing increased intracranial pressure, no relevant spectral components could be defined. Similar results were found in another series of 15 consecutive medullary brain stem infarct patients in the acute phase, but by 6 months these abnormalities had been reversed (14). In contrast to medullary infarcts, heart rate variability was not impaired in the pontine lesions. In addition to the above-mentioned methods, nonlinear analysis techniques for describing heart rate dynamics have been applied to the investigation of cardiovascular autonomic regulation in cerebral lesions. This kind of approach is considered useful as it makes it possible to have a look at random and complex patterns of heart rate dynamics, which cannot be measured with more conventional methods. A 6-month follow-up of 45 ischemic stroke patients and 30 healthy controls investigated instantaneous beat-to-beat variability and long-term continuous variability using 24-hour ambulatory ECG recordings (64). In the acute phase, traditional spectral components of heart rate variability as well as long-term continuous variability were impaired in both hemispheric and medullary brain stem infarcts but not in pontine brain stem infarcts. Reduced heart rate variability was still present 6 months after the event. The physiological circadian rhythm of heart rate variability has been reported to be distorted in cerebral lesions. A follow-up study on 24 patients with hemispheral and 8 patients with brain stem infarction showed that in the acute phase circadian oscillation of heart rate variability had been abolished in both groups with no difference between daytime and nighttime dynamics (99). This abnormality turned out to be transitory as the circadian rhythm of the stroke patients was equal to that of the healthy controls at follow-up examination 6 months later. Heart rate variability analyses provide another useful tool to detect and quantify autonomic failure and to assess sympathetic and parasympathetic balance, and they could be developed further for localizational and patient monitoring approaches. Importantly, they have given further evidence of the complexity of central cardiovascular autonomic regulation in cerebral disorders. At present, however, their importance in the clinical evaluation evaluating of patients remains

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

to be settled in cerebrovascular accidents and traumatic brain injury in contrast to their well-defined significance in cardiac diseases. The patient populations have been mostly heterogeneous, too small, and not long-lasting enough to allow evaluation of the possible prognostic significance of heart rate variability measures. 5.

Pathophysiological Aspects

The pathophysiology of localized lesions causing cardiovascular abnormalities is attributed to damage to the various autonomic regulation centers, networks, or pathways and to increased intracranial pressure with more generalized consequences, but neurohumoral mechanisms are also involved. The pathogenesis of ECG changes, arrhythmias, and myocardial damage related to acute cerebral accidents is considered to be mainly due to increased sympathetic activity. In the acute phase of cerebral damage, cardiac complications are thought to be generated by direct neural overactivation, and functionally intact efferent neural pathways are needed for transmission from the brain (66,100). In a later phase excessive amounts of catecholamines are released into circulation and may interfere with cardiac functions and cause structural damage, and this process operates without neural connection between the brain and heart. The vast literature available on the role of catecholamine release has been discussed in detail in several reviews (65,66,101–105). Regulation of blood pressure and heart rate tends to be impaired in both cortical and subcortical hemispheral lesions and even in lacunar infarcts (85,87,88). Although total cardiac autonomic innervation is reduced after cerebral accidents in general, right hemisphere lesion patients tend to have a reduction of parasympathetic activity, which is reflected in blood pressure and heart rate and arrhythmia control (84,97). Some have attributed autonomic failure particularly to lesions in the insular cortex (83,106,107). In brain stem, nucleus tractus solitarius seems to be of particular importance in terms of blood pressure regulation (86). The available clinical knowledge is fragmentary, and, several parts of the autonomic network are clearly responsible for cardiovascular control, as has been shown in experimental studies (103). B. Parkinson’s Disease and Other Degenerative CNS Diseases 1.

Parkinson’s Disease

PD may contribute to various autonomic manifestations, but the prevalence and severity of autonomic dysfunction is somewhat undetermined. Among other symptoms of autonomic failure, alterations of cardiovascular autonomic regulation have been described in PD. In several studies the most commonly reported clinical disturbances of ANS function in PD patients were postural dizziness and orthostatic hypotension (108–113).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

It is generally assumed that the supine BP level is low in parkinsonian patients, although there are contradictory results not confirming this statement (112,115). Murata et al. (116) found no difference in systolic and diastolic BP and pulse rate between PD patients and control subjects. Some degree of orthostatic hypotension (OH) has also been related to PD, reflecting impairment of baroreflex function (108,109). The mild sympathetic failure in PD is further supported by pathological BP responses to passive tilting (112,117–120) and isometric exercise (113,118,119,121–123). The causative role of antiparkinsonian drugs in the OH of PD patients has long been debated. However, the presence of OH has also been reported in untreated de novo PD patients (120). Several studies have shown that levodopa may enhance the orthostatic BP decrease (117), especially in the beginning of the treatment (108–110), whereas others have not been able to demonstrate such an effect (120,121,124,125). Appenzeller and Goss (111) showed that even though they found a profound failure in baroreceptor functions in PD, levodopa treatment did not aggravate it. Moreover, the use of dopamine receptor agonists has been associated with low BP levels (126) and pronounced OH in PD (126–130), but patients seldom considered it noteworthy (130). In a recent prospective study by Haapaniemi et al. (120), bromocriptine medication increased the systolic BP fall and presence of OH in de novo PD patients (Fig. 3). In a report by Churchyard et al. (131), based on a small PD patient group, selegiline medication combined with levodopa was associated with severe OH, and withdrawal of selegiline considerably diminished or abolished the orthostatic BP fall. On the other hand, in the prospective, long-term, and placebo-controlled trial of Turkka et al. (29), selegiline slightly diminished the sympathetic autonomic responses, but none of the patients had clinically significant OH. Accordingly, Haapaniemi et al. (120) reported a reversible increase in the BP fall upon tilting in PD patients on selegiline monotherapy, whereas the presence of OH remained the same as before the treatment (Fig. 3). Standard cardiovascular autonomic responses to physiological stimuli have provided much evidence suggesting mild ANS disturbances in PD. Patients with PD have shown diminished HR variation responses to normal breathing, deep breathing, Valsalva maneuver, and tilting together with the previously mentioned suppressed BP responses to tilting and isometric work (29,113,118,125,132), indicating a deficit in both sympathetic and parasympathetic ANS functions. Furthermore, the attenuated HR responses have been reported to correlate with the severity of clinical dysautonomia and also with the duration of the disease (113,132). A recent follow-up study by Mesec et al. (133) demonstrated deterioration in HR responses to deep breathing and Valsalva maneuver and in the HR and BP responses to the orthostatic test over a period of 3 years. The sympathetic impairment has been shown to be more prominent in akinetic-rigid than tremorakinetic-rigid PD patients, and, furthermore, the sympathetic dysfunction is more Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3 Mean systolic BP change immediately after tilting (A) and the maximum systolic BP fall after tilting (B) in PD patients randomized to levodopa, bromocriptine, or selegiline at baseline, during medication, and after the washout period. The results on medication and after washout are compared to the baseline values using the Wilcoxon signed ranks test.* p  0.05. (From Ref. 120.) Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

likely to occur early and parasympathetic dysfunction later during the course of the disease (132). In addition to the standard cardiovascular reflex test providing information obtained from responses to stimuli during short time periods, analysis of HR variability from 24-hour ECG recordings has revealed diminished SDNN and spectral components of HR variation in PD patients compared to healthy control subjects (17,134). Also the power-law relation slopes, reflecting the autonomic input to the heart, were steeper in the patients than in the controls (17). Irrespective of the findings in these studies suggesting impaired cardiovascular autonomic function in PD patients, the autonomic dysregulation is usually mild and the baroreflex function is generally preserved. The BP responses to ingestion of food have been used to reveal even latent autonomic deficits in PD. Micieli et al. (135) studied the diurnal pattern of BP, HR, and urinary catecholamine secretion and the BP and plasma catecholamine responses to tilting in 13 drug-naïve PD patients and age-matched controls. The postprandial fall of supine systolic BP was greater in 7 of the 13 PD patients than in controls, the degree of this postprandial hypotension being related to the 24hour urinary dopamine secretion. Eight of the PD patients showed OH during the tilt test both in the morning and in the postprandial phase. The authors concluded that the data suggested existence of a subtype of PD patients with widespread impairment of cardiovascular responsiveness and bordering on the specific syndromes of ANS failure. These results differ from those of Thomaides et al. (136), who investigated the effect of a balanced liquid meal on BP and HR both in a supine position and after tilting in patients with PD, multiple system atrophy (MSA), pure autonomic failure (PAF), and healthy controls. A postprandial BP fall was found in all patient groups, but not in the controls, whereas there was a postural postprandial hypotension only in the MSA and PAF patients. Hemodynamic studies on postprandial hypotension in PD have revealed a decreased vascular resistance assessed with impedance plethysmography (137). Neurohumoral factors, such as secretion of catecholamines, vasopressin, and renin-angiotensin-aldosterone, are essential in cardiovascular regulation and participate in short- and long-term BP control. Aminoff and Wilcox (110) reported an increased susceptibility to noradrenaline in PD, a finding that was recently confirmed by Niimi et al. (138). Additionally, serum noradrenaline response to standing up has been diminished in PD patients (16,138), suggesting a deficit in sympathetic ANS function. The level of 3-methoxy-4-hydroxyphenyl glycol, the main metabolite of noradrenaline, in the cerebrospinal fluid of PD patients has been low, with a correlation to the clinical severity of ANS dysfunction (113), possibly reflecting the activity of the central parts of ANS. In a recent study, Goldstein et al. (139) demonstrated decreased cardiac extraction of circulating [3H]norepinephrine, norepinephrine spillover, and virtual absence of venous-arterial increment in plasma levels of endogenous L-dopa and 3-methoxy-4-hydroxyphenyl glycol. These findings indicate a loss of cardiac sympathetic nerve terCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

minals in parkinsonian patients, an inference supported by low cardiac uptake of tracers in scintigraphy with iodine-123–labeled metaiodobenzylguanidine and PET studies with 6-[18F]fluorodopamine reflecting postganglionic cardiac sympathetic innervation (139,140). As the above-mentioned studies display, no generally accepted concept concerning the extent of cardiovascular ANS involvement in PD exists, but there are many data supporting impairment of cardiovascular autonomic responses in a noticeable number of PD patients. Theoretically this could relate to central or peripheral disorders, recent data suggesting an essential role for peripheral ANS damage. Pathologically, Parkinson’s disease exhibits progressive neuronal loss associated with Lewy bodies in the pigmented nuclei of midbrain, many subcortical nuclei, and the cortex (141). The autonomic symptoms in PD can be linked to the histopathological findings of a systematic study on the ANS by Wakabayashi and Takahashi (142). Lewy bodies were widely distributed in the hypothalamus, sympathetic system (intermediolateral nucleus of the thoracic cord and sympathetic ganglia), and parasympathetic (dorsal vagal and sacral parasympathetic nuclei) system. Furthermore, Lewy bodies were found in the enteric nervous system of the alimentary tract, cardiac plexus, pelvic plexus, and adrenal medulla. Thus, neuropathologically both the central and peripheral autonomic nervous systems are involved in the disease process of PD, corresponding to the conclusions drawn from results of studies based on functional assessment of ANS regulation. 2.

Other Degenerative CNS Diseases

Multiple system atrophy is a sporadic, adult-onset neurodegenerative disease of unknown etiology. The condition is regarded to be unique among neurodegenerative diseases due to the prominent role of oligodendroglial cells in the pathogenetic process (143). Recent contributions to the understanding of MSA have included detection of glial cytoplasmic inclusions and -synuclein accumulation. Multiple system atrophy includes syndromes of striatonigral degeneration, OPCA, and autonomic failure in several combinations. Wenning et al. (144) assessed the clinical characteristics of the disease in an extensive series of 100 patients with MSA. Autonomic symptoms were the initial feature in 41% of the cases, but they developed later on in 97% of the patients during follow-up. Progression of the disease was faster than that in PD, to such an extent that more than 40% of the subjects were markedly disabled or wheelchair bound within 5 years of the onset of the symptoms. Although the precise definition of MSA has been difficult, recently a consensus in the diagnostic criteria has been achieved (145,146). This should lead to progress in defining the underlying pathophysiology of the neuroendocrine, autonomic, and motor deficits characteristic of MSA. The diagnosis is based on the presence of autonomic failure/urinary dysfunction, parkinsonism, cerebellar Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ataxia, and corticospinal dysfunction (145). The main finding of autonomic failure (147) is orthostatic hypotension. It is usually present at the first clinical examination or at least within 1 year from the development of parkinsonism. Symptoms of orthostatic hypotension include lightheadedness, tiredness, ataxia, blurred vision, and retrocollic aching (147). Symptoms of orthostatic hypotension may worsen during exercise or standing (148), but when the condition has become chronic patients usually become very tolerant of low blood pressures. Wenning et al. (149) conducted a clinicopathological analysis of 203 cases of pathologically proven MSA from various publications. The authors confirmed the association of postural hypotension with degeneration of intermediolateral cell column of the spinal cord. This agrees with the classical concept that depletion of sympathetic preganglionic neurons in the intermediolateral cell column is thought to be the main cause of sympathetic failure in MSA, suggested by Bannister and Oppenheimer in 1972 (150). ANS functions have been assessed in other degenerative diseases of CNS. Subjects of research have included Friedreich’s ataxia, Huntington’s disease, progressive supranuclear palsy, and Alzheimer’s disease. Many pathological findings have been reported, but their clinical significance remains to be established. C. Spinal Cord Lesions Traumatic spinal cord injuries and spinal diseases are frequently manifested as various autonomic disorders affecting cardiovascular, gastrointestinal, thermoregulatory, and urogenital regulatory systems. The major clinical problems are found in patients with cervical and high thoracic spinal cord lesions, whose entire sympathetic and sacral parasympathetic outflow is separated from cerebral control as a result of an interruption of the autonomic pathways from the brain to the intermediolateral column of the spinal cord. The autonomic involvement associated with spinal cord lesion is mainly determined by the site and the extent of the lesion, but there are also certain clinically important differences between recently injured patients and those in the chronic spinal stage (151). Immediately following an acute spinal cord lesion, there is a transient period of hypoexcitability of the isolated cord called “spinal shock,” which is characterized by flaccid, areflexic paralysis of skeletal and smooth muscles (152). During this stage, spinal autonomic functions and reflexes are impaired below the level of the lesion, manifested as atonic bladder, large bowel, and low basal supine systolic and diastolic BP (often  90/50 mmHg) due to decreased sympathetic activity (153). Because these patients’ BP is mainly regulated by posture and blood volume without supraspinal control, postural hypotension is found in a majority of them (154,155). Bradycardia is another commonly found manifestation of cardiovascular autonomic dysfunction in tetraplegics, whereas tachycardia is often encountered

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

in patients with acute low spinal cord lesions (153). In most cases bradycardia, thought to be caused by a reduction in neural and hormonal sympathetically mediated influences as well as vagal overactivity (153), is mild, it may even lead to cardiac arrest as a consequence of a parasympathetic stimulation such as intubation or tracheal suction (156,157). Spinal shock normally lasts a few weeks, after which the activity of the isolated spinal cord gradually returns, leading to uncontrolled reflex activity at the spinal level and a variety of autonomic disorders. Although in chronic tetraplegics the basal HR usually returns to normal or remains only slightly lower than in normal subjects (153), the basal BP often remains low and orthostatic hypotension is a common phenomenon in chronically injured patients (153,157–159). Moreover, tetraplegics are particularly sensitive to certain drugs interfering with the cardiovascular regulatory system despite gradually developing compensational mechanisms, such as the renin-angiotensin-aldosterone system and activation of spinal and local sympathetic reflexes, to maintain BP. Even small doses of diuretics or angiotensin-converting enzyme inhibitors may cause a catastrophic BP fall (153). A study using a novel multibiological recorder suggests that the central sympathoexcitatory pathway to the upper thoracic cord plays a critical role in the maintenance of normal circadian BP rhythm in humans, whereas motor functioning and sympathoadrenal secretion are not essential to this regulation (160). Cardiovascular and thermal responses to exercise are altered in subjects with high-level spinal cord lesions, causing exercise intolerance and hyperthermia. As a result of venous pooling accompanied by reduced end-diastolic stroke volume and of incomplete ability to increase HR, an exercise-induced hypotension with subsequent exercise intolerance may develop (161,162). BP and HR responses to certain provocations such as Valsalva maneuver, cutaneous pain or cold stimulation, as well as cognitive stimulation, are also impaired in spinal subjects as a reflection of cardiovascular dysregulation. Moreover, circadian rhythm of BP is abolished in completely tetraplegic patients, while it is preserved in subjects with incomplete tetraplegia or paraplegia (163). Recent studies using spectral analysis techniques (164–166) showed significantly suppressed HR variability in both quadriplegic and paraplegic subjects compared with healthy subjects. Autonomic dysreflexia is a clinically important complication of spinal cord injury usually appearing a few months after the lesion (153,167,168). It occurs in up to 85% of individuals with spinal cord injury above the fifth thoracic segment and is defined as a massive paroxysmal reflex sympathetic activity developing in response to noxious peripheral stimuli below the level of the lesion. Although a variety of stimuli can provoke this phenomenon, bladder and bowel distension account for most episodes. The main clinical symptoms of autonomic dysreflexia are sudden headache, hypertension, bradycardia, hyperhidrosis, and piloerection. Nevertheless, the phenomenon is of clinical relevance. Mild episodes are likely to occur intermittently through the day and are of little consequences, but when pro-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

longed it may cause considerable morbidity and even mortality due to myocardial failure, retinal or intracerebral hemorrhages, seizures, and hypertensive encephalopathy. The pathophysiological mechanism of the autonomic dysreflexia is still incompletely understood, but it seems to be multifactorial, including at least a loss of supraspinal inhibitory control and denervation hypersensitivity and possibly also a formation of abnormal synaptic connections due to axonal sprouting (169,170). The most important area of treatment of autonomic dysreflexia is prevention, i.e., accounting for optimal bladder, bowel and skin care, and avoidance of other external stimuli (171). Despite several pharmacological interventions, the drug of choice for autonomic dysreflexia is still lacking. Short-acting calcium channel blockers seem to be most effective, although they may sometimes worsen orthostatic hypotension. D. Peripheral Neuropathies Autonomic disturbances commonly occur in most peripheral neuropathies, but only in a small number of conditions, such as diabetes and inflammatory polyradiculitis, are they of clinical importance (172). The autonomic nervous system is most likely affected when there is acute demyelination or damage to small myelinated and unmyelinated fibers. Autonomic dysfunction is a common and important complication of acute inflammatory polyradiculitis, or Guillain-Barré syndrome, involving cardiovascular, sudomotor, gastrointestinal, and other sympathetic and parasympathetic autonomic nervous functions (173). Clinical autonomic disorders are present in two thirds of the patients (174), whereas subclinical autonomic involvement, assessed using cardiovascular autonomic function tests, may be present in a vast majority of patients (175). Cardiovascular autonomic dysfunction associated with acute polyradiculitis manifests itself as sinus tachycardia (30%), bradycardia (8%), ECG abnormalities (33%), arrhythmias (11%), hypertension (30%), or postural hypotension (30%) (174,176–178). Hypertension is often transient, rarely lasting more than a few days, whereas postural hypotension usually lasts for several weeks or months (174,176). Microneurographic recordings have shown that tachycardia and hypertension in acute polyradiculitis result from increased sympathetic tone likely due to reduced inhibition of the vasomotor centers caused by lesions of the afferent limbs of arterial and intrathoracic baroreflexes (179). On contrary, postural hypotension is suggested to be related either to damage to efferent sympathetic supply to peripheral vascular bed leading to vasodilatation or to damage to the afferent parasympathetic fibers reducing sensitivity of baroreceptors (176).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

A few studies (174–176,180) evaluating quantitatively abnormalities of cardiovascular responses to standardized stimuli in acute polyradiculitis suggest that HR and BP responses to deep breathing, Valsalva maneuver, tilting, sustained handgrip, and cold pressor are impaired in most of patients. These abnormalities usually reach their maximum level during the first weeks after the onset of disease and seem to improve gradually over time. Recent studies (181,182) using spectral HR analysis technique have also shown that sympathovagal balance is clearly shifted to sympathetic predominance at the height of acute inflammatory polyradiculitis. However, it seems that cardiovascular autonomic disorders are only partially related to recovery of motor functions, and their prognostic value is limited (174,175). In diabetes mellitus the overall prevalence of cardiovascular autonomic dysfunction is about 10% (183), but in diabetic patients with polyneuropathy the prevalence is suggested to be over 60% (184). Most of these patients are asymptomatic, and few progress to symptomatic autonomic neuropathy, which is associated with higher mortality, mainly from renal failure (3,185). Postural hypotension, occurring in 10–43% of patients with diabetic neuropathy, is the most important clinical manifestation of cardiovascular autonomic neuropathy and may occasionally be a major problem (186–188). It is caused by degeneration of the sympathetic preganglionic and postganglionic fibers supplying mainly splanchnic mesenteric and to a lesser degree muscular resistance vessels, leading to impaired baroreflexes and reduced vasoconstriction in splanchnic and muscle blood vessels. Several quantitative analyses of cardiovascular autonomic dysfunction have recognized a high prevalence of cardiac sympathetic and parasympathetic impairment, evaluated as HR and BP responses to deep breathing, Valsalva maneuver, tilting and isometric work, even in the early phases of the disease (2,187). In addition, analyses of HR variability from 24-hour ambulatory ECG recordings using conventional time-domain and frequency-domain (spectral) analyzing techniques have shown a high prevalence of cardiovascular autonomic dysregulation in diabetes (189–194). Clinical manifestations of cardiovascular autonomic dysfunction in other peripheral neuropathies resemble those of diabetes (172). All of them may result in postural hypotension and impaired HR and BP responses in cardiovascular reflex tests as well as reduced HR variability.

IV. CARDIOVASCULAR DYSFUNCTION IN ISCHEMIC HEART DISEASE Despite impressive advances during the past three decades, ischemic heart disease remains a major health problem in western societies. Impairment of autonomic cardiovascular regulation has been observed in many disease states, including is-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

chemic coronary artery disease. The relevance of this impairment for clinical cardiology was realized in the late 1980s, when the results of impaired autonomic function were found to predict mortality among patients with coronary artery disease who had experienced acute myocardial infarction (195). For assessing cardiac autonomic regulation, analysis of heart rate variability has become an important, widely used method. HR variability has been traditionally analyzed with time and frequency domain methods. These methods measure the overall magnitude of HR fluctuation or magnitude of fluctuation in some predetermined frequencies. For uncovering subtle but important alterations and abnormalities in time series data, new methods based on the nonlinear system theory have been constantly developed (48–51). These methods differ from conventional methods in that they typically detect qualitative rather than quantitative properties of heart rate time series. Until now, measurement of HR variability has been of limited clinical value because of its poor predictive accuracy. Therefore, new methods and approach strategies providing additional information concerning abnormalities in autonomic regulation in pathological stages would be welcome. A. Heart Rate Variability in Noncomplicated Coronary Artery Disease HR variability has been shown to be altered among patients with stable coronary artery disease and has been suggested to be reduced even before the development of symptomatic coronary artery disease. Airaksinen et al. (196) were the first to report reduced vagal activity among patients with coronary artery disease manifested as reduced heart rate oscillations during deep-breathing test. Reduced highfrequency oscillations of HR, particularly during the sleeping hours, were also observed among the patients with noncomplicated coronary artery disease (8). It has also been reported that the reduction of HR variability correlates with the angiographic severity of coronary artery disease (197). Particularly, high-frequency fluctuation seems to be reduced in those with a more severe coronary artery disease, although this association is not evident in all studies (196,198). Despite the lack of consensus concerning the association between the severity of coronary artery disease and the reduction of HR variability in general, the prominent alteration detected by spectral measures is a reduction in the high-frequency component among the patients with uncomplicated coronary artery disease. Fractal scaling properties of R-R intervals have also been shown to be altered among patients with uncomplicated coronary artery disease (55). Patients with uncomplicated coronary artery disease show an enhanced regularity in HR tracings as estimated by short-term fractal scaling exponent. Furthermore, the short-term fractal exponent has performed better than other HR variability measures in detecting abnormalities in HR behavior in the group of patients with uncomplicated coronary artery disease (55).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Among patients with noncomplicated coronary artery disease, a significant reduction in the high-frequency spectral band indicates a dominant role for the low-frequency control mechanisms. The loss of high-frequency fluctuations corresponds to more regular short-term signal behavior associated with a higher short-term scaling exponent. The ratio of the low- to high-frequency spectral components correlates relatively well with the short-term fractal scaling exponent of HR behavior, particularly when breathing is controlled. This correlation becomes weaker during the “free running” situations such as ambulatory 24-hour Holter recordings. Therefore, fractal correlation properties may be more suitable to probe subtle alterations in HR dynamics than simple spectral ratios in uncontrolled ambulatory conditions. Although time domain, spectral, and fractal measurements of HR variability have been shown to be altered among patients with ischemic heart disease, the exact mechanisms of altered HR behavior are not known. The effect of the severity of coronary artery disease is controversial. Ischemia has been suggested to destroy the cardiac receptors, resulting in altered autonomic regulation (199), but aging itself also affects autonomic activity (196). Also, the significance of transient myocardial ischemia on cardiovascular autonomic regulation is not completely understood. Recent clinical data have demonstrated that myocardial ischemia caused by short-term coronary occlusion during coronary angioplasty causes divergent changes in HR variability (200). It has also been proposed that reduced HR variability may be a risk marker for the development of coronary artery disease. This observation is supported by a recent study showing that reduced HR variability predicts rapid progression of coronary atherosclerosis assessed by serial quantitative coronary angiograms (35). B. Heart Rate Variability After an Acute Myocardial Infarction As early as in 1965 it was proposed that HR fluctuations are decreased among patients with an acute myocardial infarction (201). Later several reports showed that the time domain as well as the spectral measures of HR variability are reduced after myocardial infarction (202–204). The reduction in HR variability after a myocardial infarction seems to be partly a transient phenomenon. Evidence of a recovery of HR variability after myocardial infarction has been observed (205,206), but it remains less than in healthy controls (205) late after a prior infarction. The qualitative alterations in HR dynamics seem to be clearly different among patients with uncomplicated ischemic heart disease than among patients with complicated ischemic heart disease. In patients with noncomplicated myocardial infarction, the low- to high-frequency spectral ratio is usually increased in a similar way as in the patients without a prior infarction (207). However, in patients with impaired left ventricular function after myocardial infarction, HR fluctuations are typically decreased in the low-frequency area (0.05–0.15 Hz) without

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

a prominent decrease in the high-frequency area (207). Similar reduction or absence of low-frequency spectral band of HR variability has been observed in patients with a congestive heart failure (208). This specific spectral pattern results in a lack of prominent spectral component in the high- and low-frequency areas, resulting in a reduced short-term fractal scaling exponent, indicating an increased short-term randomness in HR behavior. Also, steeper slopes in power-law slope have been observed among postinfarction patients. Steeper slopes are mainly caused by decreased values in the low- and very-low-frequency heart rate fluctuations, while ultra-low-frequency heart rate fluctuation is better preserved. C. Prognostic Value of HR Variability in Ischemic Heart Disease In 1978 Wolf et al. showed that patients with low short-term HR variability have a poor prognosis after acute myocardial infarction (209). In the late 1980s several landmark studies confirmed the predictive value of low HR variability measured by time domain indices for mortality following myocardial infarction (41,195). These observations have been confirmed in several large-scale studies. Most of the studies concerning the prognostic value of measurement of HR variability have been performed in patients after a recent myocardial infarction. Some population-based studies in subjects without myocardial infarction suggest that reduced HR variability predicts the occurrence of various cardiovascular events, such as angina pectoris, nonfatal myocardial infarction, and cardiac death. The prognostic power of methods based on nonlinear dynamics has been tested in large-scale studies recently. The ability of spectral scaling properties of long-term fluctuation to predict death after myocardial infarction was first reported by Bigger and coworkers (62). The slope of the power law relationship was found to be somewhat steeper (i.e., more negative) for myocardial infarction patients and much steeper for heart transplant patients compared with healthy subjects. Furthermore, power law slope was a powerful predictor of all-cause mortality or arrhythmic death and predicted these outcomes better than the traditional power spectral bands. In addition to patients with myocardial infarction, longterm scaling properties have been observed to predict mortality among general elderly population (57). Recently, altered short-term fractal properties of HR fluctuation have been shown to provide superior prognostic power compared to conventional measures among patients with an acute myocardial infarction and depressed left ventricular function (53–55). First, short-term fractal-like correlation properties of R-R intervals were studied in 159 patients with acute myocardial infarction and left ventricular ejection fraction of 35% with a 4-year follow-up. Among all analyzed variables, reduced scaling exponent was shown to be the best predictor of mortality. The study showed that reduction in fractal correlation properties implies more random short-term HR dynamics in patients with increased risk of death after an

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

acute myocardial infarction (53,54). More recently, in a large population of 446 survivors of acute myocardial infarction with a left ventricular ejection fraction of 35%, reduced short-term fractal exponent was the most powerful HR variability measure as a predictor of all-cause mortality. Reduced fractal exponent predicted both arrhythmic death and nonarrhythmic cardiac death. It yielded more powerful prognostic information than the traditional measures of HR variability (55). In addition to being predictors of mortality after myocardial infarction, some nonlinear measures have been shown to detect alterations in HR dynamics before the onset of arrhythmic events (51,53,54). Skinner and coworkers observed significant decline in point correlation dimension shortly prior to the onset of ventricular tachycardia (51). Altered short-term fractal scaling exponent has also been observed shortly prior ventricular fibrillation among patients who experienced ventricular fibrillation during Holter recordings performed as a part of routine investigations (53,54). In that study, the short-term fractal scaling exponent performed better than any other measurement of HR variability in detecting ventricular fibrillation patients, suggesting that dynamic analysis methods of R-R intervals could help to identify abnormalities in HR behavior before ventricular fibrillation. Also, analysis of the shape of the scatter plot pattern has been used as a potent indicator for the prediction of forthcoming ventricular tachycardia (43). Preceding the onset of ventricular tachycardia, the scatter plot most frequently tended to broaden and its length decreased. Reduced long-term R-R interval variability, associated with episodes of beat-to-beat sinus alternans, was a highly specific sign of a propensity for spontaneous onset of ventricular tachycardia, suggesting that abnormal beat-to-beat heart rate dynamics may reflect a transient electrical instability favoring the onset of ventricular tachycardia in patients conditioned by structurally abnormal hearts. D. Perspectives Research on HR variability has increased exponentially during the last decade (210) (see also Chapter xx). Methods derived from nonlinear system theory have provided new insights into the abnormalities of HR behavior in various pathological conditions, providing additional prognostic information as compared to traditional heart rate variability measures, and clearly complement conventional analysis methods. Despite statistical data suggesting the predictive power of the various HR variability indices, none are widely used in clinical practice to guide preventive therapy for individual patients because no trial has adequately linked the reliability of any of these variables of clinical outcome with intervention. Therefore, more clinical studies using new nonlinear methods will be needed before the clinical applicability of these methods can be definitively established.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

REFERENCES 1. Mathias C., Bannister R. Autonomic failure. A Textbook of Clinical Disorders of the Autonomic Nervous System. Oxford, UK: Oxford Medical Publications, 2001. 2. Ewing DJ, Campbell IW, Clarke BF. Assessment of cardiovascular effects in diabetic autonomic neuropathy and prognostic implications. Ann Intern Med 1980; 92:308–311. 3. Ewing DJ, Campbell IW, Clarke BF. Mortality in diabetic autonomic neuropathy. Lancet 1976; 1:601–603. 4. Page, M. and Watkins, P. Cardio-respiratory arrest and diabetic autonomic neuropathy. Lancet 1978; 1:14–16. 5. Lloyd-Mostyn RH, Watkins PJ. Total cardiac denervation diabetic autonomic neuropathy. Diabetes 1976; 25:748. 6. Faerman J, Faccio E, Milei J, Nunez R, Jadzinsky M, Fox D, et al. Autonomic neuropathy and paindless myocardial infarction in diabetic patients. Histologic evidence of their relationship. Diabetes 1977; 26:1147–1158. 7. Malik M, Farrel T, Camm AJ. Circadian rhythm of heart rate variability after acute myocardial infarction and its influence on the prognostic value of heart rate variability. Am J Cardiol 1990; 66:1049–1054. 8. Huikuri HV, Niemelä MJ, Ojala S, Rantala A, Ikäheimo MJ, Airaksinen KEJ. Circadian rhythms of frequency domain measures of heart rate variability in healthy subjects and patients with coronary artery disease. Effects of arousal and upright posture. Circulation 1994; 90:121–126. 9. Vanoli E, Adamson PB, Ba-Lin MPH, Pinna GD, Lazzara R, Orr WC. Heart rate variability during specific sleep stages: a comparison of health subjects with patients after myocardial infarction. Circulation 1995; 91:1918–1922. 10. Frank JI, Ropper AH, Zuñica G. Acute intracranial lesions and respiratory sinus arrhythmia. Arch Neurol 1992; 49:1200–1203. 11. Barron SA, Rogovski Z, Hemli J. Autonomic consequences of cerebral hemisphere infarction. Stroke 1994; 25:113–116. 12. Korpelainen JT, Sotaniemi KA, Suominen K, Tolonen U, Myllylä VV. Cardiovascular autonomic reflexes in brain infarction. Stroke 1994; 25:787–792. 13. Naver HK, Blomstrand C, Wallin G. Reduced heart rate variability after right-sided stroke. Stroke 1996; 27:247–251. 14. Korpelainen JT, Huikuri HV, Sotaniemi KA, Myllylä VV. Abnormal heart rate variability reflecting autonomic dysfunction in brainstem infarction. Acta Neurol Scand 1996; 94:337–342. 15. Korpelainen JT, Sotaniemi KA, Huikuri HV, Myllylä VV. Abnormal heart rate variability as a manifestation of autonomic dysfunction in hemispheric brain infarction. Stroke 1996; 27:2059–2063. 16. Turkka JT, Kuujärvi kk, Lapinlampi TO, Myllylä VV. Serum noradrenaline response to standing up in patients with Parkinson’s disease. Eur Neurol 1986; 25:355–361.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

17.

18.

19.

20. 21. 22.

23.

24. 25.

26. 27.

28. 29.

30. 31. 32. 33.

Haapaniemi TH, Pursiainen V, Korpelainen JT, Huikuri HV, Sotaniemi KA, Myllylä VV. Ambulatory ECG, and analysis of heart rate variability in Parkinson’s disease. J Neurol Neurosurg Psychiatry 2001; 70:305–310. Myllylä VV, Korpelainen JT, Tolonen U, Havanka H, Saari A. Neuropathology and cardiovascular regulation—clinical aspects. In: Horst GJ, ed. The Nervous System and the Heart. Totowa, NJ: Humana Press Inc., 2000:181–237. Huikuri HV, Koistinen MJ, Yli-Mäyry S, Airaksinen KEJ, Seppänen T, Ikäheimo MJ, Myerburg RJ. Impaired low frequency oscillations of heart rate in patients with prior acute myocardial infarction and life-threatening arrhythmias. Am J Cardiol 1995; 76:56–60. Wallin GB, Elam M. Microneurography and autonomic dysfunction. In Low PA, ed. Clinical Autonomic Disorders. Philadelphia: Lippincott-Raven, 1997:233–243. Wheeler T, Watkins PJ. Cardiac denervation in diabetes. Br Med J 1973; 4:584–586. Diehl RR, Linden D, Berlit P. Determinants of heart rate variability during deep breathing: basic findings and clinical applications. Clin Auton Res 1997; 7:131–135. Pagani M, Montano N, Porta A, Malliani A, Abboud FM, Birkett C, Somers VK. Relationship between spectral components of cardiovascular variabilities and direct measures of muscle sympathetic nerve activity in humans. Circulation 1997; 95:1441–1448. Scholz UJ, Bianchi AM, Cerutti S, Kubichi S. Vegetative background of sleep: spectral analysis of the heart rate variability. Physiol Behav 1997; 62:1037–1043. Braune S, Auer A, Schulte-Mönting J, Schwerbrock S, Luking CH. Cardiovascular parameters: sensitivity to detect autonomic dysfunction and influence of age and sex in normal subjects. Clin Auton Res 1996; 6:3–15. Suominen K. Design and implementation of a PC-based data acquisition system for measuring ECG and respiratory signals. Int J Clin Mon Com 1997; 14:225–230. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Heart rate variability: standards of measurements, physiological interpretations and clinical use. Circulation 1996; 93:1043–1065. Sundqvist G, Lilja B, Almer LO. Abnormal diastolic blood pressure and heart rate reactions to tilting in diabetes mellitus. Diabetologia 1980; 19:433–438. Turkka JT, Suominen K, Tolonen U, Sotaniemi KA, Myllylä VV. Selegiline diminishes cardiovascular autonomic responses in Parkinson’s disease. Neurology 1997; 48:662–667. Khurana RK, Nicholas EM. Head-up tilt table test: how far and how long? Clin Auton Res 1996; 6:335–341. Van Lieshout JJ, Wieling W, Karemaker JM, Eckberg DL. The vasovagal response (review). Clin Sci 1991; 81:575–586. Piha SJ. Cardiovascular autonomic reflex tests: normal responses and age-related reference values. Clin Physiol 1991; 11:277–290. Freeman R, Weiss ST, Roberts M, Zbikowski SM, Sparrow D. The relationship between heart rate variability and measures of body habitus. Clin Auton Res 1995; 5:261–266.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

34.

35.

36.

37.

38. 39. 40.

41.

42.

43.

44.

45. 46.

47.

48.

49.

Bianchi AM, Bontempi B, Cerutti S, Gianoglio P, Comi G, Natali Sora MG. Spectral analysis of heart rate variability signal and respiration in diabetic subjects. Med Biol Eng Com 1990; 28:205–211. Huikuri HV, Jokinen V, Syvänne M, Nieminen MS, Airaksinen KEJ, Ikäheimo MJ, Koistinen MJ, Kauma H, Kesäniemi YA, Majahalme S, Niemelä K, Frick H. Heart rate variability and progression of coronary atherosclerosis. Arteriosl Thromb Vasc Biol 1999; 19:1979–1985. Kamath MV, Fallen EL. Correction of heart rate variability signal for ectopics and missing beats. In: Malik M, Camm AJ, eds. Heart Rate Variability. Armonk, NY: Futura, 1995:75–86. Malik M. Effect of electrocardiogram recognition artifact in time-domain measurement of heart rate variability. In: Malik M, Camm AJ, eds. Heart Rate Variability. Armonk, NY: Futura Press, 1995:99–118. Ewing DJ, Neilson JMM, Travis P. New method for assessing cardiac parasympathetic activity using 24-hour electrocardiograms. Br Heart J 1984; 52:396–402 Kleiger RE, Stein PK, Bosner MS, Rottman JN. Time domain measurement of heart rate variability. Cardiol Clin 1992; 10:487–498 Bigger JT Jr., Kleiger RE, Fleiss JL, Rolnitzky LM, Steinman RC, Miller JP. Components of heart rate variability measured during healing of acute myocardial infarction. Am J Cardiol 1988; 61:208–215. Malik M, Farrell T, Cripps TR, Camm AJ. Heart rate variability in relation to prognosis after myocardial infarction: selection of optimal processing techniques. Eur Heart J 1989; 10:1060–1074. Woo MA, Stevenson WG, Moser DK, Middlekauff HR. Complex heart rate variability and serum norepinephrine levels in patients with advanced heart failure. J Am Coll Cardiol 1994; 23:565–569 Huikuri HV, Seppänen T, Koistinen MJ, Airaksinen KEJ, Ikäheimo MJ, Castellanos A, Myerburg RJ. Abnormalities in beat-to-beat dynamics of heart rate before the spontaneous onset of life-threatening ventricular tachyarrhythmias in patients with prior myocardial infarction. Circulation 1996; 93:1836–1844. Tulppo MP, Mäkikallio TH, Takala TES, Seppänen T, Huikuri HV. Quantitative beat-to-beat analysis of heart rate dynamics during exercise. Am J Physiol 1996; 271:H244–H252. Schechtman VL, Harper RK, Harper RM. Development of heart rate dynamics during sleep-waking states in normal infants. Pediatr Res 1993; 34:618–623. Braun C, Kowallik P, Freking A, Hadeler D, Kniffki C.D, Meesmann M. Demonstration of nonlinear components in heart rate variability of healthy person. Am J Physiol 1998; 275:H1577–H1584. Akselrod S, Gordon D, Ubel FA, Shannon DC, Barger MA, Cohen RJ. Power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control. Science 1981; 213:220–222. Peng CK, Havlin S, Stanley HE, Goldberger AL. Quantification of scaling exponents and crossover phenomena in nonstationary heartbeat time series. Chaos 1995; 5:82–87. Pincus SM, Goldberger AL. Physiologic time series analysis: What does regularity quantify? Am J Physiol 1994; 226:H1643–1656.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

50.

51.

52. 53.

54. 55.

56.

57.

58.

59. 60.

61.

62.

63. 64. 65.

Saul JP, Albrecht P, Berger RD, Cohen RJ. Analysis of long-term heart rate variability: methods, 1/f scaling and implications. In: Computers in Cardiology. Silver Spring, MD: IEEE Computer Society Press, 1987:419–422. Skinner JE, Pratt CM, Vybiral T. A reduction in the correlation dimension of heartbeat intervals precedes imminent ventricular fibrillation in human subjects. Am Heart J 1993; 125:731–743. Yamamoto Y, Hughson RL. Coarse-graining spectral analysis: new method for studying heart rate variability. J Appl Physiol 1991; 71(3):1143–1150. Mäkikallio TH, Hoiber S, Ko/ber L, Torp-Pedersen C, Peng C-K, Goldberger AL, Huikuri HV for the Trace Investigators. Fractal analysis of heart rate dynamics as a predictor of mortality in patients with depressed left ventricular function after acute myocardial infarction. Am J Cardiol 1999; 83:836–839. Mäkikallio TH, Koistinen MJ, Tulppo MP, et al. RR interval dynamics before the spontaneous onset of ventricular fibrillation. Am J Cardiol 1999; 83:880–884. Huikuri HV, Mäkikallio TH, Peng CK, Goldberger AL, Hintze U, Mo/ller M for the DIAMOND Study Group. Fractal correlation properties of R-R interval dynamics and mortality in patients with depressed left ventricular function after an acute myocardial infarction. Circulation 2000; 101:47–54. Mäkikallio TH, Ristimäe T, Airaksinen KEJ, et al. Heart rate dynamics in patients with stable angina pectoris and utility of fractal and complexity measures. Am J Cardiol 1998; 81:27–31. Huikuri HV, Mäkikallio TH, Airaksinen KEJ, Seppänen T, Puukka P, Räihä IJ, Sourander LB. Power-law relationship of heart rate variability as a predictor of mortality in the elderly. Circulation 1998; 97:2031–2036. Pikkujämsä SM, Mäkikallio TH, Sourander LB, Räihä IJ, Puukka P, Skyttä J, Peng CK, Goldberger AL, Huikuri HV. Cardiac interbeat interval dynamics from childhood to senescence: comparison of conventional and new measures based on fractals and chaos theory. Circulation 1999; 100:393–399. Pincus SM, Viscarello RR. Approximate entropy: a regularity statistic for fetal heart rate analysis. Obstet Gynecol 1992; 79:249–255. Mäkikallio TH, Seppänen T, Niemelä M, Airaksinen KEJ, Tulppo M, Huikuri HV. Abnormalities in beat-to-beat complexity of heart rate dynamics in patients with a prior myocardial infarction. J Am Coll Cardiol 1996; 28:1005–1011. Hogue CW Jr, Domitrovich PP, Stein PK, Despotis GD, Re L, Schuessler RB, Kleiger RE, Rottman JN. RR interval dynamics before atrial fibrillation in patients after coronary artery bypass graft surgery. Circulation 1998; 98:429–434. Bigger JT Jr, Steinman RC, Rolnitzky LM, et al. Power law behavior of RR-interval variability in healthy middle-aged persons, patients with recent acute myocardial infarction, and patients with heart transplants. Circulation 1996; 93:2142–2151. Korpelainen JT, Sotaniemi KA, Myllylä VV. Autonomic nervous system disorders in stroke. Clin Autonom Res 1999; 9:325–333. Korpelainen JT, Sotaniemi KA, Mäkikallio A, Huikuri HV, Myllylä VV. Dynamic behavior of heart rate in ischemic stroke. Stroke 1999; 30:1008–1013. Talman WT. Cardiovascular regulation and the lesions of the central nervous system. Ann Neurol 1985; 18:1–12.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

66.

67. 68.

69.

70. 71. 72.

73. 74. 75. 76. 77. 78. 79.

80. 81. 82.

83.

84.

Oppenheimer SM, Cechetto DF, Hachinski VC. Cerebrogenic cardiac arrhythmias: cerebral electrocardiographic influences and their role in sudden death. Arch Neurol 1990; 47:513–519. Oppenheimer SM, Hachinski VC. The cardiac consequences of stroke. Neurol Clin North Am 1992; 10:167–176. Broderick JP, Phillips SJ, O’Fallon M, Frye RL, Whisnant JP. Relationship of cardiac disease to stroke occurrence, recurrence and mortality. Stroke 1992; 23:1250–1256. Cropp GJ, Manning GW. Electrocardiographic changes simulating myocardial ischemia and infarction associated with spontaneous intracranial hemorrhage. Circulation 1969; 22:25–38. Weidler DJ. Myocardial damage and cardiac arrhythmias in patients with cerebrovascular accidents. Circulation 1969; 22:25–38. Mikolich JR, Jacobs WC, Fletcher GF. Cardiac arrhythmias in patients with acute cerebrovascular accidents. JAMA 1981; 246:1314–1317. Kono T, Morita H, Kuroiwa T, Onaka H, Takatsuka H, Fujiwara A. Left ventricular wall motion abnormalities in patients with subarachnoid hemorrhage: neurogenic stunned myocardium. J Am Coll Cardiol 1994; 24:636–640. Greenhoot JH, Reichenbach DD. Cardiac injury and subarachnoid hemorrhage, a clinical, pathological, and physiological correlation. J Neurosurg 1969; 80:521–531. Neil-Dwyer G, Walter P, Cruickshank JM. Effect of propranolol and phentolamine on myocardial necrosis after subarachnoid hemorrhage. Br J Med 1978; 2:990–992. Norris JW, Froggat GM, Hachinski VC. Cardiac arrhythmias in acute stroke. Stroke 1978; 9:392–396. Norris JW, Hachinski VC, Myers MG. Serum cardiac enzymes in stroke. Stroke 1979; 10:548–553. Valeriano J, Elson J. Electrocardiographic changes in central nervous system disease. Neurol Clin 1993; 11:257–272. Monga TK, Zimmermann KP. Autonomic nervous system function in stroke and traumatic head injury patients. Phys Med Rahabil State Art Rev 1996; 10:81–110. Adams HP, Brott TG, Crowell RM, Furlan AJ, Gomez CR, Helgason CM, Marler JR, Woolson RF, Zivin JA, Feinberg W, Mayberg M. Guidelines for the management of patients with acute ischemic stroke. Circulation 1994; 90:1588–1601. Lip GYH, Zarifis J, Farooqi IS, PageA, Sagar G, Beevers DG. Ambulatory blood pressure monitoring in acute stroke. Stroke 1997; 28:31–35. Morfis L, Schwarz RS, Poulos R, Howes LG. Blood pressure changes in acute cerebral infarction and hemorrhage. Stroke 1997; 28:1401–1405. Sander D, Klingelhofer J. Changes of circadian blood pressure patterns and cardiovascular parameters indicate lateralization of sympathetic activation following hemispheric brain infarction. J Neurol 1995; 242:313–318. Sander D, Klingelhofer J. Extent of autonomic activation following cerebral ischemia is different in hypertensive and normotensive humans. Arch Neurol 1996; 53:890–894. Robinson TG, James M, Youde J, Panerai R, Potter J. Cardiac baroreceptor sensitivity is impaired after acute stroke. Stroke 1997; 28:1671–1676.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

85.

86. 87.

88.

89. 90. 91. 92.

93.

94.

95.

96.

97. 98.

99.

100. 101.

Kukla C, Sander D, Schwarze J, Wittich I, Klingelhofer J. Changes of circardian blood pressure patterns are associated with the occurrence of lacunar infarction. Arch Neurol 1998; 55:683–688. Phillips AM, Jardine DL, Parkin PJ, Hughes T, Ikram H. Brain stem stroke causing baroreflex failure and paroxysmal hypertension. Stroke 2000; 31:1997–2001. Sander D, Winbeck K, Klingelhofer J, Conrad B. Extent of cerebral white matter lesions is related to changes of circadian blood pressure rhythmicity. Arch Neurol 2000; 57:1302–1307. Dawson SL, Evans SN, Mankletow BN, Fotherby MD, Robinson TG, Potter JF. Diurnal blood pressure change varies with stroke subtype in the acute phase. Stroke 1998; 29:1519–1524. Chesnut RM, Gautille T, Blunt BA, Klauber MR, Marshall LF. Neurogenic hypertension in patients with severe head injuries. J Trauma 1998; 44:958–963. Robinson TG, Potter JF. Postprandial and orthostatic cardiovascular changes in stroke. Stroke 1995; 26:1811–1816. Brambrink AM, Dick WF. Neurogenic pulmonary edema. Pathogenesis, clinical picture and therapy. Anaesthesist 1997; 46:953–963. Keegan MT, Lanier WL. Pulmonary edema after resection of a fourth ventricle tumor: possible evidence for a medulla-mediated mechanism. Mayo Clin Proc 1999; 74:264–268. Inobe JJ, Mori T, Ueyama H, Kumamoto T, Tsuda T. Neurogenic pulmonary edema induced by primary medullary hemorrhage: a case report. J Neurol Sci 2000; 172:72–76. Hamdy O, Nishiwaki K, Yajima M, Murakami HO, Maekawa H, Moy RT, Shimada Y, Hotta Y, Ishikawa N. Presence and quantification of neuropeptide Y in pulmonary edema fluids in rats. Exp Lung Res 2000; 26:137–147. Goldstein B, Kempski MH, DeKing D, Cox C, DeLOng DJ, Kelly MM, Woolf PD. Autonomic control of heart rate after brain injury in children. Crit Care Med 1996; 24:234–240. Goldstein B, Toweill D, Lai S, Sonnenthal K, Kimberly B. Uncoupling of the autonomic and cardiovascular systems in acute brain injury. Am J Physiol 1998; 275:R1287–1292. Klingelhofer J, Sander D. Cardiovascular consequences of clinical stroke. Clin Neurol 1997; 6:309–335. Monge Argiles JA, Palacios Ortega F, Vila Sobrino JA, Bautista Prados J, Perez Vicxente JA, Morales Ortiz A, Palao Sanchez A. Brainstem lesions decrease heart rate variability. Neurologia 2000; 15:158–163. Korpelainen JT, Sotaniemi KA, Huikuri HV, Myllylä VV. Circadian rhythm of heart rate variability is reversibly abolished in ischemic stroke. Stroke 1997; 28:2150–2154. Estanol BV, Loyo MV, Mateos JH. Cardiac arrhythmias in experimental subarachnoid hemorrhage. Stroke 1977; 8:440–447. Samuels MA. Introduction to neurocardiology. In: Mathias CJ, Bannister R, eds. Autonomic Failure. A Textbook of Clinical Disorders of the Autonomic Nervous System. 4th ed. New York: Oxford University Press, 1999: 421–427.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

102.

103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113.

114. 115. 116.

117.

118. 119. 120.

121.

Lacombe PM, MacKenzie ET. Catecholamines and their receptors. In: Welch KMA, Caplan LR, Reis DJ, Siesjö BK, Weir B, eds. Primer on Cardiovascular Diseases. San Diego: Academic Press, 1997:86–91. Benarroch EE. Central Autonomic Network: Functional Organization and Clinical Correlates. New York: Futura Publishing Company, 1977. Samuels MA. Nuerally induced cardiac damage: definition of the problem. Neurol Clin 1993; 11:273–292. Talman WT, Kelkar P. Neural control of the heart: central and peripheral. Neurol Clin 1993; 11:239–256. Oppenheimer SM, Kedem G, Martin WM. Left-insular cortex lesions perturb cardiac autonomic tone in humans. Clin Autonom Res 1996; 6:131–140. Chua HC, Sen S, Cosgriff RF, Gerstenblith G, Beauchamp NJ Jr, Oppenheimer SM. Neurogenic ST depression in stroke. Clin Neurol Neurosurg 1999; 101:44–48. Calne DB, Brennan J, Spiers ASD, Stern GM. Hypotension caused by L-dopa. Br Med J 1970; 1:474–475. McDowell FH, Lee JE. Levodopa, Parkinson’s disease, and hypotension. Ann Intern Med 1970; 72:751–752. Aminoff MJ, Wilcox CS. Assessment of autonomic function in patients with parkinsonian syndrome. Br Med J 1971; 4:80–84. Appenzeller O, Goss JE. Autonomic deficits in Parkinson’s syndrome. Arch Neurol 1971; 24:50–57. Gross M, Bannister R, Godwin-Austen R. Orthostatic hypotension in Parkinson’s disease. Lancet 1972; 1:174–176. Turkka JT. Correlation of the severity of autonomic dysfunction to cardiovascular reflexes and to plasma noradrenaline levels in Parkinson’s disease. Eur Neurol 1987; 26:203–210. Barbeau A, Gillo-Joffrey L, Boucher R, Nowaczynski W, Genest J. Renin-aldosterone system in Parkinson’s disease. Science 1969; 165:291–292. Reid JL, Calne DB. Cardiovascular effects of levodopa in parkinsonism. In: Calne DB, ed. Advances in Neurology. New York: Raven Press, 1973:223–232. Murata Y, Harada T, Ishizaki F, Izumi Y, Nakamura S. Autonomic dysfunction in Parkinson’s disease and vascular parkinsonism. Acta Neurol Scand 1997; 96:359–365. Camerlingo M, Ferraro B, Gazzaniga GC, Casto L, Cesana BM, Mamoli A. Cardiovascular reflexes in Parkinson’s disease: long-term effects of levodopa treatment on de novo patients. Acta Neurol Scand 1990; 81:346–348. Meco G, Pratesi L, Bonifati V. Cardiovascular reflexes and autonomic dysfunction in Parkinson’s disease. J Neurol 1991; 238:195–199. Van Dijk JG, Haan J, Koenderink M, Roos RAC. Autonomic nervous function in progressive supranuclear palsy. Arch neurol 1991; 48:1083–1084. Haapaniemi TH, Kallio MA, Korpelainen JT, Suominen K, Tolonen U, Sotaniemi KA, Myllylä VV. Levodopa, bromocriptine and selegiline modify cardiovascular responses in Parkinson’s disease. J Neurol 2000; 415:868–874. Sachs C, Berglund B, Kaijser L. Autonomic cardiovascular responses in parkinsonism: effect of levdodopa with dopa-decarboxylase inhibition. Acta Neurol Scand 1985; 71:37–42.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

122. 123.

124.

125. 126. 127.

128. 129.

130.

131.

132.

133. 134.

135. 136.

137.

138.

Ludin SM, Steiger UH, Ludin HP. Autonomic disturbances and cardiovascular reflexes in idiopathic Parkinson’s disease. J Neurol 1987; 235:10–15. Turkka J, Suominen K, Tolonen U, Sotaniemi KA, Myllylä VV. Selegiline diminishes cardiovascular autonomic responses in Parkinson’s disease. Neurology 1997; 48:662–667. Kuroiwa Y, Shimada Y, Toukura Y. Postural hypotension and low R-R interval variability in parkinsonism, spinocerebellar degeneration, and Shy-Drager syndrome. Neurology 1983; 33:463–467. Goetz CG, Lutge W, Tanner CM. Autonomic dysfunction in Parkinson’s disease. Neurology 1986; 36:73–85. Quinn L, Illas A, Lhermitte F, Agid Y. Bromocriptine in Parkinson’s disease: a study of cardiovascular effects. J Neurol Neurosurg Psychiatry 1981; 426–429. Greenacre JK, Teychenne PF, Petrie A, Calne DB, Leigh N, Reid JL. The cardiovascular effects of bromocriptine in parkinsonism. Br J Clin Pharmacol 1976; 3:571–574. Tanner CM, Goetz CG, Glantz RH, Glatt SL, Klawans HL. Pergolide mesylate and idiopathic Parkinson disease. Neurology 1982; 32:1175–1179. LeWitt PA, Ward CD, Larsen TA, Raphaelson MI, Newman RP, Foster N, Dambrosia JM, Calne DB. Comparison of pergolide and bromocriptine therapy in parkinsonism. Neurology 1983; 83:1009–1014. Kujawa K, Leurgans S, Taman R, Blasucci L, Goetz CG. Acute orthostatic hypotension when starting dopamine agonists in Parkinson’s disease. Arch Neurol 2000; 57:1461–1463. Churchyard A, Mathias CJ, Boonkongchuen P, Lees AJ. Autonomic effects of selegiline: possible cardiovascular toxicity on Parkinson’s disease. J Neurol Neurosurg Psychiatry 1997; 63:228–234. Mesec A, Sega S, Klauta T. The influence of the type, duration, severity and levodopa treatment of Parkinson’s disease on cardiovascular autonomic responses. Clin Auton Res 1993; 3:339–344. Mesec A, Sega S, Trost M, Pogacnik T. The deterioration of cardiovascular reflexes in Parkinson’s disease. Acta Neurol Scand 1999; 100:269–299. Mastrocola C, Vanacore N, Giovani A, Locuratolo N, Vella C, Alessandri A, Baratta L, Tubani L, Meco G. Twenty-four-hour heart rata variability to assess autonomic function in Parkinson’s disease. Acta Neurol Scand 1999; 99:245–247. Micieli G, Martignoni E, Cavallini A, Sandrini G, Nappi G. Postprandial and orthostatic hypotension in Parkinson’s disease. Neurology 1987; 37:386–393. Thomaides T, Bleasdale-Barr K, Cahudhuri KR, pavitt D, Marsden CD, Mathias CJ. Cardiovascular and hormonal responses to liquid food challenge in idiopathic Parkinson’s disease, multiple system atrophy, and pure autonomic failure: Neurology 1993; 43:900–904. Ieda T, Hirayama M, Koike Y, Hasegawa Y, Hakusui S, Takahashi A. Heodynamic study on postprandial hypotension in patients with Parkinson’s disease having autonomic failure (abstr). In: 11th Int Symp Parkinson’s disease, Rome 1994, p. 157. Niimi Y, Ieda T, Hirayama M, Koike Y, Sobue G, Hasegawa Y, Takahashi A. Clinical and physiological characteristics of autonomic failure in Parkinson’s disease. Clin Auton Res 1999; 9:139–144.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

139. 140.

141. 142. 143. 144.

145.

146. 147. 148.

149. 150. 151.

152. 153.

154. 155. 156. 157.

Goldstein DS, Holmes C, Li ST, Bruce S, Metman L, Cannon III RO. Cardiac sympathetic denervation in Parkinson’s disease. Ann Intern Med 2000; 133:338–347. Reinhardt MH, Jüngling FD, Krause TM, Braune S. Scintigraphic defferentiation between two forms of primaty dysautonomia early after onset of autonomic dysfunction: value of cardiac and pulmonaty iodine-123 MIBG uptake. Eur J Nucl Med 1999; 27:595–600. Jellinger KA. Post mortem studies in Parkinson’s disease—Is it possible to detect brain areas for spesific symptoms? J Neural Transm 1999; 56(suppl):S1–29. Wakabayashi K, Takahashi H. Neuropathology of autonomic nervous system in Parkinson’s disease. Eur Neurol 1997; 38(suppl):S2–7. Jaros E, Burn DJ. The pathogenesis of multiple system atrophy: past, present, and future. Mov Dis 2000; 15:784–788. Wenning GK, Shlomo YB, Magalhaes M, Daniel SE, Quinn NP. Clinical features and natural history of multiple system atrophy an analysis of 100 cases. Brain 1994; 117:835–845. Gilman S, Low P, Albanese A, Ben-Shlomo Y, Fowler C, Kaufmann H, Klockgether T, Lang A, Lantos P, Litvan I, Mathias C, Oliver E, Robertson D, Schazt I, Wenning G. Consensus statement on the diagnosis of multiple system atrophy. American Autonomic Society and American Academy of Neurology. Clin Auton Res 1998; 8:359–362. Kaufmann H. Multiple system atrophy. Curr Opin Neurol 1998; 11:351–355. Low PA, Bannister SR. Multiple system atrophy and pure autonomic failure. Clin Auton Disord 1997; 555–575. Smith GD, Watson LP, Pavitt DV, Mathias CJ. Abnormal cardiovascular and catecholamine responses to supine exercise in human subjects with sympathetic dysfunction. J Physiol 1995; 484:255–265. Wenning GK, Tison F, Shlomo B, Daniel SE, Quinn NP. Multiple system atrophy: a review of 203 pathologically proven cases. Move Disord 1997; 12:133–147. Bannister R, Oppenheimer DR. Degenerative diseases of the nervous system associated with autonomic failure. Brain 1972; 95:457–474. Teasell RW, Arnold JM, Krassioukov A, Delaney GA. Cardiovascular consequences of loss of supraspinal control of the sympathetic nervous system after spinal cord injury. Arch Phy Med Rehabil 2000; 8:506–516. Atkinson PP, Atkinson JL. Spinal shock. Mayo Clin Proc 1996; 71:384–389. Mathias CJ, Frankel HL. Autonomic disturbances in spinal cord lesions. In: Bannister R, Mathias CJ, eds. Autonomic Failure. 3rd ed. Oxford: Oxford University Press, 1992:839–881. Yamamoto M, Meyer JS, Sakai F, Jakoby R. Effect of differential spinal cord transection on human cerebral blood flow. J Neurol Sci 1980; 47:395–406. Rosner MJ, Elias Z, Coley I. New principles of resuscitation for brain and spinal injury. N C Med J 1984; 45:701–706. Dollfus P, Frankel HL. Cardiovascular reflexes in tracheostomised tetraplegics. Paraplegia 1965; 2:227–235. Frankel HL, Michaelis LS, Golding DR, Beral V. Blood pressure in paraplegia. I. Paraplegia 1972; 10:193–198.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

158.

159.

160.

161. 162.

163. 164.

165. 166.

167. 168.

169.

170.

171. 172. 173. 174.

Mathias CJ, Christensen NJ, Corbett JL, Frankel HL, Spalding JMK. Plasma catecholamines during paroxysmal neurogenic hypertension in quadriplegic man. Circ Res 1976; 39:204–208. Gonzalez F, Chang JY, Banovac K, Messina D, Martinez-Arizala A, Kelley RE. Autoregulation of cerebral blood flow in patients with orthostatic hypotension after spinal cord injury. Paraplegia 1991; 29:1–7. Munakata M, Kameyama J, Kanazawa M, Nunokawa T, Moriai N, Yoshinaga K. Circadian blood pressure rhythm in patients with higher and lower spinal cord injury: simultaneous evaluation of autonomic nervous system activity and physical activity. J Hypertens 1997; 15:1745–1749. Coutts KD, Rhodes EC, McKenzie DC. Maximal exercise responses of tetraplegics and paraplegics. J Appl Physiol 1983; 55:479–482. Eriksson P, Lofsröm L, Ekblom B. Aerobic power during maximal exercise in untrained and well-trained persons with quadriplegia and paraplegia. Scand J Rehabil Med 1988; 20:141–147. Nitsche B, Perschak H, Dietz V. Loss of circadian blood pressure variability in complete tetraplegia. J Human Hypertens 1996; 10:311–317. Grimm DR, Meersman RE, Almenoff PL, Spungen AM, Bauman WA. Sympathovagal balance of the heart in subjects with spinal cord injury. Am J Physiol 1997; 272:H835–842. Bunten DC, Warner AL, Brunnemann SR, Segal JL. Heart rate variability is altered following spinal cord injury. Clin Auton Res 1998; 8:329–334. Wang YH, Huang TS, Lin JL, Hwang JJ, Chan HL, Lai JS, Tseng YZ. Decreased autonomic nervous system activity as assessed by heart rate variability in patients with chronic tetraplegia. Arch Phys Med Rehabil 2000; 81:1181–1184. Colachis SC. Autonomic hyperreflexia with spinal cord injury. J Am Paraplegia Soc 1992; 15:171–186. Harati Y. Autonomic disorders associated with spinal cord injury. In: Low PA, ed. Clinical Autonomic Disorders. 2nd ed. Philadelphia: Lippincott-Raven Publishers, 1997:455–461. Arnold JM, Feng QP, Delaney GA, Teasell RW. Autonomic dysreflexia in tetraplegic patients: evidence for alpha-adrenoceptor hyper-responsiveness. Clin Autonom Res 1995; 5:267–270. Teasell RW, Arnold JMO, Delaney GA. Sympathetic nervous system dysfunction in high-level spinal cord injuries. Phys Med Rehabil State Art Rev 1996; 10:37–60. Braddon RL, Rocco JF. Autonomic dysreflexia. A survey of current treatment. Am J Phys Med Rehabil 1991; 70:234–241. McLeod JG. Invited review: autonomic dysfunction in peripheral nerve disease. Muscle Nerve 1992; 15:3–13. Zochodne DW. Autonomic involvement in Guillain-Barré syndrome: a review. Muscle Nerve 1994; 17:1145–1155. Singh NK, Jaiswal AK, Misra S, Srivastava PK. Assessment of autonomic dysfunction in Guillain-Barré syndrome and its prognostic implications. Acta Neurol Scand 1987; 75:101–105.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

175.

176. 177. 178. 179. 180. 181.

182.

183.

184.

185. 186.

187. 188.

189.

190.

191.

192.

Flachenecker P, Wermuth P, Hartung HP, Reiners K. Quantitative assessment of cardiovascular autonomic function in Guillain-Barré syndrome. Ann Neurol 1997; 42:171–179. Tuck HR, McLeod JG. Autonomic dysfunction in Guillain-Barré syndrome. J Neurol Neurosurg Psychiatry 1981; 44:983–990. Winer JB, Hughes RA. Identification of patients at risk of arrhythmia in the Guillain-Barré syndrome. QJM 1988; 68:735–739. Robber AH. The Guillain-Barré syndrome. N Engl J Med 1992; 326:1130–1136. Fagius J, Wallin G. Microneurographic evidence of excessive sympathetic outflow in the Guillain-Barré syndrome. Brain 1983; 106:589–600. Persson A, Solders G. R-R variations in Guillain-Barré syndrome: a test of autonomic dysfunction. Acta Neurol. Scand 1983; 67:294–300. Flachenecker P, Hartung HP, Reiners K. Power spectrum analysis of heart rate variability in Guillain-Barré syndrome. A longitudinal study. Brain 1997; 120:1885–1894. Annane D, Baudrie V, Blanc AS, Laude D, Raphael JC, Elghozi JL. Short-term variability of blood pressure and heart rate in Guillain-Barré syndrome without respiratory failure. Clin Sci 1999; 96:613–621. Neil HAW. The epidemiology of diabetic autonomic neuropathy. In: Bannister R, Mathias CJ, eds. Autonomic Failure. 3rd ed. Oxford: Oxford University Press, 1992:682–697. Töyry JP, Partanen JV, Niskanen LK, Länsimies EA, Uusitupa MI. Divergent development of autonomic and peripheral somatic neuropathies in NIDDM. Diabetologia 1997; 40:953–958. Orchard TJ, Lloyd CE, Maser RE, Kuller LH. Why does diabetic autonomic neuropathy predict IDDM mortality? Diabetes Res Clin Practice 1996; 34:S165–171. Mulder DW. The neuropathies associated with diabetes mellitus: a clinical and electromyographic study of 103 unselected diabetic patients. Neurology 1961; 11:275–284. Low PA, Walsh JC, Huang C-Y, McLeod JG. The sympathetic nervous system in diabetic neuropathy—a clinical and pathological study. Brain 1975; 98:341–356. Veglio M. Autonomic neuropathy in non-insulin-dependent diabetic patients: correlation with age, sex, duration and metabolic control of diabetes. Diabetes Metab 1990; 16:200–206. Ewing DJ, Borsey DQ, Bellavere F, Clarke BF. Cardiac autonomic neuropathy in diabetes: comparison of measures of R-R interval variation. Diabetologia 1981; 21:18–24. Lishner M, Akselrod S, Mor Avi V, Oz O, Divon M, Ravid M. Spectral analysis of heart rate fluctuations. A non-invasive, sensitive method for the early diagnosis of autonomic neuropathy in diabetes mellitus. J Auton Nerv Syst 1987; 19:119–125. Pagani M, Malfatto G, Pierini S, Casati R, Masu AM, Poli M, Guzzetti S, Lombardi F, Cerutti S, Malliani A. Spectral analysis of heart rate variability in assessment of autonomic diabetic neuropathy. J Auton Nerv Syst 1988; 23:143–153. Ewing DJ, Neilson JM. QT interval length and diabetic autonomic neuropathy. Diabet Med 1990; 7(1):23–26.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

193.

194.

195.

196. 197.

198.

199. 200.

201. 202.

203.

204.

205.

206.

207.

Ewing DJ, Neilson JMM, Shapiro CM, Reid W. Twenty four hour heart rate variability: effects of posture, sleep, and time of day in healthy controls and comparison with bedside tests of autonomic function in diabetic patients. Br Heart J 1991; 65:239–244. Freeman R, Saul P, Roberts MS, Berger RD, Broadbridge C, Cohen RJ. Spectral analysis of heart rate in diabetic autonomic neuropathy. Arch Neurol 1991; 48:185–190. Kleiger RE, Miller JP, Bigger JT, Moss AJ, the Multicenter Post-infarction Research Group. Decreased heart rate variability and its association with increased mortality after myocardial infarction. Am J Cardiol 1987; 59:256–262. Airaksinen KEJ, Ikäheimo MJ, Linnaluoto MK, Niemelä M, Takkunen JT. Impaired vagal heart rate control in coronary artery disease. Br Heart J 1987; 58:592–597. Hayano J, Sakakibara Y, Yamada A, Ohte N, Fujinami T, Yokoyama K, Watanabe Y, Takata K. Decreased magnitude of heart rate spectral components in coronary artery disease. Its relation to angiographic severity. Circulation 1990; 81:1217–1224. Rich MW, Saini JS, Keiger RE, Carney RM, teVelde A, Freedland KE. Correlation of heart rate variability with clinical and angiographic variables and late mortality after coronary angiography. Am J Cardiol 1988; 62:714–717. Minisi AJ, Thames MD. Effect of chronic myocardial infarction on vagal cardiopulmonary baroreflex. Circ Res 1989; 65:396–405. Airaksinen KEJ, Niemelä MJ, Ikäheimo MI, Huikuri HV, Linnaluoto MK, Takkunen JT. Effect of coronary arterial occlusion on vagal control of heart rate. Int J Cardiol 1991; 30:269–274. Schneider RA, Costiloe JP. Relationship of sinus arrhythmia to age and its prognostic significance in ischemic heart disease. Clin Res 1965; 13:219. Bigger JT Jr., Fleiss JL, Steinman RC, Rolnitzky LM, Kleiger RE, Rottman JN. Frequency domain measures of heart period variability and mortality after myocardial infarction. Circulation 1992; 85:164–171. Pipilis A, Flather M, Ormerod O, Sleight P. Heart rate variability in acute myocardial infarction and its association with infarct site and clinical course. Am J Cardiol 1991; 67:1137–1139. Valkama JO, Huikuri HV, Airaksinen KEJ, Linnaluoto MK, Takkunen JT. Determinants of frequency domain measures of heart rate variability in the acute and convalescent phases of myocardial infarction. Cardiovasc Res 1994; 28:1273–1276. Bigger JT Jr., Fleiss JL, Rolnitzky LM, Steinman RC, Schneider WJ. Time course of recovery of heart period variability after myocardial infarction. J Am Coll Cardiol 1991; 18:1643–1649. Flapan AD, Wright RA, Nolan J, Neilson JMM, Ewing DJ. Differing patterns of cardiac parasympathetic activity and their evolution in selected patients with a first myocardial infarction. J Am Coll Cardiol 1993; 21:926–931. Lombardi F, Sandrone G, Mortara A, Torzillo D, La Rovere MT, Signorini MG, Gerutti S, Malliani A. Linear and nonlinear dynamics of heart rate variability after acute myocardial infarction with normal and reduced left ventricular ejection fraction. Am J Cardiol 1996; 77:1283–1288.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

208.

Van de Borne P, Montano N, Pagani M, Oren R, Somers VK. Absence of low-frequency variability of sympathetic activity in severe heart failure. Circulation 1997; 95:1449–1454. 209. Wolf MM, Varigos GA, Hunt D, Sloman JG. Sinus arrhythmia in acute myocardial infarction: two-year follow-up. Med J Aust 1978; 2:52–53. 210. Huikuri HV, Mäkikallio T, Airaksinen KEJ, Mitrani R, Castellanos A, Myerburg RJ. Measurement of heart rate variability: a clinical tool or a research toy? J Am Coll Cardiol 1999; 34:1878–1883.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

12 Uremic Cardiac Autonomic Neuropathy Clinical Evaluation with Heart Rate Variability and Metaiodobenzylguanidine Chinori Kurata Yamaha Health Care Center, Hamamatsu, Japan

Uremic cardiac autonomic nervous system alterations have not been well described, although it is as remarkable as cardiac autonomic neuropathies observed in diabetes mellitus and congestive heart failure. Heart rate variability (HRV), one of the most promising markers of cardiac autonomic activity, has been shown to be markedly decreased in most patients with chronic renal failure (CRF). In CRF patients, a few studies have reported the prognostic value of HRV as well as several variables responsible for low HRV, both of which need to be examined by more studies in a larger sample size. Hemodialysis (HD) may remove some metabolic agents interfering with HRV, but there are not a few patients on maintenance HD who show markedly reduced HRV. On the other hand, 123Imetaiodobenzylguanidine (MIBG) has been recently developed to image cardiac sympathetic innervation. Because myocardial MIBG kinetics reflects the reuptake, storage, and release of norepinephrine in cardiac presynaptic sympathetic nerves, mainly in the left ventricular myocardium, HRV and MIBG variables reflect different aspects, that is, sinus node versus left ventricular myocardium and postsynaptic versus presynaptic side. Several studies have demonstrated markedly rapid MIBG clearance from the heart in CRF patients without diabetes mellitus, coronary artery disease, and heart failure. Thus, the abnormalities in HRV and MIBG observed in previous studies suggest that uremic autonomic neuropathy is characterized by sympathetic overactivity with parasympathetic nervous system dysfunction, which are common in congestive heart failure. Furthermore, several Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

investigators have demonstrated that kidney transplantation improves HRV and MIBG variables in most patients with CRF on dialysis. Observations about possible improvement in HRV and MIBG measures by renal transplantation may afford excellent insight into not only the pathophysiology of uremic cardiac autonomic neuropathy, but also the recovery process of cardiac autonomic nervous system in patients with congestive heart failure.

I. UREMIC AUTONOMIC NEUROPATHY Patients with chronic renal failure frequently have cardiovascular complications such as hypertension, myocardial hypertrophy, decreased left ventricular contractility, persistent hypotension, or dialysis-induced hypotension. Earlier studies (1–4) suggested that these complications may be associated with uremic cardiovascular autonomic neuropathy. For example, increased sympathetic activity contributes to hypertension in patients with CRF (5,6), while cardiovascular sympathetic nervous system function may be damaged in some CRF patients (1,2,4,7,8). Although uremic cardiac autonomic neuropathy is as remarkable as cardiac autonomic neuropathies observed in diabetes mellitus and heart failure (9), the clinical characteristics of cardiac autonomic nervous system alterations have not been well described in CRF patients. Uremic patients frequently complain of orthostatic hypotension, impotence, bowel dysfunction (diarrhea or constipation), impaired sweating, bladder problems, and so on. For an examination of uremic autonomic neuropathy, cardiovascular autonomic function has been clinically evaluated by using traditional autonomic tests, for example, heart rate and blood pressure responses to head-up tilt, standing, the Valsalva maneuver, and sustained handgrip, as well as beat-to-beat variations during quiet or deep breathing (2,10). However, the sensitivity and reproducibility of the traditional autonomic tests are insufficient (11–14), and more sensitive and reliable tests are needed for evaluation of cardiac autonomic neuropathy (see also Chapter 11). The precise pathogenesis of uremic autonomic neuropathy remains unknown, although several mechanisms have been suggested (5,15–17). Furthermore, inconsistent findings have been reported about the effects of hemodialysis, continuous ambulatory peritoneal dialysis, and kidney transplantation on uremic autonomic neuropathy (3,4,18–20). In regard to uremic sympathetic neuropathy, many investigators (4,5,21) reported an increase in plasma catecholamine concentrations in CRF patients, although it has been controversial whether plasma catecholamines are elevated or not in these patients (22,23). Reduced end-organ response to norepinephrine may be one of the factors underlying sympathetic nervous system abnormalities in uremic patients (4,7). Furthermore, it has been re-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ported that sympathetic activation observed in uremic patients may be mediated by an afferent signal arising in the failing kidneys rather than by high plasma norepinephrine concentrations (5).

II. HEART RATE VARIABILITY IN UREMIC CARDIAC AUTONOMIC NEUROPATHY Heart rate variability is one of the most promising markers of cardiac autonomic activity (24). In particular, the frequency-domain measures of HRV obtained by power spectral analysis reflect cardiac autonomic modulation: the high-frequency (HF) component reflects vagal activity, the low-frequency (LF) component (expressed in normalized units) is a marker of sympathetic modulation, and the LF/HF ratio is considered to mirror sympathovagal balance (24,25). Reduced HRV in CRF patients on conservative management, HD, or continuous ambulatory peritoneal dialysis has been demonstrated by a large number of studies (2,14,16,17,21,26–36). In uremic patients, Axelrod et al. (27) observed a strong reduction in all the frequency ranges of the power spectrum of HRV, both sympathetically and parasympathetically mediated, and Cloarec-Blanchard et al. (29) reported reduced short-term HRV and blood pressure variability. In addition, a marked decrease in the time-domain and frequency-domain measures of 24-hour HRV has been shown in CRF patients (14,17,21,28,36), and the abnormality of 24-hour HRV observed in some patients with end-stage renal failure may be the most severe in a general clinical setting (9). Some of CRF patients on HD show normal HRV measures comparable to healthy controls, although most of them show extremely abnormal HRV measures (21). Factors responsible for low HRV in uremia have not been established, but several variables have been reported to be associated with low HRV. Tamura et al. (15) investigated the determinants of 24-hour HRV (the standard deviation of RR intervals) in HD patients and found that hematocrit, body mass index before HD, and duration of HD, in addition to previously reported variables (age, diabetes mellitus, and smoking), were associated with HRV. In HD patients, however, Zoccali et al. (37) demonstrated that there was no relationship between duration of HD and HRV during deep breathing and that decreased HRV did not change in 13 HD patients who were reexamined after an interval of 4 years. In dialysis patients, Thomson et al. (28) found no significant correlation between duration of dialysis and the number of pairs of adjacent RR intervals different by more than 50 msec over the 24-hour period (an indicator of cardiac parasympathetic activity). Moreover, Roger et al. (38) reported that HRV during deep respiration did not change after correction of anemia with erythropoietin in 15 dialysis patients. We also observed that 24-hour HRV did not significantly correlate with

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

hematocrit and duration of dialysis in 44 HD patients (21). On the other hand, Steinberg et al. (16) demonstrated that the predictor variables of cardiac diseases—diabetes mellitus, hypertension, heart rate, age, gender, and smoking—explained only a small portion of markedly decreased HRV in uremic patients and suggested that uremia is an independent factor for decreased HRV. HRV may have prognostic significance in CRF patients, as in patients with cardiac disorders (24,39–42). Up to now, however, only a few reports have been published on the prognostic value of HRV in CRF patients. Hathaway et al. (43) measured 24-hour HRV in 278 patients awaiting kidney transplantation, 5 of whom experienced sudden cardiac death within 6 months of HRV measurements. They concluded that the standard deviation of all RR intervals during the 24-hour period holds the promise of identifying patients at increased risk for early death. Through a prospective follow-up of 31 HD patients, Hayano et al. (44) indicated that 24-hour HRV has an independent prognostic value in patients on maintenance HD and identifies an increased risk for all-cause and sudden death. However, more studies in a larger sample size are necessary for confirmation of the prognostic value of HRV in CRF patients.

III. EFFECTS OF HD ON HRV The adequacy of HD efficiency may be one factor affecting HRV in HD patients (20). Forsström et al. (26) evaluated HRV immediately before and after HD in patients on maintenance HD and showed an increase in HRV after HD, suggesting that some metabolic agents interfering with HRV are removed by HD. Vita et al. (45) observed the recovery of autonomic neuropathy (including HRV during deep breathing) following long-term bicarbonate HD and postulated that hypoxemia in HD patients may play a role in autonomic neuropathy. Furthermore, Rubinger et al. (17) demonstrated that reduction in 24-hour HRV was remarkable in HD patients with systemic amyloidosis and that renal transplantation normalized abnormal HRV in most HD patients, suggesting that the reduced HRV is caused by humoral factors reversed by the normalization of the renal function. The low LF/HF ratio of HRV may be associated with persistent hypotension in HD patients, and the apparent sympathetic dysfunction could result from a reduction in cardiovascular responsiveness to sympathetic stimulation (7). On the other hand, Barnas et al. (46) demonstrated that the HF power of HRV decreased and the LF power and LF/HF ratio increased up to the moment of HD-induced hypotension, but that the LF power decreased, the HF power increased, and the LF/HF ratio fell markedly at the time of hypotension. Pelosi et al. (8) also indicated that HD-induced hypotension may be associated with a deficit of increase in

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

LF/HF ratio of HRV, an index of sympathetic activity, during the late phase of HD.

IV.

123

I-METAIODOBENZYLGUANIDINE SCINTIGRAPHY

123

I-Metaiodobenzylguanidine, a radioiodinated analog of guanethidine (Fig. 1), has been developed to image cardiac sympathetic innervation (47,48). Similar to norepinephrine, MIBG enters adrenergic cells, is stored in vesicles, and is secreted (47–55) (Fig. 2).* Through the postganglionic presynaptic uptake of MIBG, scintigraphy with MIBG functions as a noninvasive method for the in vivo evaluation of the sympathetic activity. Myocardial scintigraphy with MIBG can provide indices of adrenergic neuron integrity and function in the heart, and myocardial MIBG uptake and the clearance rate of MIBG from the myocardium may reflect cardiac sympathetic innervation and nerve activity, respectively (48,50,56–58). *Some views on MIBG are not unanimous; see Chapter 9.

Figure 1 Structures of norepinephrine, guanethidine, and 123I-metaiodobenzylguanidine (MIBG).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Uptake, store, and release of MIBG: 1, specific neuronal uptake (uptake 1 by norepinephrine transporter) (49,50); 2, vesicular storage (51,52); 3, exocytotic release (48); 4, spillover to the circulating blood; 5, nonspecific neuronal uptake (passive diffusion) (48–50); 6, transport via uptake 2 (nonneuronal uptake and release) (47,50,53); 7, nonexocytotic release (via uptake-1 carrier) (54); 8, not bound to adrenergic receptors (51); 9, metabolized neither by monoamine oxidase nor by catechol-O-methyltransferase (47,55).

Table 1 MIBG Studies Utilized in Evaluation of Cardiac Sympathetic Nervous System Disorder Ischemic heart disease Cardiomyopathy Congestive heart failure Arrhythmia Hypertension Diabetes mellitus Transplanted heart Parkinson’s disease

Ref. 57,59–61 56,62–65 66–70 71–74 75–77 78–84 62,85–87 88–91

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Actually, MIBG myocardial scintigraphy has been utilized for evaluation of cardiac sympathetic nervous system in patients with various cardiac and autonomic disorders (56,57,59–91) (Table 1).

V. HRV VERSUS MIBG Both HRV and MIBG are of great clinical value because they have been demonstrated to provide prognostic information. Many investigators have already reported that low HRV is associated with increased risk for sudden death (39), allcause death (40), death in patients with heart failure (41), and coronary heart disease and death (42). On the other hand, the prognostic value of MIBG scintigraphy has been demonstrated in only a few reports. Low cardiac MIBG uptake in the late image has been reported to be associated with mortality in patients with congestive heart failure (66) and with dilated cardiomyopathy (65), cardiac death in patients with and without heart failure (68), and death or heart transplantation in patients with heart failure (70). The LF component and/or normalized LF of HRV may be a marker of sympathetic modulation on the sinus node, and the LF/HF ratio may be a marker of sympathovagal balance or sympathetic modulation (24,25). Cardiac sympathetic neuropathy as well as parasympathetic neuropathy, therefore, may be assessed with HRV analysis. However, Kingwell et al. (92) reported that both the LF power and normalized LF of HRV were not directly related to cardiac norepinephrine spillover measured using 3H-norepinephrine, a more direct measure of the sympathetic nerve-firing rate. In healthy subjects, also, none of the HRV measures including normalized LF power and LF/HF ratio comprehensively and consistently described the changes in autonomic balance (93). In dogs with healed myocardial infarctions, moreover, neither the LF component of HRV nor its normalized unit reflects changes in cardiac sympathetic activity (94). On the other hand, spectral analysis of simultaneous recordings of muscle sympathetic nerve activity and RR interval in patients with severe heart failure demonstrated that the marked sympathetic excitation was associated with reductions in LF component, normalized LF, and LF/HF ratio (95). Thus, the power spectral analysis of HRV does not provide a reliable measurement of cardiac sympathetic activity (96,97), and MIBG scintigraphy may provide information on the cardiac sympathetic nerve system that cannot be obtained from HRV analysis. Comparative studies of HRV and MIBG have been performed in healthy controls and patients with various disorders, but their findings are not uniform. The lack of correlation between MIBG and HRV measures was reported in diabetic patients (78) and in patients with recurrent reentrant tachycardia who underwent catheter ablation (98). On the other hand, Mantysaari et al. (59) reported that lower HRV during deep breathing was associated with larger MIBG defect size

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

within viable myocardium in 12 patients after myocardial infarction. We also observed that parasympathetic HRV parameters such as HF power had a negative correlation with severity of MIBG defect in the late image in 64 diabetic patients (83). Similarly, myocardial MIBG uptake was reported to correlate with HRV (triangular index) in 12 patients with dilated cardiomyopathy (64). Furthermore, in 211 patients with various disorders, we observed that the HF power of 24-hour HRV correlated positively with cardiac MIBG uptake in the late image and negatively with MIBG clearance rate from the heart, although these correlation coefficients were small (99). On the whole, abnormal MIBG measures may be associated with reduced HF component of HRV, probably because of the coexistence of sympathetic overactivity with parasympathetic dysfunction. Regarding the LF component of HRV, Morozumi et al. (100) studied the relationship between myocardial MIBG uptake and HRV in 15 normal subjects. They showed that the normalized LF of HRV correlated negatively with heart/mediastinum MIBG uptake ratio in the planar image obtained at 240 minutes after MIBG injection and positively with the washout rate of MIBG in the heart. However, it is possible that removal of only two subjects from their study population invalidates the correlation. Somsen et al. (67) also demonstrated that in 10 patients with mild to moderate heart failure, cardiac MIBG washout positively correlates with normalized LF power of 24-hour HRV, but the correlation may depend on only 2 patients and therefore might be accidental. In contrast, Miyanaga et al. (79) demonstrated a weak but significant inverse correlation between normalized LF power of HRV and severity of myocardial MIBG defect in diabetic patients and controls. Langer et al. (80) also mentioned an inverse relation between LF power of HRV and severity of regional MIBG defect (MIBG mismatch with perfusion) in 23 normal subjects and 65 asymptomatic diabetic patients, although they did not describe how they recorded and analyzed electrocardiograms. Similarly, Murata et al. (81) showed that the LF component of HRV correlated positively with regional myocardial MIBG uptake in the late image and negatively with MIBG washout rate from the heart in 42 diabetic patients. Delahaye et al. (101) also reported an inverse correlation between the LF component of HRV during the day and the washout rate of MIBG from the heart in 17 patients with familial amyloid polyneuropathy. Moreover, we observed that like the HF power, the LF power of HRV correlated positively with cardiac MIBG uptake in the late image and negatively with MIBG clearance rate from the heart in 211 patients with various disorders (99). We have also demonstrated that MIBG clearance from the heart increased and HRV, including normalized LF power, decreased with progress of congestive heart failure, although the HRV and MIBG measures did not similarly change in proportion to the severity of cardiac autonomic dysfunction (69). Thus, all of these studies indicate that reduction in LF power is associated with MIBG abnormalities; that is, a reduction in LF component of HRV may be associated with rapid MIBG washout from the heart and het-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

erogeneous myocardial MIBG distribution. However, the mechanism of the association is a matter of speculation: both changes in LF power and MIBG may reflect cardiac sympathetic overactivity, or the reduced LF power may merely reflect diminished parasympathetic modulation. From a different point of view, the above-mentioned weak correlation between MIBG and HRV variables suggests that these two indices explore different aspects of cardiac autonomic nervous system integrity. HRV represents an end-organ response localized in the sinus node, which is determined by several steps such as nerve firing, cardiac adrenergic receptor sensitivity, and postsynaptic signal transduction. On the other hand, myocardial MIBG kinetics reflects the reuptake, storage, and release of norepinephrine in cardiac presynaptic sympathetic nerves, mainly in the left ventricular myocardium (Fig. 2). Thus, HRV and MIBG variables reflect different aspects, that is, sinus node versus left ventricular myocardium and postsynaptic versus presynaptic side. The combination of HRV and MIBG scintigraphy, therefore, may allow a comprehensive assessment of both presynaptic and postsynaptic aspects of cardiac sympathetic nerve system. In regard to diagnostic ability, MIBG scintigraphy may be more sensitive than HRV analysis in some disorders. For example, Druschky et al. (102) reported that the heart/mediastinum ratio of MIBG uptake was significantly reduced in 40 patients with an early stage of amyotrophic lateral sclerosis but that HRV variables including LF component did not differ between the patients and controls. They also demonstrated that not HRV analysis but MIBG scintigraphy could differentiate between Parkinson’s disease and multiple system atrophy in early disease stages (90). Furthermore, Delahaye et al. (101) showed that in patients with familial amyloid polyneuropathy, cardiac MIBG uptake was dramatically decreased while HRV showed a considerable scatter of values. The difference in diagnostic ability may depend not only on that in sensitivity between HRV and MIBG measures, but also on that in subject for evaluation with HRV and MIBG measures.

VI. MIBG FINDINGS IN UREMIC CARDIAC AUTONOMIC NEUROPATHY Only a few reports (21,31,103) have been published on the utilization of MIBG scintigraphy for evaluating uremic sympathetic neuropathy. We studied myocardial accumulation and clearance of MIBG in 10 CRF patients on dialysis without diabetes mellitus and coronary artery disease (103). The early (15 minutes) myocardial uptake of MIBG in patients was similar to that in controls, but the clearance rate of MIBG from the heart was significantly higher and the late (150 minutes) myocardial uptake was significantly lower in patients. In addition, these changes were striking in those with left ventricular dysfunction or hypertrophy,

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

and kidney transplantation improved the clearance rate of MIBG in one patient with severe ventricular failure and in another with septal hypertrophy. Similarly, in 11 CRF patients with autonomic dysfunction (symptoms of orthostatic hypotension, hypotension during HD, or anhidrosis), Miyanaga et al. (31) demonstrated decreased heart/mediastinum MIBG ratio with rapid cardiac clearance and abnormal myocardial MIBG distribution in both initial (15 minutes) and delayed (4 hours) images. In 44 CRF patients on HD, moreover, we described slow MIBG clearance from the lung and heterogeneous myocardial MIBG distribution, as well as rapid MIBG clearance from the heart (21), and found that these MIBG variables were similar between 26 patients without and 18 with hypertension. The studies above have demonstrated that uremic cardiac sympathetic neuropathy may be characterized by rapid MIBG clearance from the heart. Several mechanisms have been proposed for the rapid clearance of MIBG from the heart (Fig. 2). First, a reduced number of myocardial sympathetic neurons or decreased neuronal uptake of MIBG may accelerate MIBG clearance, since MIBG efflux from extraneuronal sites is more rapid than that from intraneuronal sites (52). However, extraneuronal MIBG uptake may be negligible in case of MIBG of a high specific activity (104), and in humans extraneuronal MIBG uptake may account for only a minor part of the total myocardial uptake (105). Second, the rapid clearance may be due to high plasma norepinephrine levels (99,106), but plasma norepinephrine levels in most uremic patients are not high enough to explain rapid MIBG clearance (62). Third, the rapid MIBG clearance from the heart may result from increased release of MIBG from cardiac sympathetic neurons, which may be due to an impairment in vesicular storage (107), an increase in nonexocytotic release (54), or increased cardiac sympathetic nerve activity (58,62). The release of intracytoplasmic but extravesicular MIBG is more rapid than that of intravesicular MIBG (52), and impaired vesicular storage may partially explain the rapid clearance in CRF patients. At the present time there is no evidence to contradict the hypothesis that cardiac sympathetic overactivity is the main mechanism of rapid MIBG clearance from the heart in uremic patients. In addition, abnormal MIBG kinetics in the lung (21) may imply pulmonary sympathetic nervous dysfunction (108) and/or pulmonary endothelial dysfunction in uremic patients (109). Slow MIBG clearance from the lung has been also reported in diabetic patients (110,111). Prolonged pulmonary retention of MIBG may be influenced by ongoing pulmonary endothelial dysfunction in diabetes rather than by pulmonary passive congestion itself (110). Moreover, accelerated MIBG washout from the heart may lead to the decrease in lung washout of MIBG in diabetic patients (111). Comparison of MIBG with iodinated human serum albumin (112) or 201Tl (111) suggested that pulmonary uptake and retention of MIBG are independent of nonspecific residual lung activity or lung congestion. Further studies will be required to elucidate clinical implications of slow MIBG clearance from the lung in patients with CRF.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

VII.

CHANGES IN HRV AND MIBG FINDINGS BY RENAL TRANSPLANTATION

Successful kidney transplantation has been reported to ameliorate uremic autonomic neuropathy (3,113) as well as peripheral neuropathy (114). Röckel et al. (3) demonstrated that successful renal transplantation improved autonomic dysfunction assessed by the pupillary reaction to tyramine, the Valsalva maneuver and a postural tolerance test. Agarwal et al. (113) studied autonomic function tests before and after kidney transplantation in 12 CRF patients and found a significant reversal of all the abnormal parasympathetic tests (expiration/inspiration, supine/standing, and Valsalva ratios of RR interval; and baroreceptor sensitivity) 12–56 weeks after renal transplantation. Several reports (17,115–117) have demonstrated that kidney transplantation improves HRV in most patients with CRF on dialysis, suggesting that uremic cardiac autonomic neuropathy (particularly, parasympathetic neuropathy) is reversible. In 12 nondiabetic CRF patients, Strano et al. (115) observed that the LF component of short-term HRV significantly increased 8–12 months after kidney transplantation. Yildiz et al. (116) studied the time-domain and frequency-domain measures of 24-hour HRV before and after renal transplantation in 14 CRF patients with no coexistent disease such as diabetes mellitus and amyloidosis. They showed that renal transplantation may reverse the sympathetic and parasympathetic autonomic dysfunction in HD patients simultaneously and at a relatively early stage (as early as 3–6 months after transplantation). Furthermore, Cashion et al. (117) evaluated the time- and frequency-domain measures of 24-hour HRV before and again at 6 and 12 months after kidney transplantation in 90 nondiabetic CRF patients. As a result, HRV measures partially improved at 6 months after transplantation and all of them improved by 12 months. Rubinger et al. (17) examined 4 patients both during HD and (3 months) after renal transplantation, 2 of whom showed a dramatic increase in all 24-hour HRV values after transplantation, suggesting that uremic autonomic dysfunction is caused by humoral factors reversed by the normalization of the renal function. These studies thus indicate that renal transplantation can improve at least cardiac parasympathetic nerve function in CRF patients on dialysis. On the other hand, it remains unclear whether renal transplantation can also correct uremic cardiac sympathetic neuropathy. Recently, we studied analysis of 24-hour HRV and cardiac MIBG scintigraphy immediately before and 1–3 months after renal transplantation in 13 CRF patients on dialysis (118). From before to after transplant, MIBG washout rate from the myocardium significantly decreased and the heart-to-mediastinum ratio of MIBG uptake in the late image significantly increased, while the increase in HRV measures did not reach statistical significance (p 0.05). For example, in one of 13 patients, MIBG washout rate showed marked improvement at a month posttransplantation, from 78% to

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

13% (within a normal range), while the 24-hour SD of 5-minute average of RR intervals (SDANN) increased only a little from 24 msec before to 39 msec a month after transplantation. At 3.5 months after transplantation, however, the patient underwent an additional study of 24-hour HRV, which showed an increase in SDANN to 115 msec (around a lower limit). These findings suggest that renal transplantation provides an improvement of uremic cardiac sympathetic neuropathy, which may start earlier than that of cardiac parasympathetic neuropathy. The observation of possible improvement in MIBG uptake and kinetics by renal transplantation may afford excellent insight into not only the pathophysiology of uremic cardiac sympathetic neuropathy, but also the recovery process of cardiac sympathetic nervous dysfunction in patients with heart failure.

VIII. SUMMARY AND FUTURE PROSPECTS Most patients with end-stage renal failure (2,14,16,17,21,26–36) show remarkable abnormalities of HRV as in those with moderate to severe congestive heart failure, suggesting that CRF is similar in parasympathetic nervous system dysfunction to heart failure. Furthermore, if rapid MIBG clearance from the heart in end-stage renal failure, as in congestive heart failure (119,120), reflects increased neuronal release of norepinephrine, congestive heart failure and end-stage renal failure may resemble each other in pathophysiology of cardiac sympathetic nervous system. Thus, uremic autonomic neuropathy may be characterized by sympathetic overactivity with parasympathetic nervous system dysfunction, which are common in congestive heart failure. Correction of the uremic state by renal transplantation can provide normalization of left ventricular contractility in systolic dysfunction as well as regression of hypertrophy in concentric left ventricular hypertrophy (103,121,122). Renal transplantation may lead to both correction of left ventricular dysfunction and remarkable improvement of HRV and MIBG measures in dialysis patients with dilated cardiomyopathy (103,118). Therefore, observations about possible improvement in HRV and MIBG measures by renal transplantation may afford excellent insight into not only the pathophysiology of uremic cardiac autonomic neuropathy, but also the recovery process of cardiac autonomic nervous system in patients with congestive heart failure or left ventricular hypertrophy. In addition to renal transplantation, cardiac autonomic nerve function in CRF patients may be improved by an increased intake of n-3 polyunsaturated fatty acids (123), physical training (36), and treatment with an angiotensin-converting enzyme inhibitor (6). Further prospective studies using HRV analysis and MIBG scintigraphy are expected to elucidate precise mechanisms and clinical implications of uremic cardiac autonomic neuropathy and to promote the development of its treatments.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

REFERENCES 1.

2. 3.

4. 5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Kersh ES, Kronfield SJ, Unger A, Popper RW, Cantor S, Cohn K. Autonomic insufficiency in uremia as a cause of hemodialysis-induced hypotension. N Engl J Med 1974; 290:650–653. Ewing DJ, Winney R. Autonomic function in patients with chronic renal failure on intermittent haemodialysis. Nephron 1975; 15:424–429. Röckel A, Hennemann H, Sternagel-Haase A, Heidland A. Uraemic sympathetic neuropathy after haemodialysis and transplantation. Eur J Clin Invest 1979; 9:23–27. Campese VM, Romoff MS, Levitan D, Lane K, Massry SG. Mechanisms of autonomic nervous system dysfunction in uremia. Kidney Int 1981; 20:246–253. Converse RL Jr, Jacobsen TN, Toto RD, Jost CM, Cosentino F, Fouad-Tarazi F, Victor RG. Sympathetic overactivity in patients with chronic renal failure. N Engl J Med 1992; 327:1912–1918. Ligtenberg G, Blankestijn PJ, Oey PL, Klein IH, Dijkhorst-Oei LT, Boomsma F, Wieneke GH, van Huffelen AC, Koomans HA. Reduction of sympathetic hyperactivity by enalapril in patients with chronic renal failure. N Engl J Med 1999; 340:1321–1328. Takahashi H, Matsuo S, Toriyama T, Kawahara H, Hayano J. Autonomic dysfunction in hemodialysis patients with persistent hypotension. Nephron 1996; 72:418–423. Pelosi G, Emdin M, Carpeggiani C, Morales MA, Piacenti M, Dattolo P, Cerrai T, Macerata A, L’Abbate A, Maggiore Q. Impaired sympathetic response before intradialytic hypotension: a study based on spectral analysis of heart rate and pressure variability. Clin Sci (Colch) 1999; 96:23–31. Nishiyama K, Kurata C, Wakabayashi Y, Ohno R. Disorders accompanied with marked abnormality of cardiac autonomic function: 24-hour heart rate variability (abstr). J Jpn Soc Intern Med 1996; 85:99. Vita G, Dattola R, Calabro R, Manna L, Venuto C, Toscano A, Savica V, Bellinghieri G. Comparative analysis of autonomic and somatic dysfunction in chronic uraemia. Eur Neurol 1988; 28:335–340. Molgaard H, Christensen PD, Sorensen KE, Christensen CK, Mogensen CE. Association of 24-h cardiac parasympathetic activity and degree of nephropathy in IDDM patients. Diabetes 1992; 41:812–817. Stevens MJ, Raffel DM, Allman KC, Dayanikli F, Ficaro E, Sandford T, Wieland DM, Pfeifer MA, Schwaiger M. Cardiac sympathetic dysinnervation in diabetes: implications for enhanced cardiovascular risk. Circulation 1998; 98:961–968. Burger AJ, Weinrauch LA, D’Elia JA, Aronson D. Effect of glycemic control on heart rate variability in type I diabetic patients with cardiac autonomic neuropathy. Am J Cardiol 1999; 84:687–691. Vita G, Bellinghieri G, Trusso A, Costantino G, Santoro D, Monteleone F, Messina C, Savica V. Uremic autonomic neuropathy studied by spectral analysis of heart rate. Kidney Int 1999; 56:232–237.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

15.

16. 17.

18.

19. 20.

21.

22.

23.

24.

25. 26.

27.

28.

29.

30.

Tamura K, Tsuji H, Nishiue T, Yajima I, Higashi T, Iwasaka T. Determinants of heart rate variability in chronic hemodialysis patients. Am J Kidney Dis 1998; 31:602–606. Steinberg AA, Mars RL, Goldman DS, Percy RF. Effect of end-stage renal disease on decreased heart rate variability. Am J Cardiol 1998; 82:1156–1158. Rubinger D, Sapoznikov D, Pollak A, Popovtzer MM, Luria MH. Heart rate variability during chronic hemodialysis and after renal transplantation: studies in patients without and with systemic amyloidosis. J Am Soc Nephrol 1999; 10:1972–1981. Mallamaci F, Zoccali C, Ciccarelli M, Briggs JD. Autonomic function in uremic patients treated by hemodialysis or CAPD and in transplant patients. Clin Nephrol 1986; 25:175–180. Malik S, Winney RJ, Ewing DJ. Chronic renal failure and cardiovascular autonomic function. Nephron 1986; 43:191–195. Laaksonen S, Voipio-Pulkki L, Erkinjuntti M, Asola M, Falck B. Does dialysis therapy improve autonomic and peripheral nervous system abnormalities in chronic uraemia? J Intern Med 2000; 248:21–26. Kurata C, Uehara A, Sugi T, Ishikawa A, Fujita K, Yonemura K, Hishida A, Ishikawa K, Tawarahara K, Shouda S, Mikami T. Cardiac autonomic neuropathy in patients with chronic renal failure on hemodialysis. Nephron 2000; 84:312–319. Corder CN, Sharma J, McDonald RH Jr. Variable levels of plasma catecholamines and dopamine beta-hydroxylase in hemodialysis patients. Nephron 1980; 25:267–272. Musso NR, Deferrari G, Pende A, Vergassola C, Saffioti S, Gurreri G, Lotti G. Free and sulfoconjugated catecholamines in normotensive uremic patients: effects of hemodialysis. Nephron 1989; 51:344–349. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Heart rate variability: standards of measurement, physiological interpretation and clinical use. Circulation 1996; 93:1043–1065. Malliani A, Pagani M, Lombardi F, Cerutti S. Cardiovascular neural regulation explored in the frequency domain. Circulation 1991; 84:482–492. Forsström J, Forsström J, Heinonen E, Välimäki I, Antila K. Effects of haemodialysis on heart rate variability in chronic renal failure. Scand J Clin Lab Invest 1986; 46:665–670. Axelrod S, Lishner M, Oz O, Bernheim J, Ravid M. Spectral analysis of fluctuations in heart rate: an objective evaluation of autonomic nervous control in chronic renal failure. Nephron 1987; 45:202–206. Thomson BJ, McAreavey D, Neilson JM, Winney RJ, Ewing DJ. Heart rate variability and cardiac arrhythmias in patients with chronic renal failure. Clin Auton Res 1991; 1:131–133. Cloarec-Blanchard L, Girard A, Houhou S, Grünfeld JP, Elghozi JL. Spectral analysis of short-term blood pressure and heart rate variability in uremic patients. Kidney Int 1992; 37(suppl):S14–18. Enzmann G, Bianco F, Paolini F, Panzetta G. Autonomic nervous function and blood volume monitoring during hemodialysis. Int J Artif Organs 1995; 18:504–508.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

31.

32.

33.

34. 35.

36. 37. 38.

39.

40.

41.

42.

43.

44.

Miyanaga H, Yoneyama S, Kamitani T, Kawasaki S, Takahashi T, Kunishige H. Abnormal myocardial uptake and clearance of 123I-labeled metaiodobenzylguanidine in patients with chronic renal failure and autonomic dysfunction. J Nucl Cardiol 1996; 3:508–515. Kirvela M, Salmela K, Toivonen L, Koivusalo AM, Lindgren L. Heart rate variability in diabetic and non-diabetic renal transplant patients. Acta Anaesthesiol Scand 1996; 40:804–808. Yildiz A, Akkaya V, Sever MS, Bozfakioglu S, Kudat H, Demirel S, Ark E. Heart rate variability in patients with secondary amyloidosis. Isr J Med Sci 1997; 33:781–785. Kirvela M, Toivonen L, Lindgren L. Cardiac repolarization interval in end-stage diabetic and nondiabetic renal disease. Clin Cardiol 1997; 20:791–796. Tamura K, Tsuji H, Nishiue T, Tokunaga S, Yajima I, Higashi T, Iwasaka T. Determinants of ventricular arrhythmias in hemodialysis patients. Evaluation of the effect of arrhythmogenic substrate and autonomic imbalance. Am J Nephrol 1998; 18:280–284. Deligiannis A, Kouidi E, Tourkantonis A. Effects of physical training on heart rate variability in patients on hemodialysis. Am J Cardiol 1999; 84:197–202. Zoccali C, Ciccarelli M, Mallamaci F, Maggiore Q. Parasympathetic function in haemodialysis patients. Nephron 1986; 44:351–354. Roger SD, Baker LR, Raine AE. Autonomic dysfunction and the development of hypertension in patients treated with recombinant human erythropoietin (r-HuEPO). Clin Nephrol 1993; 39:103–110. Algra A, Tijssen JG, Roelandt JR, Pool J, Lubsen J. Heart rate variability from 24hour electrocardiography and the 2-year risk for sudden death. Circulation 1993; 88:180–185. Tsuji H, Venditti FJ Jr, Manders ES, Evans JC, Larson MG, Feldman CL, Levy D. Reduced heart rate variability and mortality risk in an elderly cohort. The Framingham Heart Study. Circulation 1994; 90:878–883. Nolan J, Batin PD, Andrews R, Lindsay SJ, Brooksby P, Mullen M, Baig W, Flapan AD, Cowley A, Prescott RJ, Neilson JM, Fox KA. Prospective study of heart rate variability and mortality in chronic heart failure: results of the United Kingdom heart failure evaluation and assessment of risk trial (UK-heart). Circulation 1998; 98:1510–1516. Dekker JM, Crow RS, Folsom AR, Hannan PJ, Liao D, Swenne CA, Schouten EG. Low heart rate variability in a 2-minute rhythm strip predicts risk of coronary heart disease and mortality from several causes: the ARIC Study. Circulation 2000; 102:1239–1244. Hathaway DK, Cashion AK, Milstead EJ, Winsett RP, Cowan PA, Wicks MN, Gaber AO. Autonomic dysregulation in patients awaiting kidney transplantation. Am J Kidney Dis 1998; 32:221–229. Hayano J, Takahashi H, Toriyama T, Mukai S, Okada A, Sakata S, Yamada A, Ohte N, Kawahara H. Prognostic value of heart rate variability during long-term followup in chronic haemodialysis patients with end-stage renal disease. Nephrol Dial Transplant 1999; 14:1480–1488.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

45.

46.

47.

48.

49.

50.

51.

52.

53. 54.

55.

56.

57.

58.

59.

Vita G, Savica V, Milone S, Trusso A, Bellinghieri G, Messina C. Uremic autonomic neuropathy: recovery following bicarbonate hemodialysis. Clin Nephrol 1996; 45:56–60. Barnas MG, Boer WH, Koomans HA. Hemodynamic patterns and spectral analysis of heart rate variability during dialysis hypotension. J Am Soc Nephrol 1999; 10:2577–2584. Wieland DM, Brown LE, Rogers WL, Worthington KC, Wu JL, Clinthorne NH, Otto CA, Swanson DP, Beierwaltes WH. Myocardial imaging with a radioiodinated norepinephrine storage analog. J Nucl Med 1981; 22:22–31. Sisson JC, Wieland DM, Sherman P, Mangner TJ, Tobes MC, Jacques S Jr. Metaiodobenzylguanidine as an index of the adrenergic nervous system integrity and function. J Nucl Med 1987; 28:1620–1624. Tobes MC, Jaques S, Wieland DM, Sisson JC. Effect of uptake-one inhibitors on the uptake of norepinephrine and metaiodobenzylguanidine. J Nucl Med 1985; 26:897–907. Glowniak JV, Kilty JE, Amara SG, Hoffman BJ, Turner FE. Evaluation of metaiodebenzylguanidine uptake by the norepinephrine, dopamine and serotonin transporters. J Nucl Med 1993; 34:1140–1146. Wieland DM, Brown LE, Tobes MC, Rogers WL, Marsh DD, Mangner TJ, Swanson DP, Beierwaltes WH. Imaging the primate adrenal medulla with [123I] and [131I] meta-iodobenzylguanidine: concise communication. J Nucl Med 1981; 22:358–364. Nakajo M, Shimabukuro K, Yoshimura H, Yonekura R, Nakabeppu Y, Tanoue P, Shinohara S. Iodine-131 metaiodobenzylguanidine intra- and extravesicular accumulation in the rat heart. J Nucl Med 1986; 27:84–89. DeGrado TR, Zalutsky MR, Vaidyanathan G. Uptake mechanisms of meta[123I]iodobenzylguanidine in isolated rat heart. Nucl Med Biol 1995; 22:1–12. Rose CP, Cousineau D, Goresky CA, De Champlain J. Constitutive nonexocytotic norepinephrine release in sympathetic curves of in situ canine heart. Am J Physiol 1994; 266:H1386–1394. Mangner TJ, Tobes MC, Wieland DW, Sisson JC, Shapiro B. Metabolism of iodine131 metaiodobenzylguanidine in patients with metastatic pheochromocytoma. J Nucl Med 1986; 27:37–44. Henderson EB, Kahn JK, Corbett JR, Jansen DE, Pippin JJ, Kulkarni P, Ugolini V, Akers MS, Hansen C, Buja LM. Abnormal I-123 metaiodobenzylguanidine myocardial washout and distribution may reflect myocardial adrenergic derangement in patients with congestive cardiomyopathy. Circulation 1988; 78:1192–1199. Dae MW, O’Connell JW, Botvinick EH, Chin MC. Acute and chronic effects of transient myocardial ischemia on sympathetic nerve activity, density, and norepinephrine content. Cardiovasc Res 1995; 30:270–280. Estorch M, Serra-Grima R, Carrio I, Flotats A, Lizarraga A, Berna LL, Prats T, Segura R. Influence of prolonged exercise on myocardial distribution of 123I-MIBG in long-distance runners. J Nucl Cardiol 1997; 4:396–402. Mantysaari M, Kuikka J, Hartikainen J, Mustonen J, Mussalo H, Tahvanainen K, Lansimies E, Uusitupa M, Pyorala K. Myocardial sympathetic nervous dysfunction detected with iodine-123-MIBG is associated with low heart rate variability after myocardial infarction. J Nucl Med 1995; 36:956–961.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

Sakata K, Shirotani M, Yoshida H, Kurata C. Iodine-123 metaiodobenzylguanidine cardiac imaging to identify and localize vasospastic angina without significant coronary artery narrowing. J Am Coll Cardiol 1997; 30:370–376. Hartikainen J, Mustonen J, Kuikka J, Vanninen E, Kettunen R. Cardiac sympathetic denervation in patients with coronary artery disease without previous myocardial infarction. Am J Cardiol 1997; 80:273–277. Glowniak JV, Turner FE, Gray LL, Palac RT, Lagunas-Solar MC, Woodward WR. Iodine-123 metaiodobenzylguanidine imaging of the heart in idiopathic congestive cardiomyopathy and cardiac transplants. J Nucl Med 1989; 30:1182–1191. Wakabayashi Y, Kurata C, Mikami T, Shouda S, Okayama K, Tawarahara K. Effects of cilazapril and verapamil on myocardial iodine-125-metaiodobenzylguanidine accumulation in cardiomyopathic BIO 53.58 hamsters. J Nucl Med 1997; 38:1540–1545. Lotze U, Kober A, Kaepplinger S, Neubauer S, Gottschild D, Figulla HR. Cardiac sympathetic activity as measured by myocardial 123-I-metaiodobenzylguanidine uptake and heart rate variability in idiopathic dilated cardiomyopathy. Am J Cardiol 1999; 83:1548–1551. Merlet P, Benvenuti C, Moyse D, Pouillart F, Dubois-Rande JL, Duval AM, Loisance D, Castaigne A, Syrota A. Prognostic value of MIBG imaging in idiopathic dilated cardiomyopathy. J Nucl Med 1999; 40:917–923. Merlet P, Valette H, Dubois-Rande JL, Moyse D, Duboc D, Dove P, Bourguignon MH, Benvenuti C, Duval AM, Agostini D. Prognostic value of cardiac metaiodobenzylguanidine imaging in patients with heart failure. J Nucl Med 1992; 33:471–477. Somsen GA, Szabo BM, van Veldhuisen DJ, de Milliano PA, de Groot CA, Lie KI. Comparison between iodine 123 metaiodobenzylguanidine scintigraphy and heart rate variability for the assessment of cardiac sympathetic activity in mild to moderate heart failure. Am Heart J 1997; 134:456–458. Nakata T, Miyamoto K, Doi A, Sasao H, Wakabayashi T, Kobayashi H, Tsuchihashi K, Shimamoto K. Cardiac death prediction and impaired cardiac sympathetic innervation assessed by MIBG in patients with failing and nonfailing hearts. J Nucl Cardiol 1998; 5:579–590. Kurata C, Shouda S, Mikami T, Uehara A, Ishikawa K, Tawarahara K, Nakano T, Matoh F, Takeuchi K. Metaiodobenzylguanidine and heart rate variability in heart failure. Jpn Circ J 1998; 62:770–772. Cohen-Solal A, Esanu Y, Logeart D, Pessione F, Dubois C, Dreyfus G, Gourgon R, Merlet P. Cardiac metaiodobenzylguanidine uptake in patients with moderate chronic heart failure: relationship with peak oxygen uptake and prognosis. J Am Coll Cardiol 1999; 33:759–766. Mitrani RD, Klein LS, Miles WM, Hackett FK, Burt RW, Wellman HN, Zipes DP. Regional cardiac sympathetic denervation in patients with ventricular tachycardia in the absence of coronary artery disease. J Am Coll Cardiol 1993; 22:1344–1353. Gill JS, Hunter GJ, Gane J, Ward DE, Camm AJ. Asymmetry of cardiac [123I] metaiodobenzyl-guanidine scans in patients with ventricular tachycardia and a “clinically normal” heart. Br Heart J 1993; 69:6–13.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

73.

74.

75.

76.

77. 78.

79.

80.

81.

82.

83. 84.

85.

86.

Dae MW, Lee RJ, Ursell PC, Chin MC, Stillson CA, Moise NS. Heterogeneous sympathetic innervation in German shepherd dogs with inherited ventricular arrhythmia and sudden cardiac death. Circulation 1997; 96:1337–1342. Schafers M, Wichter T, Lerch H, Matheja P, Kuwert T, Schafers K, Borggrefe M, Breithardt G, Schober O. Cardiac 123I-MIBG uptake in idiopathic ventricular tachycardia and fibrillation. J Nucl Med 1999; 40:1–5. Fujiwara Y, Hamada M, Shigematsu Y, Sumimoto T, Hamamoto K, Hiwada K. Scintigraphic assessment of cardiac adrenergic innervation in patients with essential hypertension. J Cardiovasc Pharmacol 1991; 17(suppl 2):S148–150. Kuwahara T, Hamada M, Hiwada K. Direct evidence of impaired cardiac sympathetic innervation in essential hypertensive patients with left ventricular hypertrophy. J Nucl Med 1998; 39:1486–1491. Sakata K, Shirotani M, Yoshida H, Kurata C. Cardiac sympathetic nervous system in early essential hypertension assessed by 123I-MIBG. J Nucl Med 1999; 40:6–11. Claus D, Feistel H, Brunholzl C, Platsch G, Neundorfer B, Wolf F. Investigation of parasympathetic and sympathetic cardiac innervation in diabetic neuropathy: heart rate variation versus meta-iodobenzylguanidine measured by single photon emission computed tomography. Clin Auton Res 1994; 4:117–123. Miyanaga H, Yoneyama S, Kamitani T, Kawasaki S, Takahashi T, Kunishige H. Clinical usefulness of 123I-metaiodobenzylguanidine myocardial scintigraphy in diabetic patients with cardiac sympathetic nerve dysfunction. Jpn Circ J 1995; 59:599–607. Langer A, Freeman MR, Josse RG, Armstrong PW. Metaiodobenzylguanidine imaging in diabetes mellitus: assessment of cardiac sympathetic denervation and its relation to autonomic dysfunction and silent myocardial ischemia. J Am Coll Cardiol 1995; 25:610–618. Murata K, Sumida Y, Murashima S, Matsumura K, Takeda H, Nakagawa T, Shima T. A novel method for the assessment of autonomic neuropathy in type 2 diabetic patients: a comparative evaluation of 123I-MIBG myocardial scintigraphy and power spectral analysis of heart rate variability. Diabet Med 1996; 13:266–272. Kurata C, Okayama K, Wakabayashi Y, Shouda S, Mikami T, Tawarahara K, Sugiyama T. Cardiac sympathetic neuropathy and effects of aldose reductase inhibitor in streptozotocin-induced diabetic rats. J Nucl Med 1997; 38:1677–1680. Uehara A, Kurata C, Sugi T, Mikami T, Shouda S. Diabetic cardiac autonomic dysfunction: parasympathetic versus sympathetic. Ann Nucl Med 1999; 13:95–100. Sugiyama T, Kurata C, Tawarahara K, Nakano T. Is abnormal iodine-123-MIBG kinetics associated with left ventricular dysfunction in patients with diabetes mellitus? J Nucl Cardiol 2000; 7:562–568. De Marco T, Dae M, Yuen-Green MS, Kumar S, Sudhir K, Keith F, Amidon TM, Rifkin C, Klinski C, Lau D, et al. Iodine-123 metaiodobenzylguanidine scintigraphic assessment of the transplanted human heart: evidence for late reinnervation. J Am Coll Cardiol 1995; 25:927–931. Guertner C, Krause BJ, Klepzig H, Jr., Herrmann G, Lelbach S, Vockert EK, Hartmann A, Maul FD, Kranert TW, Mutschler E. Sympathetic re-innervation after heart transplantation: dual-isotope neurotransmitter scintigraphy, norepinephrine content and histological examination. Eur J Nucl Med 1995; 22:443–452.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

87.

88.

89.

90.

91.

92.

93.

94. 95.

96.

97.

98.

99.

100.

Toba M, Ishida Y, Fukuchi K, Shimotsu Y, Takamiya M, Komamura K, Nakatani T, Ohuchi H, Ono Y, Kamiya T. Sympathetic reinnervation demonstrated on serial iodine-123-metaiodobenzylguanidine SPECT images after cardiac transplantation. J Nucl Med 1998; 39:1862–1864. Hakusui S, Yasuda T, Yanagi T, Tohyama J, Hasegawa Y, Koike Y, Hirayama M, Takahashi A. A radiological analysis of heart sympathetic functions with meta[123I]iodobenzylguanidine in neurological patients with autonomic failure. J Auton Nerv Syst 1994; 49:81–84. Satoh A, Serita T, Seto M, Tomita I, Satoh H, Iwanaga K, Takashima H, Tsujihata M. Loss of 123I-MIBG uptake by the heart in Parkinson’s disease: assessment of cardiac sympathetic denervation and diagnostic value. J Nucl Med 1999; 40:371–375. Druschky A, Hilz MJ, Platsch G, Radespiel-Troger M, Druschky K, Kuwert T, Neundorfer B. Differentiation of Parkinson’s disease and multiple system atrophy in early disease stages by means of I-123-MIBG-SPECT. J Neurol Sci 2000; 175:3–12. Takatsu H, Nishida H, Matsuo H, Watanabe S, Nagashima K, Wada H, Noda T, Nishigaki K, Fujiwara H. Cardiac sympathetic denervation from the early stage of Parkinson’s disease: clinical and experimental studies with radiolabeled MIBG. J Nucl Med 2000; 41:71–77. Kingwell BA, Thompson JM, Kaye DM, McPherson GA, Jennings GL, Esler MD. Heart rate spectral analysis, cardiac norepinephrine spillover, and muscle sympathetic nerve activity during human sympathetic nervous activation and failure. Circulation 1994; 90:234–240. Hojgaard MV, Holstein-Rathlou NH, Agner E, Kanters JK. Dynamics of spectral components of heart rate variability during changes in autonomic balance. Am J Physiol 1998; 275:H213–219. Houle MS, Billman GE. Low-frequency component of the heart rate variability spectrum: a poor marker of sympathetic activity. Am J Physiol 1999; 276:H215–223. van de Borne P, Montano N, Pagani M, Oren R, Somers VK. Absence of low-frequency variability of sympathetic nerve activity in severe heart failure. Circulation 1997; 95:1449–1454. Skyschally A, Breuer HW, Heusch G. The analysis of heart rate variability does not provide a reliable measurement of cardiac sympathetic activity. Clin Sci (Colch) 1996; 91:102–104. Notarius CF, Butler GC, Ando S, Pollard MJ, Senn BL, Floras JS. Dissociation between microneurographic and heart rate variability estimates of sympathetic tone in normal subjects and patients with heart failure. Clin Sci (Colch) 1999; 96:557–565. Psychari SN, Theodorakis GN, Koutelou M, Livanis EG, Kremastinos DT. Cardiac denervation after radiofrequency ablation of supraventricular tachycardias. Am J Cardiol 1998; 81:725–731. Kurata C, Shouda S, Mikami T, Wakabayashi Y, Nakano T, Sugiyama T, Tawarahara K, Sakata K. Comparison of [123I]metaiodobenzylguanidine kinetics with heart rate variability and plasma norepinephrine level. J Nucl Cardiol 1997; 4:515–523. Morozumi T, Kusuoka H, Fukuchi K, Tani A, Uehara T, Matsuda S, Tsujimura E, Ito Y, Hori M, Kamada T, Nishimura T. Myocardial iodine-123-metaiodobenzylguanidine images and autonomic nerve activity in normal subjects. J Nucl Med 1997; 38:49–52.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

101.

102.

103.

104. 105.

106.

107.

108.

109.

110. 111.

112. 113.

114.

115.

Delahaye N, Dinanian S, Slama MS, Mzabi H, Samuel D, Adams D, Merlet P, Le Guludec D. Cardiac sympathetic denervation in familial amyloid polyneuropathy assessed by iodine-123 metaiodobenzylguanidine scintigraphy and heart rate variability. Eur J Nucl Med 1999; 26:416–424. Druschky A, Spitzer A, Platsch G, Claus D, Feistel H, Druschky K, Hilz MJ, Neundorfer B. Cardiac sympathetic denervation in early stages of amyotrophic lateral sclerosis demonstrated by 123I-MIBG-SPECT. Acta Neurol Scand 1999; 99:308–314. Kurata C, Wakabayashi Y, Shouda S, Okayama K, Yamamoto T, Ishikawa A, Suzuki K, Ishizuka T, Sakamoto S, Tawarahara K. Enhanced cardiac clearance of iodine-123-MIBG in chronic renal failure. J Nucl Med 1995; 36:2037–2043. Mock BH, Tuli MM. Influence of specific activity on myocardial uptake of 123ImIBG in rats. Nucl Med Commun 1988; 9:663–667. Dae MW, De Marco T, Botvinick EH, O’Connell JW, Hattner RS, Huberty JP, Yuen-Green MS. Scintigraphic assessment of MIBG uptake in globally denervated human and canine hearts—implications for clinical studies. J Nucl Med 1992; 33:1444–1450. Shouda S, Kurata C, Mikami T, Wakabayashi Y. Effects of extrinsically elevated plasma norepinephrine concentration on myocardial 123I-MIBG kinetics in rats. J Nucl Med 1999; 40:2088–2093. Rabinovitch MA, Rose CP, Schwab AJ, Fitchett DH, Honos GN, Stewart JA, Chen LF, Castilla EP, Gomez AA, Abrahamowicz M. A method of dynamic analysis of iodine-123-metaiodobenzylguanidine scintigrams in cardiac mechanical overload hypertrophy and failure. J Nucl Med 1993; 34:589–600. Eisenhofer G, Smolich JJ, Esler MD. Different desipramine-sensitive pulmonary removals of plasma epinephrine and norepinephrine in dogs. Am J Physiol 1992; 262:L360–365. Slosman DO, Davidson D, Brill AB, Alderson PO. 131I-metaiodobenzylguanidine uptake in the isolated rat lung: a potential marker of endothelial cell function. Eur J Nucl Med 1988; 13:543–547. Ünlü M, Inanir S. Prolonged lung retention of iodine-123-MIBG in diabetic patients. J Nucl Med 1998; 39:116–118. Murashima S, Takeda K, Matsumura K, Yamakado K, Sakuma H, Kitano T, Nakagawa T, Ichihara T, Yamakado T, Murata K. Increased lung uptake of iodine-123MIBG in diabetics with sympathetic nervous dysfunction. J Nucl Med 1998; 39:334–338. Slosman DO, Polla BS, Donath A. 123I-MIBG pulmonary removal: a biochemical marker of minimal lung endothelial cell lesions. Eur J Nucl Med 1990; 16:633–637. Agarwal A, Anand IS, Sakhuja V, Chugh KS. Effect of dialysis and renal transplantation on autonomic dysfunction in chronic renal failure. Kidney Int 1991; 40:489–495. Bolton CF, Baltzan MA, Baltzan RB. Effects of renal transplantation on uremic neuropathy. A clinical and electrophysiologic study. N Engl J Med 1971; 284:1170–1175. Strano S, De Castro S, Urani C, Ferrucci A, Marcheselli A, Poli L, Berloco P, Calcagnini GJr, Cerutti S, Cortesini R. Power spectrum analysis of heart rate variability following kidney transplantation. Transplant Proc 1993; 25:2600–2601.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

116.

117.

118.

119.

120.

121.

122.

123.

Yildiz A, Sever MS, Demirel S, Akkaya V, Türk S, Türkmen A, Ecder T, Ark E. Improvement of uremic autonomic dysfunction after renal transplantation: a heart rate variability study. Nephron 1998; 80:57–60. Cashion AK, Hathaway DK, Milstead EJ, Reed L, Gaber AO. Changes in patterns of 24-hr heart rate variability after kidney and kidney-pancreas transplant. Transplantation 1999; 68:1846–1850. Kurata C, Uehara A, Sugi T, Yamazaki K. Improvement of uremic cardiac autonomic neuropathy by renal transplantation: metaiodobenzylguanidine and heart rate variability (abstr). Jpn Circ J 2000; 64:501. Meredith IT, Eisenhofer G, Lambert GW, Dewar EM, Jennings GL, Esler MD. Cardiac sympathetic nervous activity in congestive heart failure. Evidence for increased neuronal norepinephrine release and preserved neuronal uptake. Circulation 1993; 88:136–145. Eisenhofer G, Friberg P, Rundqvist B, Quyyumi AA, Lambert G, Kaye DM, Kopin IJ, Goldstein DS, Esler MD. Cardiac sympathetic nerve function in congestive heart failure. Circulation 1996; 93:1667–1676. Burt RK, Gupta-Burt S, Suki WN, Barcenas CG, Ferguson JJ, Van Buren CT. Reversal of left ventricular dysfunction after renal transplantation. Ann Intern Med 1989; 111:635–640. Parfrey PS, Harnett JD, Foley RN, Kent GM, Murray DC, Barre PE, Guttmann RD. Impact of renal transplantation on uremic cardiomyopathy. Transplantation 1995; 60:908–914. Christensen JH, Aaroe J, Knudsen N, Dideriksen K, Kornerup HJ, Dyerberg J, Schmidt EB. Heart rate variability and n-3 fatty acids in patients with chronic renal failure—a pilot study. Clin Nephrol 1998; 49:102–106.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

13 The Function of the Autonomic Nervous System in Hypertension Guido Grassi and Giuseppe Mancia University of Milan-Bicocca and San Gerardo Hospital, Monza (Milan), Italy

Time-honored studies of classic physiology have shown that autonomic neural factors play a fundamental role in the homeostatic control of the cardiovascular system by regulating organ perfusion and systemic blood pressure levels through changes in cardiac output and peripheral vascular resistance. Given the evidence that increased cardiac output and elevated peripheral vascular resistance represent the basic hemodynamic abnormalities of the hypertensive state, the hypothesis has been made that a dysfunction of the autonomic circulatory control might be the mechanism leading to the blood pressure elevation. Countless studies have tested this hypothesis, providing convincing evidence that an imbalance in the autonomic modulation of the cardiovascular function with resultant sympathetic activation and parasympathetic inhibition does occur at the very beginning as well as at the later stages of the hypertensive state, thus contributing, in association with other etiological factors, to the development, maintenance, and progression of the disease. The goal of this chapter is to examine the main autonomic alterations characterizing essential hypertension and to review the mechanisms through which these neurogenic derangements take place. The pivotal role of the autonomic dysfunction in the development of the metabolic, emorrheologic, and structural changes of the heart and arterial vessels observed in chronic hypertension as well as in the genesis of the hypertension-related cardiovascular risk will be finally highlighted.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

I. AUTONOMIC DYSFUNCTION IN EARLIER PHASES OF HYPERTENSION The early stages of hypertension are characterized by hyperkinetic circulation, i.e., increased cardiac output coupled with a resting tachycardia. These findings, originally reported in the late 1950s (1), have been more recently confirmed by longitudinal and cross-sectional studies (2,3), which have also shown their dependence on a reduced parasympathetic function. Indeed, intravenous atropine (the drug that selectively blocks the effects of the vagal neurotransmitter acetylcholine on cardiac muscarinic receptors) induces in young borderline hypertensives an increase in heart rate and cardiac output of lesser magnitude than that found in age-matched normotensive controls (4). Evidence has been also provided that the blunted parasympathetic tone observed in the early hypertensive phases 1) is also a hallmark of established hypertension (5) and 2) is not limited to the cardiovascular system, a reduced salivary flow (whose control is under parasympathetic influences) being reported in subjects with borderline hypertension as well (6). Taken together these findings support the concept that an autonomic dysfunction involving parasympathetic cardiovascular control characterizes at the very early beginning the hypertensive state. Does the above-mentioned dysregulation also affect sympathetic cardiovascular influences? This question has been addressed throughout the years by investigators using different methodological approaches to assess adrenergic function. Early studies performed in genetic animal models of hypertension (Dahl salt-sensitive strain of hypertensive rats, spontaneously hypertensive rats, DOCAsalt hypertension) have shown that sympathetic outflow to the kidney is enhanced and that in central areas involved in blood pressure control, the turnover of the adrenergic neurotransmitter norepinephrine is increased (7,8). In more recent studies performed in other animal models (Milan and Lyon strains of hypertensive rats), however, the evidence in favor of a sympathetic activation as a cause of hypertension has not been unequivocally demonstrated (9). The participation of sympathetic neural factors at the pathogenesis of hypertension has also been investigated in humans, and the results can be summarized as follows. First, in a metaanalysis of all published studies (10), Goldstein reported that, even accounting for some negative results, an indirect marker of sympathetic tone such as plasma norepinephrine is significantly elevated in essential hypertensive patients as compared to age-matched normotensive subjects. Second, by employing the technique based on the intravenous tracer infusion of small doses of radiolabeled norepinephrine (11), Esler and coworkers were able to show that the rate of norepinephrine spillover from the neuroeffector junctions is increased in young subjects with a borderline blood pressure elevation and that this enhanced release takes place particularly in the kidney and in the heart, i.e., two organs of key importance in blood pressure homeostatic control (12). Third,

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

direct measurement of sympathetic nerve traffic to the skeletal muscle circulation, employing tungsten microelectodes inserted in the peroneal or brachial nerves (11), has documented an increase in central sympathetic outflow in young borderline hypertensives (13). It should finally be mentioned that another abnormality involving the sympathetic neural function in young patients with mild hypertension is represented by a heightened density of -adrenergic receptors at both cardiac and vascular levels and/or by a hyperresponsiveness of these receptors to adrenergic neurotransmitters (14).

II. AUTONOMIC DYSFUNCTION IN ESTABLISHED HYPERTENSION While parasympathetic dysfunction remains stable in the hypertensive states characterized by more severe increases in blood pressure, sympathetic activation undergoes a progressive and further increase. This has recently been shown by a study performed by our group (15), in which we quantified sympathetic nerve traffic to the skeletal muscle district in three groups of age-matched subjects: with normal blood pressure, with moderate essential hypertension, and with essential hypertension of a more severe degree. As shown in Figure 1, the progressive increase in blood pressure values observed in these three conditions was paralleled by a progressive and marked elevation in sympathetic nerve traffic, suggesting the key role of adrenergic neural factors not only in the development but also in the progression of the hypertensive state. A further demonstration of this phoenomenon comes from the evidence, collected by our group, that blood pressure variability, i.e., the magnitude of the blood pressure oscillations occurring

Figure 1 Individual and average values of mean arterial pressure (MAP), heart rate (HR), and muscle sympathetic nerve traffic (MSNA) in three groups of age-matched subjects (normotensives: open circles; mild hypertensives: closed circles; more severe hypertensives: triangles). Symbols refer to the statistical significance between groups (**p  0.01, mild or severe hypertensives vs. normotensives, †p  0.02, ††d  0.01, severe vs. mild hypertensives). (Modified from Ref. 15.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

during the daytime and nighttime, which is largely dependent on adrenergic influences, undergoes an increase in hypertension and progresses as hypertension becomes more severe (16). This is illustrated in Figure 2, which refers to normotensive subjects and subjects with mild, moderate, and more severe essential hypertension who underwent 24-hour intra-arterial beat-to-beat blood pressure monitoring. The standard deviation of 24-hour average mean arterial pressure values (i.e. an index of the blood pressure variability) increased progressively from the first to the fourth group, indicating that the more elevated the daily life blood pressure values, the greater was the magnitude of the blood pressure oscillations. Two further issues related to the autonomic alterations characterizing essential hypertension deserve to be mentioned. First, a state of sympathetic hyperactivity is not only a feature of young and middle-age hypertensives, but it also occurs in elderly hypertensives, even when the blood pressure elevation selectively affects systolic values. Indeed, when sympathetic nerve traffic was recorded in elderly subjects with systo-diastolic or isolated systolic hypertension, a clear sym-

Figure 2 Standard devations of 24-hour average mean arterial pressure (MAP) in four groups of subjects (normotensives, n  10; mild hypertensives, n  31; moderate hypertensives, n  69; severe hypertensives, n  66). Asterisks (*p  0.05) refer to the statistical significance between groups. Data are shown as mean  SEM. (Modified from Ref. 16.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3 Systolic blood pressure (BP), diastolic BP, heart rate (HR), and muscle sympathetic nerve traffic (MSNA) in elderly normotensives (open histograms), and elderly patients with systo-diastolic (shaded histograms) or isolated systolic (closed histograms) hypertension. Asterisks (*d  0.05, **d  0.01) refer to the statistical significance between groups. Data are shown as mean  SEM. (Modified from Ref. 17.)

pathetic activation was observed when the values were compared to those found in elderly normotensive controls (17) (Fig. 3). Second, the hypertension-related increase in adrenergic outflow appears 1) to be specific for some cardiovascular districts, such as the heart, the kidneys, and the skeletal muscle vasculature (12,18) and, more importantly, 2) peculiar to the hypertensive state of essential nature. This is documented by the evidence that secondary forms of high blood pressure elevation caused by primary hyperaldosteronism or by renal artery stenosis appear not to be characterized by an elevated sympathetic cardiovascular drive (15,19). It is further documented by the evidence that in patients with adrenal phoechromocytoma central sympathetic outflow is not increased (20). Thus, in sharp contrast with what has been described for essential hypertension, in secondary hypertensive states the autonomic imbalance is confined to the parasympathetic control of heart rate, which appears in these conditions also to be clearly impaired (15,21).

III. MECHANISMS RESPONSIBLE FOR THE AUTONOMIC DYSFUNCTION Despite years of investigation, the origin of autonomic dysfunction in essential hypertension remains largely unknown. An attractive hypothesis is that these alterations occur because of an excessive number of and/or reactivity of stressful environmental stimuli, which lead, through frequent transient blood pressure elevations, to a permanently hypertensive state. To date, however, this hypothesis has been confirmed only in animal studies (5), with controversial or circumstantial support in humans (22). First, the cardiovascular responses to laboratory stressors, the approach that has been mostly used, have not consistently been reported to be Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

greater before or after development of hypertension than in normotension (22). Second, human reactivity to stress has been difficult to quantify precisely, because the effect of any given stress has limited reproducibility (23) and because in any given subject an increase in blood pressure response to a stressor can coexist with normal or even reduced responses to other stressors. Finally, in humans, blood pressure responses to laboratory or even more natural stressful stimuli do not bear a close relationship to 24-hour blood pressure variability (22), presumably because behavioral influences are only one of the determinants of blood pressure changes in daily life (24). Another attractive hypothesis (not exclusive of the previous one) is that the autonomic alterations originate from an impairment of the baroreflex, that is, of a major restraining mechanism of parasympathetic and sympathetic tone (21). This is supported by evidence that in congestive heart failure and other diseases, the adrenergic activity is related to a reduced sympathetic modulation by the baroreflex (25). However, in hypertension, a baroreflex impairment has been demonstrated for the parasympathetic but not for the sympathetic component of the reflex (21). The impaired parasympathetic modulation of the heart by the arterial baroreceptors occurring in hypertension has been documented by the evidence that the magnitude of the reflex heart rate changes (bradycardia and tachycardia), induced by increasing and reducing blood pressure values by intravenous injection of phenylephrine and nitroprusside, is markedly less in hypertensive patients than in normotensive individuals (15) (Fig. 4, upper panels). This baroreflex impairment has been more directly demonstrated by the use of a technique allowing study of the baroreflex control of parasympathetic tone not just in the laboratory but in daily life (24). The technique consists of scanning by computer of the intra-arterial blood pressure signal to identify the spontaneous sequences characterized by 1) a progressive increase in systolic blood pressure together with a linearly related increase in pulse interval, i.e., bradycardia, and 2) a progressive reduction in systolic blood pressure together with a linearly related shortening in pulse interval, i.e., tachycardia. These sequences, which disappear after sino-aortic denervation (a finding that documents their baroreflex nature) (26), were found to be markedly reduced in hypertensive individuals (27) (Fig. 5). This confirms that in daily life baroreflex control of heart rate is impaired. As mentioned above, this impairment does not involve the sympathetic component

Figure 5 Frequency of spontaneous sequences characterized by a progressive increase (PI/SBP) or by a progressive decrease (PI/SBP) in pulse interval (PI) and systolic blood pressure (SBP) observed in the 24-hour period in normotensive subjects (open histograms) and in age-matched essential hypertensive patients (closed histograms). Asterisks refer to the statistical significance (*d 0.05) between groups. Data are shown as mean  SEM. (Modified from Ref. 15.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 4 Sensitivity of the baroreflex-heart rate (HR/MAP, upper panels) and muscle sympathetic nerve traffic (MSNA/MAP, lower panels) in normotensive subjects (open histograms) and in mild (dotted histograms) and in more severe (dashed histograms) essential hypertension. Asterisks (**d  0.01) refer to the statistical significance between groups. Data are shown as mean  SEM. (Modified from Ref. 17.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

of the baroreflex function. Indeed, as shown in the lower panels of Figure 4, both the sympathoinhibitory and the sympathoexcitatory responses to intravenous infusions of phenylephrine and nitroprusside were virtually superimposable in essential hypertensive and in normotensive subjects (15), a finding that speaks against the hypothesis that a baroreflex impairment is responsible for the sympathetic activation in hypertension. These data do not rule out the possibility that reflex cardiovascular regulation may contribute to this phoenomenon. First, although unimpaired, the baroreflex is reset towards elevated blood pressure values in hypertension, which means that its influence preserves rather than suppresses the increased sympathetic activity. Second, sympathoinhibitory influences stemming from another reflexogenic area of major importance in controlling circulating blood volume and release of vasoactive substances (such as atrial natriuretic peptides, vasopressin and renin), the so-called cardiopulmonary receptors (21), appear to be slightly reduced in hypertension and more so when the hypertensive state is accompanied by cardiac hypertrophy (28) (Fig. 6). It is thus likely that reflex mechanisms contribute to the sympathetic activation and parasympathetic inhibition occurring in hypertension, although their effects on autonomic function (particularly the adrenergic one) appear to be late and nonspecific. Another hypothesis advanced in recent years claims that the sympathetic activation and parasympathetic inhibition seen in hypertension depend on a metabolic alteration (i.e., hyperinsulinemia and the related insulin resistance) accompanying the hypertensive condition. This hypothesis comes from the evidence that in experimental animals and in humans, acute infusion of insulin, without altering glycemic levels (the so-called euglycemic clamp infusion technique), markedly stimulates the sympathetic nervous system (29). This finding is of particular relevance when one takes into account that a large proportion of hypertensive patients ( 40%) displays elevated insulin levels and an insulin-resistance state. This means that hyperinsulinemia and related insulin-resistance conditions may represent one of the mechanisms responsible for the sympathetic activation that characterizes essential hypertension. It should be mentioned, however, that this effect is reciprocal, namely that a state of sympathetic activation may cause insulin resistance as well (30). It should be finally mentioned that, according to the “epinephrine hypothesis,” this adrenergic neurotransmitter may act as an amplifier of sympathetic activity, at both the central and peripheral levels, thus being hypothetically responsible for the heightened sympathetic cardiovascular drive documented in hypertension (31) (Fig. 7.). Although widely debated, this hypothesis appears to be confirmed by the evidence that an infusion of epinephrine at subpressor doses potentiates in borderline hypertensives (but not in normotensive individuals) the

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 6 Reflex changes in forearm vascular resistance (FVR), plasma norepinephrine (NE), and plasma renin activity (PRA) induced by cardiopulmonary receptor deactivation and stimulation in normotensive subjects (open histograms) and in age-matched essential hypertensives without (shaded histograms) and with (dashed histograms) left ventricular hypertrophy. Asterisks (*p  0.05; **p  0.01) refer to the statistical significance between groups. Data are shown as mean  SEM. (Modified from Ref. 28.)

pressor and sympathoexcitatory responses to adrenergic stimuli (32). It is further confirmed by the evidence that in essential hypertensive patients (but not in healthy subjects with normal blood pressure) epinephrine may be released not only from the adrenal medulla but also from extra-adrenal areas (e.g., the heart) at a rate closely related to the one characterizing the cardiac spillover of norepinephrine (33).

Figure 7 Mechanisms responsible for the atherogenic effects of an increase in sympathetic cardiovascular drive (SNA).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

IV. ADVERSE HEMODYNAMIC AND NONHEMODYNAMIC EFFECTS OF THE AUTONOMIC DYSFUNCTION IN HYPERTENSION Conclusive evidence is now available that sympathetic activation directly promotes cardiac and vascular alterations that lead to the hypertension-related morbidity and mortality independent of blood pressure elevation. In experimental settings this has been shown to occur for left ventricular hypertrophy, which 1) can be induced by subpressor doses of adrenergic agents (34), 2) can be prevented only if blood pressure reduction is not accompanied by excessive reflex cardiac sympathetic stimulation (19,30), and 3) is accompanied by increased cardiac norepinephrine release (35) and by reduced reuptake of this adrenergic neurotransmitter from cardiac sympathetic nerve terminals, as directly quantified by sympathoneuronal imaging techniques employing positron emission tomography coupled with 6-[18F]-fluorodopamine (36). It has also been shown to occur for arteriolar wall hypertrophy or remodeling (37), a structural abnormality that can be found at an early stage of hypertension and help maintain blood pressure elevation (38). Animal and human evidence has recently been obtained showing that sympathetic influences also stiffen the large artery walls. This has an unequivocal pathophysiological implication, because in stiffer arteries the traumatic effect of intravascular pressure is greater and the progression of atherosclerosis probably faster (39). Recent data from a study in rats showed that carotid artery distensibility, as measured by sonographic assessment of arterial diameter over the diastolosystolic pressure range, is markedly increased following sympathectomy (40). Also, radial artery distensibility assessed by the above-mentioned technique is markedly increased following anesthesia of the brachial plexus in humans undergoing surgery for a Dupuytren disease (41). Thus, the stiffening influence of the sympathetic nervous system involves the wall of both elastic-type and muscletype arteries and is visible in both normal and diseased vessels. The mechanism involved is likely to be smooth muscle contraction, because contracting muscle tissue is obviously less distensible than relaxed muscle. Other adverse consequences of the autonomic dysfunction in hypertension should briefly be highlighted. These include an increase in blood viscosity due to the elevated hematocrit level not infrequently displayed by hypertensive patients and ascribed to an -adrenergic–mediated translocation of plasma onto the interstitium (42). They also include a reduction in the arrhythmogenic threshold due to increased heart rate and reduced coronary perfusion triggered, respectively, by the parasympathetic inhibition and the sympathetic activation characterizing the hypertensive state (19,30). It should be mentioned that the sympathetic activation and the concomitant parasympathetic inhibition characterizing the hypertensive state are markedly en-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 8 Values of mean arterial pressure (MAP), body mass index (BMI), heart rate (HR), and muscle sympathetic nerve traffic (MSNA) in lean normotensives (open histograms), lean hypertensives (shaded histograms), obese normotensives (closed histograms), and obese hypertensives (dashed histograms). Asterisks (*p  0.01) refer to the statistical significance between groups. Data are shown as mean  SEM. (Modified from Ref. 45.)

hanced when hypertension is associated with other cardiovascular diseases, such as congestive heart failure (43) or cardiovascular risk factors, such as smoking or obesity (44,45) (Fig. 8). These positive feedback relationships with several factors or conditions further enhance the already impaired autonomic function observed in hypertension, thus increasing the overall risk profile of the patient. V. CONCLUSIONS The data reviewed in this chapter underscore the pathophysiological and clinical relevance of the sympathetic activation in the hypertensive state. They also pro-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

vide a solid background for the use, in the therapeutic approach of hypertension, of drugs with sympathomodulating properties. This would make it possible to achieve better blood pressure control and favor the regression of end-organ damage associated with the hypertensive state.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

8.

9. 10. 11. 12. 13.

14. 15.

16.

Widimsky J, Fejfarova HM, Fejfar Z. High blood pressure in youth (in German). Arch Kreislaufforschung 1958; 28:100–124. Lund-Johansen P. Hemodynamics in early essential hypertension. Acta Med Scand 1967; 482:1–105. Julius S, Conway J. Hemodynamic studies in patients with borderline blood pressure elevation. Circulation 1968; 38:282–288. Julius S, Pasqual AV, London R. Role of parasympathetic inhibition in the hyperkinetic type of borderline hypertension. Circulation 1971; 44:413–418. Folkow B. Physiological aspects of primary hypertension. Physiol Rev 1982; 62:347–504. Bohm R, Van Baak M, Van Hooff M, Moy J, Rahn KH. Salivary flow in borderline hypertension. Klin Wochenschr 1985; 63(suppl 3):154–156. Takeshita A, Mark AL. Neurogenic contribution to hindquarters vasoconstriction during high sodium intake in Dahl strain genetically salt-sensitive rats. Hypertension 1981; 3(suppl. 1):135–141. Judy WJ, Watanabe AM, Murphy WR, Aprison BS, Yu PL. Sympathetic nerve activity and blood pressure in normotensive backcross rats genetically related to the spontaneously hypertensive rat. Hypertension 1979; 1:598–604. Oparil S. The sympathetic nervous system in clinical and experimental hypertension. Kidney Int 1986; 30:437–452. Goldstein DS. Plasma catecholamines and essential hypertension: an analytical review. Hypertension 1983; 5:86–99. Grassi G, Esler MD. How to assess sympathetic activity in humans. J Hypertens 1999; 17:719–734. Esler M, Lambert G, Jennings G. Regional norepinephrine turnover in human hypertension. Clin Exp Hypertens 1989; 11(suppl 1):75–89. Anderson EA, Sinkey CA, Lawton WJ, Mark AL. Elevated sympathetic nerve activity in borderline hypertensive humans: evidence from direct intraneural recordings. Hypertension 1988; 14:1277–1283. De Champlain J. Pre- and post-synaptic adrenergic dysfunctions in hypertension. J Hypertens 1990; 8(suppl 7):s77–s85. Grassi G, Cattaneo BM, Seravalle G, Lanfranchi A, Mancia G. Baroreflex control of sympathetic nerve activity in essential and secondary hypertension. Hypertension 1998; 81:68–72. Mancia G, Ferrari A, Gregorini L, Parati G, Pomidossi G, Bertinieri G, Grassi G, Di Rienzo M, Pedotti A, Zanchetti A. Blood pressure and heart rate variabilities in normotensive and hypertensive human beings. Circ Res 1983; 53:96–104.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

17.

18.

19. 20. 21.

22.

23.

24.

25.

26.

27.

28.

29. 30.

31. 32.

Grassi G, Seravalle G, Bertinieri G, Turri C, Dell’Oro R, Stella ML, Mancia G. Sympathetic and reflex alterations in systo-diastolic and systolic hypertension of the elderly. J Hypertens 2000; 18:587–593. Grassi G, Colombo M, Seravalle G, Spaziani D, Mancia G. Dissociation between muscle and skin sympathetic nerve activity in essential hypertension, obesity and congestive heart failure. Hypertension 1998; 81:64–67. Grassi G. Role of the sympathetic nervous system in human hypertension. J Hypertens 1998; 16:1979–1987. Grassi G, Seravalle G, Turri C, Mancia G. Sympathetic nerve traffic responses to surgical removal of pheochromocytoma. Hypertension 1999; 84:461–465. Mancia G, Grassi G, Ferrari AU. Reflex control of the circulation in experimental and human hypertension, In: Zanchetti A, Mancia G, eds. Handbook of Hypertension, Vol. 17: Pathophysiology of Hypertension. Amsterdam: Elservier Science, 1997:586–601. Mancia G, Parati G. Reactivity to physical and behavioural stress and blood pressure variability in hypertension. In: Julius S, Bassett DR, eds. Handbook of Hypertension, Vol 9: Behavioural Factors in Hypertension. Amsterdam: Elsevier Science, 1987:104–122. Parati G, Pomidossi G, Ramirez AJ, Cesana B, Mancia G. Variability of hemodynamic responses to laboratory tests employed in assessment of neural cardiovascular regulation in man. Clin Sci 1985; 69:533–540. Mancia G, Parati G, Di Rienzo M, Zanchetti A. Blood pressure variability. In: Zanchetti A, Mancia G, eds, Handbook of Hypertension, Vol. 17: Pathophysiology of Hypertension. Amsterdam: Elsevier Science, 1997:117–169. Grassi G, Seravalle G, Cattaneo BM, Lanfranchi A, Vailati S, Giannattasio C, Del Bo A, Sala C, Bolla G, Pozzi M, Mancia G. Sympathetic activation and loss of reflex sympathetic control in mild congestive heart failure. Circulation 1995; 92:3206–3211. Bertinieri G, Di Rienzo M, Cavallazzi A, Ferrari AU, Pedotti A, Mancia G. Evaluation of baroreceptor reflex by blood pressure monitoring in unanesthetized cats. Am J Physiol 1988; 254:H377–H383. Parati G, Di Rienzo M, Bertinieri G, Pomidossi G, Casadei R, Groppelli A, Pedotti A, Zanchetti A, Mancia G. Evaluation of the baroreceptor-heart rate reflex by 24-h intra-arterial blood pressure monitoring in humans. Hypertension 1988; 12:214–222. Grassi G, Giannattasio C, Cleroux J, Cuspidi C, Sampieri L, Bolla GB, Mancia G. Cardiopulmonary reflex before and after regression of left ventricular hypertrophy in essential hypertension. Hypertension 1988; 12:227–237. Scherrer U, Sartori C. Insulin as a vascular and sympathoexcitatory hormone. Circulation 1997; 96:4104–4113. Mancia G, Grassi G, Giannattasio C, Seravalle G. Sympathetic activation in the pathogenesis of hypertension and progression of organ damage. Hypertension 1999; 34:724–728. Floras JS. Epinephrine and the genesis of hypertension. Hypertension 1992; 19:1–18. Grassi G, Rea RF, Somers VK, Abboud FM, Mark AL. Epinephrine acts centrally to facilitate reflex sympathetic nerve response in borderline hypertension (abstr). Hypertension 1988; 12:355.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

33. Rumantir MS, Jennings GL, Lambert GW, Kaye DM, Seals DR, Esler M. The “adrenaline hypothesis” of hypertension revisited: evidence for adrenaline release from the heart of patients with essential hypertension. J Hypertens 2000; 18:717–723. 34. Sen S, Tarazi RC, Khairallah P, Bumpus M. Cardiac hypertrophy in spontaneously hypertensive rats. Circ Res 1974; 35:775–781. 35. Kelm M, Schafer S, Mingers S, Heydtheusen M, Vogt M, Motz W, Strauer BE. Left ventricular mass in linked to cardiac noradrenaline in normotensive and hypertensive patients. J Hypertens 1996; 14:1357–1364. 36. Li ST, Tack CJ, Fananapazir L, Goldstein DS. Myocardial perfusion and sympathetic innervation in patients with hypertrophic cardiomiopathy. J Am Coll Cardiol 2000; 35:1867–1873. 37. Heagerty AM. Structural changes in resistance arteries in hypertension. In: Zanchetti A, Mancia G, eds. Handbook of Hypertension, Vol. 17, Pathophysiology of Hypertension. Amsterdam: Elsevier Science, 1997:426–437. 38. Folkow B. The Fourth Volhard Lecture: cardiovascular structural adaptation; its role in the initiation and maintenance of primary hypertension. Clin Sci 1978; 4(suppl 3):3s–22s. 39. Bernini F, Corsini A, Raiteri M, Soma MR, Paoletti R. Effects of lacidipine on experimental models of atherosclerosis. J Hypertens 1993; 11(suppl 1):s61–s66. 40. Mangoni AA, Mircoli L, Giannattasio C, Mancia G, Ferrari AU. Effects of sympathectomy on mechanical properties of carotid and femoral arteries. Hypertension 1997; 80:1085–1088. 41. Grassi G, Giannattasio C, Failla M, Pesenti A, Peretti G, Marinoni E, Mancia G. Sympathetic modulation of radial artery compliance in congestive heart failure. Hypertension 1995; 26:348–354. 42. Cohn JN. Relationship of plasma volume changes to resistance and capacitance vessel effects of sympathomimetic amines and angiotensin in man. Clin Sci 1966; 30:267–278. 43. Grassi G, Dell’Oro R, Turri C, Magnoni M, Bertinieri G, Seravalle G, Mancia G. Sympathetic and reflex abnormalities in hypertensive heart failure patients (abstr). Eur Heart J 1999; 20(suppl):224. 44. Grassi G, Seravalle G, Calhoun DA, Bolla G, Giannattasio C, Marabini M, Del Bo A, Mancia G. Mechanisms responsible for sympathetic activation by cigarette smoking in humans. Circulation 1994; 90:248–253. 45. Grassi G, Seravalle G, Dell’Oro R, Turri C, Bolla GB, Mancia G. Adrenergic and reflex abnormalities in obesity-related hypertension. Hypertension 2000; 36:538–542.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

14 Autonomic Control of the Airways Peter J. Barnes National Heart & Lung Institute, Imperial College, London, United Kingdom

Autonomic nerves regulate airway caliber, blood flow and secretions. They play an important role in the pathophysiology of airway diseases, such as asthma and chronic obstructive pulmonary disease, modulating the inflammatory process and the airway response to inflammation. Cholinergic nerves play a major role in regulating airway caliber in humans, whereas adrenergic nerves are less important. Afferent nerves are important in mediating responses to inhaled irritants and other stimuli. Many neuropeptides are expressed in airway nerves and may play an important modulating role in disease. Several treatments for airway disease interact with autonomic control mechanisms.

I. OVERVIEW OF AIRWAY INNERVATION Airway nerves regulate the caliber of the airways and control airway smooth muscle tone, airway blood flow, and mucus secretion. They may also influence the inflammatory process and play an integral role in host defense. Neural control of airway function is more complex than previously recognized. Many neurotransmitters are now identified and these act on a multitude of autonomic receptors. Three types of airway nerve are recognized: parasympathetic nerves that primarily release acetylcholine (ACh), sympathetic nerves that primarily release (norepinephrine), and afferent (sensory nerves) whose primary transmitter may be glutamate. In addition to these classical transmitters, multiple neuropeptides have now been localized to airway nerves and may have potent effects on airway function (1). All of these neurotransmitters act on autonomic receptors that are expressed

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

on the surface of target cells in the airway. It is increasingly recognized that a single transmitter may act on several subtypes of receptor, which may lead to different cellular effects mediated via different second messenger systems. Several neural mechanisms are involved in the regulation of airway caliber, and abnormalities in neural control may contribute to airway narrowing in disease (Fig. 1). Neural mechanisms may be involved in the pathophysiology of airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD), contributing to the symptoms and possibly to the inflammatory response (2). There is a close interrelationship between inflammation and neural responses in the airways, since inflammatory mediators may influence the release of neurotransmitters via activation of sensory nerves leading to reflex effects and via stimulation of prejunctional receptors that influence the release of neurotransmitters (3). In turn, neural mechanisms may influence the nature of the inflammatory response, either reducing inflammation or exaggerating the inflammatory response. A. Neural Interactions Complex interactions between various components of the autonomic nervous system are now recognized. Adrenergic nerves may modulate cholinergic neurotransmission in the airways, and sensory nerves may influence neurotransmission in parasympathetic ganglia and at postganglionic nerves. This means that changes in the function of one neural pathway may have effects on other pathways.

Figure 1 Autonomic control of airway smooth muscle tone. There are neural mechanisms resulting in bronchoconstriction (B/C) and bronchodilatation (B/D). ACh  acetylcholine; NA  noradrenaline, A  adrenaline, VIP  vasoactive intestinal peptide, NO  nitric oxide, NK  neurokinin.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

B. Cotransmission Almost every nerve contains multiple transmitters. Thus airway parasympathetic nerves, in which the primary transmitter is ACh, also contain the neuropeptides vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine/methionine (PHI/M), pituitary adenylate cyclase–activating peptide (PACAP), helodermin, galanin, and nitric oxide (NO) (Fig. 2). These cotransmitters may have either facilitatory or antagonistic effects on target cells, or they may influence the release of the primary transmitter via prejunctional receptors. Thus, VIP modulates the release of ACh from airway cholinergic nerves. Sympathetic nerves, which release norepinephrine, may also release neuropeptide Y (NPY) and enkephalins, whereas afferent nerves may contain a variety of peptides including substance P (SP), neurokinin A (NKA), calcitonin gene–related peptide (CGRP), galanin, VIP, and cholecystokinin. The physiological role of neurotransmission may be in “fine-tuning” of neural control. Neuropeptides may be preferentially released by high-frequency firing of nerves, and their effects may therefore only become manifest under condition of excessive nerve stimulation. Neuropeptide neurotransmitters may also act on target cells different from the primary transmitter, resulting in different physiological effects. Thus in airways ACh causes bronchoconstriction, but VIP that is

Figure 2 Neurotransmitters and cotransmitters in airway nerves. SP  substance P, NKA  neurokinin A, CGRP  calcitonin gene–related peptide, Gal  galanin, VIP  vasoactive intestinal peptide, PHI/PHM  peptide histidine isoleucine/methionine, NPY  neuropeptide Y.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

coreleased may have its major effect on bronchial vessels, thus increasing blood flow to the airways. In chronic inflammation the role of cotransmitters may be increased by alterations in the expression of their receptors or by increased synthesis of transmitters via increased gene transcription.

II. AFFERENT NERVES The sensory innervation of the respiratory tract is mainly carried in the vagus nerve. The neuronal cell bodies are localized to the nodose and jugular ganglia and input to the solitary tract nucleus in the brain stem. A few sensory fibers supplying the lower airways enter the spinal cord in the upper thoracic sympathetic trunks, but their contribution to respiratory reflexes is minor, and it is uncertain whether they are represented in humans. There is a tonic discharge of sensory nerves that has a regulatory effect on respiratory function and also triggers powerful protective reflex mechanisms in response to inhaled noxious agents, physical stimuli, or certain inflammatory mediators. At least three types of afferent fiber have been identified in the lower airways (4) (Fig. 3). Most of the information on their function has been obtained from studies in anesthetized animals, so it is difficult to know how much of the information obtained in anesthetized animals can be extrapolated to human airways.

Figure 3 Afferent nerves in airways. Slowly-adapting receptors (SAR) are found in airway smooth muscle, whereas rapidly adapting myelinated (RAR) and unmyelinated Cfibers are present in the airway mucosa.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

A. Slowly Adapting Receptors Myelinated fibers associated with smooth muscle of proximal airways are probably slowly adapting (pulmonary stretch) receptors (SAR), which are involved in reflex control of breathing. Activation of SARs reduces efferent vagal discharge and mediates bronchodilatation. During tracheal constriction the activity of SARs may serve to limit the bronchoconstrictor response (4). SARs may play a role in the cough reflex since when these receptors are destroyed by high concentrations of SO2 the cough response to mechanical stimulation is lost. B. Rapidly Adapting Receptors Myelinated fibers in the epithelium, particularly at the branching points of proximal airways, show rapid adaptation. Rapidly adapting receptors (RAR) account for 10–30% of the myelinated nerve endings in the airways (5). These endings are sensitive to mechanical stimulation and to mediators such as histamine. The response of RAR to histamine is partly due to mechanical distortion consequent to bronchoconstriction, although if this is prevented by pretreatment with isoproterenol the RAR response is not abolished, indicating a direct stimulatory effect of histamine. It is likely that mechanical distortion of the airway may amplify irritant receptor discharge. RAR with widespread arborizations are very numerous in the area of the carina, where they have been termed “cough receptors,” as cough can be evoked by even the slightest touch in this region. RAR respond to inhaled cigarette smoke, ozone, serotonin, and prostaglandin F2, although it is possible that these responses are secondary to the mechanical distortion produced by the bronchoconstrictor response to these irritants. Neurophysiological studies using an in vitro preparation in guinea pig trachea and bronchi show that a majority of afferent fibers are myelinated and belong to the A -fiber group. Although these fibers are activated by mechanical stimulation and low pH, they are not sensitive to capsaicin, histamine, or bradykinin (6,7). C. C-Fibers There is a high density of unmyelinated fibers (C-fibers) in the airways, and they greatly outnumber myelinated fibers. In the bronchi C-fibers account for 80–90% of all afferent fibers in cats. C-fibers play an important role in the defense of the lower respiratory tract (8). C-fibers contain neuropeptides, including SP, NKA, and CGRP, that confer a motor function on these nerves (9). Bronchial C-fibers are insensitive to lung inflation and deflation but typically respond to chemical stimulation. In vivo studies suggest that bronchial C-fibers in dogs respond to the inflammatory mediators histamine, bradykinin, serotonin, and prostaglandins (8).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

They are selectively stimulated by capsaicin given either intravenously or by inhalation and are also stimulated by SO2 and cigarette smoke. Since these fibers are relatively unaffected by lung mechanics, it is likely that these agents act directly on the unmyelinated endings in the airway epithelium. In the in vitro guinea pig trachea preparation, C-fibers are stimulated by capsaicin and by bradykinin, but not by histamine, serotonin, or prostaglandins (with the possible exception of prostacyclin) (6). Both RARs and C-fibers are sensitive to water and hyperosmotic solutions, with RARs showing a greater sensitivity to hypotonic and C-fibers to hypertonic saline. In the in vitro guinea pig trachea preparation, A fibers and C-fibers are stimulated by water and by hyperosmolar solutions; a small proportion of A fibers are also stimulated by low-chloride solutions, whereas the majority of Cfibers are activated (10). D. Cough Cough is an important defense reflex, which may be triggered from either laryngeal or lower airway afferents (11). There is debate about which are the most important afferents for initiation of cough, and this may be dependent on the stimulus. Thus, RARs are activated by mechanical stimuli (e.g., particulate matter), bronchoconstrictors, and hypotonic saline and water, whereas C-fibers are more sensitive to hypertonic solutions, bradykinin, and capsaicin. In normal humans inhaled capsaicin is a potent tussive stimulus, and this is associated with a transient bronchoconstrictor reflex that is abolished by an anticholinergic drug. It is not certain whether this is due to stimulation of C-fibers in the larynx, but as these are very sparse it is likely that bronchial C-fibers are also involved. Citric acid is commonly used to stimulate coughing in experimental challenges in human subjects; it is likely that it produces cough by a combination of low pH (which stimulates C-fibers) and low chloride (which may stimulate laryngeal and lower airway afferents). Inhaled bradykinin causes coughing and a raw sensation retrosternally, which may be due to stimulation of C-fibers in the lower airways. Bradykinin appears to be a relatively pure stimulant of C fibers (6). Prostaglandins E2 and F2 are potent tussive agents in humans and also sensitize the cough reflex (12,13). E. Afferent Nerves in Airway Disease Airway afferent nerves may become sensitized in inflammatory airway diseases, resulting in increased symptoms, such as cough and chest tightness. Cough is a prominent symptom of asthma and COPD, and there is evidence that cough sensitivity is increased (14). This may be due to sensitization of afferent nerves in the airways as a result of inflammatory mediators produced during asthma and COPD. PGE2 is a potent sensitizer of airway sensory nerves and is increased in

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

asthma and COPD. Bradykinin is also a potent afferent nerve sensitizer (7). Chronic inflammation may lead to neural hyperesthesia through mechanisms that may involve cytokines and neurotrophins (15). There may be changes in ion channels in sensory nerves in response to inflammation, which increases neural sensitivity (16). Neurotrophins, such as nerve growth factor and ciliary neurotrophic factor, may result in proliferation of airway sensory nerves and a change in the nerve phenotype, with a reduced threshold of activation and increased expression of neuropeptides (17). The role of neurotrophins in airway diseases has not yet been explored (18). III. CHOLINERGIC NERVES Cholinergic nerves are the major neural bronchoconstrictor mechanism in human airway and are the major determinant of airway caliber. A. Cholinergic Control of Airways Cholinergic nerve fibers arise in the nucleus ambiguous in the brain stem and travel down the vagus nerve and synapse in parasympathetic ganglia, which are located within the airway wall. From these ganglia short postganglionic fibers travel to airway smooth muscle and submucosal glands (Fig. 4). In animals, elec-

Figure 4 Cholinergic control of airway smooth muscle. Preganglionic and postganglionic parasympathetic nerves release acetylcholine (ACh) and can be activated by airway and extrapulmonary afferent nerves.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

trical stimulation of the vagus nerve causes release of ACh from cholinergic nerve terminals, with activation of muscarinic cholinergic receptors on smooth muscle and gland cells, which results in bronchoconstriction and mucus secretion. Prior administration of a muscarinic receptor antagonist, such as atropine, prevents vagally induced bronchoconstriction. A novel aspect of cholinergic control is the demonstration that human airway epithelial cells may release Ach as a result of upregulation of choline acetytransferase in response to inflammatory stimuli (19). The contribution of extraneuronal ACh to cholinergic responses in the airways is currently unknown. B. Muscarinic Receptors Of the five know subtypes of muscarinic receptor, four have been identified by binding studies and pharmacologically in lung (20). The muscarinic receptors that mediate bronchoconstriction in human and animal airways belong to the M3-receptor subtype, whereas mucus secretion appears to be mediated by M1- and M3receptors. M1-receptors are also localized to parasympathetic ganglia, where they facilitate the neurotransmission mediated via nicotinic receptors (Fig. 5). Inhibitory muscarinic receptors (autoreceptors) have been demonstrated on cholinergic nerves of airways in animals in vivo and in human bronchi in vitro (21). These prejunctional receptors inhibit ACh release and may serve to limit vagal bronchoconstriction. Autoreceptors in human airways belong to the M2-receptor subtype, whereas those on airway smooth muscle and glands belong to the M3-receptor subtype (22). Drugs such as atropine and ipratropium bromide, which block both prejunctional M2-receptors and postjunctional M3-receptors on smooth muscle with equal efficacy, therefore increase ACh release, which may then over-

Figure 5 Muscarinic receptor subtypes in airways. M2-receptors on postganglionic cholinergic nerve terminals inhibit the release of acetylcholine (ACh), thus reducing the stimulation of postjunctional M3-receptors which constrict airway smooth muscle.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

come the postjunctional blockade. This means that such drugs will not be as effective against vagal bronchoconstriction as against cholinergic agonists, and it may be necessary to reevaluate the contribution of cholinergic nerves when drugs that are selective for the M3-receptors are developed for clinical use. The presence of muscarinic autoreceptors has been demonstrated in human subjects in vivo (23). A cholinergic agonist, pilocarpine, that selectively activates M2-receptors inhibits cholinergic reflex bronchoconstriction induced by sulfur dioxide in normal subjects, but such an inhibitory mechanism does not appear to operate in asthmatic subjects, suggesting that there may be dysfunction of these autoreceptors. Such a defect in muscarinic autoreceptors may then result in exaggerated cholinergic reflexes in asthma, since the normal feedback inhibition of ACh release may be lost. This might also explain the sometimes catastrophic bronchoconstriction that occurs with -blockers in asthma, which, at least in mild asthmatics, appears to be mediated by cholinergic pathways (24). Antagonism of inhibitory -receptors on cholinergic nerves would result in increased release of ACh, which could not be switched off in the asthmatic patient. This explains why anticholinergics prevent -blocker–induced asthma. The mechanisms which lead to dysfunction of prejunctional M2-receptors in asthmatic airways are not certain, but it is possible that M2-receptors may be more susceptible to damage by oxidants or other products of the inflammatory response in the airways. Experimental studies have demonstrated that influenza virus infection and eosinophils in guinea pigs may result in a selective loss of M2-receptors compared with M3-receptors, resulting in a loss of autoreceptor function and enhanced cholinergic bronchoconstriction. Cholinergic innervation is greatest in large airways and diminishes peripherally, although in humans muscarinic receptors are localized to airway smooth muscle in all airways (25). In humans, studies that have tried to distinguish large and small airway effects have shown that cholinergic bronchoconstriction predominantly involves larger airways, whereas -agonists are equally effective in large and small airways. This relative diminution of cholinergic control in small airways may have important clinical implications, since anticholinergic drugs are likely to be less useful than -agonists when bronchoconstriction involves small airways. Normal human subjects also have resting bronchomotor tone, since atropine causes bronchodilatation. C. Cholinergic Reflexes A wide variety of stimuli are able to elicit reflex cholinergic bronchoconstriction through activation of sensory receptors in the larynx or lower airways. Activation of cholinergic reflexes may result in bronchoconstriction and an increase in airway mucus secretion through the activation of muscarinic receptors on airway smooth muscle cells and submucosal glands. Cholinergic reflexes may also be activated from extrapulmonary afferents, and these reflexes may also contribute to

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

airway defenses. Esophageal reflux may be associated with bronchoconstriction in asthmatic patients. In some patients this may be due to aspiration of acid into the airways, in other cases acid reflux into the esophagus activates a reflex cholinergic bronchoconstriction (the “reflux reflex”) (26). D. Modulation of Cholinergic Neurotransmission Many agonists may modulate cholinergic neurotransmission via prejunctional receptors on postganglionic nerves (3,27). Some receptors increase (facilitate), whereas others inhibit the release of ACh. Inflammatory mediators may influence cholinergic neurotransmission via prejunctional receptors. For example, thromboxane and prostaglandin (PG)D2 facilitate ACh release from postganglionic nerves in the airways. Facilitation may also occur at parasympathetic ganglia in the airways; these structures are surrounded by inflammatory cells and have an afferent neural input. Electrophysiological recordings show a prolonged potentiation of neurotransmission in ganglia after allergen exposure in sensitized guinea pigs (28). E. Role in Asthma Many of the stimuli that produce bronchospasm in asthma activate sensory nerves and reflex bronchoconstriction in animals, so it was logical to suggest that asthma may be due to exaggerated cholinergic reflex mechanisms. There is some evidence that cholinergic tone is increased in asthmatic airways (29). There are several mechanisms by which cholinergic tone might be increased in asthma. An increase in cholinergic tone could arise via several mechanisms: Increased afferent receptor stimulation by inflammatory mediators, such as histamine or prostaglandins, which may be released from mast cells and other inflammatory cells in the asthmatic airway or from bradykinin formed from precursors in exuded plasma. Increased release of ACh from cholinergic nerve terminals by an action on cholinergic nerve endings themselves, or by an increase in nerve traffic through cholinergic ganglia (local airway reflex) (3). Abnormal muscarinic receptor expression, via either an increase in M3-receptors or a reduction in M2-receptors. There is no evidence for increased M1 or M3 receptor expression in asthmatic lungs (30), but there is functional evidence for a defect in M2-receptor function that may be secondary to the inflammatory process, as discussed above. Decrease in the neuromodulators (VIP, NO) that have a “braking” effect on neurotransmission (see below). The effect of ACh on asthmatic airways is exaggerated, as a manifestation of nonspecific hyperresponsiveness of the airways that is so characteristic of asthma. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

However, asthmatic airways are hyperresponsive to many spasmogens in addition to ACh, mediators such as histamine, leukotrienes, and prostaglandins have a direct contractile effect on bronchial smooth muscle, which is not blocked by anticholinergic drugs. Anticholinergic agents will only counteract the cholinergic reflex component of bronchoconstriction, which may be less prominent in human airways than animal studies had indicated. By contrast 2-agonists reverse bronchoconstriction irrespective of the mechanism, since they act as functional antagonists. In patients with chronic asthma that has been poorly controlled, there is a progressive decline in lung function over the years (31), which presumably results from chronic inflammation. Vagal tone increases the airway narrowing further and for geometric reasons will have a greater effect on airway resistance in narrowed airways. This may explain why anticholinergics are often of greater use in chronic asthmatics with a major element of fixed airway obstruction.

F. Role in COPD The structural narrowing of the airways in COPD means that even normal vagal tone will exert a greater effect on airway caliber than in normal airways. This may account for the efficacy of anticholinergics as bronchodilators in COPD, as cholinergic tone is the only reversible element. In addition, cholinergic mechanisms may account for the mucus hypersecretion of chronic bronchitis.

IV. ADRENERGIC CONTROL The airways are also under adrenergic control, which includes sympathetic nerves (which release norepinephrine), circulating catecholamines (predominantly epinephrine), and - and -adrenoceptors (Fig. 6). The fact that -adrenergic antagonists cause bronchoconstriction in asthmatic patients, but not in normal individuals, suggests that adrenergic control of airway smooth muscle may be abnormal in asthma.

A. Sympathetic Innervation Although sympathetic bronchodilator nerves have been demonstrated in several species, including cats, dogs, and guinea pigs, most evidence suggests that adrenergic nerves do not control human airway smooth muscle directly. Sympathetic nerves may influence cholinergic tone of airway smooth muscle via adrenoceptors localized to parasympathetic ganglia and prejunctionally on postganglionic nerves, however (3). Sympathetic nerves may play an important role in the regulation of airway blood flow and in mucus secretion. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 6 Adrenergic control of airway smooth muscle. Sympathetic nerves release norepinephrine (NE), which may modulate cholinergic nerves at the level of the parasympathetic ganglion or postganglionic nerves, rather than directly at smooth muscle in human airways. Circulating epinephrine (E) is more likely to be important in adrenergic control of airway smooth muscle.

B. ␤-Adrenoceptors -Adrenoceptors regulate many aspects of airway function, including airway smooth muscle tone, mast cell mediator release, and plasma exudation (32). The possibility that -receptors are abnormal in asthma has been extensively investigated. The suggestion that there is a primary defect in -receptor function in asthma has not been substantiated, and any defect in -receptors is likely to be secondary to the disease, perhaps as a result of inflammation or as a consequence of adrenergic therapy. Some studies have demonstrated that airways from asthmatic patients fail to relax normally to isoproterenol, suggesting a possible defect in -receptor function in airway smooth muscle (33). Whether this is due to a reduction in -receptors, a defect in receptor coupling, or some abnormality in the biochemical pathways leading to relaxation is not yet known, although the density of -receptors in airway smooth muscle appears to be normal (34), and there is no reduction in the density of 1- or 2-receptors in asthmatic lung, at either the receptor or the mRNA level (30). There is some evidence that proinflammatory cytokines may affect 2-receptor function. IL-1 reduces the bronchodilator effect of isoproterenol in vitro and in vivo, and this appears to be due to uncoupling of 2-receptors due to increased expression of the inhibitory G protein, Gi (35,36). Studies of 2-receptor Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

expression in asthmatic airways obtained by biopsy have demonstrated only small defects in coupling after local allergen challenge, however (37). C. ␣-Adrenoceptors -Receptors that mediate bronchoconstriction have been demonstrated in airways of several species and may only be demonstrated under certain experimental conditions. There is now considerable doubt about the role of -receptors in the regulation of tone in human airways, however, since it has proved difficult to demonstrate their presence functionally or by autoradiography (38), and -blocking drugs do not appear to be effective as bronchodilators. It is possible that -receptors may play an important role in regulating airway blood flow, which may indirectly influence airway responsiveness, and there is some evidence that -agonists may reduce airway narrowing in exercise-induced asthma (39).

V. NANC NERVES AND NEUROPEPTIDES Neural responses that are not blocked by a combination of adrenergic and cholinergic antagonists are known as nonadrenergic noncholinergic (NANC) nerves. These NANC responses appear to be due to the release of neurotransmitters from classical autonomic nerves, which include neuropeptides, nitric oxide (NO), and adenosine triphosphate (ATP). In the airways both inhibitory NANC (bronchodilator, i-NANC) and excitatory NANC (bronchoconstrictor, e-NANC) nerves have been described. A. i-NANC Nerves i-NANC nerves that mediate bronchodilatation have been described in may species, including humans, in whom they are of particular importance in the absence of any direct sympathetic innervation of airway smooth muscle (40). The neurotransmitter for these nerves in some species, including guinea pigs and cats, is vasoactive intestinal polypeptide (VIP) and related peptides. The i-NANC bronchodilator response is blocked by -chymotrypsin, an enzyme that very efficiently degrades VIP, and by antibodies to VIP. However, although VIP is present in human airways and VIP is a potent bronchodilator of human airways in vitro, there is no evidence that VIP is involved in neurotransmission of i-NANC responses in human airways, and -chymotrypsin that completely blocks the response to exogenous VIP has no effect on neural bronchodilator responses (41). It is likely that VIP and related peptides may be more important in neural vasodilatation responses and may result in increased blood flow to bronchoconstricted airways. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The predominant I-NANC neurotransmitter of human airways is NO. NO synthase inhibitors, such as NG-L-arginine methyl ester, virtually abolish the iNANC response (42). This effect is more marked in proximal airways, consistent with the demonstration that nitrergic innervation is greatest in proximal airways. NO appears to be a cotransmitter with ACh, and NO acts as a “braking” mechanism for the cholinergic system by acting as a functional antagonist to ACh at airway smooth muscle (43) (Fig. 7). B. Airway Neuropeptides Many neuropeptides are localized to sensory, parasympathetic, and sympathetic neurons in the human respiratory tract. These peptides have potent effects on bronchomotor tone, airway secretions, the bronchial circulation, and inflammatory and immune cells (1). Although the precise physiological roles of each peptide are not yet fully understood, clues are provided by their localization and functional effects. Recently the development of specific neuropeptide receptor antagonists has provided important new insights into the roles of these neurotransmitters. Many of the inflammatory and functional effects of neuropeptides

Figure 7 Nitric oxide (NO) and vasoactive intestinal peptide (VIP) may modulate cholinergic neural effects mediated via acetylcholine (ACh). In inflammation NO may be removed by superoxide anions (O\2) generated from inflammatory cells and VIP by mast cell tryptase, therefore diminishing their “braking” effects, resulting in exaggerated cholinergic bronchoconstriction.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

are relevant to asthma, and there is compelling evidence for the involvement of neuropeptides in the pathophysiology and symptomatology of asthma and COPD. Although classically neuropeptides are released from autonomic nerves, there is increasing evidence that these peptides may be synthesized and released from inflammatory and nonneural structural cells, particularly in disease. Inflammatory cytokines may increase the expression of neuropeptide genes in inflammatory cells, so that inflammatory become a major source of the neuropeptide at the inflammatory site. For example, both VIP and substance P have been localized to human eosinophils and substance P to macrophages (44,45). C. Neuropeptides and Airway Inflammation Neuropeptides have multiple inflammatory and immune effects on the airways, thereby intensifying the ongoing inflammation (46). In turn, inflammatory mediators may amplify or sometimes dampen neuropeptide effects. Inflammatory mediators may increase the release of neuropeptides from sensory and other nerves, may increase the expression of neuropeptide genes in neural and inflammatory cells, may increase the expression of neuropeptide receptors, and may decrease the degradation of neuropeptides. Neuropeptides may be involved in the recruitment and activation of inflammatory cells, such as eosinophils (47). D. VIP and Related Peptides VIP-immunoreactive nerves are widely distributed throughout the respiratory tract in humans, and there is also evidence for the presence of several closely related peptides: peptide histidine methionine, peptide histidine valine, helodermin, helospectins I and II, and pituitary adenylate cyclase activating peptide (PACAP38) and PACAP-27, which have similar functional effects. VIP may be localized to parasympathetic and sensory nerves. It is a potent vasodilator, a bronchodilator, increases mucus secretion, and may have anti-inflammatory effects. In some species it is a mediator of neurogenic bronchodilatation, but this is not the case in human airways. A defect in VIP has been proposed in asthma, but there is little evidence for this (48). E. Tachykinins Substance P (SP) and neurokinin A (NKA), but not NKB, are localized to C-fibers are abundant in rodent airways, but are sparse in human airways (49). Tachykinins are also be expressed human macrophages, which also express tachykinin receptors. Tachykinins have many different effects on the airways that may be relevant to asthma, and these effects are mediated via NK1-receptors (preferentially activated by SP) and NK2-receptors (activated by NKA) (Table 1). Tachykinins con-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Table 1 Effects of Tachykinins on Airways Effect Bronchoconstriction (small large airways) Plasma exudation Bronchial vasodilatation ↑ Neurotransmission (ganglia, cholinergic nerves) Cough (peripheral and central mechanisms) Mucus secretion (submucosal glands and goblet cells) ↑ Adhesion molecules (ICAM-1, E-selectin) Activation of inflammatory cells (macrophages, T lymphocytes, eosinophils) Angiogenesis Fibroblast activation

Neurokinin receptor NK2, NK1 NK1 NK1 NK1, NK2, NK3 NK1, NK2, NK3 NK1 NK1 NK1, NK2 NK1 NK1, NK2

strict smooth muscle of human airways in vitro via NK2-receptors. Tachykinin receptors are widely distributed in human airways, with localization predominantly to airway smooth muscle, mucus-secreting cells, and bronchial cells (50). NKA causes bronchoconstriction after both intravenous and inhaled administration in asthmatic subjects. Mechanical removal of airway epithelium potentiates the bronchoconstrictor response to tachykinins, largely because the neutral endopeptidase (NEP), which is a key enzyme in the degradation of tachykinins in airways, is strongly expressed on epithelial cells (51). SP stimulates mucus secretion from submucosal glands in human airways in vitro and is a potent stimulant to goblet cell secretion in guinea pig airways via activation of NK1-receptors. NK1-receptors also mediate the increased plasma exudation and the vasodilator response to tachykinins. Tachykinins may also interact with inflammatory and immune cells, although whether this is of pathophysiological significance remains to be determined (52). SP degranulates skin mast cells and eosinophils through a non–receptor-mediated mechanism. Tachykinins also enhance eosinophil chemotaxis. Tachykinins may activate alveolar macrophages and monocytes to release inflammatory cytokines, such as IL-6. SP stimulates proliferation of blood vessels (angiogenesis) and may therefore be involved in the new vessel formation that is found in asthmatic airways. SP and NKA also stimulate the proliferation and chemotaxis of human lung fibroblasts, suggesting that tachykinins may contribute to the fibrotic process in chronic asthma (53). Tachykinins also enhance cholinergic neurotransmission by facilitating acetylcholine release at cholinergic nerve terminals and by enhancing ganglionic transmission. Tachykinins are subject to degradation by at least two enzymes, angiotensin-converting enzyme (ACE) and NEP. ACE is predominantly localized to vascular endothelial cells and therefore degrades intravascular peptides, whereas Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

NEP is important for degrading tachykinins in the airways. The activity of NEP may therefore determine tachykinin responsiveness in the airways. Inhibition of NEP by phosphoramidon or thiorphan markedly potentiates bronchoconstriction in vitro in animal and human airways and after inhalation in vivo (54). The activity of NEP is reduced by mechanical removal of the epithelium, some virus infections, cigarette smoke, and hypertonic saline. Several of the stimuli known to induce bronchoconstrictor responses in asthmatic patients have been found to reduce the activity of airway NEP (55).

F. Calcitonin Gene–Related Peptide CGRP-immunoreactive nerves are abundant in the respiratory tract of several species and is costored and colocalized with SP in afferent nerves. CGRP is a potent vasodilator, which has long-lasting effects and potently dilates bronchial vessels in vitro and in vivo. It is possible that CGRP may be the predominant mediator of arterial vasodilatation and increased blood flow in response to sensory nerve stimulation in the bronchi. CGRP may be an important mediator of airway hyperemia in asthma. CGRP has variable effects on airway smooth muscle tone and appears to act indirectly through the relapse of other constrictors, such as endothelin. Like tachykinins, CGRP is chemotactic for eosinophils.

G. Neurogenic Inflammation in Airway Disease Sensory nerves may be involved in inflammatory responses through the antidromic release of neuropeptides from nociceptive nerves or C-fibers via a local (axon) reflex (56) (Fig. 8). The phenomenon is well documented in several organs, including skin, eye, gastrointestinal tract, and bladder (57). There is increasing evidence that neurogenic inflammation occurs in the respiratory tract and that it may contribute to the inflammatory response in asthma. Neurogenic inflammation has been well documented in the airways of rodents, and there is good evidence that tachykinins contribute to the airway hyperresponsiveness in several animal models of asthma, using capsaicin depletion or specific tachykinin antagonists. However, although it was proposed several years ago that neurogenic inflammation and peptides released from sensory nerves might be important as an amplifying mechanism in asthmatic inflammation, there is little evidence to date to support this idea, despite the extensive work in rodent models. There is some evidence in support of a role for tachykinins in asthma: An increase in SP-immunoreactive nerves has been described in patients with severe asthma (58). Substance P and NKA levels are increased in bronchoalveolar lavage fluid of asthmatic patients (59) and in induced sputum of patients with asthma Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 8 Axon reflex mechanisms. Possible neurogenic inflammation in asthmatic airways via retrograde release of peptides from sensory nerves via an axon reflex. Substance P (SP) causes vasodilatation, plasma exudation, and mucus secretion, whereas neurokinin A (NKA) causes bronchoconstriction and enhanced cholinergic reflexes and calcitonin gene–related peptide (CGRP) vasodilatation.

(60). There is increased expression of SP in airway epithelial cells of patients with asthma (61). There is increased expression of NK1- and NK2-receptors in asthmatic lungs and airways (61–63). A tachykinin antagonist inhibits bradykinin-induced bronchoconstriction and cough in asthmatic patients (64). However, this is more evidence against a role for tachykinins: SP-immunoreactive nerves are sparse in human airways and are not increased in lungs and biopsies from asthmatic patients (48). Capsaicin has no effect on human airways in vitro, whereas it potently constructs guinea pig airways. Similarly, inhaled capsicum causes cough and transient bronchoconstriction, but not prolonged bronchoconstriction as in rodents (65) NEP inhibitors have no different effects in patients with asthma than normal subjects (66). Tachykinin antagonists that are NK1-selective have so far been found to be ineffective in asthma (67,68). It is possible that it will be important to block NK2-receptors in addition, and several nonselective tachykinin an-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

tagonists are now in development. It is possible that some effect might be seen in more severe asthma or in patients with virally induced exacerbations, but such studies have not yet been reported Neurogenic inflammation may also be important in COPD. SP levels are elevated in induced sputum of patients with COPD (60). Cigarette smoke activates C-fibers in airways and may result in mucus hypersecretion and goblet cell discharge (69), and tachykinins are potent stimuli of mucus secretion in human airways (70). H. Other Neuropeptides Several other neuropeptides have been identified in human airways (Table 2) (71), but their function is even less well defined that the neuropeptides discussed.

Table 2 Neuropeptides in the Respiratory Tract Neuropeptide Vasoactive intestinal peptide Peptide histidine isoleucine/methionine Peptide histidine valine-42 Parasympathetic Helodermin Helospectins I and II PACAP-27 Galanin Substance P Neurokinin A Neuropeptide K Calcitonin gene-related peptide Gastrin-releasing peptide Secretoneurin? Nociceptin

Localization

( Afferent)

Afferent ( C-fibers)

Neuropeptide Y Opioids?

Sympathetic

Somatostatin Enkephalin Endomorphins Cholecystokinin octapeptide

Afferent/uncertain

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

8. 9. 10.

11. 12.

13.

14.

15. 16. 17. 18. 19.

20.

Barnes PJ, Baraniuk J, Belvisi MG. Neuropeptides in the respiratory tract. Am Rev Respir Dis 1991; 144:1187–1198 (part 1), 1391–1399 (Part 2). Barnes PJ. Autonomic Control of the Respiratory System. London: Harwood, 1997. Barnes PJ. Modulation of neurotransmission in airways. Physiol Rev 1992; 72:699–729. Karlsson J-A, Sant’Ambrogio G, Widdicombe JG. Afferent neural pathways in cough and reflex bronchconstriction. J Appl Physiol 1988; 65:1007–1023. Sant’Ambrogio G, Widdicombe J. Reflexes from airway rapidly adapting receptors. Respir Physiol 2001; 125:33–45. Fox AJ, Barnes PJ, Urban L, Dray A. An in vitro study of the properties of single vagal afferents innervating guinea-pig airways. J Physiol 1993; 469:21–35. Fox AJ, Lalloo UG, Belvisi MG, Bernareggi M, Chung KF, Barnes PJ. Bradykininevoked sensitization of airway sensory nerves: a mechanism for ACE-inhibitor cough. Nature Med 1996; 2:814–817. Coleridge HM, Coleridge JCG. Afferent nerves in the airways. In: Barnes PJ, ed. Autonomic Control of the Respiratory System. London: Harwood, 1997:39–58. Maggi CA, Meli A. The sensory efferent function of capsaicin sensitive sensory nerves. Gen Pharmacol 1988; 19:1–43. Fox AJ, Barnes PJ, Dray A. Stimulation of afferent fibres in the guinea pig trachea by non-isomotic and low chloride solutions and its modulation by frusemide. J Physiol 1995; 482:179–187. Lalloo UG, Barnes PJ, Chung KF. Pathophysiology and clinical presentations of cough. J Allergy Clin Immunol 1996; 98:S91–97. Choudry NB, Fuller RW, Pride NB. Sensitivity of the human cough reflex: effect of inflammatory mediators prostaglandin E2, bradykinin and histamine. Am Rev Respir Dis 1989; 140:137–141. Nicholson CD, Challiss RAJ, Shahid M. Differential modulation of tissue function and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes. Trends Pharmacol Sci 1991; 12:19–27. Doherty MJ, Mister R, Pearson MG, Calverley PM. Capsaicin responsiveness and cough in asthma and chronic obstructive pulmonary disease. Thorax 2000; 55:643–649. Dray A, Urban L, Dickenson A. Pharmacology of chronic pain. Trends Pharmacol Sci 1994; 15:190–197. Carr MJ, Undem BJ. Ion channels in airway afferent neurons. Respir Physiol 2001; 125:83–97. Carr MJ, Hunter DD, Undem BJ. Neurotrophins and asthma. Curr Opin Pulm Med 2001; 7:1–7. Braun A, Lommatzsch M, Renz H. The role of neurotrophins in allergic bronchial asthma. Clin Exp Allergy 2000; 30:178–186. Wessler I, Kirkpatrick CJ, Racke K. Non-neuronal acetylcholine, a locally acting molecule, widely distributed in biological systems: expression and function in humans. Pharmacol Ther 1998; 77:59–79. Barnes PJ. Muscarinic receptor subtyes in airways. Life Sci 1993; 52:521–528.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

21. 22.

23.

24. 25. 26.

27. 28. 29.

30. 31. 32. 33. 34. 35.

36.

37.

38. 39.

Fryer AD, Adamko DJ, Yost BL, Jacoby DB. Effects of inflammatory cells on neuronal M2 muscarinic receptor function in the lung. Life Sci 1999; 64:449–455. Patel HJ, Barnes PJ, Takahashi T, Tadjkarimi S, Yacoub MH, Belvisi MG. Characterization of prejunctional muscarinic autoreceptors in human and guinea-pig trachea in vitro. Am J Respir Crit Care Med 1995; 152:872–878. Minette PAH, Lammers J, Dixon CMS, McCusker MT, Barnes PJ. A muscarinic agonist inhibits reflex bronchoconstriction in normal but not in asthmatic subjects. J Appl Physiol 1989; 67:2461–2465. Ind PW, Dixon CMS, Fuller RW, Barnes PJ. Anticholinergic blockade of betablocker induced bronchoconstriction. Am Rev Respir Dis 1989; 139:1390–1394. Mak JCW, Baraniuk JN, Barnes PJ. Localization of muscarinic receptor subtype mRNAs in human lung. Am J Respir Cell Mol Biol 1992; 7:344–348. Herve P, Denjean A, Jian R, Simmoneau G, Duroux P. Intraesophageal perfusion of acid increases the bronchomotor response to methacholine and to isocapnic hyperventilation in asthmatic subjects. Am Rev Respir Dis 1986; 139:986–989. Spina D. Modulation of sensory nerve function in the airways. Pulm Pharmacol Ther 1998; 11:319–330. Undem BJ, Riccio MM, Weinreich D, Ellis JL, Myers AC. Neurophysiology of mast cell-nerve interactions in the airways. Int Arch Allergy Immunol 1995; 107:199–201. Molfino NA, Slutsky AS, Julia-Serda G, Hofstein V, Szalai JP, Chapman KR, Rebuck AS, Zamel N. Assessment of airway tone in asthma. Am Rev Respir Dis 1993; 148:1238–1243. Haddad E-B, Mak JCW, Barnes PJ. Expression of -adrenergic and muscarinic receptors in human lung. Am J Physiol 1996; 270:L947–L953. Lange P, Parner J, Vestbo J, Schnohr P, Jensen G. A 15-year follow-up study of ventilatory function in adults with asthma. N Engl J Med 1998; 339:1194–1200. Barnes PJ. Beta-adrenergic receptors and their regulation. Am J Respir Crit Care Med 1995; 152:838–860. Bai TR. Abnormalities in airway smooth muscle in fatal asthma: a comparison between trachea and bronchus. Am Rev Respir Dis 1991; 143:441–443. Spina D, Rigby PJ, Paterson JW, Goldie RG. Autoradiographic localization of adrenoceptors in asthmatic human lung. Am Rev Respir Dis 1989; 140:1410–1415. Hakonarson H, Herrick DJ, Serrano PG, Grunstein MM. Mechanism of cytokine-induced modulation of -adrenoceptor responsiveness in airway smooth muscle. J Clin Invest 1996; 97:2593–2600. Koto H, Mak JCW, Haddad E-B, Xu WB, Salmon M, Barnes PJ, Chung KF. Mechanisms of impaired -adrenergic receptor relaxation by interleukin-1 in vivo in rat. J Clin Invest 1996; 98:1780–1787. Penn RB, Shaver JR, Zangrilli JG, Pollice M, Fish JE, Peters SP, Benovic JL. Efects of inflammation and acute -agonist inhalation on b2-AR signaling in human airways. Am J Physiol 1996; 271:L601–L608. Spina D, Rigby PJ, Paterson JW, Goldie RG. -Adrenoceptor function and autoradiographic distribution in human asthmatic lung. Br J Pharmacol 1989; 97:701–708. Dinh-Xuan AT, Chaussain M, Regnard J, Lockart A. Pretreatment with an inhaled 1-adrenergic agonist, methoxamine, reduces exercise-induced asthma. Eur Respir J 1989; 2:409–414.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

40. 41.

42. 43.

44.

45.

46. 47. 48.

49. 50.

51.

52. 53.

54.

55. 56. 57.

Lammers JWJ, Barnes PJ, Chung KF. Non-adrenergic, non-cholineergic airway inhibitory nerves. Eur Respir J 1992; 5:239–246. Belvisi MG, Stretton CD, Miura M, Verleden GM, Tadjarimi S, Yacoub MH, Barnes PJ. Inhibitory NANC nerves in human tracheal smooth muscle: a quest for the neurotransmitter. J Appl Physiol 1992; 73:2505–2510. Belvisi MG, Ward JR, Mitchell JA, Barnes PJ. Nitric oxide as a neurotransmitter in human airways. Arch Int Pharmacodyn Ther 1995; 329:111–120. Ward JK, Belvisi MG, Fox AJ, Miura M, Tadjkarimi S, Yacoub MH, Barnes PJ. Modulation of cholinergic neural bronchoconstriction by endogenous nitric oxide and vasoactive intestinal peptide in human airways in vitro. J Clin Invest 1993; 92:736–743. Ho WZ, Lai JP, Zhu XH, Uvaydova M, Douglas SD. Human monocytes and macrophages express substance P and neurokinin-1 receptor. J Immunol 1997; 159:5654–5660. Germonpre PR, Bullock GR, Lambrecht BN, Van DV, V, Luyten WH, Joos GF, Pauwels RA. Presence of substance P and neurokinin 1 receptors in human sputum macrophages and U-937 cells. Eur Respir J 1999; 14:776–782. Barnes PJ. Neurogenic inflammation in the airways. Respir Physiol 2001; 125:145–154. Dunzendorfer S, Wiedermann CJ. Neuropeptide-induced chemotaxis of eosinophils in pulmonary diseases. Ann Med 2000; 32:429–439. Lilly CM, Bai TR, Shore SA, Hall AE, Drazen JM. Neuropeptide content of lungs from asthmatic and non-asthmatic patients. Am J Respir Crit Care Med 1995; 151:548–553. Joos GF, Germonpre PR, Pauwels RA. Role of tachykinins in asthma. Allergy 2000; 55:321–337. Mapp CE, Miotto D, Braccioni F, Saetta M, Turato G, Maestrelli P, Krause JE, Karpitskiy V, Boyd N, Geppetti P, Fabbri LM. The distribution of neurokinin-1 and neurokinin-2 receptors in human central airways. Am J Respir Crit Care Med 2000; 161:207–215. Frossard N, Rhoden KJ, Barnes PJ. Influence of epithelium on guinea pig airway responses to tachykinins: role of endopeptidase and cyclooxygenase. J Pharmacol Exp Ther 1989; 248:292–298. Daniele RP, Barnes PJ, Goetzl EJ, Nadel J, O’Dorisio S, Kiley J, Jacobs T. Neuroimmune interactions in the lung. Am Rev Respir Dis 1992; 145:1230–1235. Harrison NK, Dawes KE, Kwon OJ, Barnes PJ, Laurent GJ, Chung KE. Effects of neuropeptides in human lung fibroblast proliferation and chemotaxis. Am J Physiol 1995; 12:L278–283. Cheung D, Bel EH, den Hartigh J, Dijkman JH, Sterk PJ. An effect of an inhaled neutral endopeptidase inhibitor, thiorphan, on airway responses to neurokinin A in normal humans in vivo. Am Rev Respir Dis 1992; 145:1275–1280. Di Maria GU, Bellofiore S, Geppetti P. Regulation of airway neurogenic inflammation by neutral endopeptidase. Eur Respir J 1998; 12:1454–1462. Barnes PJ. Asthma as an axon reflex. Lancet 1986; i:242–245. Maggi CA, Patacchini R, Santicioli P, Giuliani S, del Bianco E, Gepetti P, Meli A. The “efferent” function of capsaicin-sensitive nerves: ruthenium red discriminates between different mechanisms of activation. Eur J Pharmacol 1989; 170:167–177.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

58. 59.

60.

61.

62.

63.

64.

65. 66.

67.

68.

69. 70. 71.

Ollerenshaw SL, Jarvis D, Sullivan CE, Woolcock AJ. Substance P immunoreactive nerves in airways from asthmatics and non-asthmatics. Eur Respir J 1991; 4:673–682. Nieber K, Baumgarten CR, Rathsack R, Furkert J, Oehame P, Kunkel G: Substance P and b-endorphin-like immunoreactivity in lavage fluids of subjects with and without asthma. J Allergy Clin Immunol 1992; 90:646–652. Tomaki M, Ichinose M, Miura M, Hirayama Y, Yamauchi H, Nakajima N, Shirato K. Elevated substance P content in induced sputum from patients with asthma and patients with chronic bronchitis. Am J Respir Crit Care Med 1995; 151:613–617. Chu HW, Kraft M, Krause JE, Rex MD, Martin RJ. Substance P and its receptor neurokinin 1 expression in asthmatic airways. J Allergy Clin Immunol 2000; 106:713–722. Adcock IM, Peters M, Gelder C, Shirasaki H, Brown CR, Barnes PJ. Increased tachykinin receptor gene expression in asthmatic lung and its modulation by steroids. J Mol Endocrinol 1993; 11:1–7. Bai TR, Zhou D, Weir T, Walker B, Hegele R, Hayashi S, McKay K, Bondy GP, Fong T. Substance P (NK1)- and neurokinin A (NK2)-receptor gene expression in inflammatory airway diseases. Am J Physiol 1995; 269:L309–L317. Ichinose M, Nakajima N, Takahashi T, Yamauchi H, Inoue H, Takishima T. Protection against bradykinin-induced bronchoconstriction in asthmatic patients by a neurokinin receptor antagonist. Lancet 1992; 340:1248–1251. Fuller RW, Dixon CMS, Barnes PJ. The bronchoconstrictor response to inhaled capsaicin in humans. J Appl Physiol 1985; 85:1080–1084. Cheung D, Timmers MC, Zwinderman AH, den Hartigh J, Dijkman JH, Sterk PJ. Neonatal endopeptidase activity and airway hyperresponsiveness to neurokinin A in asthmatic subjects in vivo. Am Rev Respir Dis 1993; 148:1467–1473. Fahy J, Wong HH, Geppetti P, Reiss JM, Harris SC, MacLean DB, Nadel JA, Boushey HA. Effect of an NK1 receptor antagonist (CP-99,994) on hypertonic salineinduced bronchoconstriction and cough in male asthmatic subjects. Am J Respir Crit Care Med 1995; 152:879–884. Ichinose M, Miura M, Yamauchi H, Kageyama N, Tomaki M, Oyake T, Ohuchi Y, Hida W, Miki H, Tamura G, Shirato K. A neurokinin 1-receptor antagonist improves exercise-induced airway narrowing in asthmatic patients. Am J Respir Crit Care Med 1996; 153:936–941. Kuo H-P, Rohde JAL, Barnes PJ, Rogers DF. Cigarette smoke induced goblet cell secretion: neural involvement in guinea pig trachea. Eur Respir J 1990; 3:1895. Rogers DF, Aursudkij B, Barnes PJ. Effects of tachykinins on mucus secretion on human bronchi in vitro. Eur J Pharmacol 1989; 174:283–286. Uddman R, Hakanson R, Luts A, et al. Distribution of neuropeptides in airways In: Barnes PJ, ed. Autonomic Control of the Respiratory System. London: Harwood Academic, 1997: 21–37.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

15 The Parasympathetic Nervous System in the Pathophysiology of the Gastrointestinal Tract Yvette Taché CURE: Digestive Diseases Research Center, VA Greater Los Angeles Healthcare System, and David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A.

The influence of the parasympathetic nervous system on digestive function was experimentally established beginning with Pavlov’s work early in the last century. During the past few decades, advances in methodological approaches, including single unit electrical recording of autonomic nerve activity, novel anatomical tracing strategies combined with ultrastructural analysis, and immunohistochemistry have provided new insights into the central organization and mechanisms through which preganglionic parasympathetic neurons are regulated. Characterization of autonomic nerve endings and the interface with gut effector cells have also been gained. The discovery that neuropeptides and transmitters act in the brain to regulate gut function through modulation of autonomic nervous system activity provided novel understanding of the chemicals at play in the regulation of autonomic outflow. Focus has then been on these signals and their role in the normal regulation of gastrointestinal function. It is now increasingly recognized that brain-gut interactions play an important role in the physiology and pathophysiological regulation of gastrointestinal tract.

I. INTRODUCTION Neuroscience and gastroenterology developed independently for many years despite early clinical reports by Cabanis (1) and Beaumont (2) followed by Pavlov’s experimental work (3) demonstrating that the brain influences gut function. The

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

concept of the autonomic nervous system derived largely from Langley (4), particularly the notion that the enteric nervous system (ENS) embedded within the gut wall has features that distinguish it from the components of the parasympathetic and sympathetic divisions. The last two decades have witnessed an explosive growth of interdisciplinary research on brain-gut interactions, and the pathophysiological relevance of these interconnected systems is emerging (5,6). Advances in more precise anatomical and electrophysiological techniques and the characterization of many neuropeptides and their receptors in the brain provided novel insight into the mechanisms through which the brain regulates gastrointestinal (GI) function. In addition, the demonstration that peptides exert potent actions in the brain to influence GI function through vagal pathways (7) and act peripherally on vagal afferents (8) renewed interest in the implications of the autonomic nervous system in the cross-talk between the brain and the gut. This review described the pathophysiology of the autonomic nervous system to the gut from the viewpoint of the parasympathetic nervous system innervation of the GI tract. We shall first outline novel anatomical aspects of the vagal innervation of the digestive tract and brain nuclei established to regulate gut function through alterations of vagal outflow to the gut. We will also review current concepts on how specific neuropeptides, namely thyrotropin-releasing hormone (TRH) and corticotropin-releasing factor (CRF), act at specific brain nuclei to regulate gut function through neural pathways. The implications of sensory vagal fibers sending information to the brain in the modulation of gut function will be also outlined. Lastly, we will survey the clinical implications of disregulation of parasympathetic pathways and possible emerging therapeutic venues based on these recently unraveled mechanisms. Detailed accounts of the different aspects of the autonomic nervous system in the pathophysiology of the GI tract can also be found in previous books (9,10).

II. FUNCTIONAL ANATOMY OF THE PARASYMPATHETIC INNERVATION OF THE GUT The parasympathetic innervation to the alimentary tract is provided mainly by the vagus nerve. In addition, the sacral parasympathetic component selectively innervates the distal colon and is issued from sacral parasympathetic preganglionic neurons (SPN) with efferent axons traveling within the pelvic nerve. The efferent parasympathetic innervation is involved in transmitting the neural information from the brain to the gut. The extrinsic afferent fibers innervating the gut send sensory information from the GI tract to the brain and could also exert peripheral efferent function by releasing neuropeptides acting locally (11).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

A. The Vagus Nerve 1.

General Distribution in the Gastrointestinal Tract

The vagus nerve is composed of motor axons that originate from cell bodies located in specific brainstem nuclei, namely the dorsal motor nucleus of the vagus (DMN) and nucleus ambiguus (NA) and afferent fibers originating from the nodose ganglia. The right and left cervical vagi emerge from the brain medulla at the jugular foramen extend via the nodose ganglia into the neck and course along the esophagus. They enter the diaphragm as ventral and dorsal trunks. The basic branching pattern of vagal trunks has been most studied in rats, but it is largely similar across species, including humans (12). The vagal trunk innervating the gut is composed mainly of unmyelinated fibers (conducting impulses at less than 2.55 m/sec) with afferent fibers being the major component (over 80%) while vagal efferents encompass less than 20% (12). The ventral and dorsal vagal trunks divide into five branches: the ventral and dorsal gastric branches, the ventral and dorsal celiac branches, and a single hepatic branch derived from the ventral trunk (Fig. 1). The ventral gastric branch of the vagus supplies the ventral part of the stomach, the pyloric sphincter, and also the proximal duodenum. The dorsal gastric branch enters near the cardia and innervates the dorsal part of the stomach as well as the proximal duodenum through transpyloric fibers. The ventral and dorsal celiac branches of the vagus course along the celiac artery and near the celiac ganglia distribute to innervate the small intestine and the proximal and descending colon by traveling along the superior mesenteric artery (Fig. 1). The common hepatic branch contains about 3000 fibers, of which 800 are efferent fibers (12). This branch divides into the hepatic branch proper that innervates the liver and the gastroduodenal/pyloric branch that innervates the gastric antrum, pylorus, duodenum, and pancreas (13). While the vagus nerve in itself is merely a wire, the most prominent features are its central interface with neurons in the dorsal vagal complex (DVC) composed of the nucleus tractus solitarius (NTS) and DMN and its peripheral endings on sensor and effector cells within the gut wall. 2.

Vagal Efferent Innervation of the GI Tract

a. Organization of Preganglionic Motoneurons in the DMN and NA. Tracing studies by Powley and Berthoud (14,15) contributed largely to our understanding of the relationship between the preganglionic motoneurons located within the DMN and vagal efferent fibers innervating the GI tract. Preganglionic somata in the DMN exhibit a viscerotopic organization with distinctive rostrocaudally symmetrical pairs of medial and lateral longitudinal columns. The anterior and posterior gastric vagal branches originate from ipsilateral projections of

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 1 Schematic representation of parasympathetic innervation of the gastrointestinal tract.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

neurons located within the medial part of the left and right DMN respectively (14,16) (Fig. 2). Another pair of symmetrical compact columns situated laterally within the left and right DMN projects ipsilaterally forming the anterior and posterior celiac branches, respectively (Fig. 2). The preganglionic neurons of the hepatic common branch are distributed in a sparse but distinctive columnar pattern located exclusively throughout the left DMN (13). The duodenum, unlike the other

Figure 2 Schematic representation of projections of preganglionic motoneurons in the dorsal motor nucleus of the vagus (DMN) providing vagal efferent innervation of the gastrointestinal tract and preganglionic motoneurons in the nucleus ambiguus providing vagal innervation of the striated tissue of the esophagus. Dendritic projections of DMN motoneurons into the nucleus tractus solitarius are also shown. AP: area postrema; CV: cervical vagus; NA: nucleus ambiguus; NTS: nucleus tractus solitarius. (Adapted from Ref. 15.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

parts of the gut, receives vagal efferent innervation from both the medial and lateral right and left DMN columns (17). Tracing studies showed that all regions of the colon except the rectum received efferent innervation from the celiac and accessory celiac branches of the vagus nerve (18). By contrast to the other parts of the alimentary tract, the esophagus innervation, namely the vagal efferent fibers to the striated muscle fibers, originates mainly from the NA, more specifically from a distinct group of neurons localized in the compact formation of the NA (19,20). (Fig. 2). b. Characteristics of DMN Preganglionic Vagal Motoneurons. Quantatitive and regional differences in the morphology, electrophysiology, and dendritic arborization of DMN neurons within the columnar organization have been reported (21,22). In particular, the medial column innervating the stomach has a higher number of preganglionic neurons than the lateral column (18,23). Within the median column, site-specific organization of motoneurons has been delineated, with those projecting to the fundus being more laterally located than those innervating the antrum/pylorus which are more medial (16). With respect to the morphology, within the lateral column, DMN neurons projecting to the intestine have the largest size while neurons in the medial column are smaller (22). Morphological diversity of DMN neurons has also been reported in humans (24). Consistent with the size principle whereby neurons with the smallest cell bodies have the lowest threshold for synaptic activation, electrophysiological studies established that DMN neurons projecting to the stomach have higher input resistance, smaller and shorter after-hyperpolarization, and higher frequency of action potential firing than cecal projecting neurons (22). These features lend support to a differential modulation of DMN motoneurons based on their electrophysiological and morphological characteristics. Neurons in the DMN display dendritic arborization predominantly spreading into the horizontal plane (21). The dendritic field of preganglionic motoneurons extends preponderantly in the rostral and middle DMN compared with the caudal part. However, an interesting feature relates to the dendritic fields associated with each column, which is spatially separated and provides anatomical substrata for selective regional regulation (23). In addition, the potential for interactions between DMN motoneurons of the lateral and medial columns is supported by the presence of cecal motoneuron dendrites projecting into the medial column neurons (18, 23). Such projections may provide an anatomical basis for gastrocolonic response to a meal that is mediated primarily by vagal pathways (25). However, although the majority of dendrites are confined within the DMN, a small percentage also extends beyond the cytoarchitectural boundaries of the DMN into surrounding brainstem regions allowing possible access to humoral input from the cerebrospinal fluid. There is also evidence that dendrites of DMN neurons reach the overlying NTS (26). The highest density of vagal motoneurons dendrites is localized within the subnucleus gelatinosus of the dorsal medial NTS

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

just rostral to the obex where gastric vagal afferent fibers project (26) (Fig. 1). Ultrastructural analysis revealed many asymmetrical contacts between labeled gastric vagal sensory afferents and labeled motoneuron dentrites selectively in the subnucleus gelatinosus (26). The axodendritic contacts are organized to allow for organ-specific monosynaptic interactions supporting neuroanatomic substrate for gastric vago-vagal reflex (18,23,26). A similar possible cecovago-vagal reflex has also been suggested (18). Within the NA, the extensive bundling of motoneuronal dendrites supports the hypothesis that these structures serve as networks for the generation of complex motor activities, such as swallowing (19). c. Neurochemistry of Vagal Preganglionic Neurons. A number of studies have characterized the neurochemical phenotypes of neurons contained in the DMN. However, only a few have ascertained their efferent vagal projections to the gut. Acetylcholine is a major transmitter of preganglionic parasympathetic motoneurons in the DMN. Intense choline acetyltransferase (ChAT) gene expression has been localized in the DMN showing marked representation of cholinergic neurons (27). Retrograde labeled cells following subdiaphragmatic vagal injection in mice showed that the majority (52%) of labeled cells are ChAT positive (20). Nitric oxide (NO) is recognized as a neuronal messenger molecule, and NO-synthesizing neurons are identified by the detection of NO synthase (NOS) (28). Double labeling studies showed that 13% of retrolabeled neurons from the vagal trunks contained both ChAT and NOS and 9% are NOS positive/ChAT negative in mice (20). Likewise in rats, in the medial portion of caudal DMN, around 12% of NOSpositive neurons, project preferentially to the gastric fundus (29). They are postulated to play a role in the vagally mediated gastric accommodation (29). In the caudal DMN, another subpopulation of tyrosine hydroxylase (TH)–immunoreactive (IR) neurons (~14%) not colocalized with NOS neurons project selectively to the gastric corpus (30). Other studies established that TH-positive neurons in the DMN, retrogradely labeled from the subdiaphragmatic trunk, are primarily dopaminergic neurons, which are the source of catecholaminergic fibers in the vagus (31). Their possible role in gastric protection based on the important role of dopamine in gastrointestinal protection of the mucosa (32) needs to be explored. In the NA, 96% of cells innervating the esophagus striated muscles show both ChAT- and calcitonin gene–related peptide (CGRP) immunoreactivity and 4% displays CGRP immunoreactivity alone, while no NOS-IR cell bodies were observed (20). d. Central Input to DMN Neurons and Influence on GI Function. The DMN receives input from various brain centers. Detailed tracing studies revealed that the parvocellular part of the paraventricular nucleus of the hypothalamus (PVN), the central amygdala, and bed nucleus stria terminali represent an interconnected continuum of “prevagal neurons” (33–35). The DMN also receives inputs from the caudal raphe nuclei, including the raphe pallidus, raphe obscurus,

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

and parapyramidal region (36). Consistent with these efferent projections to the DMN, neuropharmacological studies showed that activation of these brain sites influence gastrointestinal function through vagal dependent pathways (36–43). e. Characterization of Vagal Efferent Fibers Innervating the GI Tract. The end target of vagal preganglionic neurons originating from DMN neurons is the myenteric plexus located between the gut muscular layers (44). Recent advances in tracing studies provided novel information how the vagal efferent fibers and terminals interface within the ENS. The prevailing view that vagal efferents project to selected “mother cells/command neurons” located within the ENS (45) has been challenged by the recent demonstration that individual vagal efferent axons ramify widely to produce extensive collaterals enveloping virtually all myenteric plexus with pearl string–like ending (46,47). Electrophysiological studies also support the anatomical evidence that a high percentage of gastric myenteric neurons receive direct synaptic fast excitatory postsynaptic potentials (EPSPs) input from vagal fibers (48). In the stomach, in particular, the vagal efferent terminals encircle or make putative contacts with all the myenteric ganglia as well as the submucosal plexus, although to a lesser extent. In the pyloric sphincter, small myenteric ganglia are embedded within the circular sphincter muscle. Vagal efferent preganglionic fibers display also high varicose nerve endings on the ganglionic cells as well as on single neurons scattered in the thin pyloric submucosal layer (49). In the duodenum, the hepatic and gastric branches innervate almost 70% of myenteric ganglia, suggesting a dual input with only a minor contribution of the celiac branch (50). In the intestine and proximal colon, the patterns of the vagal efferent network, which derived exclusively from the celiac branches, resembled that of the stomach with a lesser density (46,50,51). Advances have also been made in unraveling the neurochemical make-up and axonal projections of myenteric neurons, which are the final targets of the preganglionic vagal input (52). Termination of vagal preganglionic fibers have been established on serotonin (51), vasoactive intestinal peptide (VIP) (51), NOS (47), gastrin-releasing peptide (GRP), and GRP/VIP (52)–containing neurons in gastric myenteric ganglia. The recent cloning of a splice variant of the ChAT mRNA expressed selectively in the peripheral nervous system (pChAT) (53) has allowed investigators to establish that 60–70% of the myenteric neurons in the GI tract are pChAT-IR and surrounded by pChAT-positive fibers (54). This is indicative of the large cholinergic input to ENS neurons. The intrinsic or vagal efferent extrinsic origin of these pChAT fibers, however, needs to be further assessed. Vagal efferent neurons originating from the NA directly innervate the striated esophageal muscles and not the myenteric ganglia as in the other parts of the GI tract (20,44). Vagal efferent terminals to the esophageal striated muscle contained both cholinergic markers and CGRP (20). These neuroanatomic features indicate that vagal efferent fibers directly control the peristalsis of striated muscles in the esophagus through cholinergic/CGRP transmission. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

3.

Vagal Afferent Fibers Innervating the GI Tract

a. Cells of Origin in Nodose Ganglia and Neurochemistry. Neural tracers injected directly into the nodose ganglia provided a comprehensive account of gut structures innervated by vagal afferent fibers. The left nodose ganglion is the source of vagal afferents within the ventral gastric, common hepatic, and ventral celiac branches, while the right nodose ganglion provides vagal afferents to the dorsal gastric and celiac branches (13). In the nodose ganglia there is no strict somatotopic organization of the cell bodies innervating the gut. However, some tendency toward this organization has been observed as shown by the rostal location of perikaria innervating the esophagus, while projections to the stomach and pancreas are located more caudally (55). So far there are no immunohistochemical markers selective for vagal afferents innervating the gut, unlike splanchnic afferents (20). ChAT-positive neurons are not observed in the nodose ganglia, and only a small proportion (~3%) contained CGRP or NOS (20). A small number of retrograde labeled truncal vagal afferents also contain TH-IR (20). Several types of receptors have been localized in the nodose ganglia, of which 5-HT3, leptin, and cholecystokinin (CCK)A and CCKB receptors (55–58) have relevance in the modulation of GI vagal afferents (59–61). b. Central Projections of GI Vagal Afferents. The primary recipient of vagal afferents innervating the gut is the NTS (62). Detailed studies by Miselis et al. (63) have outlined a rostrocaudal viscerotopic representation from the tongue to the cecum and the segregation of cytoarchitectonically distinct subnuclei based on their function. For instance, vagal afferents innervating the esophagus, stomach, and colon terminate, respectively, in the centralis, gelatenosus, and commissural subnuclei of the NTS (18,23,63). Central terminals of vagal primary afferents from the duodenum are found mainly in the subpostrema/commissural region of the NTS and to a smaller extent in the medial and gelatinous subnuclei (17). Such a segregation of sensory vagal information entering the brain medulla provides a basis for information processing adapted to the peripheral origin of the afferents. These neurons receiving synaptic input from vagal afferents innervating the gut may influence secondary sensory neurons, which themselves send axonal projections to third order neurons. Major output from the NTS includes nearby projections to medullary motor nuclei namely the DMN, specific subdivision of the NA as well as the rostroventrolateral medulla (RVLM), the A1 noradrenergic group, and the reticular formation as well as ascending projections to various parts of the brain in particular, the PVN, the central amygdala, bed nucleus stria terminalis, and insular cortex (62,64–66). Esophageal vagal afferents have been shown to end on NOS interneurons in the NTS, which make direct synaptic contacts with esophageal motoneurons in the NA providing a basis for vago-vagal esophageal Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

reflex (67). There are also circumscribed projections from cell body neurons in the central NTS to the NA that contain somatostatin-28-IR. It has been suggested that these interneurons play a role in vagal reflex of esophageal motility (68). c. Gastrointestinal Projections of Vagal Afferents. Vagal afferent innervation of the gut exhibits regional variations with a more dense representation rostrally (esophagus, antrum, pylorus, and proximal duodenum) than caudally (distal small and large intestine) (18,49,69). Vagal afferent fibers and endings display a lateralization of their projections from the esophagus to the duodenum with the ventral and dorsal parts of the stomach innervated by the left and right vagal gastric branches, respectively (69). In addition, the hepatic branch provides afferent innervation to the ventral wall of the stomach and a few bundles to the pylorus. In the intestine, the densest representations from the hepatic afferent terminals are within the first 3 cm from the pylorus with declining innervation distally with a sparse innervation in the cecum (13). The celiac branches provide bilateral afferent innervation of the intestine with an asymmetrical predominance from the left vagus particularly at the level of the proximal duodenum (69). Within the GI smooth muscle wall, vagal afferent endings produce abundant collaterals and terminals specialized in two distinct structures, the intraganglionic laminar endings (IGLEs) and the intramuscular array (IMAs) (70) (Fig. 3). Information on these afferent terminals with respect to their target tissues, terminal architecture, and regional distribution has recently been reviewed (70). IGLEs are

Figure 3 Schematic representation of vagal efferent and efferent terminals innervating the gastrointestinal tract. Vagal efferents originate from the dorsal motor nucleus of the vagus and project exclusively to the myenteric nervous system embedded between the longitudinal and circular muscle layers of the gut wall. Vagal afferents originate from the nodose ganglion and project centrally to the nucleus tractus solitarius and peripherally to the longitudinal muscle layers forming the intramuscular arrays, in the circular muscles forming the intraganglionic lamina endings and the mucosa. (Adapted from Ref. 47.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

named after the location of their endings exclusively in the myenteric ganglia and their morphological features (lamina of puncta), are the most prominent type. IMAs are located within the smooth muscles and display arrays of rectilinear terminals paralleling the longitudinal and circular muscle fibers (47,70). Both vagal afferent endings are located in specific parts of the GI tract (13,71). The highest density of IGLEs occurs in the esophagus, gastric corpus, and duodenal bulb with a decrease in the arboral direction (44,69,70). In the esophagus in particular, IGLEs could be observed in 50% of myenteric ganglia (44). By contrast, IMAs are less globally distributed and are predominantly in the regions of the lower esophagus, forestomach, sphincters, duodenum, and colon. They appear to have an important interconnection with the interstitial cells of Cajal (13,69,70). Some sites, like the upper esophagus, are innervated selectively by the IGLEs, while others such as the lower esophageal sphincter (LES) and pyloric sphincter are only innervated by IMAs (70). The dissimilar morphology, localization with a restricted gut layer, divergent regional distribution, and density within the GI tract lend support to the notion that these two vagal afferent endings serve different functions, which are yet to be fully elucidated. In the liver, the sites of termination of vagal afferent axons are the connective tissue mainly around the intrahepatic blood vessels where terminal-like arborizations can be seen in the walls of arteries and bile ducts. In several species, except rats, there is also a rich lobular parenchymal vagal afferent innervations (72). Additional types of nodosal afferents innervate the submucosa and mucosa of the GI tract. Mucosal vagal afferents are observed as free endings at the base of the epithelial cells throughout the GI tract (69,73) (Fig. 3). They are found in greater abundance in the proximal duodenum with a scarce occurrence in the distal small intestine (73). By contrast, a recent report shows their presence as far as the distal colon, where they appear as free endings at the basis of epithelial cells (69). They form networks of multiple branching axons within the lamina propria, around the crypts, and to the tip of the villi (74). d. Functional Implications of Gastrointestinal Vagal Afferents. It is generally accepted that low threshold functional information is conveyed to the brain by vagal afferents (75). The mucosal vagal afferents innervating the gut are mainly polymodal fibers and can sense mechanical, chemical, and temperature changes (76). The electrophysiological characteristics of mucosal mechanoreceptors are their low threshold for mechanical stimuli and rapid adaptation to ongoing stimuli. The terminal axons in close contact with the lamina propria provide anatomical support to receive chemical information’s from both epithelial cells and other nerves coursing through the lamina propria. Special considerations have been given related to the anatomical relationship between the mucosal masts cells or CCK-containing entero-endocrine cells and vagal afferents providing support for paracrine modulations of vagal afferents by transmitters released by these cells Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

(73,77). Indeed these mucosal vagal afferents respond to a variety of luminal environments (acid, osmolarity, and temperature) as well as CCK, bradykinin, interleukin 1, and 5-HT (74,78,79). The transduction mechanisms triggering vagal afferent sensor responses involve direct activation of nerve endings through specific receptors present in vagal afferents such as CCKA and 5-HT3 receptors (57,58,80) and/or mechanosensitive ion channels in addition to indirect activation through products of taste receptor cells (81). Recent evidence also suggests that the activation of vagal afferents induced by changes in luminal osmolarity after a carbohydrate load is dependent upon the release of 5-HT from the EC cells acting on 5-HT3 receptors located on vagal afferents (79). The ubiquitous pattern of distribution and close association of IGLEs with myenteric ganglia suggests that they function as tension receptors capable of transducing active and passive variations of shearing forces associated with the movement of circular and longitudinal muscle layers. The current view is that they operate like Golgi tendons with increased activity during both distention and isometric contraction. They are also involved in initiating reflexes that promote rhythmic motor programs such as peristalsis and other intrinsic motility patterns of the gut (swallowing, antral pumping, migrating motor complex) (70). They could operate both locally by way of axon collaterals acting upon neurons in myenteric plexus as well as by conventional vago-vagal reflexes with a medullary relay (70). The vagal afferent IMA terminals are also viewed as mechanoreceptors specially involved in the detection of stretch (82). This inference is based on the IMAs’ elongated processes that may transduce changes in the length of muscle layers and detect distension associated with the degree of filling of the viscus (13). In the pyloric muscle, the IMAs ending in hepatic afferent branches may detect passive distention during the passage of ingesta and sphincter constriction. 4.

Sacral Parasympathetic Innervation of the Colon

a. Anatomy of Spinal Parasympathetic Nervous System to the Colon. The sacral parasympathetic efferent innervation of the distal colon comprises three serial chains of neurons located in the sacral spinal cord, pelvic plexus, and myenteric plexus by contrast to the vagal innervation, which involves two serial neurons. The preganglionic cell bodies lie in the intermedio-lateral column of the spinal cord mainly at the S1 level in the rat forming the sacral parasympathetic nucleus (SPN) (83). However, major species differences have been reported regarding the spinal segmental origin of preganglionic parasympathetic neurons (42,84). The sacral parasympathetic preganglionic neurons send their axons via the ventral roots forming the pelvic nerve, which provides innervation to the pelvic plexus. The postganglionic axons project to the distal colon and rectum through the rectal nerve as the primary route with the majority of pelvic projections innervating only the myenteric plexus (85). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

b. Sacral Parasympathetic Motoneurons: Characteristic and Central Input. The SPN innervating the rat colon corresponds to an aggregate of around 550 triangular or spindle-shaped neurons of medium size mainly located within the intermediolateral region of the gray matter within the lamina V (86). In cats, a viscerotopic representation has been shown between the SPN innervating the colon (dorsal band) and the bladder (lateral band) (87). The dendritic pattern of SPN neurons is similar across species with extensive dendritic projections into the dorsolateral funiculus, allowing descending influence from pontine nuclei, as well as medially directed along the lateral marginal zone of the dorsal horn and into the dorsal gray commissure (86,88). There are also large numbers of varicose axoncollateral projections to the lateral lamina I, V, and VII in proximity to visceral sensory axons and second order interneurons (89). These neurons, which represent the first component of the two-neuron efferent pathways delivering the central parasympathetic input to the distal colon, may have the potential of broad synaptic influence within the spinal cord. Morphological evidence for a direct input to SPN from sacral sensory fibers allowing direct spinal afferent-efferent interactions have also been reported (90). Transneuronal tracing from the distal colon combined with spinal cord section showed that synaptically linked central autonomic circuits to the SPN through bulbospinal pathways include the Barrington nucleus, previously known as a pontine micturition center, the subcoeruelus, Edinger-Westphal nucleus, periaqueductal gray and red nucleus in the midbrain, dorsal region of the PVN, and the medial frontal lobe of the cortex (83,84). Emotional and cognitive stimuli impact on pelvic function including colonic motility, and these transynaptically connected cortical and limbic areas provide anatomical support for the modulation of SPN outflow colonic motility by these brain nuclei and colonic motor function changes (91). c. Morphological and Chemical Properties of Pelvic Postsynaptic Ganglia. Retrograde tracing studies identified that postganglionic pelvic neurons projecting to the distal colon are mainly located near the entrance of the pelvic nerve (92). The pelvic neurons have weak dendritic arborization with a mean number of 1.5 primary axonal and dendritic processes (93,94). Although the pelvic ganglia received unmyelinated inputs from both the sympathetic hypogastric nerve and parasympathetic pelvic nerve, there are no pelvic ganglionic cells receiving a convergence from both nerves. Intracellular recording also established that each cell responds by one large amplitude excitatory postsynpatic potential, which translates into an action potential (94). These morphological and electrophysiological characteristics indicate a limited capacity of synaptic modulation and integration in this ganglion, suggesting that pelvic neurons may function mainly as a relay between the spinal cord and the viscera (94). The chemical phenotype of postganglionic pelvic projections to the distal colon is cholinergic and also includes VIPpositive fibers (93). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

d. Functional Significance. Activation of the sacral parasympathetic nervous system influences distal colonic and rectal motility and is essential for evacuative functions. The pelvic nerves convey both cholinergic and noncholinergic excitatory, as well as nonadrenergic, noncholinergic inhibitory influence to the distal colon. In particular, electrical stimulation of pelvic nerves at different intensities revealed the recruitment of three different mechanisms: 1) low stimulation–evoked distal colonic and rectal contractions mediated by the activation of nicotinic/muscarinic pathways, 2) high-intensity stimulation–induced contractions, which are resistant to nicotinic/muscarinic blockade, and 3) intermediate intensity stimulation, which results in distal and rectal relaxation mediated by nicotinic/nonmuscarinic pathways (95). e. Spinal Afferents. The primary afferent innervation of the colon arises mainly from cell bodies located within the dorsal root ganglia exclusively in the L6 and S1 segments. Around 1500 cells, usually small and oval in shape, are retrograde labeled via the pelvic nerve in rats (86). The central distribution pattern of rat pelvic afferents includes a prominent lateral pathway along the lateral margin of the dorsal horn that intermingled with neurons and dendrites of the SPN in laminae V and VII and lesser medial pathways with terminals in the dorsal gray commissure (86,96). These overlaps between pelvic afferents, SPN, and interneurons in the S1 provide an anatomical basis for polysynaptic parasympathetic reflex associated with excretory function. A large proportion of primary afferent neurons supplying the colon contained CGRP in rats (86).

III. BRAIN REGULATION OF AUTONOMIC OUTFLOW TO THE GUT The central nervous system exerts powerful control over GI function, mainly through the vagus nerve. While the biochemical coding involved in preganglionic motor neuron activation in the DMN was little known two decades ago, a better understanding of the transmitters at play to regulate vagal activity has recently emerged. Early on we identified a variety of neuropeptides acting in the brain to regulate gastric function through autonomic pathways. These peptides include somatostatin, bombesin, TRH, substance P, CRF, and adrenomedullin (7,97). Further studies provided insight into their mechanisms of action. Among them, CRF and TRH have emerged as candidates for regulating autonomic outflow to the gut. Recent anatomical and functional studies also support a possible role of substance P in the vagal regulation of gastric function (36,91). A. Medullary TRH: Role in Vagal Activation of Gut Function We previously reported that intracisternal injection of TRH acts in the brain to stimulate gastric acid secretion through vagal atropine-sensitive pathways in rats, Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

providing the first evidence that a peptide acts centrally to influence gastric function through autonomic pathways (98). Now, compelling anatomical, electrophysiological, and pharmacological evidence has established that medullary TRH plays a role in the vagal regulation of gut function by activating preganglionic neurons in the DMN (99). The use of TRH injected intracisternally also provides a physiologically relevant tool to selectively activate vagal efferent discharges and to probe the vagal regulation of gut function. Indeed, the previous assessment of vagal influence on gastric function derived mainly from electrical stimulation of the cervical vagus or field stimulation of the stomach, which induces a nonselective activation of both afferent and efferent fibers. 1.

Anatomical Evidence

The DVC, including the DMN and NTS, is densely innervated by TRH-containing fibers and nerve terminals, which synapse on dendrites of DMN neurons projecting to the stomach in experimental animals (100,101). In humans as well, the most prominent network of fibers in the DMN was found to be TRH-IR compared with 12 other neuropeptides studied (102). TRH-IR fibers in the DVC originate exlusively from cell bodies located in the raphe pallidus, raphe obscurus, and parapyramidal region (103,104). Two TRH receptor subtypes 1 and 2 (TRH-R1 and TRH-R2) have been cloned (105,106). Comparative brain distribution of these two receptors indicates that the DMN expresses solely TRH-R1 mRNA (107,108). In addition, the highest concentration of TRH-binding sites was observed in the medial column of the DMN (109), consistent with marked effects of TRH on gastric function (99). 2.

Electrophysiological Evidence

In vivo and in vitro electrophysiological studies established that TRH stimulates the firing rate of DMN neurons (110–112). This is achieved through a direct postsynaptic excitatory action on DMN neurons by increasing an inward cationic current and reducing calcium dependence after hyperpolarizing current. In addition, TRH inhibits the neuronal activity of NTS gastric distention–responsive cells (110). Consistent with the activation of preganglionic vagal motoneuron activity, intracisternal injection of TRH or a stable TRH analog, RX 77368, induces a sustained activation of efferent activity as monitored in the gastric or cervical branch of the vagus (113–115). The activation of vagal efferent outflow translates into a nicotinic-dependent widespread activation of myenteric neurons of the corpus and antrum as shown by Fos expression (116), a marker of neuronal activation (117). 3.

Functional Evidence

A plethora of studies showed that TRH or TRH analogs, namely, RX 77368, injected into the cerebrospinal fluid (CSF) induces a vagal dependent stimulation of Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

gut functions (99). Central TRH stimulates gastric acid secretion, pepsin (118) and mucus (119), gastric motility and transit (120,121), and gastric and hepatic mucosal blood flow in various animal species through vagal cholinergic pathways (122–125). Central injection of TRH also stimulates duodenal bicarbonate secretion (126), ileal and jejunal water secretion (127), pancreatic exocrine secretion (128), and colonic and anorectal motility (129,130) through vagal pathways. Consistent with TRH action on preganglionic motoneurons, microinjection of TRH or RX-77368 directly into the DMN stimulates gastric secretory and motor function as well as blood flow and pancreatic exocrine secretion (128). Studies on the peripheral mechanisms involved in TRH-induced vagal stimulation of gastric function showed an interplay between various transmitters released simultaneously by vagal cholinergic pathways (Fig. 3). In addition, depending upon the intensity of the vagal activation, the resulting endpoint on effector cells varies. Central injection of TRH increases the release of gastric prostaglandins (131), histamine (132,133), gastrin (134), NO (124,135), serotonin (136), and CGRP (137). With regard to the gastric hyperemia induced by TRH injected intracisternally at a dose subthreshold to increase acid secretion, the response results from vagal cholinergic-dependent activation of capsaicin-sensitive CGRP/NO pathways, while prostaglandins do not play a role (119,138,139). By contrast, at the maximal vagal cholinergic stimulation, the increase in gastric mucosal blood flow is mainly mediated by the activation of cholinergic NO pathways independently from capsaicin-sensitive mechanisms or other established vasoactive substances, namely VIP, prostaglandins, or histamine (122,124,140,141). Likewise, the absence of acid response to low doses of TRH analog (injected intracisternally) in the presence of increased vagal gastric efferent discharge and gastric mucosal blood flow (113,138) results from the antisecretory effects of vagally released gastric prostaglandins, CGRP, and serotonin (131,138,142,143). However, at higher doses of TRH or TRH analogs, the stimulation of acid secretion by histamine and acetylcholine in urethane-anesthetized rats (132,133) along with gastrin in conscious rats (134) is no longer influenced by the inhibitory effects of prostaglandins, CGRP, or serotonin (131,142,143). Under these conditions, the sustained vagal activation induced prolonged stimulation of gastric acid, pepsin, and motility leading to the development of gastric hemorrhagic lesions in 24-hour fasted rats (144). With regard to the peripheral mediators involved in the intestinal responses, serotonin plays an important role in intracerebroventricular TRH-induced vagal cholinergic-dependent stimulation of GI motility (145), while VIP contributes to the stimulation of intestinal secretion (127). 4.

Physiological Role

Important tools to assess the physiological relevance of transmitters are the blockade of their receptors by selective antagonists. However, so far there are no specific TRH receptor antagonists. Alternative strategies were thus intended to block Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

endogenous brain medullary TRH or TRH receptors using TRH antibody or TRH receptor antisense oligodeoxynucleotides targeted to inhibit TRH-R1 expression (146,147). For instance, chemical activation of cell bodies in the raphe pallidus, raphe obscurus, and parapyramidal region, which contain TRH neurons projecting to the DMN (103), results in a vagal cholinergic stimulation of gastric secretory and motor functions, an increase in gastric mucosal blood flow, and alterations of the resistance of the gastric mucosa to injury in rats. These gastric responses are abolished by bilateral microinjections of TRH antibody into the DVC or pretreatment with oligodeoxynucleotide antisense against the TRH-R1, indicating that medullary TRH plays a primary role in the gastric vagal response to activation of raphe nuclei (36,43,147–152). Using bilateral microinjections of TRH antibody into the DVC or the cisterna magna to immunoneutralize endogenous TRH released in response to stimuli, several reports established that medullary TRH plays a physiological role in the vagal-dependent gastric responses to 2-deoxy-D-glucose, sham feeding, and the adaptive gastric protection induced by mild followed by a strong irritants (acid or ethanol) (153–155). In addition, environmental stimuli that activate these raphe-DVC TRH pathways stimulate gut function through vagal pathways. In particular, acute cold exposure is well known to trigger the increased synthesis and release of hypothalamic TRH and stimulation of pituitary TSH secretion (156,157). In addition, we established that TRH-synthesizing neurons in the raphe pallidus, raphe obscurus, parapyramidal region are also activated, as shown by the induction of Fos expression (158,159) and increased proTRH mRNA expression in these nuclei (43,160). Consistent with this medullary TRH activation, acute cold exposure induces a sustained vagal cholinergic stimulation (161), activation of gastric myenteric neurons as shown by Fos expression (162), and vagal-dependent stimulation of gastric and motor function leading to gastric hemorrhagic lesions in fasted rats (160,163,164). We also showed that pretreatment with oligodeoxynucleotide antisense against the TRH-R1 receptor abolished cold exposure–induced vagal cholinergic-dependent stimulation of gastric emptying, whereas the mismatch antisense had no effect (165,166). B. Brain CRF and Modulation Autonomic Outflow to the GI Tract The characterization of the 41-amino-acid peptide corticotropin-releasing factor (CRF) and related family members, urocortin, urocortin II, and urocortin III (167,168) and the cloning of CRF receptor subtypes 1 (CRF-R1) and 2 (CRF-R2) and the development of specific CRF-R1/CRF-R2 receptor antagonists, namely, -helical CRF9–41, [D-Phe12,Nle21,38]h/rCRF12–41, and more recently, astressin, cyclo(30–33)[D-Phe12,Nle21,38,Glu30,Lys33]CRF12–41 (169), provided key tools to unravel the neurochemical basis of the stress response. Evidence has emerged that the activation of CRF receptors in the brain mediates almost the entire repertoire Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

of behavioral, neuroendocrine, autonomic, immunological, and visceral responses characteristic of stress in rodents and primates (170–172). In particular, the activation of brain CRF receptors modulates autonomic outflow and plays a role in stress-related autonomic alterations of gut function (91). 1. Brain CRF Inhibits Parasympathetic Vagal Efferent Activity to the Stomach The centrally mediated actions of CRF or urocortin to influence gastric function, including the inhibition of gastric emptying and motility (5,173–177), somatostatin release (178), and acid secretion (179), are mediated exclusively or in part by vagal pathways. Consistent with a role in vagal mechanisms, the brain sites of action of CRF to inhibit gastric motor function are found in hypothalamic (PVN) as well as medullary (DVC) nuclei regulating vagal outflow to the stomach (5,180). Although the exact cellular mechanisms of action are to be further established, there is evidence that central CRF inhibits DMN neurons activated by exogenous or endogenous TRH (159,180). The role of the vagus was further established electrophysiologically by the demonstration that intracisternal injection of CRF induced a dose-related inhibition of gastric vagal efferent discharge (181). In addition, the CRF-related peptide sauvagine injected under the same conditions was found more potent than CRF (181). Since sauvagine has greater affinity to the CRF-R2 than CRF-R1 and both peptides have nearly similar affinity to the CRFR1 (182), CRF action in the medulla to decrease vagal outflow to the stomach may be primarily mediated by the CRF-R2. These results are also consistent with functional studies showing the medullary urocortin-induced inhibition of gastric motor function is mediated by CRF-R2 as shown by the use of selective CRF receptor antagonists (183). In addition, mapping studies of the distribution of CRF receptor gene expression in the medulla showed the presence of CRF-R2, particularly in the NTS (184). There is also a network of CRF-IR fibers (102) suggesting that CRF may act through activation of NTS inhibitory input to the DMN preganglionic neurons. 2.

Brain CRF Activates Sacral Parasympathetic Activity to the Colon

CRF injected into the CSF increases colonic motility, decreases colonic transit time, and induces fecal excretion reproducing colonic motor response to various stressors (91). Indirect evidence indicates that the stimulatory action on the colon may be mediated by the activation of the SPN. CRF brain sites of action to stimulate colonic motor function are the PVN and locus coeruleus complex (5,91), which have direct projections to the SPN as shown by transneuronal retrograde tracing studies (84). In addition, the colonic response to central CRF is unchanged by hypophysectomy, adrenalectomy, and noradrenergic blockade and abolished by ganglionic and muscarinic blockade (185–188). Recent studies also showed

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

that Lewis rats, which have a lowered mounting of hypothalamic CRF released in response to stress (189), displayed an attenuated activation of colonic motor response to water-avoidance stress. This was associated with a decreased activation of sacral parasympathetic neurons, as shown by Fos expression in the SPN (190). Pharmacological characterization of CRF receptor subtypes using selective nonpeptide CRF-R1 antagonists, namely CP-154,526 and NBI-27914, indicates that CRF-R1 is primarily involved in the stimulation of colonic propulsion induced by central administration of CRF (191). 3.

Brain CRF Stimulates Sympathetic Outflow to the GI Tract

The activation of the sympathetic nervous system in response to central injection of CRF has been firmly established by pharmacological, biochemical, and direct electrophysiological assessment (192–196). Earlier reports established that CRF injected into the CSF of conscious rats induces a sustained increase in norepinephrine and a shorter-lasting rise in circulating epinephrine, which was abolished by ganglionic blockade (192,197). Direct measurements of adrenal nerve activity and catecholamine concentrations in the adrenal vein revealed that sympathoadrenal medullary functions are activated by intracerebroventricular injection of CRF independent of its action on the pituitary in rats (193,196). Central injection of CRF also increased spontaneous splenic sympathetic nerve activity (195) and splenic norepinephrine release from sympathetic nerve terminals (198). Central CRF exerts a stimulatory effect on the sympathetic efferent activity innervating other organs, including the interscapular brown adipose tissue and the kidney (193,194). The action of central CRF on sympathetic outflow is receptor mediated, and recent study indicates a role of hypothalamic CRF-R1 in CRF- and urocortin-induced activation of sympathetic-adrenomedullary outflow (196,199). Although direct monitoring to the sympathetic outflow to the gut in response to central CRF has not yet been investigated, functional studies indicate that CRF acts in the brain to influence specific gut function through sympathetic dependent pathways, namely the exacerbation of acute liver injury induced by carbon tetrachloride (200), the stimulation of ileal water absorption (201), the inhibition of canine gallbladder contraction and exocrine pancreatic secretion (202), and gastric acid secretion (179,203,204).

C. Substance P Action on Gastric Preganglionic Gastric Neurons 1. Anatomical Distribution of Substance P and Neurokinin-1 Receptor on Identified Gastric DMN Neurons Convergent findings support a direct action of substance P on preganglionic vagal motor neurons innervating the gut. The entire length of the DMN and the NTS is

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

innervated by substance P–immunoreactive fibers in rats (205) and humans (102). Retrograde labeling from the gastric corpus with immunostaining showed that all the retrograde neurons mainly at the rostral level of the obex were in contact with terminals containing substance P fibers (205,206). The localization of neurokinin1 receptor (NK1) on some gastric projecting preganglionic vagal motoneurons (207) and within the medial NTS where there is monosynaptic connections between gastric afferents and drendrites of gastric efferents provides the basis for a role of substance P in vago-vagal gastric reflexes. 2. Substance P in the DMN Inhibits Gastric Function Through Vagal Pathways We initially reported that direct microinjection of substance P in the DVC dampened the stimulatory excitatory action of TRH to induce a vagal-dependent stimulation of gastric acid secretion through neurokinin-1 receptor–mediated action while not influencing peripherally stimulated acid secretion (208). Other studies also showed that substance P microinjected into the DMN decreases intragastric luminal pressure through cholinergic vagal pathways (209,210). However, further studies are required to establish the role of medullary substance P in the vagal regulation of GI function.

IV. VAGAL REGULATION OF GI FUNCTION: PATHOPHYSIOLOGICAL RELEVANCE Parasympathetic vagal neurons form synapses directly within the myenteric plexus with excitatory or inhibitory postganglionic neurons. Depending upon their connections, activation of preganglionic vagal neurons can lead to either stimulatory or inhibitory functional responses in the gut. It is well established that central vagal activation through postganglionic muscarinic activation evokes stimulation of digestive function including gastrointestinal secretion, propulsive motility, absorption, blood flow, and influences the resistance of the gastric mucosa to injury as outlined with the central injection of TRH (42,144,211,212) (Fig. 4). With respect of inhibitory response, pharmacological evidence supports the view that the relaxation of the lower esophageal sphincter (LOS) is mediated in part by vagal dependent stimulation of nonadrenergic-noncholinergic NO-dependent mechanisms in a variety of species including humans (213). Likewise, the gastric receptive relaxation, which refers to the capacity of the stomach to receive a large volume during filling with a minimal increase in gastric pressure, is mediated by vagal cholinergic stimulation of inhibitory gastric innervation (214). Anatomical and functional bases for this vagal inhibitory pathway have been established by the demonstration that vagal efferent terminals contact NOS neurons in the Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 4 Summary of experimental studies showing the central vagally mediated changes in gastric transmitters and function induced by TRH acting on dorsal motor nucleus neurons. CGRP: calcitonin gene–related peptide; EC: enterochromaffin cell; ENS: enteric nervous system; GMBF: gastric mucosal blood flow; Hist: histamine; NO: nitric oxide.

gastric myenteric ganglia (47), vagal stimulation increases gastric NO oxide release (215), and pharmacological blockade of this pathway prevents adaptive relaxation (47,215). In addition, within the DMN excitatory and inhibitory control of the LES, intragastric pressure and pylorus activity have been located in two distinct rostral and caudal areas of the DMN, respectively (216). Advances in the understanding of the autonomic regulation of gut function provide important insights into pathophysiological processes and possible clinical applications are emerging. A. GI Pathophysiology Linked with Parasympathetic Efferents 1. Dual Role of Vagal Efferent Pathways in the Resistance of the Gastric Mucosa to Injury a. Protection of the Gastric Mucosa by Low Vagal Efferent Activity. The gastric vagal innervation plays an important role in the resistance of the gastric mucosa to withstand damaging agents. Experimental studies established that low vagal activation by central injection of small doses of TRH protects the gastric mucosa against ethanol-induced gastric lesions (144). The gastric protection is Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

brought out by vagal cholinergic-dependent release of gastric prostaglandins, NO as well as activation of local effector function of capsaicin-sensitive splanchnic afferents containing CGRP (217–219) (Fig. 4). These vagal gastric protective pathways are involved in the mechanisms of adaptive gastric protection (whereby a mild gastric irritant protects against the damaging effect of a strong irritant) and activated by the release of medullary TRH medullary (153,220). In this regard, the cephalic phase, which induces low vagal stimulation, may have beneficial effects on the gastric mucosa by triggering protective mechanisms while deficiencies of these vagal protective mechanisms enhanced the damaging effect of ulcerogenic stimuli (144). b. Gastric Erosions Induced by Sustained Vagal Efferent Activity. By contrast, maximal vagal stimulation results in the occurrence of gastric hemorrhagic erosions in 24-hour fasted rats (144,217,218). Likewise, sustained activation of DMN neurons by exogenous or endogenous TRH or other cell body activators such as kainate lead to gastric erosions through activation of vagal cholinergic pathways (150,160,161,221). Therefore, overstimulation of the vagal activity can cause the development of gastric lesions. These data established the dual actions of central vagal stimulation on the gastric mucosa either protective or damaging depending upon the intensity and chronicity of the vagal stimulation (144). 2. Role of Medullary TRH in Autonomic-Related GI Disorders During Hypothyroidy Hypothyroidism is associated with autonomic nervous system–related disorders characterized by pathophysiological changes in the GI tract, namely gastric ulcer formation occurs under hypothyroidic conditions (222). Yang et al. (223) demonstrated that TRH mRNA expression is increased in the brain medulla by hypothyroidy mainly in TRH-containing neurons of the raphe pallidus and raphe obscurus. Hypothyroidy removes the negative feedback of thyroid hormones on TRH gene expression in medullary raphe neurons. These result in the activation of medullary TRH containing neurons projecting to the DMN (224). These mechanisms may have implications in the understanding and possible treatment of autonomic-related GI dysfunction during hypothyroidy. 3. Impairment of Vagal Efferent-Induced Esophageal Sphincter and Gastric Relaxation Functional studies indicate that vagal input to the LOS plays a key role in maintaining LOS tone and relaxation associated with swallowing, belching, and reflux secondary esophageal peristalsis. A recent clinical study showed that patients with non-cardiac chest pain and symptoms of gastroesophageal reflux disease (GERD) display a high incidence of abnormal vagal function (225). The vagally mediated Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

receptive gastric relaxation is an important process as intragastric and pyloric sphincter pressure are codeterminant of gastric emptying of liquids. Impairment of this reflex results in accelerated gastric emptying, and the vagus has been involved in the pathogenesis of the dumping syndrome (226). Malfunctioning of the accommodation reflex may also play a role in GERD, and vagal efferent dysfunction has been implicated as a primary etiological factor in GERD causing the sensation of heartburn or chest pain (225,227,228). 4.

Stress-Related Autonomic Alterations of GI Motor Function

Stress induces autonomic mediated disturbances in GI motor function. Convergent findings point to a role of brain CRF receptors in the autonomic mediated alterations of gut motor function evoked by stress (5,91,229). a. Various Stressors Alter GI Motor Function. Acute stress induces differential motor effects in the upper and lower GI tract. Delayed gastric emptying is commonly induced by various acute stressors, including operant avoidance, water avoidance, radiation, handling, acoustic stimulation, hemorrhage, abdominal or cranial surgery, tail shock, trunk clamping, wrap restraint at room temperature, swimming, and anesthetic exposure in experimental animals (mice, rats, guinea pigs, dogs, and/or monkeys) (5,230,231). In healthy subjects, anger, fear, labyrinthine stimulation, painful stimuli, preoperative anxiety, or intense exercise results in a slowing of gastric emptying (5,230,231). Acute exposure to wrap restraint, cold water swim, or ether also slows down intestinal transit in the rat (232). By contrast, colonic motor function (motility, transit and/or defecation) are stimulated by conditioned fear to inescapable foot shocks and exposure to water avoidance, tail shock, loud noise, open field test, and restraint at room temperature or in cold environment in experimental animals (230,231). Similarly, in healthy volunteers painful stimuli, dichotomous listening test, fear, anxiety, anger, or a stressful interview enhances colonic motor activity (233). b. Role of Brain CRF Receptors in the GI Motor Response to Stress. The implication of CRF receptors in the stress response is shown by the demonstration that CRF receptor antagonists, -helical CRF9–41, [D-Phe12,Nle21,38]h/rCRF12–41, or astressin, injected into the cerebrospinal fluid or the PVN at doses preventing the delayed gastric emptying to centrally injected CRF, blocked the gastric stasis induced by various forms of stress, either immunological (intravenous or intracisternal injection of interleukin-1), physico-psychological (partial restraint, forced swimming), chemical (ether), or visceral (abdominal or cranial surgery or peritoneal irritation induced by intraperitoneal injection of acetic acid) (5,91). Likewise, the role of CRF receptors in mediating stress-related activation of colonic motor function is supported by the demonstration that nonselective CRFR1/CRF-R2 peptide antagonists such as -helical CRF9–41 or astressin (234) injected intracerebroventricularly, or into specific brain nuclei, the PVN, or locus Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

coeruleus complex, abolished partial wrap restraint and water avoidance–induced stimulation of colonic transit and defecation as well as conditioned fear-induced increase in cecal and colonic spike burst frequency (186,190,229,235–237). Central injection of interleukin-1, known to activate hypothalamic CRF release (238), stimulated colonic motor function through activation of central CRF receptors (239). Although the mechanisms through which CRF receptors antagonize stressinduced alterations of gut motor function are still to be defined, it is likely that prevention of autonomic alterations may play a role. Central CRF inhibits gastric vagal efferent outflow to the stomach and is involved in the related gastric stasis (174). There is evidence that central injection of CRF receptor antagonists prevent restraint stress–induced increase in TH mRNA in the locus coeruleus and the rise in circulating epinephrine and dopamine, supporting the notion that central CRF receptor activation plays a role in the sympathoadrenal response to stress (240). c. Clinical Implications of CRF-R1 in Irritable Bowel Syndrome. The role of activation of CRF-R1 in the activation of sacral parasympathetic outflow to the colon leading to stimulation of colonic motor activity and diarrhea has been recently reviewed (91,241). Preclinical studies provide convergent evidence to support the concept that hyperactivity of CRF-R1 pathways may contribute to the comorbidity of anxiety/depression in irritable bowel syndrome patients with enhanced bowel movements and diarrhea-predominant GI symptoms (241). CRFR1 antagonists may provide potential novel therapeutic intervention by inhibiting CRF-R1–mediated anxiogenic behavior and activation of locus coeruleus/Barrington nucleus projecting to the SPN and related stimulating colonic motor function (241). B. Pathophysiology Linked with Parasympathetic Afferents 1.

Role of GI Vagal Afferent in Emetic Response

Cytotoxic antineoplastic drugs and x-ray radiation are potent inducers of nausea and vomiting in humans and retching animals. Convergent findings conclusively established that the vagus is a major afferent pathway involved in the mechanism of the emetic response induced by anticancer drugs (242,243). Electrical stimulation of the abdominal vagal afferents can induce emesis, and cisplatin-induced emesis is prevented by vagotomy and associated with increased abdominal vagal afferent activity (243). Likewise, x-ray irradiation of the abdomen increases neuronal activity in NTS as shown by Fos expression in subdiaphragmatic vagal afferent terminal fields, and the response is lowered by vagotomy in rats (244). The pivotal role played by 5-HT and 5-HT3 receptors was shown by the release of 5-HT from the EC cells in the gut mucosa by emetic drugs, the presence of 5-HT3–binding sites on the nodose ganglia, and subnucleus gelatinosus of the Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

NTS, the stimulation of vagal afferent activity by 5-HT, and the prevention of the vagal excitatory and emetic responses to cytotoxic drugs and x-ray by 5-HT3 receptor antagonists in experimental animals and humans (243–245). These observations provided important insights into the vagal mechanisms involved in cytotoxic and radiation-induced emesis and revolutionized the treatment of acute chemotherapy–evoked emesis (242). 2.

Role of GI Vagal Afferents in the Regulation of Ingestive Behavior

A subset of vagal afferents in the stomach and intestine has been implicated as a key negative feedback in the control of meal size (246,247). The neuroanatomical, neurophysiological, molecular, and behavioral evidence in support of such a role of GI vagal afferent pathways has recently been reviewed (247). Knockout experimental models with selective deficiency in intestinal IGLEs display an increase in ingestion of solid food and meal size of liquid food indicative that that these vagal afferent mechanoreceptors play a role in short-term satiety (248). The pathophysiological significance of targeting such pathways to reduce food intake may have important clinical applications. V. CONCLUSIONS The parasympathetic nervous system innervating the gut is composed of preganglionic neurons located in the medulla oblongata mainly in the DMN and in the sacral spinal cord in the SPN. DMN neurons send signals by an axonal process forming the vagus innervating the whole GI tract except the rectum, and the SNP innervates the distal colon. The vagal efferent axons synapse directly within the ENS of the gut. The pelvic efferent fibers originating from the SPN synapse with neurons of the pelvic ganglia and postganglionic neurons innervate the ENS of the distal colon and rectum. Major advances have been made in our understanding of the role and mechanisms through which the parasympathetic nervous system influences GI function. Disregulation of these autonomic regulatory mechanisms could lead to disease states including GERD, irritable bowel syndrome or gastric ulcers, emesis, and altered feeding behavior. The recent use of transneuronal labeling from distinct parts of the GI tract yields relevant neuroanatomical information regarding the synaptically linked central autonomic circuits that control gut function. Multilabel fluorescence immunohistochemistry, confocal microscopy, and three-dimensional reconstruction techniques will allow investigators to examine in the near future the spatial relationship between nerve terminals and neurons characterized at the levels of their receptor and phenotype as well as their projections. TRH and CRF are two examples of brain peptides that exert a powerful influence on GI functions through alterations of the autonomic nervous system. Both peptides, based on Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

experimental studies, have physiological relevance in the regulation of GI function in response to stress. Evolving concepts point to the role of CRF-R1 as potential target for alleviating stress-related autonomic disregulation of GI function.

ACKNOWLEDGMENTS The author’s work was supported by the National Institute of Diabetes and Digestive and Kidney Disease Grants DK-33061, DK 30110, and DK 41301 (Animal Core). The author thanks Dr. Jean Rivier (Salk Institute, La Jolla, CA) for providing peptides and Paul Kirsch for his help in preparing the manuscript.

REFERENCES 1. 2.

3. 4. 5. 6. 7.

8. 9. 10. 11.

12. 13.

Cabanis PJG. In: More G, ed. On the Relation Between the Physical and Moral Aspects of Man. Baltimore: John Hopkins University Press, 1981. Beaumont W. In: Osler W, ed. Experiments and Observations on the Gastric Juice and the Physiology of Digestion. New York: Dover Publications Inc., 1833, pp 103–116. Pavlov I. The Work of the Digestive Glands (trans. Thompson WH). London: C. Criffin & Co, 1910. Langley JN. The Autonomic Nervous System, Part I. Cambridge: Heffer, 1921. Taché Y, Martinez V, Million M, Rivier J. Corticotropin-releasing factor and the brain-gut motor response to stress. Can J Gastroenterol 1999; 13(suppl A):18A–25A. Schmulson MJ. Brain-gut interaction in irritable bowel syndrome: new findings of a multicomponent disease model. Isr Med Assoc J 2001; 3:104–110. Taché Y. Peptidergic activation of brain gut efferent pathways. In: Nerves and the gastrointestinal tract. Singer MV, Goebell H, eds. Lancaster: Kluwer Academic Publishers, 1989:315–331. Schwartz GJ, Moran TH. Duodenal nutrient exposure elicits nutrient-specific gut motility and vagal afferent signals in rat. Am J Physiol 1998; 274:R1236–R1242. Taché Y, Wingate D. Brain-Gut Interactions. Boca Raton, FL: CRC Press, 1991. Taché Y, Wingate DL, Burks TF. Innervation of the Gut. Boca Raton, FL: CRC Press, 1994. Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neurotransmitters. Neuroscience 1988; 24:739–768. Prechtl JC, Powley TL. The fiber composition of the abdominal vagus of the rat. Anat Embryol 1990; 181:101–115. Phillips RJ, Baronowsky EA, Powley TL. Afferent innervation of gastrointestinal tract smooth muscle by the hepatic branch of the vagus. J Comp Neurol 1997; 384:248–270.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

14.

15.

16.

17. 18. 19.

20. 21. 22.

23.

24. 25. 26.

27.

28. 29. 30.

31.

Fox EA, Powley TL. Longitudinal columnar organization within the dorsal motor nucleus represents separate branches of the abdominal vagus. Brain Res 1985; 341:269–282. Powley TL, Berthoud H-R, Prechtl JC, Fox AE. Fibers of the vagus regulating gastrointestinal function. In: Taché Y, Wingate D, eds. Brain-Gut Interactions. Boca Raton, FL: CRC Press, 1991:73–82. Okumura T, Namiki M. Vagal motor neurons innervating the stomach are sitespecifically organized in the dorsal motor nucleus of the vagus nerve in rats. J Auton Nerv Syst 1990; 29:157–162. Zhang X, Renehan WE, Fogel R. Vagal innervation of the rat duodenum. J Auton Nerv Syst 2000; 79:8–18. Altschuler SM, Escardo J, Lynn RB, Miselis RR. The central organization of the vagus nerve innervating the colon of the rat. Gastroenterology 1993; 104:502–509. Broussard DL, Altschuler SM. Brainstem viscerotopic organization of afferents and efferents involved in the control of swallowing. Am J Med 2000; 108(suppl 4a):79S–86S. Sang Q, Young HM. The origin and development of the vagal and spinal innervation of the external muscle of the mouse esophagus. Brain Res 1998; 809:253–268. Fox EA, Powley TL. Morphology of identified preganglionic neurons in the dorsal motor nucleus of the vagus. J Comp Neurol 1992; 322:79–98. Browning KN, Renehan WE, Travagli RA. Electrophysiological and morphological heterogeneity of rat dorsal vagal neurones which project to specific areas of the gastrointestinal tract. J Physiol 1999; 517(Pt 2):521–532. Altschuler SM, Ferenci DA, Lynn RB, Miselis RR. Representation of the cecum in the lateral dorsal motor nucleus of the vagus nerve and commissural subnucleus of the nucleus tractus solitarii in rat. J Comp Neurol 1991; 304:261–274. Huang XF, Tork I, Paxinos G. Dorsal motor nucleus of the vagus nerve: a cyto- and chemoarchitectonic study in the human. J Comp Neurol 1993; 330:158–182. Dapoigny M, Cowles VE, Zhu Y-R, Condon RE. Vagal influence on colonic motor activity in conscious nonhuman primates. Am J Physiol 1992; 262:G231–G236. Rinaman L, Card JP, Schwaber JS, Miselis RR. Ultrastructural demonstration of a gastric monosynaptic vagal circuit in the nucleus of the solitary tract in rat. J Neurosci 1989; 9:1985–1996. Oh JD, Woolf NJ, Roghani A, Edwards RH, Butcher LL. Cholinergic neurons in the rat central nervous system demonstrated by in situ hybridization of choline acetyltransferase mRNA. Neuroscience 1992; 47:807–822. Bredt DS, Hwang PM, Snyder SH. Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 1990; 347:768–770. Zheng ZL, Rogers RC, Travagli RA. Selective gastric projections of nitric oxide synthase-containing vagal brainstem neurons. Neuroscience 1999; 90:685–694. Guo JJ, Browning KN, Rogers RC, Travagli RA. Catecholaminergic neurons in rat dorsal motor nucleus of vagus project selectively to gastric corpus. Am J Physiol 2001; 280:G361–G367. Yang M, Zhao X, Miselis RR. The origin of catecholaminergic nerve fibers in the subdiaphragmatic vagus nerve of rat. J Auton Nerv Syst 1999; 76:108–117.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

32. 33.

34.

35.

36. 37.

38.

39.

40.

41. 42.

43.

44.

45. 46.

47.

Glavin GB, Murison R, Overmier JB, Pare WP, Bakke HK, Henke PG, et al. The neurobiology of stress ulcers. Brain Res Rev 1991; 16:301–343. Petrov T, Krukoff TL, Jhamandas JH. Convergent influence of the central nucleus of the amygdala and the paraventricular hypothalamic nucleus upon brainstem autonomic neurons as revealed by c-fos expression and anatomical tracing. J Neurosci Res 1995; 42:835–845. Veening JG, Swanson LW, Sawchenko PE. The organization of projections from the central nucleus of the amygdala to brainstem sites involved in central autonomic regulation: a combined retrograde transport-immunohistochemical study. Brain Res 1984; 303:337–357. Pluzhnichenko EB. Spatial organization of hypothalamic neurons projecting to the “gastric” region of the vagosolitary complex. Neurosci Behav Physiol 1997; 27:688–691. Taché Y, Yang H, Kaneko H. Caudal raphe-dorsal vagal complex peptidergic projections: role in gastric vagal control. Peptides 1995; 16:431–435. Morrow NS, Hodgson DM, Garrick T. Microinjection of thyrotropin-releasing hormone analogue into the central nucleus of the amygdala stimulates gastric contractility in rats. Brain Res 1996; 735:141–148. Rogers RC, Hermann GE. Oxytocin, oxytocin antagonist, TRH, and hypothalamic paraventricular nucleus stimulation effects on gastric motility. Peptides 1987; 8:505–513. Zhang X, Fogel R, Renehan WE. Stimulation of the paraventricular nucleus modulates the activity of gut-sensitive neurons in the vagal complex. Am J Physiol 1999; 277:G79–G90. Mönnikes H, Schmid BG, Taché Y. Psychological stress-induced accelerated colonic transit in rats involves hypothalamic corticotropin-releasing factor. Gastroenterology 1993; 104:716–723. Hermann GE, McCann MJ, Rogers RC. Activation of the bed nucleus of the stria terminalis increases gastric motility in the rat. J Auton Nerv Syst 1990; 30:123–128. Gillis RA, Quest JA, Pagani FD, Norman WP. Control centers in the central nervous system for regulating gastrointestinal motility. In: Wood JD, Schultz SC, eds. Handbook of Physiology—The Gastrointestinal System, Vol. 1. Motility and Circulation. New York: Oxford University Press, 1989:621–683. Yang H, Yuan PQ, Wang L, Taché Y. Activation of the parapyramidal region in the ventral medulla stimulates gastric acid secretion through vagal pathways in rats. Neuroscience 2000; 95:773–779. Neuhuber WL, Kressel M, Stark A, Berthoud HR. Vagal efferent and afferent innervation of the rat esophagus as demonstrated by anterograde DiI and DiA tracing: focus on myenteric ganglia. J Auton Nerv Syst 1998; 70:92–102. Wood JD. Enteric neurophysiology. Am J Physiol 1984; 247:G585–G598. Holst MC, Kelly JB, Powley TL. Vagal preganglionic projections to the enteric nervous system characterized with Phaseolus vulgaris-leucoagglutinin. J Comp Neurol 1997; 381:81–100. Berthoud HR. Anatomical demonstration of vagal input to nicotinamide acetamide dinucleotide phosphate diaphorase-positive (nitrergic) neurons in rat fundic stomach. J Comp Neurol 1995; 358:428–439.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

48. 49.

50. 51.

52.

53.

54.

55. 56.

57. 58.

59.

60. 61.

62.

63.

64.

Schemann M, Grundy D. Electrophysiological identification of vagally innervated enteric neurons in guinea pig stomach. Am J Physiol 1992; 263:G709–G718. Kressel M, Berthoud HR, Neuhuber WL. Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy. Cell Tissue Res 1994; 275:109–123. Berthoud HR, Carlson NR, Powley TL. Topography of efferent vagal innervation of the rat gastrointestinal tract. Am J Physiol 1991; 260:R200–R207. Kirchgessner AL, Gershon MD. Identification of vagal efferent fibers and putative target neurons in the enteric nervous system of the rat. J Comp Neurol 1989; 285:38–53. Berthoud H-R. Morphological analysis of vagal input to gastrin releasing peptide and vasoactive intestinal peptide containing neurons in the rat glandular stomach. J Comp Neurol 1996; 370:61–70. Tooyama I, Kimura H. A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers. J Chem Neuroanat 2000; 17:217–226. Nakajima K, Tooyama I, Yasuhara O, Aimi Y, Kimura H. Immunohistochemical demonstration of choline acetyltransferase of a peripheral type (pChAT) in the enteric nervous system of rats. J Chem Neuroanat 2000; 18:31–40. Zhuo H, Ichikawa H, Helke CJ. Neurochemistry of the nodose ganglion. Prog Neurobiol 1997; 52:79–107. Buyse M, Ovesjo ML, Goiot H, Guilmeau S, Peranzi G, Moizo L, Walker F, Lewin MJ, Meister B, Bado A. Expression and regulation of leptin receptor proteins in afferent and efferent neurons of the vagus nerve. Eur J Neurosci 2001; 14:64–72. Corp ES, McQuade J, Moran TH, Smith GP. Characterization of type A and type B CCK receptor binding sites in rat vagus nerve. Brain Res 1993; 623:161–166. Moran TH, Norgren R, Crosby RJ, McHugh PR. Central and peripheral vagal transport of cholecystokinin binding sites occurs in afferent fibers. Brain Res 1990; 526:95–102. Wang YH, Taché Y, Sheibel AB, Go VLW, Wei JY. Two types of leptin-responsive gastric vagal afferent terminals: an in vitro single-unit study in rats. Am J Physiol 1997; 273:R833–R837. Blackshaw LA, Grundy D. Effects of cholecystokinin (CCK-8) on two classes of gastroduodenal vagal afferent fibre. J Aut Nerv Syst 1990; 31:191–202. Yoshida-Yoneda E, O-Lee TJ, Wei JY, Vigna SR, Taché Y. Peripheral bombesin induces gastric vagal afferent activation in rats. Am J Physiol 1996; 271:R1584–R1593. Rogers RC, McCann MJ. Intramedullary connections of the gastric region in the solitary nucleus: a biocytin histochemical tracing study in the rat. J Aut Nerv Syst 1993; 42:119–130. Miselis RR, Rinaman L, Altschuler SM, Bao X, Lynn RB. Medullary viscerotopic representation of the alimentary canal innervation in the rat. In: Taché Y, Wingate D, eds. Brain-Gut Interactions. Boca Raton, FL: CRC Press, 1991:3–21. Aicher SA, Kurucz OS, Reis DJ, Milner TA. Nucleus tractus solitarius efferent terminals synapse on neurons in the caudal ventrolateral medulla that project to the rostral ventrolateral medulla. Brain Res 1995; 693:51–63.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

65. Herbert H, Moga MM, Saper CB. Connections of the parabrachial nucleus with the nucleus of the solitary tract and the medullary reticular formation in the rat. J Comp Neurol 1990; 293:540–580. 66. Petrov T, Krukoff TL, Jhamamdas JH. Branching projections of catecholaminergic brainstem neurons to the paraventricular hypothalamic nucleus and the central nucleus of the amygdala in the rat. Brain Res 1993; 609:81–92. 67. Hyland NP, Abrahams TP, Fuchs K, Burmeister MA, Hornby PJ. Organization and neurochemistry of vagal preganglionic neurons innervating the lower esophageal sphincter in ferrets. J Comp Neurol 2001; 430:222–234. 68. Cunningham ET, Sawchenko PE. A circumscribed projection from the nucleus of the solitary tract to the nucleus ambiguus in the rat: anatomical evidence for somatostatin-28-immunoreactive interneurons subserving reflex control of esophageal motility. J Neurosci 1989; 9:1668–1682. 69. Wang FB, Powley TL. Topographic inventories of vagal afferents in gastrointestinal muscle. J Comp Neurol 2000; 421:302–324. 70. Phillips RJ, Powley TL. Tension and stretch receptors in gastrointestinal smooth muscle: re-evaluating vagal mechanoreceptor electrophysiology. Brain Res Brain Res Rev 2000; 34:1–26. 71. Berthoud HR, Patterson LM, Neumann F, Neuhuber WL. Distribution and structure of vagal afferent intraganglionic laminar endings (IGLEs) in the rat gastrointestinal tract. Anat Embryol (Berl) 1997; 195:183–191. 72. Tiniakos DG, Lee JA, Burt AD. Innervation of the liver: morphology and function. Liver 1996; 16:151–160. 73. Berthoud HR, Patterson LM. Anatomical relationship between vagal afferent fibers and CCK-immunoreactive entero-endocrine cells in the rat small intestinal mucosa. Acta Anat (Basel) 1996; 156:123–131. 74. Berthoud HR, Neuhuber WL. Functional and chemical anatomy of the afferent vagal system. Auton Neurosci 2000; 85:1–17. 75. Grundy D, Richards W. Vagal and spinal afferent innervation: role in sensation and reflex regulation of upper gastrointestinal function. In: Mayer AE, Raybould HE, eds. Basic and Clinical Aspects of Chronic Abdominal Pain. Amsterdam: Elsevier Science Publishers, 1993:37–43. 76. Davidson JS. Response of single vagal afferent fibres to mechanical and chemical stimulation of the gastric and duodenal mucosa in cats. Q J Exp Physiol 1972; 57:405–416. 77. Williams RM, Berthoud HR, Stead RH. Vagal afferent nerve fibres contact mast cells in rat small intestinal mucosa. Neuroimmunomodulation 1997; 4:266–270. 78. Hillsley K, Grundy D. Serotonin and cholecystokinin activate different populations of rat mesenteric vagal afferents. Neurosci Lett 1998; 255:63–66. 79. Zhu JX, Zhu XY, Owyang C, Li Y. Intestinal serotonin acts as a paracrine substance to mediate vagal signal transmission evoked by luminal factors in the rat. J Physiol 2001; 530:431–442. 80. Rosenberg M, Pie B, Cooper E. Developing neonatal rat sympathetic and sensory neurons differ in their regulation of 5-HT3 receptor expression. J Neurosci 1997; 17:6629–6638.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

81. 82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94. 95.

96.

Hofer D, Asan E, Drenckhahn D. Chemosensory perception in the gut. News Physiol Sci 1999; 14:18–23. Berthoud H-R, Powley TL. Vagal afferent innervation of the rat fundic stomach: morphological characterization of the gastric tension receptor. J Comp Neurol 1992; 319:261–276. Valentino RJ, Kosboth M, Colflesh M, Miselis RR. Transneuronal labeling from the rat distal colon: anatomic evidence for regulation of distal colon function by a pontine corticotropin-releasing factor system. J Comp Neurol 2000; 417:399–414. Vizzard MA, Brisson M, de Groat WC. Transneuronal labeling of neurons in the adult rat central nervous system following inoculation of pseudorabies virus into the colon. Cell Tissue Res 2000; 299:9–26. Luckensmeyer GB, Keast JR. Projections of pelvic autonomic neurons within the lower bowel of the male rat: an anterograde labelling study. Neuroscience 1998; 84:263–280. Nadelhaft I, Booth AM. The location and morphology of preganglionic neurons and the distribution of visceral afferents from the rat pelvic nerve: a horseradish peroxidase study. J Comp Neurol 1984; 226:238–245. Leedy MG, Bresnahan JC, Mawe GM, Beattie MS. Differences in synaptic inputs to preganglionic neurons in the dorsal and lateral band subdivisions of the cat sacral parasympathetic nucleus. J Comp Neurol 1988; 268:84–90. Nadelhaft I, Booth AM. The location and morphology of preganglionic neurons and the distribution of visceral afferents from the rat pelvic nerve: a horseradish peroxidase study. J Comp Neurol 1984; 226:238–245. Morgan CW, de Groat WC, Felkins LA, Zhang SJ. Axon collaterals indicate broad intraspinal role for sacral preganglionic neurons. Proc Natl Acad Sci USA 1991; 88:6888–6892. Mawe GM, Bresnahan JC, Beattie MS. A light and electron microscopic analysis of the sacral parasympathetic nucleus after labelling primary afferent and efferent element with HRP. J Comp Neurol 1986; 250:33–57. Taché Y, Martinez V, Million M, Wang L. Stress and the gastrointestinal tract III. Stress-related alterations of gut motor function: role of brain corticotropin-releasing factor receptors. Am J Physiol 2001; 280:G173–G177. Keast JR, Booth AM, de Groat WC. Distribution of neurons in the major pelvic ganglion of the rat which supply the bladder, colon or penis. Cell Tissue Res 1989; 256:105–112. Luckensmeyer GB, Keast JR. Immunohistochemical characterisation of sympathetic and parasympathetic pelvic neurons projecting to the distal colon in the male rat. Cell Tissue Res 1995; 281:551–559. Tabatai M, Booth AM, de Groat WC. Morphological and electrophysiological properties of pelvic ganglion cells in the rat. Brain Res 1986; 382:61–70. Hedlund H, Fandriks L, Delbro D, Fasth S. On the transmission of sacral parasympathetic nervous influence on distal colonic and rectal motility in the cat. Acta Physiol Scand 1985; 125:225–234. Morgan C, Nadelhaft I, de Groat WC. The distribution of visceral primary afferents from the pelvic nerve to the Lissauer’s tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus. J Comp Neurol 1981; 201:415–440.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

97.

98. 99.

100.

101.

102. 103.

104.

105. 106.

107.

108.

109.

110.

111.

112.

Taché Y. Central regulation of gastric acid secretion. In: Johnson LR, Christensen J, Jackson M, Jacobson ED, Walsh JH, eds. Physiology of the Gastrointestinal Tract. New York: Raven Press, 1987:911–930. Taché Y, Vale W, Brown M. Thyrotropin-releasing hormone-CNS action to stimulate gastric acid secretion. Nature 1980; 287:149–151. Taché Y, Yang H. Role of medullary TRH in the vagal regulation of gastric function. In: Taché Y, Wingate DL, Burks TF, eds. Innervation of the Gut: Pathophysiological Implications. Boca Raton, FL: CRC Press, 1993:67–80. Rinaman L, Miselis RR. Thyrotropin-releasing hormone-immunoreactive nerve terminals synapse on the dentrites of gastric vagal motoneurons in the rat. J Comp Neurol 1990; 294:235–251. Rinaman L, Miselis RR, Kreider MS. Ultrastructural localization of thyrotropin-releasing hormone immunoreactivity in the dorsal vagal complex in rat. Neurosci Lett 1989; 104:7–12. Fodor M, Pammer C, Gorcs T, Palkovits M. Neuropeptides in the human dorsal vagal complex: an immunohistochemical study. J Chem Neuroanat 1994; 7:141–157. Lynn RB, Kreider MS, Miselis RR. Thyrotropin-releasing hormone-immunoreactive projections to the dorsal motor nucleus and the nucleus of the solitary tract of the rat. J Comp Neurol 1991; 311:271–288. Bayliss DA, Viana F, Kanter RK, Szymeczek-Seay CL, Berger AJ, Millhorn DE. Early postnatal development of thyrotropin-releasing hormone (TRH) expression, TRH receptor binding, and TRH responses in neurons of rat brainstem. J Neurosci 1994; 14:821–833. Gershengorn MC, Osman R. Molecular and cellular biology of thyrotropin-releasing hormone receptors. Physiol Rev 1996; 76:175–191. Cao J, O’Donnell D, Vu H, Payza K, Pou C, Godbout C, Jakob A, Pelletier M, Lembo P, Ahmad S, Walker P. Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. J Biol Chem 1998; 273:32281–32287. Wu W, Elde R, Wessendorf MW, Hokfelt T. Identification of neurons expressing thyrotropin releasing-hormone receptor mRNA in spinal cord and lower brainstem of rat. Neurosci Lett 1992; 142:143–146. Heuer H, Schafer MK, O’Donnell D, Walker P, Bauer K. Expression of thyrotropinreleasing hormone receptor 2 (TRH-R2) in the central nervous system of rats. J Comp Neurol 2000; 428:319–336. Manaker S, Rizio G. Autoradiographic localization of thyrotropin-releasing hormone and substance P receptors in the rat dorsal vagal complex. J Comp Neurol 1989; 290:516–526. McCann MJ, Hermann GE, Rogers RC. Thyrotropin-releasing hormone: effects on identified neurons of the dorsal vagal complex. J Auton Nerv Syst 1989; 26:107–112. Travagli RA, Gillis RA, Vicini S. Effects of thyrotropin-releasing hormone on neurons in the rat dorsal motor nucleus of the vagus, in vitro. Am J Physiol 1992; 263:G508–G517. Livingston CA, Berger AJ. Response of neurons in the dorsal motor nucleus of the vagus to thyrotropin-releasing hormone. Brain Res 1993; 621:97–105.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

113. 114.

115.

116.

117. 118.

119.

120.

121. 122.

123.

124.

125.

126.

127. 128.

129.

O-Lee TJ, Wei JY, Taché Y. Intracisternal TRH and RX 77368 potently activate vagal efferent discharge in rats. Peptides 1997; 18:213–219. Taché Y, Goto Y, Hamel D, Pekary A, Novin D. Mechanisms underlying intracisternal TRH-induced stimulation of gastric acid secretion in rats. Regul Pept 1985; 13:21–30. Somiya H, Tonoue T. Neuropeptides as central integrators of autonomic nerve activity: effects of TRH, SRIF, VIP and bombesin on gastric and adrenal nerves. Regul Pept 1984; 9:47–52. Miampamba M, Yang H, Sharkey KA, Taché Y. Intracisternal TRH analog induces Fos expression in gastric myenteric neurons and glia in conscious rats. Am J Physiol Gastrointest Liver Physiol 2001; 280:G979–G991. Krukoff TL. Expression of c-fos in studies of central autonomic and sensory systems. Mol Neurobiol 1993; 7:247–263. Taché Y, Goto Y, Lauffenburger M, Lesiege D. Potent central nervous system action of p-Glu-His-(3,3-dimethyl)-Pro NH2, a stabilized analog of TRH, to stimulate gastric secretion in rats. Regul Pept 1984; 8:71–78. Tanaka S, Taché Y, Kaneko H, Guth PH, Kaunitz JD. Central vagal activation increases mucus gel thickness and surface cell intracellular pH in rat stomach. Gastroenterology 1997; 112:409–417. Garrick T, Stephens R, Ishikawa T, Sierra A, Avidan A, Weiner H, Taché Y. Medullary sites for TRH analogue stimulation of gastric contractility in the rat. Am J Physiol 1989; 256:G1011–G1015. Taché Y, Stephens RL, Ishikawa T. Central nervous system action of TRH to influence gastrointestinal function and ulceration. Ann NY Acad Sci 1989; 553:269–285. Király Á, Sütó G, Guth PH, Taché Y. Peripheral mediators involved in gastric hyperemia to vagal activation by central TRH analog in rats. Am J Physiol 1998; 274:G170–G177. Tamori K, Yoneda M, Nakamura K, Makino I. Effect of intracisternal thyrotropinreleasing hormone on hepatic blood flow in rats. Am J Physiol 1998; 274:G277–G282. Kato S, Hirata T, Kitamura M, Takeuchi K. Gastric hyperemic response during vagally mediated acid secretion by TRH analog in rats. J Pharmacol Exp Ther 1997; 282:1351–1357. Feng HS, Lynn RB, Han J, Brooks FP. Gastric effects of TRH analogue and bicuculline injected into dorsal motor vagal nucleus in cats. Am J Physiol 1990; 259:G3210–G326. Lenz HJ, Vale WW, Rivier JE. TRH-induced vagal stimulation of duodenal HCO3 mediated by VIP and muscarinic pathways. Am J Physiol 1989; 257:G677–G682. Lenz HJ, Silverman TA. Effects of cerebral TRH on intestinal water transport: role of vagal, muscarinic, and VIP pathways. Am J Physiol 1995; 269:G138–G143. Okumura T, Taylor IL, Pappas TN. Microinjection of TRH analogue into the dorsal vagal complex stimulates pancreatic secretion in rats. Am J Physiol 1995; 269:G328–G334. LaHann TR, Horita A. Thyrotropin releasing hormone: centrally mediated effects on gastrointestinal motor activity. J Pharmacol Exp Ther 1982; 222:66–70.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

130.

131. 132. 133.

134.

135.

136.

137.

138.

139.

140. 141. 142.

143.

144.

145.

Holmes GM, Rogers RC, Bresnahan JC, Beattie MS. Thyrotropin-releasing hormone (TRH) and CNS regulation of anorectal motility in the rat. J Auton Nerv Syst 1995; 56:8–14. Yoneda M, Taché Y. Vagal regulation of gastric prostaglandin E2 release by central TRH in rats. Am J Physiol 1993; 264:G231–G236. Yanagisawa K, Taché Y. Intracisternal TRH analog RX 77368 stimulates gastric histamine release in rats. Am J Physiol 1990; 259:G599–G604. Yanagisawa K, Yang H, Walsh JH, Taché Y. Role of acetylcholine, histamine and gastrin in the acid response to intracisternal injection of TRH analog, RX 77368, in the rat. Regul Pept 1990; 27:161–170. Song M, Yang H, Walsh JH, Ohning G, Wong H, Taché Y. Intracisternal TRH analog increases gastrin release and corpus histidine decarboxylase activity in rats. Am J Physiol 1999; 276:G901–G908. Saperas E, Mourelle M, Santos J, Moncada S, Malagelada J-R. Central vagal activation by an analogue of TRH stimulates gastric nitric oxide release in rats. Am J Physiol 1995; 268:G895–G899. Yoneda M, Taché Y. Serotonin enhances gastric acid response to TRH analog in dorsal vagal complex through 5-HT2 receptors in rats. Am J Physiol 1995; 269:R1–R6. Király Á, Sütó G, Livingston EH, Guth PH, St. Pierre S, Taché Y. Central vagal activation by TRH induces gastric hyperemia: role of CGRP in capsaicin-sensitive afferents in rats. Am J Physiol 1994; 267:G1041–G1049. Király Á, Sütó G, Guth PH, Taché Y. Mechanisms mediating gastric hyperemic and acid responses to central TRH analog at a cytoprotective dose. Am J Physiol 1997; 273:G31–G38. Király Á, Sütó G, Guth PH, Taché Y. Ketotifen prevents gastric hyperemia induced by intracisternal thyrotropin-releasing hormone at a low dose. Eur J Pharmacol 1997; 334:241–247. Tanaka T, Guth P, Taché Y. Role of nitric oxide in gastric hyperemia induced by central vagal stimulation. Am J Physiol 1993; 264:G-280–G-284. Király Á, Sütó G, Guth P, Taché Y. Influence of [4Cl-D-Phe6,Leu17]VIP on VIPand central TRH-induced gastric hyperemia. Peptides 1997; 18:1321–1325. Stephens RL, Garrick T, Weiner H, Taché Y. Serotonin depletion potentiates gastric secretory and motor responses to vagal but not peripheral gastric stimulants. J Pharmacol Exp Ther 1989; 251:524–530. Cardin S, Soll AH, Taché Y. Different effects of indomethacin and nabumetone on prostaglandin-mediated gastric responses to central vagal activation in rats. J Pharmacol Exp Ther 1995; 275:667–673. Taché Y, Yoneda M. Central action of TRH to induce vagally mediated gastric cytoprotection and ulcer formation in rats. J Clin Gastroenterol 1993; 17(suppl 1):S58–S63. Horita A, Carino MA. Centrally administered thyrotropin-releasing hormone (TRH) stimulates colonic transit and diarrhea production by a vagally mediated serotonergic mechanism in the rabbit. J Pharmacol Exp Ther 1982; 222:367–371.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

146.

147.

148. 149.

150.

151.

152.

153.

154. 155.

156.

157.

158. 159.

160.

161.

Suzuki S, Pilowsky P, Minson J, Arnolda L, Llewellyn-Smith I, Chalmers J. Antisense to thyrotropin releasing hormone receptor reduces arterial blood pressure in spontaneously hypertensive rats. Circ Res 1995; 77:679–683. Sivarao DV, Krowicki ZK, Abrahams TP, Hornby PJ. Intracisternal antisense oligonucleotides to TRH receptor abolish TRH-evoked gastric motor excitation. Am J Physiol 1997; 272:G1372–G1381. Yang H, Ohning G, Taché Y. TRH in dorsal vagal complex mediates acid response to excitation of raphe pallidus neurons in rats. Am J Physiol 1993; 265:G880–G886. Kaneko H, Yang H, Ohning G, Taché Y. Medullary TRH is involved in gastric protection induced by low dose of kainic acid into the raphe pallidus. Am J Physiol 1995; 268:G548–G552. Kaneko H, Taché Y. TRH in the dorsal motor nucleus of the vagus is involved in gastric erosion induced by excitation of raphe pallidus in rats. Brain Res 1995; 699:97–102. Garrick T, Prince M, Yang H, Ohning G, Taché Y. Raphe pallidus stimulation increases gastric contractility via TRH projections to the dorsal vagal complex in rats. Brain Res 1994; 636:343–347. Kaneko H, Kaunitz J, Taché Y. Vagal mechanisms underlying gastric protection induced by chemical activation of the raphe pallidus in rats. Am J Physiol 1998; 275:G1056–G1062. Kaneko H, Kato K, Ohning G, Taché Y. Medullary thyrotropin-releasing hormone mediates vagal-dependent adaptive gastric protection induced by mild acid in rats. Gastroenterology 1995; 109:861–865. Barrachina MD, Wu V, Taché Y. Central TRH receptors are involved in the gastric secretory response to sham-feeding in rats. Gastroenterology 1997; 112:A1130. Okumura T, Taylor IL, Ohning G, Taché Y, Pappas TN. Intracisternal injection of TRH antibody blocks gastric emptying stimulated by 2-deoxy-D-glucose in rats. Brain Res 1995; 674:137–141. Lin MT, Wang PS, Chuang J, Fan LJ, Won SJ. Cold stress or a pyrogenic substance elevates thyrotropin-releasing hormone levels in the rat hypothalamus and induces thermogenic reactions. Neuroendocrinology 1989; 50:177–181. Arancibia S, Rage F, Astier H, Tapia-Arancibia L. Neuroendocrine and autonomous mechanisms underlying thermoregulation in cold environment. Neuroendocrinology 1996; 64:257–267. Bonaz B, Taché Y. Induction of Fos immunoreactivity in the rat brain after cold-restraint induced gastric lesions and fecal excretion. Brain Res 1994; 652:56–64. Wang L, Cardin S, Martinez V, Taché Y. Intracerebroventricular CRF inhibits cold restrain-induced c-fos expression in the dorsal motor nucleus of the vagus and gastric erosions in rats. Brain Res 1996; 736:44–53. Yang H, Wu SV, Ishikawa T, Taché Y. Cold exposure elevates thyrotropin-releasing hormone gene expression in medullary raphe nuclei: relationship with vagally mediated gastric erosions. Neuroscience 1994; 61:655–663. Cho CH, Qui BS, Bruce IC. Vagal hyperactivity in stress induced gastric ulceration in rats. J Gastroenterol Hepatol 1996; 11:125–128.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

162.

163. 164.

165.

166. 167.

168.

169. 170. 171. 172.

173.

174.

175. 176. 177.

Yuan PQ, Taché Y, Miampamba M, Yang H. Acute cold exposure induces vagally mediated Fos expression in gastric myenteric neurons in conscious rats. Am J Physiol 2001; 281:G560–G568. Garrick T, Buack S, Bass P. Gastric motility is a major factor in cold restraint-induced lesion formation in rats. Am J Physiol 1986; 250:G191–G199. Ephgrave KS, Cullen JJ, Broadhurst K, Kleiman-Wexler R, Shirazi SS, SchulzeDelrieu K. Gastric contractions, secretions and injury in cold restraint. Neurogastroenterol Motil 1997; 9:187–192. Martinez V, Wu SV, Taché Y. Intracisternal antisense oligodeoxynucleotides to the thyrotropin-releasing hormone receptor blocked vagal-dependent stimulation of gastric emptying induced by acute cold in rats. Endocrinology 1998; 139:3730–3735. Martinez V, Wang L, Taché Y. Central TRH receptor 1 antisense blocks cold-induced gastric emptying but not brain c-Fos induction. Peptides 2001; 22:81–90. Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, Arias CA, et al. Urocortin II: A member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci USA 2001; 98:2843–2848. Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, et al. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 2001; 98:7570–7575. Perrin MH, Vale WW. Corticotropin releasing factor receptors and their ligand family. Ann NY Acad Sci 1999; 885:312–328. Koob GF, Heinrichs SC. A role for corticotropin releasing factor and urocortin in behavioral responses to stressors. Brain Res 1999; 848:141–152. Webster EL, Elenkov IJ, Chrousos GP. The role of corticotropin-releasing hormone in neuroendocrine-immune interactions. Mol Psychiatry 1997; 2:368–372. Habib KE, Weld KP, Rice KC, Pushkas J, Champoux M, Listwak S, et al. Oral administration of a corticotropin-releasing hormone receptor antagonist significantly attenuates behavioral, neuroendocrine, and autonomic responses to stress in primates. Proc Natl Acad Sci USA 2000; 97:6079–6084. Broccardo M, Improta G. Pituitary-adrenal and vagus modulation of sauvagine- and CRF-induced inhibition of gastric emptying in rats. Eur J Pharmacol 1990; 182:357–362. Taché Y, Maeda-Hagiwara M, Turkelson CM. Central nervous system action of corticotropin-releasing factor to inhibit gastric emptying in rats. Am J Physiol 1987; 253:G241–G245. Lee C, Sarna SK. Central regulation of gastric emptying of solid nutrient meals by corticotropin releasing factor. Neurogastroenterol Mot 1997; 9:221–229. Taché Y. Cyclic vomiting syndrome: the corticotropin-releasing-factor hypothesis. Dig Dis Sci 1999; 44:79S–86S. Kihara N, Fujimura M, Yamamoto I, Itoh E, Inui A, Fujimiya M. Effects of central and peripheral urocortin on fed and fasted gastroduodenal motor activity in conscious rats. Am J Physiol 2001; 280:G406–G419.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

178.

179.

180.

181.

182.

183.

184.

185.

186.

187.

188. 189.

190.

191. 192.

Smedh U, Uvnas-Moberg K. Intracerebroventricularly administered corticotropinreleasing factor releases somatostatin through a cholinergic, vagal pathway in freely fed rats. Acta Physiol Scand 1994; 151:241–248. Taché Y, Goto Y, Gunion MW, Vale W, Rivier J, Brown M. Inhibition of gastric acid secretion in rats by intracerebral injection of corticotropin-releasing factor. Science 1983; 222:935–937. Heymann-Mönnikes I, Taché Y, Trauner M, Weiner H, Garrick T. CRF microinjected into the dorsal vagal complex inhibits TRH analog- and kainic acid-stimulated gastric contractility in rats. Brain Res 1991; 554:139–144. Kosoyan HP, Wei JY, Taché Y. Intracisternal sauvagine is more potent than corticotropin-releasing factor to decrease gastric vagal efferent activity in rats. Peptides 1999; 20:851–858. Perrin MH, Sutton SW, Cervini LA, Rivier JE, Vale WW. Comparison of an agonist, urocortin, and an antagonist, astressin, as radioligands for characterization of CRF receptors. J Pharmacol Exp Ther 1999; 288:729–734. Chen C-Y, Million M, Adelson DW, Martinez V, Rivier J, Taché Y. Intracisternal urocortin inhibits vagally stimulated gastric motility:role of CRF2. Br J Pharmacol 2002; 136:237–247. Van Pett K, Viau V, Bittencourt JC, Chan RK, Li HY, Arias C, et al. Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse. J Comp Neurol 2000; 428:191–212. Lenz HJ, Burlage M, Raedler A, Greten H. Central nervous system effects of corticotropin-releasing factor on gastrointestinal transit in the rat. Gastroenterology 1988; 94:598–602. Gué M, Junien JL, Buéno L. Conditioned emotional response in rats enhances colonic motility through the central release of corticotropin-releasing factor. Gastroenterology 1991; 100:964–970. Mönnikes H, Schmidt BG, Raybould HE, Taché Y. CRF in the paraventricular nucleus mediates gastric and colonic motor response to restraint stress. Am J Physiol 1992; 262:G137–G143. Sarna SK. Physiology and pathophysiology of colonic motor activity part two of two. Dig Dis Sci 1991; 36:998–1018. Sternberg EM, Young III WS, Bernardini R, Calogero AE, Chrousos GP, Gold PW, et al. A central nervous system defect in biosynthesis of corticotropin-releasing hormone is associated with susceptibility to streptococcal cell wall-induced arthritis in Lewis rats. Proc Natl Acad Sci USA 1989; 86:4771–4475. Million M, Wang L, Martinez V, Taché Y. Differential Fos expression in the paraventricular nucleus of the hypothalamus, sacral parasympathetic nucleus and colonic motor response to water avoidance stress in Fischer and Lewis rats. Brain Res 2000; 877:345–353. Martinez V, Taché Y. Role of CRF receptor 1 in central CRF-induced stimulation of colonic propulsion in rats. Brain Res 2001; 893:29–35. Brown MR, Fisher LA, Spiess J, Rivier C, Rivier J, Vale W. Corticotropin-releasing factor: action on the sympathetic nervous system and metabolism. Endocrinology 1982; 111:928–931.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

193.

194. 195.

196.

197.

198.

199.

200.

201. 202.

203. 204. 205.

206.

207. 208. 209.

Kurosawa M, Sata A, Swenson RS, Takahashi Y. Sympatho-adrenal medullary functions in response to intracerebroventricularly injected corticotropin-releasing factor in anesthetized rats. Brain Res 1986; 367:250–257. Egawa M, Yoshimatsu H, Bray GA. Preoptic area injection of corticotropin-releasing hormone stimulates sympathetic activity. Am J Physiol 1990; 259:R799–R806. Katafuchi T, Ichijo T, Hori T. Sequential relationship between actions of CRF and PGE2 in the brain on splenic sympathetic nerve activity in rats. J Auton Nerv Syst 1997; 67:200–206. Yokotani K, Murakami Y, Okada S, Hirata M. Role of brain arachidonic acid cascade on central CRF1 receptor-mediated activation of sympatho-adrenomedullary outflow in rats. Eur J Pharmacol 2001; 419:183–189. Brown MR, Fisher LA, Rivier J, Spiess J, Rivier C, Vale W. Corticotropin-releasing factor: effects on the sympathetic nervous system and oxygen consumption. Life Sci 1982; 30:207–210. Shimizu N, Hori T, Nakane H. An interleukin-1 beta-induced noradrenaline release in the spleen is mediated by brain corticotropin-releasing factor: an in vivo microdialysis study in conscious rats. Brain Behav Immun 1994; 8:14–23. Nijsen MJ, croiset G, Stam R, Bruijnzeel A, Diamant M, De Wied D, et al. The role of the CRH type 1 receptor in autonomic responses to corticotropin-releasing hormone in the rat. Neuropsychopharmacology 2000; 22:388–399. Yokohama S, Yoneda M, Nakamura K, Makino I. Effect of central corticotropin-releasing factor on carbon tetrachloride-induced acute liver injury in rats. Am J Physiol 1999; 276:G622–G628. Lenz HJ. Regulation of small intestinal and pancreaticobiliary functions by CRF. Ann NY Acad Sci 1993; 697:254–259. Lenz HJ, Messmer B, Zimmerman FG. Noradrenergic inhibition of canine gallbladder contraction and murine pancreatic secretion during stress by corticotropin-releasing factor. J Clin Invest 1992; 89:437–443. Druge G, Raedler A, Heiner G, Lenz J. Pathways mediating CRF-induced inhibition of gastric acid secretion in rats. Am J Physiol 1989; 256:G214–G219. Lenz HJ, Hester SE, Brown MR. Corticotropin-releasing factor mechanisms to inhibit gastric acid secretion in conscious dogs. J Clin Invest 1985; 75:889–895. Buchan AM, Kwok YN, Pederson RA. Anatomical relationship between neuropeptide-containing fibers and efferent vagal neurons projecting to the rat corpus. Regul Pept 1991; 34:1–12. Ladic LA, Buchan AM. Association of substance P and its receptor with efferent neurons projecting to the greater curvature of the rat stomach. J Auton Nerv Syst 1996; 58:25–34. Manaker S, Zucchi PC. Effects of vagotomy on neurotransmitter receptors in the rat dorsal vagal complex. Neuroscience 1993; 52:427–441. Yang H, Taché Y. Substance P in the dorsal vagal complex inhibits medullary TRHinduced gastric acid secretion in rats. Am J Physiol 1997; 272:G987–G993. Spencer SE, Talman WT. Central modulation of gastric pressure by substance P: a comparison with glutamate and acetylcholine. Brain Res 1986; 385:371–374.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

210.

211. 212.

213. 214.

215.

216.

217.

218.

219.

220. 221.

222. 223.

224. 225.

Krowicki ZK, Hornby PJ. Substance P in the dorsal motor nucleus of the vagus evokes gastric motor inhibition via neurokinin 1 receptor in rat. J Pharmacol Exp Ther 2000; 293:214–221. Taché Y. Vagal regulation of gastric secretion. In: Mignon M, Galmiche J-P, eds. Control of Acid Secretion. Paris: John Libbey Eurotext, 1988:13–25. Thiefin G, Raybould HE, Leung FW, Taché Y, Guth PH. Capsaicin-sensitive afferent fibers contribute to gastric mucosal blood flow response to electrical vagal stimulation. Am J Physiol 1990; 259:G1037–G1043. Hornby PJ, Abrahams TP. Central control of lower esophageal sphincter relaxation. Am J Med 2000; 108(suppl 4a):90S–98S. Arakawa T, Uno H, Fukuda T, Higuchi K, Kobayashi K, Kuroki T. New aspects of gastric adaptive relaxation, reflex after food intake for more food: involvement of capsaicin-sensitive sensory nerves and nitric oxide. J Smooth Muscle Res 1997; 33:81–88. Takahashi T, Owyang C. Vagal control of nitric oxide and vasoactive intestinal polypeptide release in the regulation of gastric relaxation in rat. J Physiol 1995; 484:481–492. Rossiter CD, Norman WP, Jain M, Hornby PJ, Benjamin S, Gillis RA. Control of lower esophageal sphincter pressure by two sites in dorsal motor nucleus of the vagus. Am J Physiol 1990; 259:G899–G906. Taché Y, Kaneko H, Kawakubo K, Kato K, Kiraly A, Yang H. Central and peripheral mechanisms involved in gastric protection against ethanol injury. J Gastroenterol Hepatol 1998; 13 (suppl):S214–S220. Taché Y, Yoneda M, Kato K, Király Á, Sütó G, Kaneko H. Intracisternal thryrotropin-releasing hormone-induced vagally mediated gastric protection against ethanol lesions: central and peripheral mechanisms. J Gastroenterol Hepatol 1994; 9:S29–S35. Adelson DW, Wei JY, Yashar M, Lee TJ, Taché Y. Central autonomic activation by intracisternal TRH analogue excites gastric splanchnic afferent neurons. J Neurophysiol 1999; 81:682–691. Kato K, Yang H, Taché Y. Low doses of TRH analogue act in the dorsal motor nucleus to induce gastric protection in rats. Am J Physiol 1995; 269:R1301–R1307. Okumura T, Okamura K, Kitamori S, Hara H, Shibata Y, Namiki M. Gastric lesions induced by kainic acid injection into the dorsal motor nucleus of the vagus nerve in rats. Scand J Gastroenterol 1989; 14(suppl 162):15–18. Hernandez DE, Walker CH, Mason GA. Influence of thyroid states on stress gastric ulcer formation. Life Sci 1988; 42:1757–1764. Yang H, Yuan P, Wu V, Taché Y. Feedback regulation of thyrotropin-releasing hormone gene expression by thyroid hormone in the caudal raphe nuclei in rats. Endocrinology 1999; 140:43–49. Yuan PQ, Yang H. Hypothyroidism induces Fos-like immunoreactivity in ventral medullary neurons that synthesize TRH. Am J Physiol 1999; 277:E927–E936. Pirtniecks A, Smith LF, Thorpe JA. Autonomic dysfunction in non-specific disorders of oesophageal motility. Eur J Cardiothorac Surg 2000; 17:101–105.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

226.

227.

228. 229.

230.

231. 232.

233.

234.

235.

236.

237.

238. 239.

240.

241.

Tanabe S. An experimental study of the role of the vagus nerve in the pathogenesis of the dumping syndrome—hemodynamic aspects in the microcirculation of the intestine. Nippon Geka Hokan 1978; 47:280–301. Heatley RV, Collins RJ, James PD, Atkinson M. Vagal function in relation to gastro-oesophageal reflux and associated motility changes. Br Med J 1980; 280:755–757. Smart HL, Atkinson M. Abnormal vagal function in irritable bowel syndrome. Lancet 1987; 2:475–478. Lenz HJ, Raedler A, Greten H, Vale WW, Rivier JE. Stress-induced gastrointestinal secretory and motor responses in rats are mediated by endogenous corticotropin-releasing factor. Gastroenterology 1988; 95:1510–1517. Taché Y. Stress-induced alterations of gastric emptying. In: Buéno L, Collins S, Junien JL, eds. Stress and Digestive Motility. Montrouge: John Libbey Eurotext, 1989:123–132. Enck P, Holtmann G. Stress and gastrointestinal motility in animals: a review of the literature. J Gastrointest Motil 1992; 1:83–90. Williams CL, Burks TF. Stress, opioids, and gastrointestinal transit. In: Taché Y, Morley J, Brown MR, eds. Hans Selye Symposia on Neuroendocrinology and Stress: Neuropeptides and Stress. New York: Springer-Verlag, 1989:175–187. Rao SS, Hatfield RA, Suls JM, Chamberlain MJ. Psychological and physical stress induce differential effects on human colonic motility. Am J Gastroenterol 1998; 93:985–990. Gulyas J, Rivier C, Perrin M, Koerber SC, Sutton S, Corrigan A, et al. Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor. Proc Natl Acad Sci USA 1995; 92:10575–10579. Martinez V, Rivier J, Wang L, Taché Y. Central injection of a new corticotropin-releasing factor (CRF) antagonist, astressin, blocks CRF- and stress-related alterations of gastric and colonic motor function. J Pharmacol Exp Ther 1997; 280:754–760. Bonaz B, Taché Y. Water-avoidance stress-induced c-fos expression in the rat brain and stimulation of fecal output: role of corticotropin-releasing factor. Brain Res 1994; 641:21–28. Mönnikes H, Schmidt BG, Taché Y. Psychological stress-induced accelerated colonic transit in rats involves hypothalamic corticotropin-releasing factor. Gastroenterology 1993; 104:716–723. Turnbull AV, Rivier CL. Regulation of the hypothalamic-pituitary-adrenal axis by cytokines: actions and mechanisms of action. Physiol Rev 1999; 79:1–71. Fargeas M-J, Fioramonti J, Buéno L. Central action of interleukin-1 on intestinal motility in rats: mediation by two mechanisms. Gastroenterology 1993; 104:377–383. Jezova D, Ochedalski T, Glickman M, Kiss A, Aguilera G. Central corticotropin-releasing hormone receptors modulate hypothalamic-pituitary-adrenocortical and sympathoadrenal activity during stress. Neuroscience 1999; 94:797–802. Heinrichs SC, Taché Y. Therapeutic potential of CRF receptor antagonists: a gutbrain perspective. Expert Opin Investig Drugs 2001; 10:647–659.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

242.

243.

244.

245. 246.

247. 248.

Morrow GR, Andrews PL, Hickok JT, Stern R. Vagal changes following cancer chemotherapy: implications for the development of nausea. Psychophysiology 2000; 37:378–384. Endo T, Minami M, Hirafuji M, Ogawa T, Akita K, Nemoto M, et al. Neurochemistry and neuropharmacology of emesis—the role of serotonin. Toxicology 2000; 153:189–201. Yamada Y, Tsukamoto G, Kobashi M, Sasaki A, Matsumura T. Abdominal vagi mediate c-Fos expression induced by x-ray irradiation in the nucleus tractus solitarii of the rat. Auton Neurosci 2000; 83:29–36. Lindley C, Blower P. Oral serotonin type 3-receptor antagonists for prevention of chemotherapy-induced emesis. Am J Health Syst Pharm 2000; 57:1685–1697. Schwartz GJ, Netterville LA, McHugh PR, Moran TH. Gastric loads potentiate inhibition of food intake produced by a cholecystokinin analogue. Am J Physiol 1991; 261:R1141–R1146. Schwartz GJ. The role of gastrointestinal vagal afferents in the control of food intake: current prospects. Nutrition 2000; 16:866–873. Fox EA, Phillips RJ, Baronowsky EA, Byerly MS, Jones S, Powley TL. Neurotrophin-4 deficient mice have a loss of vagal intraganglionic mechanoreceptors from the small intestine and a disruption of short-term satiety. J Neurosci 2001; 21:8602–8615.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

16 The Autonomic Nervous System in the Normal Control and Pathophysiology of the Exocrine–Endocrine Pancreas Osvaldo M. Tiscornia University of Buenos Aires, and José de San Martin Clinical Hospital, Capital Federal Buenos Aires, Argentina

I. INTRODUCTION Hormones and the autonomic nervous system (ANS) play, through complex interaction, a crucial role in the control of exocrine pancreatic secretion (EPS) via “pancreon” units and of the endocrine pancreas by the Langerhans islets (1,2). The higher brain centers regulate both somatic and visceral functions. Interposed in the efferent peripheral pathway between the central system (CNS) and the visceral structures is an aggregation of nerve cells known as the autonomic ganglia. The latter may be classified as paravertebral, prevertebral, and terminal. To the classic main divisions of the ANS—the sympathetic or thoraco-lumbar system and the parasympathetic or cranio-sacral system—we should add the peptidergic system. The latter lies immersed in the three innervation complex systems that control the pancreatic gland: the splanchnic-celiac, the vagal, and the entero-pancreatic (4). The neural networks for control of digestive function are positioned in the brain, spinal cord, and prevertebral ganglia and in the wall of specialized organs that make up the digestive system. Control involves an integrated hierarchy of neural centers (5). Starting at the level of the gut-pancreas, four levels of integrative organization are recognized. Level I is composed of the enteric nervous system (ENS), which we have proposed to be described as a neural plexual freeway (NPF), and of the intrapancre-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 1 Different levels of neural control of the pancreatic gland.

atic ganglia (6). These two components integrate functions independent of higher centers. Level II occurs in the prevertebral ganglia, where peripheral reflex pathways are influenced by preganglionic sympathetic fibers coming from the spinal cord. Levels III and IV are within the CNS (Fig. 1).

II. MAIN FEATURES OF PANCREATIC GLAND INNERVATION A. General Considerations That the ENS (or the NPF) projects to the pancreas was first demonstrated by our group through anatomical macroscopic dissections in human cadavers, rats, and the opossum (7–22). That, indeed, many nerve fibers jump the duodeno-pancreatic cleft was later confirmed by histochemical observations (23). Kirchgessner et al. (24–26) have shown that many axons observed in the connective tissue between the gut and pancreas are serotonergic. Using different types of tracers, they were able to demonstrate that the nerves running between the duodenum and pan-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

creas are continuous with the myenteric plexus of the duodenum and with the pancreatic ganglia. Many axons that jump the duodeno-pancreatic cleft are of the sensory type. Following the pathways of either the vagal or splanchnic-celiac ganglia, they reach the CNS. An anatomical-physiological feature to be stressed is that of the peri-Vaterian-duodenum (PV-D), the most sensitive area of what we have conceived, pedagogically, as the “trigger” of an imaginary “pancreatic revolver” (6,10,14–16, 18,19). In the course of “lanes” through which flow the nerve impulses in the NPF, two regions, one at the gastric antrum and the other at the duodenum, deserve to be considered as having a higher hierarchy, that of a real autonomic nervous brain: the antral (AANB) and duodenal (DANB). Both possess, besides many nerve fibers, primary cholinergic and peptidergic neurons. From these two autonomic nervous structures, several varied influences are exerted not only upon the exocrine-endocrine pancreas, but also on the gallbladder and the gastric fundus (7–22). The DANB plays a pivotal role in the intestinal phase of the EPS and in what has been described as the “entero-insular-axis.” B. Macroscopic Innervation of the Pancreatic Gland The parasympathetic, adrenergic, and peptidergic divisions of the ANS participate in the innervation of the pancreatic gland. The parasympathetic fibers run mainly in the vagi and secondarily in the splanchnics; only a few are found in the vagi. Both vagus nerves and the celiac ganglion complex in which they synapse with preganglionic adrenergic fibers are the main source of the pancreatic gland innervation. The vagal fibers enter the pancreas partly directly and partly indirectly passing through the celiac ganglion without making synapsis in them. In both instances, the vagal fibers contribute to the neural plexuses that course along the different branches of the celiac and mesenteric arteries and, intermingled with the adrenergic and peptidergic fibers, arrive at the pancreatic gland. The macroscopic pancreatic innervation is primarily concentrated in the pancreatic head and isthmus. Faradaic stimulation of the dog’s pancreatic surface close to the pylorus, the accessory and the main pancreatic duct ending in the duodenum, evokes, contrary to what is observed with excitation of the splenic end, a marked secretory response (7–22). C. Microscopic Innervation of the Pancreatic Gland Large nerve bundles are present in the interlobar septa near the arteries. Parasympathetic preganglionic fibers become slender and unmyelinated in the interlobar septa and terminate in small ganglia. Postganglionic fibers are more slender than other neural elements. Between the pancreatic acini, small nerve bundles are abundant. (23–26) Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Along the course of the branches, small knoblike enlargements are seen. These lie on the surface of the acinar cells. Branches pass from the small nerve bundles between the pancreatic acini into the pancreatic islets. They course over the surface of the islets cells and form small networks with knob-like thickenings comparable to those related to the exocrine cells of the gland. Close to the Langerhans islets there exists a peculiar structure that Van Campenhout called “complexe sympatho-insulaire.” This structure consists of nerve cells and islets cells in juxtaposition to each other. According to De Giorgio et al. (28) nine different neuropeptides can be detected in the intrapancreatic ganglia: CCK, CGRP, GRP, ENK, Gal, NPY, NT, SP, and VIP. Adrenergic fibers are frequently adjacent to the islets of Langerhans. Many branches form a varicose network around them. Fibers extend to the center of the islet, where many appear associated with blood vessels and some are in actual contact with the islet cells. The anatomo-histological characteristics of adrenergic innervation suggest that influences of this system on the control of EPS are exerted mainly through changes in blood flow within the gland and by modulating the cholinergic impulses through the intrapancreatic ganglia (29–37). D. Ultramicroscopy Analysis of the types of vesicles present in the nerve endings permitted identification of the division of the ANS to which the fibers belong. At the level of the intrapancreatic ganglion cells, most of the nerve endings contain agranular synaptic vesicles (type 1a) or both agranular and some large cored vesicles (type 1b) or some large granular vesicles (LGV), suggesting a cholinergic type of terminal. The nerve endings in close association with the blood vessels contain vesicles with bar-shaped crystalloids (type 2b) or with dense round cores (type 2a). The nerve endings terminating in the acinar and ductular cells are almost all cholinergic (types 1a and 1b). Thus, ultrastructural analysis confirms the light microscopic findings that the “pancreon” unit is innervated mainly by the parasympathetic nerves, although a few adrenergic endings are observed in contact with acinar cells. The nerve endings within the islets are of types 1a, 1b, and 2a. Both A and B cells have cholinergic receptors that can be blocked by atropine. They are also under the influence of adrenergic impulses. E. Celiac Ganglion Complex In the celiac ganglion complex, 99% of the neurons reveal the presence of norepinephrine (NE). Among them, 20% are also reactive to SOM (NE/SOM) and 50% colocalize the NPY peptide (NE/NPY). Moreover, 1% disclose the presence of DYN, NPY, and VIP. Many peptidergic nerve fibers run among the neuron cells, such as CCK, ENK, GRP, and VIP. The NE/SOM neurons are linked pref-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

erentially with the submucous enteric plexus. The latter, according to our studies, display the greatest density of neuron cells at the level of the PV-D, the main “trigger” zone of the pancreatic gland. Richins (38) showed in cats that stimulation of the adrenergic component of the celiac ganglia resulted in vasoconstriction in the pancreas. The decreased capillary blood flow correlated with the inhibition of EPS reported by Kuntz and Richins (39) in dogs under similar experimental conditions. In contrast, stimulation of the cholinergic component resulted in vasodilatation, closing of the arterio-venous shunts and increased blood flow through the capillary beds. This is consistent with the augmented EPS due to cholinergic stimulation. Varga et al. (40) have also shown in dogs that electrical stimulation of the left celiac ganglia significantly diminishes blood flow through the gland due to constriction of the blood vessels. This procedure increses the mortality of acute pancreatitis induced by the intraductal injection of bile fourfold (41,42). F. Intrapancreatic Cholinergic Tone Concept While theoretically one should also take into account an adrenergic and a peptidergic tone, from a practical point of view one may accept the concept of an intrapancreatic cholinergic tone as representing the final result of a continuous and changing interaction of cholinergic, adrenergic, and peptidergic impulses (30–37). The element playing the central role in the integration of these different influences is the intrapancreatic ganglion. It is the embryonic equivalent of Auerbach’s and Meissner’s plexus in the intestine and functions as an integration center to process and distribute local nervous information rather than as a simple relay system. There appears to be a correlation between the number of ganglia in the pancreatic gland and the degree of the “pancreon” EPS response to vagal stimulation. It is suggestive that the salivary glands, which contain numerous ganglia, readily respond to vagal stimulation, while the liver, which does not contain demonstrable ganglia, does not respond to vagal excitation. G. The Gut-Pancreas and ANS Afferent Component The vagal and spinal afferent innervation mediates the gut-pancreas visceral sensation and is involved in multiple reflex loops. Sensory input is modulated peripherally, at the afferent nerve terminals, at the level of the prevertebral ganglia, the spinal cord, and the brainstem. The cell bodies of vagal afferents lie in the nodose ganglion, and the cell bodies of spinal afferents lie in the dorsal root ganglia. The central processes of vagal afferent fibers terminate in the nucleus of the solitary tract (NTS) in the brainstem and pass to the periphery in the vagus nerve. The central processes of spinal afferent fibers terminate in the dorsal horn of the spinal cord

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

and pass to the periphery in the main sympathetic nerve trunks. The gut-pancreas sensory receptors do not show any morphological specialization and are generally considered to be free nerve endings. They show sensitivity to a variety of stimuli, including mechanical, chemical, thermal, and osmotic. Those sensitive to glucose and acid do show a high degree of specificity for that particular stimulus. Neuron cell bodies of the nodose ganglion have been stained in immunohistochemical studies with antibodies to SP, CCK, VIP, SOM, and CGPR. These peptides accumulate above a ligature of the vagus nerves. In the dorsal root ganglia, CCK, SP, VIP, bombesin/GRP, and CGRP have been demonstrated with immunoreactivity. Their release from peripheral endings may cause them to function as neuromodulators or as mediators of local tissue response. The concept of afferent endings subserving a local effector function was first evaluated in skin and later shown in the gastrointestinal tract and pancreas. Excitation spreads to nerve endings not directly stimulated via an axon reflex that releases neuropeptides and produces local tissue effects. The sensory fibers that take part in these local effector systems are nonmyelinated peptide-containing neurons, formerly called nonadrenergic noncholinergic (NANC) neurons. The afferent component of the ANS is at the basis of physiological arc reflexes to both the exocrine and endocrine pancreas from the gastrointestinal tract. These physiological reflexes are the consequence of excitation, defined as a stimulus that brings about a physiological reaction in the body. On the other hand, when sensory neurons are subjected to irritation, or a stimulus of an intensity that goes beyond a physiological range and causes pathological lesions in the body, they evoke overreactive autonomic-arc reflexes (O-AAR). We have ascribed to them responsibility for biliary acute pancreatitis (BAP) episodes. This latter entity fits within the group of diseases that have been encompassed under the denomination of syndrome d’irritation neurovegetative, or Reilly’s phenomena (21,27). The afferent neurons that are involved are sensitive to capsaicin. Low doses of this red pepper ingredient induce transient excitation of thin primary afferent neurons, while systemic administration of high doses causes long-lasting damage to these neurons. The fine afferent neurons contain a number of bioactive peptides, such as SP, CGRP, VIP, SOM, DYN, bombesin/GRP, CCK, GAL, leucine-enkephalin, neurokinin-A, and corticotropin-releasing factor. Besides being the main transmitter of pancreatic pain through the splanchnic-celiac complex, SP induces hyperemia, which occurs by dilatation of arterioles, and protein leakage resulting from an increase in the permeability of postcapillary venules. Endothelin-derived nitric oxide (NO) is also involved in these changes. In the closely related mast cells, SP induces the release of histamine and other mediators. Concerning CGRP, this peptide exerts both proinflammatory and anti-inflammatory effects. The former is reflected by the peptide’s ability to enhance protein leakage. The anti-inflammatory effect is due to its modulation of the exudation process. At the level of the pancreas, this 33-amino-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

acid peptide induces an inhibitory influence on EPS by an indirect mechanism through an increase of plasma SOM. By means of the latter and other regulatory peptides, such as enkephalins, the afferent component of the ANS exerts a cytoprotective effect on the gastrointestinal mucosa and pancreas. This explains the fact that capsaicin-sensitive nerve degeneration by capsaicin pretreatment augments the ethanol-induced gastric damage. We have been able to demonstrate the involvement of capsaicin-sensitive neurons in two experimental models, respectively, at the gastric and pancreas level. In the former, when a transection and reanastomosis of the gastric wall is performed at the antral-fundic junction (17), an antral ulcer develops after a delay of several weeks. This lesion is restricted to the gastric side in which the cicatricial barrier of the NPF was induced. The release impairment at the antrum level of cytotrophic agents, e.g., CGRP, SOM, ENK, PG, surely plays a role. At the pancreas level, we showed in rats (unpublished observations) that in animals subjected to long-term (1–2 months) bilateral splanchnicectomy, the histopathological score of the acute pancreatitis lesions induced by the Pfeffer method (closed-duodenal-loop) was markedly aggravated in respect to control rats. As a result of experience obtained in different organs, one may conclude that capsaicin-sensitive nerves normally exert a tonic trophic action. This may depend upon the ability of released neuropeptides to increase local blood flow and activate a variety of cellular functions, such as phagocytosis, chemotaxis, and mitosis, which are important in determining tissue reaction and repair to injury. It has been postulated that the trophic action of sensory transmitters upon the tissues represents a particular form of efferent function. This system could operate tonically at a low level in such a way that normal sensory stimuli produce a continuous outflow of sensory transmitters whose actions maintain integrity of the tissue. When the stimuli are particularly intense (“irritation”), the reaction, carried to a very high degree, takes the form of neurogenic inflammation. The responsible O-AAR is the “pseudo-axonic.” In the two experimental models above described, the loss of a trophic tone due to the impairment of neuropeptides release (stomach and pancreas, respectively) might lead to an adverse condition. Indeed, the normal balance between “aggressive” and “protective” influence would be tilted in favor of the former.

III. PHASES CONTROLLING EPS The control of EPS may be divided, for descriptive purposes, into three phases according to the site at which the stimulus for secretion starts. It must, however, always be kept in mind that these phases are not separate, but overlap one another in the time sequence of digestion.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

A. Cephalic Phase This involves the secretion that can be elicited from the pancreas before food enters the mouth. The conditioned reflexes of sight and smell and the auditory phenomena of preparing an appetizing meal combine with the unconditioned stimulus of taste to excite efferent fibers lying in the vagus nerves to produce a small viscous secretion of pancreatic juice. The magnitude of the volume response in the dog has been estimated to be about one-tenth that produced by a meal, but the enzymatic component is about 25% (8,9,11,43–45). The main purpose of this phase of secretion is to mobilize the pancreatic enzymes, i.e., extrude them from the acinar cell into the duct lumen so that when electrolyte secretion becomes prominent in the intestinal phase, the enzymes are flushed into the duodenum to mix the partially digested food from the stomach. Involved in this phase is VIP (atropine-resistant hydrelatic secretion). Bombesin/GRP, a peptide released from the intermediate autonomic nervous structures—AANB, DANB, and celiac and/or the pancreas itself—has an ecbolic capacity that is atropine resistant. B. Gastric Phase This phase begins when food starts entering the stomach. The starting point of the reflex can be the gastric fundus (fundus-pancreatic reflex), identified by Harper and White (46,47). In order to interrupt this it is necessary to cut the vagi and splanchnic nerves. The secondary reflex zone is the stomach antrum. This is more important than the reflex arising from the fundus. Indeed, distention of an antral pouch with an acid solution with the aim of preventing gastric release induces an ecbolic EPS equivalent to 45% of the response obtained by a maximal CCK stimulation. In contrast, the hydrelatic component is much less, being 5% of that obtained with a maximal secretin stimulation. In this same animal it has been shown that the early EPS response elicited by a test meal is dependent primarily on antral reflexes and only secondarily on fundus-pancreatic reflexes. These data were obtained by analyzing the EPS results before and after selective antral vagotomy and truncal vagotomy (48–50). These responses suggest that the cephalic and the fundus-pancreatic reflexes have a relative participation in respect to those that arise from the antrum in the early EPS response that follows an ingested test meal. The foregoing results are consistent with the findings of Gayet and Guillaumie (51,52), who incised the mesentery of the lesser curvature at different levels. The effect of these incisions on the test meal–induced EPS was almost identical to that obtained with selective antral vagotomy. This indicates that the early EPS following a test meal results from a reflex that originates in antral afferent pathways triggered by gastric distention. Thus, the involvement of the AANB in the control of EPS is clear. Also interesting is the finding of the Dreiling group (37) that an Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

intragastric ethanol infusion (50 mL, 30%) results in an increase of intrapancreatic duct pressure (55%), which is abolished by cervical vagotomy. C. Intestinal Phase In this phase of EPS, the arc reflexes start in the duodenum, primarily from its proximal segment, in the region between the pylorus and the papilla of Vater. These reflex arcs were originally proposed by Popielski (8,9,11,12), a member of Pavlov’s school. Later, Gayet and Guillaumie (51,52) described the nervous connections between the duodenum and pancreas. Following a period in which these reflexes were disregarded, they were rerecognized by Thomas (53), but their physiological importance was not appreciated until the series of experiments by Thambugala and Baron (54), by our group (10–14,16,19), and those carried out by Grossman’s group (55–60). Recently it has been reported that activation of receptors of volume and osmolarity in the duodenum in humans excites an EPS that is blocked by atropine. Intraduodenal ethanol infusion, in contrast to an intragastric one, induces a depression of intrapancreatic duct pressure (50%), which is prevented by previous cervical vagisection (37). We have reported that the anatomical pathways of the duodeno-pancreatic reflexes are of three different types: short, intermediate, and long (1,2,8–12,16,19). In the short pathway, the impulses originating in the neural activity of the parietal plexus are transmitted directly either to the pancreon units and/or the intrapancreatic ganglia. The activity of the latter contributes to the intrapancreatic cholinergic tone. The intermediate pathway, originating from the same source, is integrated in the celiac ganglion complex. In the long pathway, the reflexes, originating in the digestive tract, go to the hypothalamic-bulbar centers, from which they return to the pancreas level. The main anatomical pathway, afferent and efferent, is the vagal nerve. Grossman’s group (55–60) hypothesized that a positive arc reflex could be initiated paracrinically from the duodenal wall enhancing the intrapancreatic cholinergic tone. This phenomenon is exacerbated by alcohol feeding.

IV. NEUROENDOCRINE BASIS OF THE FAIL-SAFE OR BRAKE SYSTEM CONTROLLING EPS The peptidergic component of the ANS is the source of what we have called a “failsafe” or “brake” mechanism, which normally prevents the supranormal ecbolic stimulation of the pancreon’s acinar segment with its deleterious consequences (30–36,61–78) (Fig. 2). Pancreatic polypeptide (PP) is the main agent, with SOM, enkephalins, and perhaps other regulatory peptides as secondary participants. PP, Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 The main features that characterize the involvement of pancreatic polypeptide (PP) in the physiology of the pancreatic gland.

a 36-amino-acid polypeptide structurally related to peptides that include peptide YY (PYY) and neuropeptide Y (NPY), is released from the PP cells. These are normal constituents of the Langerhans islets, precisely of those derived embryologically from the ventral pancreatic bud. PP secretion is stimulated by “chew” and “spit” sham feeding. The response is abolished by vagotomy and atropine. Hypoglycemia, whether induced by insulin or tolbutamide, strongly stimulates PP secretion. But the most powerful stimulus for the release of this regulatory peptide is acetylcholine (Ach). Peptides such as CCK, bombesin, and neurotensin may activate receptors on nerve cells and/or fibers and stimulate PP cells (79–88). Distention in the stomach and duodenum in humans is an efficacious stimulus to PP release. A chemical stimulation is also operative. This has been shown by the presence of food in the gut and by infusion of bile and pancreatic proteases into the duodenal lumen. The latter constitutes the basis of an entero-pancreaticinhibitory reflex, in which, according to Owyang et al. (85), PP plays a pivotal role (Figs. 2–5). These authors also stated that bursts of pancreatic and biliary secretion into the duodenum during late phase 2 and early phase 3 of the intestinal migrating complex and the associated increase in plasma PP are causally related. This contention is supported by their finding that perfusion of the duodenum with bile-pancreatic juice stimulates the release of PP into the circulation. This regulatory peptide might be responsible for the marked suppression of pancreatic and bile acid output during phase 4 of the intestinal migrating motor complex. Owyang et al. (85) concluded that an undefined factor in bile-pancreatic secretion, unrelated to pH, osmolarity, or protein, is responsible for PP release. According to Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3 Participation of PP in the short neuroendocrine reflex (Sh-NER), the basis of the negative duodeno-pancreatic reflex or entero-pancreatic inhibitory reflex. The main result is a decrease in intrapancreatic cholinergic tone.

Figure 4 The involvement of PP in the long neuroendocrine pancreatic reflex (L-NER). This induces a decrease in intrapancreatic cholinergic tone blocking the cholinergic nerve impulses from the dorsal vagal nuclei in the brainsteam.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 5 Consequences of the participation of PP—by defect or excess—in some acute pancreatitis variants.

their view, a cholinergic reflex is involved. Our group (89) also postulated, in rats, that the inhibition of bile-pancreatic secretion induced by its infusion into the duodenum was the result, at least partially, of an inhibitory duodeno-pancreatic reflex. It has been demonstrated that PP exerts its action through neural elements— that it acts, essentially, on postganglionic cholinergic neurons. The site of inhibition of the cholinergic transmission is presynaptic, between neuron and acinar cells. These effects of PP are, in fact, the first demonstration of a hormone suppressing pancreon enzyme secretion by interfering with intrapancreatic cholinergic transmission. The latter effects are duplicated by pancreastatin, methionineenkephalin, and SOM (87) (Fig. 3). Adler et al. (79) stressed that somatostatin-28, like PP, participates in the braking action on the physiological interplay that modulates the EPS process. It should be stressed that SOM released from the islet’s mantle D-cells, as PP is secreted by the PP cells in the Langerhans islets’ periphery, helps to modulate the pancreon’s secretory process through influences exerted primarily upon the periinsular pancreocytes.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

It should be emphasized that during an episode of acute pancreatitis, the pancreatic gland acts to prevent overstimulation of the acinar cells. This is supported by a progressive increment in the PP plasma values during the first week of an evolving, acute inflammatory episode (88). The foregoing indicates that the main features of the short neuroendocrine reflex (Sh-NER) are in fact equivalent to those that characterized our negative duodeno-pancreatic reflex (89) and to the one postulated by Owyang et al. (85) as the entero-pancreatic inhibitory reflex (Fig. 3). It remains to analyze the “long neuroendocrine reflex” (L-NER), which consists of two distinct hemiloops. The first runs from the vagal nuclear complex in the brainstem up to the PP cells, which are located in the periphery of the Langerhans islets and scattered in the exocrine pancreas. The hemiloop that completes the feedback circle pivots around the PP flow in the bloodstream from the pancreatic gland up to the vagal nuclear complex. The nerve impulses that flow through the former hemiloop run, initially, in the gastric branches of both vagal trunks. Subsequently, after traversing the pylorus, the neural vagal impulses follow the nerve fibers that inhabit, in rich density, the duodeno-pancreatic cleft. This duodeno-pancreatic linking takes place primarily in the region between the pylorus and the PV-D. That this is precisely the pathway followed by the first hemiloop of the L-NER is confirmed by the experimental observation, in dogs, that duodeno-pancreatic disconnection, or duodenectomy, diminishes, on the one hand, hypoglycemia-induced PP release and, on the other, hormonal PP release in response to a meal. As for the second hemiloop of the L-NER, the one centered on the bloodstream PP flow from the pancreatic gland up to the vagal nuclear complex in the brainstem, the group of Taylor (90) has given evidence that this regulatory peptide does indeed come into contact with the vagal nuclear complex. The latter is achieved at the level of the area postrema and the tractus solitarius nucleus, two regions of the CNS that lack a blood barrier. Once in the vagal nuclear complex, PP binds to specific receptors and inhibits the neuron discharge of the dorsal motor nucleus. The consequence is a depression of EPS due to a decrease in intrapancreatic cholinergic tone, which normally interacts with the hormonal system upon the pancreon secretory process. From all the preceding facts, it is evident that the long as well as the short NER are important components of a fail-safe or brake mechanism, which under physiological circumstances prevents the deleterious consequences for the acinar pancreocytes of abrupt episodes of supranormal ecbolic stimulation. When the above-depicted fail-safe or brake system is overpowered, as for example following a large meal rich in proteins, fats, and alcohol, an episode of acute pancreatitis can be evoked. In the 1960s we characterized this clinical entity as an acute pancreatitis due to an overreaction of the trigger zone (PV-D) of the pancreatic gland. Concerning the interactions between ethanol intoxication

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

and the above-depicted NER, a first physiopathogenic feature to emphasize is that of the probable involvement of L-NER in the mechanism of EPS depression induced by an acute intravenous (i.v.) ethanol injection. This phenomenon was unexpectedly observed by our group in a long series of tests performed in humans, dogs, and rats (32,61–78). As this ethanol-elicited inhibition of secretin or secretin  CCK was prevented by previous vagotomy or atropinization, we hypothesized that ethanol was essentially an activator of the Pavlov fibers (vagal inhibitory). A remarkable subsequent observation was that chronic ethanol intoxication induced a reversal of the EPS inhibitory response evoked during the nonalcoholic stage by i.v. ethanol (32). This finding was interpreted by us as the consequence of an alcohol-elicited impairment of the normal physiological brake system in the control of EPS by the pancreon units (61–78). The presumption was that an ethanol-induced vagal neuropathy was responsible for this change (91), preferentially impinging upon the Pavlov inhibitory fibers, which control the release of PP and perhaps other members of the pancreon fail-safe or brake mechanisms, such as -endorphin, SP, SOM, or pancreastatin (87,92–100). As to the postulation of an alcohol-evoked impairment of the Pavlov vagal inhibitory fibers, the vagal neuropathy described in humans by Duncan et al. (91) supports this contention. Moreover, secretory studies in dogs by Schmidt et al. (92) provide further confirmation. Another relevant finding is reported by Witt et al. (99) in diabetic patients. In those with signs of autonomic neuropathy, there is an impairment of PP release following insulin-induced hypoglycemia. Another supportive report is that of Hajnal et al. (93) of human chronic alcoholics. They found a lack of PP response following an acute ethanol or wine administration along with a test meal. We concluded that a loss of involvement of the Pavlov vagal inhibitory fibers induces, through the mechanism of neural decentralization, an enhanced reactivity of both the AANB and DANB. This is supported by the findings of Brugge et al. (94). These authors observed in humans that 1) basal duodenal trypsin output and interdigestive duodenal contraction rate are higher in chronic alcoholics; 2) chronic alcoholics have an increased postprandial trypsin secretion compared to nonalcoholics; 3) alcohol-fed and nonalcoholics show similar postprandial increments in plasma levels of gastrin and CCK; 4) when compared to nonalcoholics, alcohol-fed patients display a remarkable lack of PP increments in respect to a test meal or test solutions, e.g., glucose, ethanol, wine. The alcohol-induced impairment of the pancreon brake system might also explain the Yamasaki et al. (101) findings in monkeys characterized by papillary dysfunction and exocrine pancreas hypersecretion. A similar sequence of events develops in uremia. Indeed, Owyang et al. (102) have shown in patients with chronic renal insufficiency that both duodenal mannitol and i.v. CCK evoke an

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ecbolic hyperresponse of the exocrine pancreas. A similar finding has been reported in rats in which the renal mass had been 85% surgically reduced (103). Another observation to be stressed is that the same decrease in PP secretion found in chronic alcoholic patients has been shown in patients with cystic fibrosis (95) and in dogs with pancreatic fibrosis following ductal ligation (96). A feature to be emphasized in all the above-mentioned entities is the association of a depressed PP release with an augmented number of PP cells in both the islets and the pancreatic parenchyma. The foregoing prompts the speculation whether in the chronically inflamed gland the PP cells produce less hormone than normally or, in contrast, release less hormone in response to different stimuli. A functional feature to be stressed is that in chronic alcoholics NPF suffers the loss of the modulatory influences exerted normally by the CNS autonomic centers, primarily upon the AANB and DANB. The enhanced reactivity of these two intermediate autonomic nervous systems leads to an increased intrapancreatic cholinergic tone. This change is associated with an elevated acinar sensitivity to CCK and results in exocrine pancreas hypersecretion. From a large group of tests in both animals and humans (31–35, 61–75,77,78,91–94,101,104), we have concluded that alcohol feeding induces changes at two poles: on pancreon units (hypersecretion) and on Oddi’s sphincter (hypertension). Both conditions are the source of the patient’s symptomatology and the pathophysiology of the pancreatic gland.

V. ADRENERGIC INNERVATION AND INSULO-PANCREON AXIS INTERACTION Several studies have shown that -blockade inhibits PP release while, in contrast, -blockade enhances PP secretion. The raised PP plasma values observed after celiac ganglionectomy might be related to the interruption of -adrenergic influences. According to Larson et al. (97), celiacectomy may be considered a sort of -adrenergic blockade. In relation to the previously mentioned innervation features, it should be pointed out that celiac ganglionectomy does indeed affect the nerve terminals and the catecholamine content of the pancreas. Thus, norepinephrine drops 90%, while epinephrine and dopamine fall 50 and 70%, respectively. Blocking of the celiac ganglia by local anesthetic might trigger a rise in PP values. If this is confirmed, it would support use of this procedure in cases of acute pancreatitis. In this clinical setting, celiac ganglion anesthesia would constitute not only a therapeutic measure to relieve pain but also an efficacious method to reduce intrapancreatic cholinergic tone.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

VI. HYPOGLYCEMIA-REACTIVE EPISODES RELATED AND UNRELATED TO ALCOHOLISM Concerning the interrelationship between alcohol feeding and the insulo-pancreon axis, Patto et al. (104) found that chronic alcoholic patients reveal a significant depression of insulin release following an oral i.v. glucose tolerance test. They favor the hypothesis that this is triggered by a -cell dysfunction. They presume a direct toxic effect of ethanol but also a probable pathogenic role of an enhanced intrapancreatic cholinergic tone evoked by alcohol feeding. The latter would favor the -cell exhaustion. A phenomenon observed in acute alcoholism is that of reactive hypoglycemia episodes. Hypoglycemia-reactive episodes unrelated to fasting may be categorized as either early (2–3 h) or delayed (3–5) following an ingested meal. The early hypoglycemia reactive episode may be the consequence of a previous subtotal gastrectomy or, on the other hand, may be purely functional. The former is the result of rapid gastric emptying. This elicits an overstimulation of vagal receptors, subsequent hyperinsulinism, followed by hypoglycemia. The clinical manifestations are the result of the counterresponse of the adrenergic system. The hypoglycemia functional type pivots around the ingestion of carbohydrates characterized by rapid gastric emptying. Ethanol ingestion associated with sweetened beverages may evoke acute reactive hypoglycemia episodes of either the early or the delayed type. Its physiopathogenesis is centered on the potentiation by ethanol of insulin release induced by oral glucose ingestion. The delayed hypoglycemia reactive episode is generally evident during the first stage of a diabetic syndrome. Its physiopathogenesis is related to a delayed insulin release by the  cell. The initial hyperglycemia is subsequently followed by an exaggerated insulin secretion, in general 4–5 hours after the carbohydrate ingestion. This sequence is common in obese patients with a diabetic family background.

VII.

OVERREACTIVE AUTONOMIC ARC REFLEXES AND BILIARY ACUTE PANCREATITIS

In respect to the afferent component of pancreas innervation, its nerve fibers are immersed in the vagal, the splanchnic-celiac, and the entero-pancreatic complex innervation systems. These duodeno-pancreatic afferent nerve fibers, when activated, are the basis of the already described short, intermediate, and long duodeno-pancreatic reflexes. These play, on the one hand, an important role in the gastric and intestinal phases of EPS control. On the other, tonic influences exerted through the activity of the thin, capsaicin-sensitive nerve fibers contribute to cytoprotective effects on the gastrointestinal mucosa and the pancreatic gland (105–107).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The concept of overreactive autonomic arc reflexes (O-AAR) arose from our observations in canines equipped with duodenal Thomas cannulae and studied in a conscious state. Indeed, when intemperate maneuvers are employed, especially if either liquid or air is injected abruptly into the main pancreatic duct (distention), a sudden arrest of EPS is observed. This reflex can be elicited from other segments of the digestive tract, e.g., from the ileum and colon (108–110). This neural-centered EPS inhibition should be distinguished from that elicited by fats (oleic acid) instilled into the colon (pancreotone or PYY) (111). This type of O-AAR is equivalent to that described by Owyang and co-workers (85,112–116) as inhibitory-entero-pancreatic. According to Ohshio et al. (117), in rats a short-term pancreatic duct obstruction interferes with the acinar cell secretory process downstream of hormone receptor binding, intracellular Ca2+, release and protein-kinase activation. Our thinking is that this secretory-inhibitory reflex is related to the one we have already described as NER (Fig. 3) and linked to the intrapancreatic release of PP. The latter, as is now well known, acts, on the one hand, inhibiting directly the acinar cell secretory process, and, on the other, depressing Ach release from the postganglionic cholinergic fibers (decrease in intrapancreatic cholinergic tone). During the clinical and surgical circumstances that normally evoke an episode of BAP, e.g., a stone that migrates into the duodenum or is impacted in the distal end of the common bile duct, endoscopic maneuvering (sphincterotomy, sphinctero-manometry, retrograde cholangic-pancreatography), surgical manipulation in or close to the duodeno-pancreas, percutaneous liver-associated hemobilia, a second type of O-AAR, arising from the irritation of receptors in the PVD, elicit a type of arc reflex that we have described as sympatho-ischemic. The afferent limb arising in the duodeno-pancreas becomes integrated in the celiac ganglia complex. The efferent limb induces in the pancreatic gland the opening of arterio-venous shunts and, consequently, capillary ischemia and the depression of the pancreon’s secretory process. The third type of O-AAR is the pseudo-axonic. This leads to what is known as neurogenic inflammation (6,118–121). This reflex is primarily initiated by the irritation of the thin, unmyelinated, capsaicin-sensitive type C fibers. In their course to the nodose ganglion of the vagus nerves or to the dorsal root ganglia of the splanchnic-celiac innervation complex, they send collaterals, and in the pancreatic gland endings, through the antidromic release of a series of regulatory peptides, e.g., SP, CGRP, VIP, SOM, DYN, and neurokinin-A, they induce an acute inflammation process: vasodilatation extravasation of plasma proteins, etc. A fact to take into account is that SP and other sensory peptides are capable of activating mast cells to secrete histamine and other factors. Furthermore, leukocytes, particularly neutrophils, granulocytes, monocytes, and lymphocytes, are stimulated to adhere to the vascular endothelium and to migrate to the surrounding tissue. Once neurogenic inflammation is triggered, monocytes release prostaglandins, thromboxane, and cytokines. Some of the latter agents are va-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

soactive by themselves and, in addition, can activate afferent nerve endings and thereby provide a positive feedback loop, which reinforces the mutual stimulation of the afferent component of the ANS. Another interesting physiopathogenic feature is that the simultaneous sympathetic hypertonus significantly enhances the sensitivity of the afferent component and potentiates the acute pancreatic inflammation. In view of the above facts from the literature and from our clinical experience, we are convinced that BAP, which undoubtedly pivots around O-AAR, is another example that can be encompassed within that entity conceived by Reilly (122,123) many decades ago and that is essentially centered on a neurovegetativeirritation mechanism.

VIII. AUTONOMIC NERVOUS SYSTEM AND PANCREATIC REGENERATION CAPACITY That the ANS plays an important role in the regenerative capacity of the pancreatic gland is shown in a series of observations of our group. Indeed, it was shown in rats (124) that truncal vagotomy unexpectedly induced a marked increased (~100%) of the wet weight of the pancreatic gland, primarily of its body-tail segment. This trophic change is prevented by a simultaneous bilateral splanchnicectomy. As a consequence, a “great trophic circuit” was conceived (125). Interruption of its afferent limb (vagotomy) might trigger the release of trophic agents from the CNS. These would reach the pancreatic gland passing through the brainstem, spinal cord, and splanchnic nerves. In another series of tests it was found (125) that cicatricial interruption of the NPF (transection and reanastomosis) at different levels, primarily at the PV-D segment, induced an increase of the wet weight of the pancreas, mainly at the head segment. Suggestively, this change is abolished by a simultaneous antrectomy. Thus, a “short trophic circuit” was considered between the DANB, the AANB, and the pancreas. The loss, by the cicatricial barrier, of restraining influences on the AANB coming from the lower segments of the intestinal tract might be the basis of the trophic changes. Recently we have also speculated on the probable involvement of PP, supported by the fact that PP exerts a local trophic effect (83). This conviction has arisen from observation at an autopsy level. Indeed, in insulin-dependent diabetics it was remarkable that the pancreas was markedly decreased but at the expense of an almost complete atrophy of the lobe poor in PP cells. These findings are in keeping with those of Greenberg et al. (126) in rats in the sense that PP increases DNA synthesis in pancreatic acinar cells. Our experimental studies in dogs (127–129) have shown that the pancreas has a remarkable ability to regenerate its parenchyma and to recover its exocrine secretory capacity and that restoration of duct to intestinal continuity or relief of

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ductual obstruction is important to trigger this capacity. Regeneration appears to derive from the ductular system and progresses to restoration of acinar tissue and islets. The foregoing has been confirmed by our clinical experience. Indeed, in several alcoholic chronic pancreatitis patients subjected to a Puestow operation (ductal jejunum anastomosis), we were able to confirm, by morphological and secretory studies, a recovery of both the exocrine and endocrine components of the pancreatic gland. A remarkable certification of the latter was seen recently in a 57year-old woman who had been subjected to a left subtotal pancreatectomy to remove a mucinous cystoadenoma. Surprisingly, 4 months later, CT scan proved the complete regeneration of the body and tail of the gland as well as a normalization of its diabetic syndrome (130). That the latter is feasible is shown by our recent findings of remarkable trophic effects on the remaining pancreatic segment following a Foglia operation (95% pancreatectomy) in rats. This was associated with increased plasma insulin levels and a reduction in hyperglycemia (131). The possibility of a sympatho-vagal reflex linked to the duodeno-pancreas is shown by findings reported by Sarles and colleagues (132–134). They showed significant biliary hypertension in dogs subjected previously (3 months) to chronic irritation of the right splanchnic nerve with pumice powder. This phenomenon was triggered by a chronic inflammatory lesion at the level of Oddi’s sphincter. These changes can be prevented by a simultaneous bilateral truncal vagotomy or by an isolated interruption of the left or anterior vagus nerve.

IX. PROBABLE INTERACTIONS IN THE MODULATION OF PANCREON REACTIVITY BETWEEN CHOLINERGIC AND STEROID INTRAPANCREATIC TONE Physiological secretory studies performed by our group in dogs equipped with Thomas cannula (135) confirmed previous reports that hypophysectomy results in pancreatic atrophy and suppression of pancreatic enzyme secretion. In addition, our data indicate a simultaneous depression of secretion of fluid and electrolytes by the pancreon’s centroacinar ductal segment. The prompt reversion to normal of all secretory parameters in hypophysectomized dogs off ACTH following the administration of a variety of adrenal steroids (cortisone, decadron) and the prompt return to depressed levels following withdrawal of these steroids lends strong support to the hypothesis that the effect of hypophysectomy on pancreatic secretion is adrenally mediated. This hypothesis is supported by the data offered by the tests performed in bilaterally adrenalectomized dogs (136). It is our conviction that when considering the reactivity of both the exocrine and endocrine pancreas, one has to admit that this is the result of a complex interaction between hormones (gastrointestinal and steroids) and the ANS (intrapan-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

creatic cholinergic tone). To complete the above picture one should also take into consideration that recent studies in rats (137) and menopausal women (138,139) indicate that bile-pancreatic secretion in the rat and the secretin-evoked EPS in humans are responsive to interactions triggered by the activation of estrogen receptors in the pancreocytic cytosol. It is our conviction that, normally, the pancreocyte estrogen receptors interact with those of the secretin (cell membrane) and an ANS background (intrapancreatic cholinergic tone). The aforementioned interplay explains that the decline of plasma estradiol in menopausal women is associated with a decrease in the pancreon’s hydrelatic response in the secretin test. This is confirmed when the results are compared with those from young women or from men both older and younger than 45 years (140).

X. INTEGRATIVE SYNTHESIS OF NEUROENDOCRINE IMMUNE INTERACTIONS WITH EPICENTER IN THE PANCREATIC GLAND When adopting a global encompassing view of mechanisms that modulate EPS or Langerhans islet response, one should consider that acting from the upper gastrointestinal tract and discharging its influences through the vagal innervation complex prevents an exaggerated ecbolic stimulation of the pancreon units. Its main physiological foundation lies, as it has been already discussed, in the failsafe or brake system (Pavlov vagal inhibitory fibers) and the short and long NER (Figs. 2–5). A counterpart of the above is the neuroendocrine physiological loop, which, having as a starting point the lower intestinal tract and a discharge path through the splanchnic-celiac and the collateral secretion of glucagon, helps inhibit EPS once the digestive process has been completed (141). A gut neuroendocrine pancreatic loop, besides the classical entero-insular axis associated with insulin secretion through the intermediation of GIP, is that which links by an arc reflex, after carbohydrate ingestion, the duodenum with the lower intestinal tract. This elicits the release of another peptide, GLIP, which seems to be the real trigger for the secretion of insulin by the  cells of the Langerhans islets. Our finding in rats (20) of depressed insulin blood levels, both basally and post–glucose tolerance test, following a neural duodeno-pancreatic disconnection supports the concept that an autonomic nervous link plays a role in the physiology of the entero-insular axis. Another neuro-hormonal loop on the EPS modulation, but this one in close interaction with the immune system, enters into the scene during an acute pancreatitis episode, primarily in its biliary type variant (BAP). Indeed, in this illustrative example the concerted effects of different O-AAR concentrate primarily on the pancreon’s acinar segment. The prevalent insulting agents are the free oxygen

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

radicals, especially during the reperfusion phase that follows the initial ischemic period, and the regulatory peptides released antidromically from the capsaicinsensitive thin-afferent nerve fibers. Among the latter, primarily SP exerts changes in the pancreatic tissue both directly and indirectly through the degranulation of the mast cells. Following the foregoing insult, the acinar pancreocytes immediately reorganize their genetic program. As a result they start to send signals to the gland’s immune system through the mediation of chymokines (TNF-), PAF (platelet-activating factor), and PAP (protein-associated protein). The synthesis of the above factors is made possible by previous acinar cells ecbolic synthesis arrest. Simultaneously, there is an enhancement of the apoptotic process, which diminishes the noxious consequences associated with enzymatic cell necrosis. A feature to be stressed in the above reactions is that the extension and intensity of the acute inflammatory response is genetically determined (4,6). A crucial notion is that once the above-described pancreonal immune axis is activated, another one follows. The latter, widely known as the Selye stress alarm reaction, implies the involvement of the hypothalamic-pituitary-adrenal axis. Its participation allows the completion of a crucial feedback loop (142). The main CNS nucleus involved in the regulation of the HPAA is the paraventricular nucleus (PVN) of the hypothalamus. This is the principal source of the 41-amino-acid peptide corticotropin-releasing factor (CRF), which is the major regulator of the pituitary ACTH release hormone that induces the secretion of glucocorticoids. Activation of the HPAA is not restricted to cytokines produced by myeloid (e.g., monocyte/macrophage) cells; those originating in the lymphoid system (T lymphocytes) also participate. Similarly, TNF- synergistically enhances ACTH release by IL-1. Although the majority of cytokines exert a stimulatory action in the HPAA, other cytokines (IL-4, IFN-) inhibit HPAA. Cytokines, elaborated quickly (in minutes) in response to a stimulus, play a physiological role in the secretory activity of the HPAA. They also produce a systemic acute-phase response: fever, increased heart rate, and activation of the sympathetic nervous system. Levels of cytokines in resting, healthy, unstressed animals or humans are low, but they increase dramatically during injury, infectious disease, or tissue stress. It should be taken into account that the vagus is a neural afferent route by which inflammation in the abdominal cavity can influence the brain. The pro-inflammatory cytokines (e.g., TNF- and IL-1) stimulate the production of other cytokines, including IL-6, IL-8, IL-9. In contrast, anti-inflammatory cytokines (e.g., IL-4, IL-10, and IL-13) abrogate the production of pro-inflammatory cytokines. Peripheral cytokines stimulate ACTH release from the anterior pituitary. Hypothalamic arginine-vasopressin can act as costimulator with corticotropin-releasing hormone of ACTH release. Pituitary ACTH stimulates the

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

adrenal glands to release glucocorticoids, which suppress inflammation, completing the counterregulatory feedback loop (143). Corticosteroids elevate C1-esterase inhibitor levels, which suppresses trypsin activation. Lipomodulin, induced by corticosteroids, inhibits phospholipase A2. As a consequence, the synthesis of prostaglandins and leukotrienes is inhibited. Steroids also have a protective and stabilizing effect on lysosomal membranes and inhibit complement-induced aggregation of granulocytes and of plateletes. Corticosteroids enhance tissue and cellular protection through heat shock protein 72 (hsp-72). This agent stabilizes cytoskeleton proteins under conditions of thermal- and oxidation-induced stress. A notion to have in mind is that at the duodeno-pancreas a bidirectional communication occurs between the immune and the neuroendocrine system. That is why the triggering of severe acute pancreatitis is enhanced when the HPAA is somewhat blunted. Similarly, a deleterious consequence may occur if the degree of immunocyte overreaction to tissue injury makes the HPAA unable to attenuate the pancreatic inflammation response. A fundamental and practical concept is that anesthetizing the PV-D, the main starting source of the O-AAR, may inhibit the inflammatory cascade described above (4). Impairment of the normal interrelationships of peptide-hormones and the ANS in the feedback mechanisms that control EPS explains the induction of pain in chronic pancreatitis patients. Indeed, malabsorption resulting from defective bicarbonate secretion by the pancreatic gland leads to reduced intraduodenal bile acid. The consequence is an increase in plasma CCK levels resulting from the loss of the normal regulation exerted by bile acids on CCK endocrine cells. This peptide, through a paracrine-neural mechanism, is the triggering agent of the pain sensation (144,145). Another interesting indirect proof that pancreon units are normally subjected to tonic neuroendocrine restraining influences (pancreotone, PYY, adrenergic tone) (111,112) is offered by findings of a significant enhancement of both the trophism of the pancreatic gland and of the EPS ecbolic component (112,141) following a right hemicolectomy. REFERENCES 1.

Tiscornia OM. Concepto de Pancreón. In: Perez V, De Larrechea I, Arabehety J, Tiscornia OM, eds. Gastroenterología. El Ateneo, 1971:470–484. 2. Dreiling D, Tiscornia OM. Tests of pancreatic function. In: Sircus W, ed. Scientific Foundation of Gastroenterology. London: W. Heinemann Medical Book, 1980:591–601. 3. Carpenter M, Sutin J. The autonomic nervous system. In: Human Neuroanatomy. London: Williams & Wilkins, 1983:209–231.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

4. Tiscornia OM, Hamamura S, Lehmann ES, Otero G, Waisman H, TiscorniaWasserman P, Bank S. Biliary acute pancreatitis: a review. World J Gastroenterol 2000; 6:157–168. 5. Wood JD, Alpers DH, Andrews PLR. Fundamentals of neurogastroenterology. Gut 1999; 45(suppl II):II6–II16. 6. Tiscornia OM, Hamamura S, Lehmann ES de, González E, Vaccaro MI, Otero G, Cerini C, Waisman H. La inervación autonómica gastro-entero-bilio-pancreática. El concepto de “pista” pleuxal entérica. Pren Méd Argent 1999; 86:129–139. 7. Tiscornia OM, Martínez JL, Sarles H. Some aspects of human and canine macroscopic pancreatic innervation. Am J Gastroenterol 1976; 66:353–361. 8. Tiscornia OM. Controle nerveux cholinergique du pancréas. Biol Gastroenterol (Paris) 1976; 9:255–270. 9. Tiscornia OM. The neural control of exocrine and endocrine pancreas. Am J Gastroenterol 1977; 67:541–560. 10. Tiscornia OM. Importancia de la región Vateriana en la patología bilio-pancreática. Puesta en evidencia de reflejos duodeno-pancreáticos. Rev Argent Cirug 1979; 36:232–239. 11. Tiscornia OM, Dreiling D, Yacomoti J, Kurtzbart R, De La Torre A, Farache S. Neural control of the exocrine pancreas. An analysis of the cholinergic, adrenergic and peptidergic pathway and their positive and negative components. I. Neural mechanisms. Mt Sinai J Med 1986; 54:366–383. 12. Tiscornia OM, Dreiling D, Yacomoti J, Kurtzbart R, De La Torre A, Farache S. Neural control of the exocrine pancreas. II. Integration of neural and hormonal mechanisms. Mt Sinai J Med 1988; 55:126–131. 13. Tiscornia OM, Cresta MA, Negri G, Lehmann ES de, Vaccaro MI, Resnik R, Celener D, Hamamura S, Mora MI, Bustos Fernández LM. Sistema nervioso autónomo y páncreas exocrino-endocrino en la rata. Acta Gastroenterol Latino Am 1991; 21:204. 14. Tiscornia OM, Cresta MA, Celener D, Hamamura S, De Paula J, Celener P, Farache S, Negri G. Centro neural peri-Vateriano en la rata. Evidencias indirectas brindadas por la exclusión Vateriana, la anestesia papilar, la solarectomía y la vaguectomía troncular bilateral distal. Acta Gastroenterol Latino-Am 1991; 21:204. 15. Tiscornia OM, Hamamura S, Celener D, Cresta MA, Negri G, González E, Lehmann ES de, Tiscornia-Wasserman PG. An overview of gastro-duodeno-pancreas innervation in the rat. Emphasis on some disregarded anatomical structures (abstr). Am J Gastroenterol 1993; 88:1544. 16. Tiscornia OM, Hamamura S, Cresta MA, Lehmann ES de, Celener D, Negri G, González E, Tiscornia-Wasserman PG. Duodenal peri-Vaterian autonomic nervous center in the rat. Indirect evidence given by Vaterian papillary anesthesia, Vaterian exclusion, supra and infra-Vaterian transection and reanastomosis, celiac ganglionectomy and distal bilateral truncal vagotomy. Am J Gastroenterol 1993; 38:1565. 17. Tiscornia OM, De Paula J, Celener D, Schiffrin E, Nakasato O, Crista MA, Waisman H, Bustos Fernández L, Dreiling D. A laboratory preparation mimicking human peptic ulceration. (A chronic antral peptic ulcer. Postsection and reanastomosis at the antral fundic junction in the rat.) Mt Sinai J Med 1988; 553:357–361.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

18. Tiscornia OM, Tiscornia-Wasserman PG, Hamamura S, Cresta M, Negri G, Lehmann ES de, De Paula J, Yacomotti J, Farache S. Síntesis conceptual de la inervación macroscópica gastro-duodeno pancreática. Revisión centrada en una investigación anatómica en la rata. Rev Hosp Clín Bs As 1992; 6:28–29. 19. Tiscornia OM, Sarles H, Voirol M. Evidences for duodeno-pancreatic reflexes and an anti-CCK factor with lidocaine infused intravenously and sprayed topically in pancreatic papilla in non-alcoholic and alcohol-fed dogs. Am J Gastroenterol 1976; 66:221–240. 20. Tiscornia OM, Lehmann ES de, Hamamura S, Otero G, Tiscornia-Wasserman PG. Neural duodeno-pancreatic disconnection depresses blood insulin level, both basally and following an oral glucose tolerance test. Am J Gastroenterol 1988; 93:1662. 21. Tiscornia OM, García H, Affanni JM, Otero G, Tiscornia-Wasserman PG. Blood changes in acute pancreatitis induced by balloon distention of the peri-Vaterian duodenum in the opossum and the effects of previous truncal vagotomy and bilateral splanchnicectomy. Am J Gastroenterol 1999; 94:2638(A). 22. García H, Tiscornia OM, Iodice O. Algunas consideraciones sobre la utilización del opossum sudamericano (zarigüeya), como modelo experimental en la pancreatitis aguda (Segunda Parte). Pren Méd Argent 1998; 85:917–922. 23. Anglade P, Michel C, Rozé C. Intrinsic nerves of the pancreas after celiac and superior mesenteric ganglionectomy in rats. A morphologic study of acetylcholinesterase activity and catecholamine histofluorescence. Pancreas 1977; 2:568–577. 24. Kirchgessner AL, Gershon MD. Innervation of the pancreas by neurons in the gut. J Neurosci 1900; 10:1626–1642. 25. Kirchgessner AL, Mawe GM, Gershon MD. Evaluation of the activity of chemically identified enteric neurons through the histochemical demonstration of cytochrome oxidase. J Comp Neurol 1990; 10:1–14. 26. Kirchgessner AL, Garshon MD. Presynaptic inhibition by serotonin of nerve mediated secretion of pancreatic amylase. Am J Physiol 1995; 268:G339–G345. 27. Tiscornia OM, Lehmann ES de, Hamamura S, Otero G, Waisman H, TiscorniaWasserman PG. “Short-term,” “closed duodenal-loop” in the rat. A suitable model to elicit autonomic arc reflexes and mimick human biliary acute pancreatitis. Beneficial effects of previous intraduodenal lidocaine instillation. Am J Gastroenterol 1999; 94:2638(A). 28. De Giorgio R, Sternini C, Brecha NC, Widdison AL, Karangia ND, Eber H, Go VLM. Patterns of innervation of vasoactive intestinal polypeptide, neuropeptide Y and gastrin-releasing peptide immunoreactive nerves in the feline pancreas. Pancreas 1992; 7:376–384. 29. Höfelt T, Johansson O, Ljungdahl A, Lundberg JM, Schultzberg M. Peptidergic neurons. Nature 1980; 284:515–520. 30. Tiscornia OM, Gullo L, Sarles H. The inhibition of canine exocrine pancreatic secretion by intravenous ethanol. Digestion 1973; 9:231–240. 31. Tiscornia OM, Hage H, Palasciano G, Brasca A, Devaux MA, Sarles H. The effects of pentolinium and vagotomy on the inhibition of canine exocrine pancreatic secretion by intravenous ethanol. Biomedicine 1973; 18:159–163.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

32.

33. 34. 35.

36. 37.

38. 39. 40. 41.

42.

43. 44. 45. 46. 47. 48. 49. 50.

Tiscornia OM, Palasciano G, Dzieniszewski J, Sarles H. Simultaneous changes in pancreatic and gastric secretion induced by acute intravenous ethanol infusion. Am J Gastroenterol 1975; 63:389–395. Sarles H, Midejean A. Electromyographic study of the action of alcohol upon the Oddi’s sphincter. Digestion 1973; 9:93–94. Capitaine Y, Sarles H. Action d l’ethanol sur le tonus du sphincter d’ Oddi chez l’homme. Biol Gastroenterol (Paris) 1971; 3:231–236. Sarles JC, Midejean N, Devaux MA, Castagnini A. Electromyographic du sphincter d’ Oddi chez le lapin: action de la vagotomie, de l’ hexamethonium et de l’ alcohol. Biol Gastroenterol (Paris) 1976; 9:25–32. Sarles JC, Sarles H, Devaux MA. Experimental odditis and cholelithiasis in the dog: role of autonomic nervous system. Am J Gastroenterol 1975; 63:147–154. Chen-Hsi Su, Koller M, Toth L, Dreiling D. Enteropancreatic reflexes: the effect of duodenal distention on pancreatic exocrine secretion. Mt Sinai J Med 1986; 53:332–336. Richins CA. Effect of sympathetic stimulation on blood flow and secretion in the pancreas of the cat. Am J Physiol 1953; 173:467–470. Kuntz A, Richins CA. Effects of direct and reflex nerve stimulation on the exocrine secretory activity of pancreas. J Neurol Physiol 1949; 12:29–35. Varga B, Folly G, Papp M. L’ effet de l’excitation eléctrique du ganglion coalique sur le débit sanguin du pancréas. Lyon Chirurg 1974; 70:168–170. Papp M, Ungvari G, Nemeth PE, Munkacse I, Zubek L. The effect of bile-induced pancreatitis on the intrapancreatic vascular pattern in dogs. Scand J Gastroenterol 1969; 4:681–689. Tiscornia OM, Waisman H, Hamamura S, Lehmann ES de, García H, Otero G, Tiscornia-Wasserman PG, Bank S. Pancreatitis aguda biliar: Revisión fisiopatogénica. Rol de los reflejos autonómicos. Propuesta preventivo-terapéutica de la anestesia del duodeno peri-Vateriano (“Gatillo” Pancreonal). Rev Argent Cirug 2000; 78:65–69. Sarles H, Dani R, Prezelin G, Souville C, Figarella C. Cephalic phase of pancreatic secretion in man. Gut 1968; 9:214–221. Novis BH, Bank S, Marks IN. The cephalic phase of pancreatic secretion in man. Scand J Gastroenterol 1971; 6:417–422. Anagnostides AA, Maton PN, Chadwick VS. Cephalic phase of pancreatic secretion in man: effects of sham feeding with and without atropine. Gut 1982; 23:A880. White TT, Lundh G, Magee DF. Evidence for the existence of a gastropancreatic reflex. Am J Physiol 1960; 198:725–728. White TT, Toyoaki T, Magee DF. Alternate nervous pathways for the gastropancreatic reflex. Gastroenterology 1960; 39:615–618. Debas HT, Konturek S, Grossman MI. Effect of extragastric and truncal vagotomy on pancreatic secretion of the dog. Am J Physiol 1975; 228:1172–1177. Debas HT, Yamagishi T. Evidence for pyloropancreatic reflex for pancreatic exocrine secretion. Am J Physiol 1978; 234:E468–E471. Debas HT, Taylor IL, Seal AM, Passaro EP. Evidence for vagus-dependent pancreatic polypeptide-releasing factor in the antrum: studies with the autotransplanted dog pancreas. Surgery 1982; 92:309–314.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

51. 52. 53. 54. 55. 56. 57. 58.

59. 60. 61. 62. 63. 64.

65. 66. 67.

68.

69.

70.

Gayet R, Guillaumie M. La sécrétion provoquée par excitation directe du pancréas en certains points d’ election. C R Soc Biol 1930; 103:992–994. Gayet R, Guillaumie M. Sur le trajet des fibres excito-sécrétoires fournies au pancréas par les nerfs vagues. C R Soc Biol 1930; 103:989–994. Thomas JE. The functional innervation of the pancreas. Rev Gastroenterol 1948; 15:813–820. Thambugala KL, Baron JH. Pancreatic secretion after selective and truncal vagotomy in the dog. Br J Surg 1971; 58:839–844. Singer MV, Solomon TE, Grossman MI. Effect of atropine on secretion from intact and transplanted pancreas in dog. Am J Physiol 1980; 238:G18–G22. Singer MV, Solomon TE, Wood J, Grossman MI. Latency of pancreatic enzyme response to intraduodenal stimulants. Am J Physiol 1980; 238:G23–G29. Singer MV, Solomon TE, Rammert H. Effect of atropine on pancreatic response to HCL and secretin. Am J Physiol 1981; 240:G376–G380. Singer MV, Niebel W, Flashoff J, Grossman MI. Does basal cholinergic activity potentiate exogenous secretin for stimulation of pancreatic bicarbonate output in dog? Digestion 1982; 24:209–214. Solomon TE, Grossman MI. Cholecystokinin and secretin release are not affected by vagotomy or atropine. Gastroenterology 1977; 72:111–113. Solomon TE, Grossman MI. Vagal control of pancreatic exocrine secretion. In: Brooks F, ed. Nerves and the Gut. New York: Ch.B. Slack, 1977:119–132. Tiscornia OM, Palasciano G, Sarles H. Effects of chronic ethanol administration on canine exocrine pancreatic secretion (further studies). Digestion 1974; 11:172–182. Sarles H, Figarella C, Tiscornia OM. Alcoholic pancreatitis. Mt Sinai J Med 1975; 42:540–551. Tiscornia OM, Palasciano G, Sarles H. Atropine and exocrine pancreatic secretion in alcohol-fed dogs. Am J Gastroenterol 1975; 63:33–36. Celener D., Lechene de La Porte, Tiscornia OM, Sarles H. Histochemical study of cholinergic activity in the exocrine pancreas of dogs. Modifications related to chronic alcoholism. Biomedicine 1977; 27:161–165. Sarles H, Tiscornia OM. Chronic alcoholism and canine exocrine pancreatic secretion. A long-term follow-up study. Gastroenterology 1977; 72:238–243. Tiscornia OM. Pancreatitis crónica: etanol y desequilibrio neuro-endocrino. Medicina (Bs.As.) 1977; 37:187–190. Perec C, Celener D, Tiscornia OM, Baratti C. Effects of chronic ethanol administration on the autonomic innervation of salivary glands, pancreas and heart. Am J Gastroenterol 1979; 7:46–59. Baratti C, Rubio MC, Perec C, Tiscornia OM. Effect of chronic alcohol feeding on adrenergic and cholinergic neurotransmission mechanism. Am J Gastroenterol 1980; 73:21–27. Tiscornia OM, Celener D, Perec C, Lehmann ES, Cresta MA, Dreiling D. Physiopathogenic basis of alcoholic pancreatitis. The effects of elevated cholinergic tone and increased “pancreon” ecbolic response to CCK. Mt Sinai J Med 1983; 50:369–387. Perec C, Rubio M, Baratti C, Tiscornia OM. Effects of chronic ethanol feeding on sympathetic innervated organs. Alcohol Clin Exp Res 1984; 8:37–41.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

71.

72. 73.

74.

75.

76. 77. 78.

79. 80. 81.

82. 83.

84.

85. 86. 87.

Perec C, Tiscornia OM, Baratti C, Tumilasci O, Dreiling D. Trophic, biochemical and functional changes in submaxillary glands and pancreas induced by chronic feeding as indirect effects induced by parasympathetic autonomic centers. Mt Sinai J Med 1984; 51:664–674. Vaccaro MI, Tiscornia OM, Calvo E, Celener D. Effect of ethanol intake on pancreatic exocrine secretion in mice. Scand J Gastroenterol 1992; 27:783–786. Tiscornia OM, Perec C, Celener D, Cresta MA, Tumilasci O, Lehmann ES, Dreiling D. The relationship of hyperactivity of the duodenal autonomic nervous brain and enhanced “pancreon” secretory response to CCK in chronic alcoholism. Mt Sinai J Med 1984; 51:650–663. Tiscornia OM, Dreiling D, Vaccaro MI, Negri G, Celener D, Calvo E, Cresta MA, Perec C. Hipótesis fisiopatogénica de la pancreatitis alcohólica. Medicina (Bs.As.) 1986; 46:616–624. Tiscornia OM, Dreiling D. Supranormal ecbolic stimulation of the pancreon units secondary to the loss of the negative component of pancreatic innervation. Pancreas 1987; 2:604–612. Tiscornia OM, Celener D, Vaccaro MI, Cresta MA, Waisman H. Pancreatitis aguda. Hipótesis fisiopatogénica de la necrosis grasa. Medicina (Bs.As.) 1988; 48:530–542. Tiscornia-Wasserman PG, Tiscornia OM, Rybak BJ, Dreiling D. Acute pancreatitis in a patient treated for alcohol hepatitis. Int J Pancreatol 1989; 4:345–352. Tiscornia OM, Celener D, Cresta MA, Negri G, Vaccaro MI, Bustos Fernández L. El alcoholismo crónico descentraliza autonómicamente el páncreas e incrementa la reactividad de los centros neurales periféricos que modulan su secreción exocrina. Arch Arg Enf Ap Digest 1991; 5:143–172. Adler G., Nelson DK, Katschinski M, Beglinger Ch. Neurohormonal control of human pancreatic secretion. Pancreas 1995; 10:1–13. Schwartz TW. Pancreatic polypeptide: a hormone under vagal control. Gastroenterology 1983; 85:1411–1425. Putnam WS, Liddle RA, Williams JA. Inhibitory regulation of a rat exocrine pancreas by peptide YY and pancreatic polypeptide. Am J Physiol 1983; 256:G698–G703. Sundler F, Bötcher G. Islet innervation with special reference to neuropeptides. In: Samols E, ed. The Endocrine Pancreas New York: Raven Press, 1991:29–52. Raher J, Wallon S, Lefevre A, Gepts W, Haot J. The pancreatic polypeptide cells in the human pancreas. The effects of age and diabetes. J Chem Endocr Metab 1983; 56:441–444. Prinz RA, El Sabbagh H, Adrian T, Bloom S, Gardner L, Polak J, Inoukuchi J, Bishop AF, Welbourn R. Neural regulation of pancreatic polypeptide release. Surgery 1983; 94:1011–1018. Owyang C, Achen-Karam SP, Vinik A. Pancreatic polypeptide and intestinal migratory complex in humans. Gastroenterology 1983; 84:10–17. Jung G, Louie DS, Owyang C. Pancreatic polypeptide inhibits pancreatic enzyme secretion via a cholinergic pathway. Am J Physiol 1987; 253:G706–G719. Karkl-Heinz H, Louie DS, Tatemoto K, Owyang C. Pancreastatin inhibits pancreatic enzyme secretion by presynaptic enzyme secretion by presynaptic modulation of acetylcholine release. Am J Physiol 1992; 262:G113–G117.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

88.

89. 90.

91. 92.

93. 94. 95.

96.

97. 98.

99.

100. 101.

102. 103. 104.

105.

De Diego-Carmona JA, Molina-Trigueros L, Hamelin M, Sanchez A, López J, Correas L, Represa J, Velazquez L. Comportamiento de la secreción de polipéptido pancreático por la célula PP durante la pancreatitis aguda. Rev Esp Enf Ap Digest 1985; 67:160–164. Tiscornia OM, Dreiling D. Is basal bile-pancreatic juice influenced by gastric juice diversion in the rat? Mt Sinai J Med 1986; 53:368–376. Okumura T, Pappas Th, Taylor L. Pancreatic polypeptide microinjection into the dorsal motor nucleus inhibits pancreatic secretion in rats. Gastroenterology 1995; 108:1517–1525. Duncan G, Lambee DG, Johnson RH, Whiteside EA. Evidence of vagal neuropathy in chronic alcoholics. Lancet 1980; 1053–1057. Schmidt N, Devaux MA, Bradzinski TM, Sarles H. Disappearance of an inhibitory factor of exocrine pancreas secretion in chronic alcoholic dogs. Scand J Gastroenterol 1982; 17:761–768. Hajnal F, Flores C, Valenzuela JE. Pancreatic secretion in chronic alcoholics. Effects of acute alcohol or wine on response to a meal. Dig Dis Sci 1993; 78:12–17. Brugge WR, Burice CA, Brand DL, Chey W. Increased interdigestive pancreatic trypsin secretion in alcoholic pancreatic disease. Dig Dis Sci 1985; 30:431–439. Stern H, Davidson GP, Kirubakaran CP, Reutasch J, Hansky J. Pancreatic polypeptide secretion as a marker for disturbed pancreatic function in cystic fibrosis. Dig Dis Sci 1983; 28:870–873. Inoue K, Watson LC, Thompson J. Reduction of post-prandial release of pancreatic polypeptide after development of pancreatic fibrosis. Surg Gynecol Obst 1982; 154:699–703. Larson GM, Sullivan HW, O’Dorisio T. Surgical sympathectomy increases pancreatic polypeptide response to food. Surgery 1985; 98:236–242. Brunicardi F Ch, Druck P, Seymour NF, Su Sun Y, Gingerich R, Darlush E, Andersen DK. Splanchnic neural regulation of pancreatic polypeptide release in the isolated perfused human pancreas. Am J Surg 1989; 157:52–56. Witt NS, Vinick AL, Sive A, Van Tonder S, Lund A. Impaired pancreatic polypeptide responses to insulin-induced hypoglycemia in diabetic neuropathy. J Clin Endocrinol Metab 1980; 50:445–449. Berger Z, Feher E. Degeneration of intrapancreatic nerve fibers after chronic alcohol administration in mice. Int J Pancreatol 1997; 21:165–171. Yamasaki K, Ckasaki K, Sakamoto Y, Yamamoto Y, Okada T. Effects of ethanol on the motility of papillary sphincter and exocrine pancreas in the monkey. Am J Gastroenterol 1993; 88:2078–2083. Owyang C, Miller LJ, Di Magno EP, Mitchell JC, Go VL. Pancreatic exocrine function in severe human chronic renal failure. Gut 1982; 23:357–361. Fleischer K, Kasper H. Exocrine pancreatic function in uremic rats. Acta Hepat Gastroenterol 1974; 21:398–403. Patto RJ, Russo EK, Borges D, Neves M. The entero-insular axis and endocrine pancreatic function in chronic alcoholic consumers. Evidence for early beta-cell hypofunction. Mt Sinai J Med 1993; 60:317–320. Sosa Gallardo C, Kesner L, Ferraris A, Herrero A. Contribución clínico-experimental a la patogenia del infarto segmentario idiopático del epiplón mayor. Bol y Trab Soc Arg Cirug 1960; 21:5–28.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

106. 107. 108. 109. 110. 111.

112.

113. 114. 115. 116.

117.

118.

119. 120. 121. 122.

123.

124.

Barnes PJ, Belvisi MG, Rogers DF. Modulation of neurogenic inflammation. Novel approaches to inflammatory disease. Trends Pharmacol 1990; 11:185–190. Salazar JR. Pancreatitis Aguda. In: Cátedra de Cirugía. Facultad de Ciencias Médicas de la Universidad de Córdoba, Córdoba, Argentina, 1988. Harper AA, Hood JC, Mushens J, Smy JR. Inhibition of external pancreatic secretion by intracolonic and intraileal infusion in the cat. J Physiol 1979; 292:445–454. Owyang C, Green T, Rader P. Colonic inhibition of pancreatic and biliary secretion. Gastroenterology 1983; 84:470–475. Salinas A, Klein E, Toth L, Dreiling D. Enteropancreatic reflexes. The effect of jejunal distension. Mt Sinai J Med 1985; 52:331–335. Hage G, Tiscornia OM, Palasciano G, Sarles H. Inhibition of pancreatic exocrine secretion by intra-colonic oleic acid infusion in the dog. Biomedicine 1974; 21:263–267. Adrian TE, Savage AP, Tuessl HS, Wolfe K, Besterman HS, Bloom SR. Release of peptide YY (PYY) after resection of small bowel, colon or pancreas in man. Surgery 1987; 101:715–719. Kim CD, Lee KY, Chang TM, Chey WY. Negative feedback regulation of pancreatic exocrine secretion in guinea-pigs. Pancreas 1995; 10:173–179. Liddle RA. Regulation of cholecystokinin secretion by intraluminal releasing factors. Am J Physiol 1995; 269:G310–G327. Mizutani S, Miyata M, Izukura M, Tanaka Y, Matsuda H. Role of bile and trypsin in the release of cholecystokinin in humans. Pancreas 1995; 10:194–199. Miyasaka K, Funakoshi A. Involvement of gene expressions of cholecystokinin and secretin in luminal feedback regulation in conscious rats. Pancreas 1995; 10:200–209. Ohshio G, Okada N, Manabe T, Imamura M. Pancreatic exocrine secretion in shortterm pancreatic duct obstruction induced acute pancreatitis in rats. An in-vivo and in-vitro study. Digestion 1994; 55:200–207. Jäning W. Pain and the sympathetic nervous system. Pathophysiologic mechanisms. In: Bannister R, Mathias C, eds. Autonomic Failure. Oxford: Oxford University Press, 1992:231–251. Kowalski M, Kaliner MA. Neurogenic inflammation, vascular permeability and mast cells. J Immunol 1988; 140:3905–3911. Payan D. Substance P. A modulator of neuroendocrine-immune function. Hosp Pract 1986; 24:63–76. Kiernan J. Neurogenic inflammation (Interaction between mast cells and nerves) Trends Pharmacol Sci 1990; 11:316–320. Iovino P, Azpiroz T, Domingo F, Malagelada JR. The sympathetic nervous system modulates perception and reflex responses to gut distention in humans. Gastroenterology 1995; 108:680–686. Reilly J, Darnis F, Rivalier R, Tournier P, Du Buit H, Tsuchiya M, Delorme M. Le syndrome d’irritation des voies biliaires extrahépatiques. Acta Gastroenterol Belg 1959; 9:677–797. Tiscornia OM, Perec CJ, Celener D, Lehmann ES de, Caro L, De Paula J, Baratti O, Martínez JL, Dreiling D. Chronic truncal vagotomy: its effects on the weight and function of the rat’s pancreas. Mt Sinai J Med 1981; 48:295–303.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

125.

126. 127. 128.

129. 130.

131.

132. 133.

134. 135. 136. 137.

138. 139.

140. 141.

142.

Tiscornia OM, Celener D, Cresta MA, Perez CJ, Tumilasci O, Dreiling D. Trophic and antitrophic circuit controlling pancreatic weight in the rat. Mt Sinai J Med 1986; 53:343–355. Greenberg GR, Mitznegg P, Bloom SR. Effect of pancreatic polypeptide on DNA synthesis in the pancreas. Experientica 1997; 33:1332–1334. Tiscornia OM, Dreiling D. Does the pancreatic gland regenerate? Gastroenterology 1966; 51:267–271. Tiscornia OM, Jacobson JH, Dreiling D. Microsurgery of the canine pancreatic duct. Experimental studies and review of previous approaches to the management of pancreatic duct pathology. Surgery 1965; 58:58–72. Tiscornia OM, Dreiling D. Recovery of pancreatic secretory capacity following prolonged ductal obstruction. Ann Surg 1966; 169:267–276. Schlegel RD, Tiscornia OM, Vedia y Mitre E, Lembeye AR, Cogni R, Maccagno D, Cravino T, Waisman H. Existe la regeneración pancreática? Certificación morfológica y funcional luego de una esplenopancreatectomía córporo-caudal. Acta Gastroenterol Latino Am 2000; 30:107–113. Tiscornia OM, Lehmann ES de, Hamamura S, Negri G, Otero G, Waisman H. Sistema nervioso autónomo y páncreas. Análisis de la influencia de diversos tipos de denervación autonómica en los fenómenos de regeneración glandular y en las interacciones del Eje Endocrino-Exocrino. A Ge La 2000; 30:253–265. Sarles H. Etude des facteurs nerveux centraux et periphériques au cours des dyskinesis biliaires. Arch Mal App Dig 1954; 45:712. Lamy J, Sarles H, Micotey G, Sarles JC. Oddite expérimentale chez le chien par irritation chronique du nerf splanchnique droite. Etude manométrique, radiologique et anatomique. Rev Fr Etud Clin Biol 1966; 63:147–154. Sarles JC, Sarles H, Devaux MA. Experimental Odditis and cholelithiasis in the dog. Role of the autonomic nervous system. Am J Gastroenterol 1975; 63:147–154. Tiscornia OM, Dreiling DA. The effect of hypophysectomy on canine pancreatic function. Surgery 1966; 60:883–890. Tiscornia OM, Hansky J, Janowitz H, Dreiling D. The adrenal cortex and external pancreatic secretion in the dog. Mt Sinai J Med 1965; 32:551–561. Tiscornia OM, Cresta MA, Celener D. Estrogen effects on basal bile-pancreatic secretion and the exocrine-endocrine pancreatic gland in the rat. Mt Sinai J Med 1986; 53:462–469. Tiscornia OM, Cresta MA, Lehmann ES de, Celener D, Dreiling D. Sex, age and pancreas. Mt Sinai J Med 1986; 53:452–461. Tiscornia OM, Cresta MA, Lehmann ES de, Belardi G, Dreiling D. Estrogen effects on the exocrine pancreatic secretion in menopausal woman. A hypothesis for menopausal-induced chronic pancreatitis. Mt Sinai J Med 1986; 53:356–360. Tiscornia OM, Cresta MA, Lehmann ES de, Celener D, Dreiling D. Effects of sex and age on pancreatic secretion. Int J Pancreatol 1986; 1:95–118. Tiscornia OM, Bustos D, Negri G, De Paula J, Nakasato O, Cresta MA, Bustos Fernández L. ¿Es la resección del hemicolon derecho inductora de una hipersecreción pancreática exocrina en la rata? Medicina (Bs.As.) 1986; 17:59. Sternberg EM. Perspectives series: cytokines and the brain. (Neural-immune interactions in health and disease.) J Clin Invest 1997; 100:2641–2647.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

143.

Abe R., Shimosegawa T, Kimura K, Abe T, Kashimura J, Koizumi M, Toyota T. The role of endogenous glucocorticoids in rat experimental models of acute pancreatitis. Gastroenterology 1995; 109:933–943. 144. Garces CM, Gómez-Cerezo J, Codoceo AR. Relationship of basal and postprandial intraduodenal bile acid concentrations and plasma cholecystokinin levels with abdominal pain in patients with chronic pancreatitis. Pancreas 1998; 17:397–401. 145. Pitchumoni CS. Better understanding of the pain of pancreatitis. Am J Gastroenterol 1999; 94:1700–1701.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

17 Diabetes and the Autonomic Nervous System Maria Grazia Natali-Sora and Guido Pozza Vita-Salute San Raffaele University, Milan, Italy

I. INTRODUCTION Diabetic neuropathy is the most common neuropathy in developed countries. Diabetes is characterized by a series of hormone-induced metabolic abnormalities and by a series of long-term complications that lead to considerable morbidity and premature mortality. Diabetes is the most common of the serious metabolic diseases of humans. While some patients may never develop these problems and others note their onset early, on average symptoms develop 10–15 years after the onset of disease. A single problem may dominate the picture, or the patient may experience several complications simultaneously. Diabetes may affect any part of the nervous system. Diabetic neuropathy is rarely a direct cause of death, but it is a major cause of morbidity. Distinct syndromes may be recognized, and often several different types of neuropathy are present in the same patient. The most common picture is that of peripheral polyneuropathy. Mononeuropathy and radiculopathy are less common. Autonomic neuropathy may present in a variety of ways. It usually occurs in association with distal symmetric polyneuropathy. Occasionally it may constitute the major neurological dysfunction. It is probably widely underdiagnosed by clinicians because symptoms and signs are often vague and nonspecific. The gastrointestinal tract, the cardiovascular system, and the genitourinary system are primary targets. II. FUNCTIONAL ANATOMY The autonomic nervous system (ANS) provides a rapidly responding mechanism to control a wide range of functions. Cardiovascular, respiratory, gastrointestinal,

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

renal, endocrine, and other systems are regulated by either the sympathetic or the parasympathetic nervous system. The ANS innervates every visceral organ of the body. Body functions, which can proceed independently of volition activity, are regulated at least in part by reflex mechanisms served by afferents, efferents, and central integrating structures included in the ANS. The ANS has a complex neural organization in the brain, spinal cord, and periphery. The hypothalamus can be considered the highest level of integration of autonomic function. It is under the influence of the cortex and structures of the limbic system, which includes the hippocampus and amygdaloid complex, the olfactory areas, the cingulate cortex, and the septal area. The limbic system function is believed to be concerned with lev-

Figure 1 Sympathetic and parasympathetic autonomic nervous system.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Autonomic and visceral afferent neurons: presynaptic sympathetic neurons with cell bodies in the lateral columns of the spinal cord pass along the spinal cord and along a white ramus to reach the sympathetic chain, where they either synapse or pass along the splanchnic nerve to synapse in a prevertebral ganglion. The postganglionic neurons contact the effector organs.

els below cognitive behavior; it analyzes the significance of the input of sensation to the organism in relation to instinctive drives. It is also concerned with maintaining homeostasis in light of a changing environment. The essence of its function is choice of patterns of behavior based on sensory input. It overlaps with both sensory and motor systems and is essential for many aspects of memory and learning. The hypothalamus controls the ANS by means of the pituitary and hence other endocrine glands and by direct descending nervous pathways. The peripheral ANS is an efferent system. Both sympathetic and parasympathetic systems have preganglionic neurons in the brain and spinal cord. Sympathetic innervation is derived from the efferent preganglionic fibers, whose cell bodies lie in the intermediolateral column of the spinal cord at the level of the thoracic and upper lumbar roots. The afferent limbs of autonomic reflexes may lie in any afferent nerve. The preganglionic sympathetic fibers are myelinated and leave the spinal roots as white rami and synapse in the ganglia. Unmyelinated postganglionic fibers rejoin the anterior spinal roots, and some sympathetic fibers cross the ganglia and synapse in more peripheral ganglia. The preganglionic neurons are cholinergic or use acetylcholine as a transmitter, while the neurotransmitter for postganglionic sympathetic nerves is noradrenaline with the exception of sweat glands, which afCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ter birth are switched from adrenergic to cholinergic sympathetic innervation, some vasodilator fibers to muscle, and the adrenal medulla, which is innervated by preganglionic cholinergic fibers and secretes both adrenaline and noradrenaline. The adrenal medulla can be considered as a modified sympathetic ganglia. It exerts circulating humoral actions. The different actions of noradrenaline and adrenaline are caused by effects on different receptors. In the early 1970s the best indicator of sympathetic nerve activity was plasma levels of norepinephine (1). In the diagnosis of diabetic autonomic neuropathy, for the majority of patients plasma catecholamine kinetics are no more sensitive than supine plasma norepinephrine. Unlike sympathetic neurotransmitters, the parasympathetic neurotransmitter acetylcholine cannot be measured in peripheral venous blood, primarily because of the ubiquitous presence of high intrinsic activity of cholinesterase. Other biochemical markers of parasympathetic neuropathy are indirect. Pancre-

Figure 3 Autonomic innervation of the heart.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

atic polypeptide is secreted from the pancreatic polypeptide cells on vagal stimulation (2). Pancreatic polypeptide response to hypoglycemia and to a mixed meal is blunted even in patients with diabetes of short duration who have no other detectable signs of neuropathy, including abnormal results of cardiovascular reflex test (3). Alpha receptors mediate vasoconstriction, intestinal relaxation, and dilatation of the pupil; alpha receptors may be postsynaptic (1) or presynaptic (2). Beta receptors mediate vasodilataion, especially in muscles, increase the rate and force of heart, and cause bronchial relaxation. They are further subdivided into 1receptors, mediating the chronotropic cardiac action, and 2-receptors, which are responsible for most of the peripheral effects of -adrenergic stimulation. The au-

Figure 4 The autonomic nerves of the thorax. The sympathetic trunk and the vagus nerves bring sympathetic and parasympathetic innervation, respectively, to the thoracic viscera.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

tonomic ganglia also contain many peptides, e.g., vasoactive intestinal peptide (VIP), substance P, encephalins, and somatostatin. These substances are thought to act as modulators and transmitters at synaptic ends. From a functional point of view there are two main effector limbs of the ANS. Postganglionic parasympathetic fibers from the vagus innervate the cardiac, pulmonary, and upper gastrointestinal systems, while pelvic organs are innervated by the sacral parasympathetic outflow. The anatomical pathways of the central connections of baroreceptor reflex have been described (4). Blood pressure homeostasis is maintained by a negative feedback mechanism that is under the control of the ANS. Changes in blood pressure are detected by baroreceptors in the heart, carotid sinus, aortic arch, and other large vessels. Afferent impulses are transmitted from these structures via the carotid sinus nerve and the glossopharyngeal and vagus nerves to the brainstem. The carotid sinus nerve terminates in two areas, which play a major role in blood pressure control: the nuclei of the tractus solitarius (NTS) and the paramedian nucleus. From the NTS neuronal connections are made both with efferent pathways, which are the secondary neurons on the baroreceptor reflex arc, and with ascending neurons, which carry information to higher structures in the brain. The dorsal vagal nucleus, the nucleus reticularis lateralis, and the nucleus reticularis medullae oblongatae project directly to the interomediolateral nucleus of the spinal cord, and baroreflex information may reach the preganglionic sympathetic cells of the spinal cord by multisynaptic pathways. The precise neuroanatomical pathways

Figure 5 Innervation of the gastrointestinal tract.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

connecting the NTS to higher cardiovascular control centers are still unknown. Higher brain centers seems actively involved in the modulation of the lower cardiovascular reflex control areas.

III. AUTONOMIC NERVOUS SYSTEM AND ITS PATHOPHYSIOLOGY The most common type of diabetic neuropathy is a distal symmetrical, predominantly sensory polyneuropathy (Table 1). There are indications that small fiber sensory modalities are affected earlier. Minor distal motor involvement may coexist. Severe autonomic neuropathy is present in type I diabetic patients, but mild or moderate involvement is frequent in both type I and type II patients. It is still unknown whether the mechanism for distal sensory symmetrical polyneuropathy is a direct metabolic effect or whether it is secondary to hypoxia from microvascular disease (5). The results of the Diabetes Control and Complications Trial (6) have demonstrated that strict control of blood glucose concentrations significantly reduced the rate of developing neuropathy. It seems unlikely that hypoxia is the major cause of distal symmetrical sensory polyneuropathy, as in other situations nerve ischemia gives rise to predominant motor involvement and not to sensory/autonomic neuropathy (5). The underlying pathology in this type of neuropathy has been shown to consist of a distal axonal degeneration of the dyingback type (7). It would be difficult to explain the occurrence of a central peripheral distal axonopathy on an ischemic basis. Microvascular disease is often present in diabetic polyneuropathy, and a distally accentuated sensorimotor neuropathy can

Table 1 Classification of Diabetic Neuropathies Hyperglycemic neuropathy Generalized neuropathies Sensorimotor polyneuropathy Autonomic neuropathy Acute painful sensory neuropathy Focal and multifocal neuropathies Cranial neuropathies Thoracoabdominal radiculoneuropathy Focal limb neuropathies Proximal diabetic neuropathy Superimposed chronic inflammatory demyelinating polyneuropathy Source: Ref. 47.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

result from the accumulation of multiple proximal nerve trunk lesions (8). For such cases an ischemic basis can be postulated. In taking account of possible metabolic causes for polyneuropathy, a major metabolic abnormality in nerve cells is the accumulation of sorbitol because of increased flux in the polyol pathway secondary to hyperglycemia (9). Glucose is converted to sorbitol by enzyme aldose reductase. The quantities of sorbitol present in diabetic nerve cells are insufficient to produce osmotic damage, but it is possible that they may have deleterious effects on neural metabolism (5). Trials with aldose reductase inhibitors to reduce the production of sorbitol have so far failed to show any substantial effects on diabetic polyneuropathy. Reduced nerve myoinositol concentrations have been implicated in a series of changes via reduced NaK-ATPase activity, leading to nerve fiber degeneration. The reduction of nerve myoinositol concentrations found in diabetic rats has not been confirmed in human diabetic nerve tissue. Attention has also been directed toward alterations in the metabolism of essential fatty acids, necessary for the maintenance of normal cell membrane structure. Treatment of human diabetic neuropathy by the administration of -linoleic acid has not resulted in substantial beneficial effects on neuropathy (5). Axonal proteins have been shown to be abnormally glycated in the diabetic patient, but whether their effects are important in the pathogenesis of diabetic neuropathy has not been established. There is experimental evidence that insulin-like growth factor I (IGF-I) may improve regeneration in animal models of diabetes. Insulin antibodies may cross-react with growth factor, leading to sympathetic nerve damage. Because of similar antigenic determinants on growth factor and insulin, insulin antibodies may cross-react and lead to sympathetic damage. An important aspect of diabetic sensory polyneuropathy is a failure of axonal regeneration (10). This is initially diffuse, but later it fails. This probably explains the lack of reversibility of the neuropathy once it is established, even with good glycemic control (5). The role of immunological factors in the pathogenesis of diabetic neuropathy has generated much interest in recent years. Much evidence suggests that type I diabetes mellitus is an autoimmune disorder associated with antibody- and cell-mediated immunity directed against islet cells of the pancreas. Islet cells and the nervous system share many antigenic similarities, such as specific gangliosides, sulfatides, and glutamic acid decarboxylase (11). Antineuronal antibodies have been reported in patients with different types of neuropathy, including subjects with diabetic neuropathy and their first-degree relatives (12). The degree to which antineural antibodies are pathogenic in neuropathy is controversial. Direct evidence showing the ability of these antibodies to cause clinical pathology is limited. The presence of antibodies directed against ANS structures is associated with changes in autonomic function (13). Type I diabetes often occurs as a component of the type II polyglandular autoimmune syndrome, which consists of several associated autoimmune glandular and nonglandular disorders. Evidence shows that many patients have polyneuroendocrine autoimmunity (14).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

IV. DIAGNOSTIC AND CLINICAL IMPLICATIONS The first complete description of autonomic dysfunction in diabetic patients was written by Rundles in 1945 (15). Since that time many studies have been performed to estimate incidence and prevalence of diabetic autonomic neuropathy. Data vary greatly in the literature because of widely differing standards of diagnosis, but prevalence is somewhere between 30 and 60% depending on the diagnostic criteria (16–19). Although not always clinically evident, disordered autonomic function can be detected throughout the body if techniques are sensitive enough. Little is known about the natural history of diabetic autonomic neuropathy. Clinical or laboratory evidence of autonomic involvement is usually absent in the first 10 years of the disease; later signs of autonomic neuropathy are present in one third of insulin-dependent diabetic patients (20). Diabetic neuropathy is the most common cause of autonomic neuropathy, causing functional defects and symptoms in a wide variety of systems. The gastrointestinal tract, the genitourinary system, the heart, and blood vessels are all affected; there are abnormalities of sweating, pupillary defects, and a wide variety of metabolic disorders. The main characteristic features are due to the early and extensive involvement of small nerve fibers. Early small-fiber damage is manifested by impairment of vagally controlled heart rate variability, while diminished peripheral sympathetic tone leads to increased blood flow, which is detectable before there is clinical evidence of neuropathy (21). Reduced sweating is very common (22), and thermal sensation (a small-fiber modality) is lost before vibration sensation (a large-fiber modality) (23). A. Cardiovascular System The most common cardiovascular abnormalities are resting tachycardia and postural hypotension. Symptoms are initially vague and nonspecific and often poorly appreciated by patient and physician. Most pictures of autonomic failure are insidious in their onset, with mild symptoms for years because of autonomic compensatory mechanisms. Patients may start with mild symptoms of vague weakness, postural dizziness, or faintness, which can very easily be overlooked or result in erroneous referral to a psychiatrist rather than a neurologist. The clinician’s ability to diagnose autonomic neuropathy has been enhanced by the introduction of noninvasive tests of autonomic function. The central point of the diagnosis is the measurement of blood pressure when standing rather than lying, which can reveal a much more complex underlying autonomic disturbance. Cardiovascular system/cardiovascular responses never occur in isolation but accompany the process of exercise, digestion, sexual function, and temperature regulation. Orthostatic hypotension is a relatively frequent symptom of autonomic diabetic neuropathy. In some studies orthostatic hypotension was found as a frequent symp-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

tom—43% (24). Veglio et al. (25) reported orthostatic intolerance in 34% of a group of patients with non–insulin-dependent diabetes mellitus (NIDDM), while Young et al. (26), in a study performed on teenagers, found no signs of autonomic failure. The prevalence of symptomatic orthostatic hypotension is less than 1%. The underlying mechanism involves degeneration of the sympathetic preganglionic and postganglionic fibers supplying the splanchnic mesenteric bed. Autonomic denervation of the muscular resistance bed is present and is implicated but less important in causing orthostatic hypotension. It has been shown that damage to the sympathetic and parasympathetic pathways to the heart is particularly dangerous to the patient. There is an increased incidence of myocardial infarction in patients with diabetic autonomic neuropathy; myocardial infarction may not be associated with typical cardiac pain. Sudden cardiorespiratory arrest has been described in diabetes with autonomic neuropathy. Diabetic subjects with damage to the autonomic pathways might not respond normally to hypoxia, and this could lead to cardiorespiratory arrest under conditions such as chest infections or surgery. Cardiac arrhythmia and sleep apnea have also been proposed as causes of sudden unexpected death in diabetics (27). The true importance of abnormal ventilatory responses as a cause of respiratory arrest has yet to be established. Some authors (28) found a defective response to hypoxia, while others found normal increased ventilatory response to transient hypoxia. Both normal and reduced ventilatory responses have been reported (27). Some authors assessed the integrity of the respiratory reflexes, mainly vagal, which determine bronchomotor tone. This has been found to be reduced in diabetics with autonomic neuropathy. The autonomic function in clinical practice is currently assessed by a cardiovascular reflex test that measures variation of heart rate or blood pressure in response to a given stimulus. There are few controversies about exactly how tests should be performed and what measurement should be made to assess the integrity of the response. Cardiovascular heart rate tests have been conducted in diabetic patients since the 1970s (16). The most widely used test is the heart rate response to deep breathing (16,29) followed by the heart rate response to Valsalva maneuver (30) and to standing (31). Other tests of cardiovascular function used in the past, such as heart rate responses to apneic face immersion and mental stress (32), are no longer used in routine clinical practice. To record heart rate, electrodes are best placed at sites where movement artifacts are minimal during autonomic maneuvers. The rate of breathing has a profound influence on R-R variations. The variation is maximal at a breathing rate of 6 breaths per minute (33,34). The patient is asked to breath maximally at a rate of 6 breaths per minute for 1 minute, while the heart rate is recorded by ECG (Fig. 6). The maximum and minimum R-R intervals during each breathing cycle are measured and converted to beats per minute. The mean difference between maximum and minimum heart rate is calculated (16). The expiration-inspiration ratio can alternatively be calculated. Some factors affect the heart rate responses to deep breathing and must be taken into account—

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 6 Deep breathing in (a) a normal subject and (b) a diabetic patient with autonomic neuropathy.

among them, the patient’s age. There is a progressive reduction in forced sinus arrhythmia, quantified as a mean range of heart rate response to deep breathing, with increasing age. Other factors are the rate of breathing, analytical methods, hypocapnia, the influence of sympathetic activity, the position of the subject, concomitant medications, and obesity (35). Lying to standing: ECG is recorded while the patient, after 5–10 minutes of recumbency, changes position from supine to standing (Fig. 7). The immediate response in a healthy subject is characterized by a sharp decrease in BP and total systemic resistance (5–10 seconds) followed by a rapid rebound and overshoot. A ratio is calculated dividing the R-R interval at the 30th beat by the R-R interval at the 15th beat (16). Valsalva ratio: The patient is asked to blow through a mouthpiece attached to a manometer, maintaining 40 mmHg pressure for 15 seconds under continuous ECG monitoring (Fig. 8) (the effort must be done with an open glottis). A pressure of 20 is inadequate, and a pressure 60 results in less reproducibility. The Valsalva ratio is calculated dividing the longest R-R interval after strain release by the shortest R-R interval dur-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ing the strain period. In dysautonomic patients there is usually a loss of blood pressure overshoot and the reflex bradycardia. Factors that may influence the Valsalva ratio are age, gender, position of the subject, respiratory pressure, duration of effort, inspiration volume, and medications. Blood pressure response to standing: Blood pressure is measured with the patient supine after 10 minutes of recumbency at 30 seconds and 1 and 3 minutes after standing. Blood pressure response to sustained hand grip: The maximum voluntary contraction with the dominant arm is calculated; then the handgrip is maintained steadily at 30% of this power for 3 minutes. Blood pressure is measured on the not exercising arm at rest and at 1-minute intervals during hand grip. If the heart rate is monitored during a 24-hour period, the major fluctuations in rate seen in normal subjects may be absent in patients with autonomic neuropathy.

Figure 7 Lying to standing in (a) a normal subject and (b) a diabetic patient with autonomic neuropathy.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 8 Valsalva ratio in (a) a normal subject and (b) a diabetic patient with autonomic neuropathy.

More recently, heart rate changes in the frequency domain have been studied. Previous cardiovascular tests were designed to measure changes in heart rate induced by a standard stimulus. Spontaneous fluctuations of R-R intervals over a short period of time or over 24 hours have been analyzed (36–39). Spectral analysis of short-term variability is now used as a method for the study of cardiocirculatory reflexes. This noninvasive method allows one to evaluate the interactions between parasympathetic and sympathetic nervous systems in the control of heart rate. In normal subjects, spectral analysis of R-R variability shows two major peaks: one at low frequency (LF) (~0.1 Hz) and one at high frequency (HF) (0.2–0.3 Hz) (Fig. 8). Cross-spectral analysis of tachogram and spirogram demonstrate that the HF peak is an expression of the respiratory arrhythmia. During active tilt, a sympathetic stimulation, the power of the HF component decreases and the power of the LF component increases, suggesting that the LF peak is predominantly the expression of R-R variability induced by the sympathetic system, as indicated by pharmacological studies (36). HF peak, due to respiratory arrythmia, is

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 9 Spectral analysis of R-R variability in a normal subject (a) and in a diabetic patient (b) with autonomic neuropathy in three different epochs: (top) at rest, (middle) standing, and (bottom) during controlled respiration.

the expression of heart rate modifications under the control of the parasympathetic system. In fact, during controlled respiration (12 breaths/min) there is a marked enhancement of the HF peak, with a decrease of the LF peak. In diabetic patients with neuropathy, LF and HF were decreased (40). Power spectral analysis makes it possible to analyze the sympathetic-parasympathetic balance. In diabetic autonomic neuropathy sympathetic and parasympathetic involvement appear to be similar in degree. The HF component was found to be a a significant predictor for the lying-to-standing and the Valsalva ratio. The LF component was a poor predictor for blood pressure response to sustained handgrip test or orthostatic hypotension.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

B. Gastrointestinal System Delayed gastric emptying, diarrhea, and constipation are the major symptoms of gastrointestinal involvement in diabetics with autonomic neuropathy. Gastrointestinal symptoms are common in patients with type I diabetes. Vomiting is a rare symptom; gastroparesis is probably due to vagal degeneration. It is characterized by food residues, absent peristalsis, and failure to empty the stomach. Liquids and solids are emptied through the stomach at different rates and by different mechanisms (41). Gastric emptying of digestible and indigestible solids is abnormal in patients with diabetes mellitus and gastroparesis. Studies have demonstrated a reduction of antral contractions in the distal portion of the stomach during fasting postprandially. Many studies have examined the role of gastrointestinal hormones and catecholamines in diabetic autonomic neuropathy. Autonomic modulation influences the secretion of polypeptides active in the gastrointestinal system. Perhaps impairment of cholinergic innervation to the gut precedes cardiovagal impairment. The blunted pancreatic polypeptide response to hypoglycemia and to a mixed meal has been shown in diabetics with short duration of disease who have no other measurable signs of neuropathy, and it may be the first evidence of parasympathetic neuropathy in diabetic patients. Both somatostatin and glucagon responses to hypoglycemia have been shown to be reduced in diabetic autonomic neuropathy. Gastric inhibitory peptide secretion after a meal is diminished in diabetics with autonomic damage. This may be secondary to delayed gastric emptying. Motilin may play a key role in regulating gastric motility and intestinal transit. It is probably modulated primarily by vagal fibers, and its release is abnormal in diabetics with autonomic neuropathy. Unfortunately, investigations for assessing autonomic dysfunction in the gastrointestinal system are not noninvasive. In the diagnostic evaluation of gut dysautonomia, traditional gastrointestinal radiographs are important to obtain as a first step to exclude mechanical obstruction, mucosal disease, or an infiltrative process affecting gut, smooth muscle, or supporting connective tissues. Radionuclide studies are employed in several useful capacities. In research laboratories they allow calculation of gastric emptying and secretion rates of trypsis, bicarbonate, and bile acids into the duodenum. Esophageal function can be assessed by manometry or scintiscanning. Multipeaked esophageal pressure waves are detected in most diabetics with neuropathy. Scintiscanning techniques measuring both solid and liquid transit times may show abnormal motility patterns in diabetics with autonomic neuropathy. These patients show reduced or abnormal esophageal peristalsis and delayed esophageal emptying. Isotopic gastric scanning techniques have confirmed that liquid emptying is normal but solid emptying is delayed in diabetics with other evidence of autonomic neuropathy. Vagally mediated gastric acid secretion in response to sham feeding is reduced. Diarrhea is a very uncomfortable symptom of autonomic neuropathy in diabetic patients. Patients complain of abdominal discomfort and rum-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

bling, which precede attacks of osmotic diarrhea, without pain or bleeding. Fecal incontinence is common at night. Anal-rectal manometry studies have shown reduced basal anal sphincter smooth muscle tone in incontinent diabetics. The threshold to conscious rectal distension is higher in incontinent patients. Symptoms last for hours or days, after which patients return to a normal bowel function or sometimes to constipation. This last condition is frequent but not severe. Diarrhea or fecal incontinence may result from several mechanisms: dysfunction of anorectal sphincter or abnormal rectal sensation, abnormality of small bowel motility, decreased gut transit time, bacterial overgrowth due to small bowel stasis or uncoordinated peristalsis, and bile salt malabsorption. Rarely an associated gluten-sensitive enteropathy or pancreatic exocrine insufficiency is present. These associated conditions are potentially reversible. The diagnosis of diabetic diarrhea must be confirmed by the presence of autonomic neuropathy and excluding other causes of diarrhea. C. The Pupil The size of the pupil is regulated by the musculature of the iris, the dilator and sphincter of the pupil. The sphincter may shorten more than any other muscle of the body. The sphincter muscle of the pupil is supplied by crossed and uncrossed parasympathetic fibers, which reach it through the 3rd nerve and have their origin in the paired nuclei of Edinger-Westpal. The dilator muscle of the pupil is supplied by sympathetic fibers. These two muscles are thought to be antagonistic in activity, but the sphincter seems more powerful. The pupil may also dilate due to relaxation of the constrictor of the pupil. The normal pupil is 2.5–5.5 mm in diameter. The pupil is smaller in infancy, widens progressively until adolescence, when it reaches its largest diameter, and becomes narrower again in old age. Unequal pupils, or anisocoria, may be a reaction to varied illumination of the eye or may be a physiological variant, rather than an indication of disease. Hippus, a continuous rhythmic variation in pupil size under constant illumination, which can be observed in some subjects, is considered a variant of normal pupil unrest, but it has been compared to physiological tremor or to imbalance between sympathetic and parasympathetic activity of the ANS. Pupil function is affected by central, sympathetic, and parasympathetic damage in diabetes. A mild sympathetic deficit appears to be the earliest sign; the pupil showing a partial Horner syndrome is common, while signs suggesting an acute Adie’s pupil are rare. Diabetic patients usually have small pupils (15,42). About 20% of diabetic patients have pupils that fail to dilate normally in darkness. Significant associations between small pupils and cardiovascular autonomic dysfunction (43), peripheral sensory loss (44), retinopathy, and nephropathy have been reported. Pupil function can be investigated with noninvasive techniques. Pupil diameter is measured using an infrared pupillometer or a simple polaroid pupillometer. Pupil size is best measured in

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

darkness. Pupil cycle time is another index of sympathetic function. It was found to be prolonged in diabetics with autonomic neuropathy, presumably due to parasympathetic dysfunction. D. Thermoregulation and Sweating Diabetic anhidrosis is a condition in which sweating is absent in the lower limbs and trunk of patients with diabetic neuropathy. Patients with this disorder are intolerant to heat and may also complain of excessive perspiration on the head, face, and neck. This hyperhidrosis may be compensatory thermoregulatory sweating. In diabetic neuropathy sympathetic denervation occurs predominantly in the lower limbs and is evidenced by reduced or absent sweating and disturbed vasomotor control. Sweating during eating has been found in diabetic patients. Sweating is confined to the territory of the superior cervical ganglion and is a feature of autonomic neuropathy. Patients who experience this symptom have abnormal cardiovascular tests. Local sympathetic sweat production can be measured quantitatively. The number of sweat glands secreting after iontophoresis of pilocarpine on 1 cm2 of skin is determined with a special Silastic material spread on the skin. As sweat is produced by the sweat ducts it pushes into the Silastic material, which form an impression of the sweat droplets. The hardened mold is removed from the skin and viewed under a dissecting microscope to count the number of droplets per cm2 of skin (45). A quantitative sudomotor axon reflex test (Q-SART), in which local sweating is stimulated by acetylcholine iontophoresis, has been described by Low (22). He found abnormal Q-SART in the foot and abnormal heart rate variation in more or less equal numbers of patients with diabetic neuropathy. Moreover, Q-SART is more frequently abnormal than orthostatic hypotension. This suggests that abnormalities of distal sympathetic function occur early in the natural history of diabetic autonomic neuropathy. E. Genitourinary System Bladder function tests are commonly abnormal in neuropatic diabetics, but symptoms from neurogenic bladder in diabetics usually occur in patients who already have advanced complications. Impairment of bladder function is mainly the result of afferent sensory abnormalities and results in impaired sensation of bladder filling and subsequently leads to detrusor areflexia. Measurements of urine flow show that the peak flow rate is reduced and that duration of flow is increased. Pudendal innervation of perineal and periurethral striated muscle is usually unaffected. In the early stages patients are asymptomatic; later they experience hesitancy during micturition and develop the need to strain and a feeble stream. Intervals between micturitions are longer, and a sensation of inadequate bladder emptying may be present. In later stages residual urine volume increases and se-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

vere bladder retention occurs, leading to overflow incontinence. The most important complication of urine retention is urinary tract infections. Repeated infections may worsen renal function, particularly if nephropathy is present, and may contraindicate transplantation. Differential diagnosis with prostatic obstruction must be considered in men. Rarely in diabetic autonomic neuropathy hydroureter and hydronephrosis develop. Ultrasound examination provides much information about bladder emptying. Cystoscopy and urine flow-rate measurements are sometimes needed. Most patients with neurogenic bladder are also impotent. The onset is usually gradual, but complete erectile failure is usually achieved within 2 years of onset of symptoms. Autonomic neuropathy is still considered the main etiological factor. Erectile failure is due to damage of parasympathetic and sympathetic innervation of the corpora cavernosa. Failure to achieve erection may also result from the concomitant sensory deficit of the dorsal nerve of the penis. Psychogenic impotence can be differentiated because it begins suddenly and nocturnal erection is preserved. Another etiological factor of impotence in diabetic patients is the vascular occlusion of the branches of the internal pudendal artery. Diagnosis of neuropathic impotence may be difficult with anamnestic data alone. Several advanced techniques are available. Quantitation of erectile function with the measurement of continuous nocturnal penile expansion and concomitant rigidity during sleep provides information to differentiate a psychogenic cause from organic damage. Vasculogenic impotence can be suspected with the measurement of penile blood supply. Autonomic function tests may yield more information about the presence or not of autonomic neuropathy in the cardiovascular system, but do not allow one to conclude about the presence of autonomic damage in the genitourinary system. Electrophysiological assessment of reflex sexual pathways can give more information if a neuropathic cause is suspected. Conduction velocity of the dorsal nerve of the penis is reduced and the latency of the bulbocavernous reflex is prolonged. F. The Neuropathic Foot A special problem in diabetics is the development of ulcers of the lower extremities, namely the feet. The pathogenesis is not completely known and probably is due to a combination of different mechanisms secondary to somatic and autonomic neuropathy and microvascular lesion. Peripheral neuropathy to the foot leads to both somatic and autonomic damage. Small fiber loss may predominate, leading to loss of pain and thermal sensation before light touch and vibration senses are blunted (23). Small fiber loss leads to sympathetic denervation, which is characteristic of diabetic neuropathy. Sympathetic nerve endings to small arterioles in the diabetic limb are entirely absent or are found distant from effector sites. In the neuropathic foot blood flow is increased with arteriovenous shunting, and the peripheral arteries are dilated and stiff. These abnormalities may be par-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

tially responsible for the pathogenesis of the neuropathic complications of the diabetic foot and may also be important in acute, painful neuropathy. Autonomic denervation may be responsible for abnormalities in blood flow, with an increased velocity of forward flow in dilated peripheral arteries, increased peripheral blood flow, and a fixed increased resting blood flow (21) in patients assessed in the supine position. In normal subjects standing causes precapillary vasoconstriction in the foot, reducing the increment in capillary pressure and reducing blood flow. This response is dependent upon a sympathetic axon reflex. The postural control of blood flow is impaired in patients with diabetic neuropathy, with loss of sympathetic vascular tone. Rigid dilated arteries and shunting lead to a rapid increase of flow with short-circuiting of capillaries and distal ischemia. Autonomic neuropathy may be responsible for two other abnormalities in the foot, namely disordered thermal regulation and disturbances in sweating. Increased blood flow leads to raised skin temperature in the resting neuropathic limb (21). Vasoconstriction in response to local cold is often prolonged. An autonomic unbalance with unopposed sympathetic nervous activity may be responsible for such vasospasm; alternatively, a sympathetic denervation hypersensitivity may be the cause. Autonomic neuropathy also predisposes to ulceration by other mechanisms. Abnormal pressure distribution secondary to diabetic neuropathy and eventually the presence of bony distortion are potentially harmful for callus formation. Alternatively, the ulcer may be initiated by ill-fitting shoes, which cause blister formation in patients whose sensory deficits alter pain sensations. Abnormal responses to temperature changes, in particular prolonged vasoconstriction on exposure to cold, could be harmful. Loss of sweating leads to a dry skin with thick plaques of hard callus, which readily crack. This can lead to fissuring of the skin and eventual ulceration. Although less frequent, the Charcot joint can be a devastating complication of the neuropathic foot. Circulatory changes can lead to abnormalities in bone structure, with a reduction in bone cortical thickness rendering the foot susceptible to even minor trauma. The combination of somatic and autonomic neuropathy allows an abnormal mechanical stress to occur, resulting in fracture and finally in bone and joint disorganization.

V. THERAPEUTICS Treatment of diabetic neuropathy can be considered as prevention, treatment of neuropathy, or treatment of complications. It is now clear that strict control of glycemia by intensive insulin treatment will prevent or even improve neuropathy. In the Diabetic Control and Complication Trial (6), which studied 1441 patients with IDDM for 10 years, the patients with multiple insulin injections after 3 years had a reduction of clinical neuropathy by 60%. This treatment is applicable only to patients with type I insulin-dependent diabetes. The maintenance of eu-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

glycemia, associated with the control of hypertension, avoidance of smoke, maintenance of ideal weight, normal lipid levels, and adequate exercise, is recommended. Prevention of complications includes avoidance of abuse of joints with impaired sensation, avoidance of trauma to skin, and avoidance of cold and heat injury. Trials with aldose reductase inhibitors to reduce the production of sorbitol have so far failed to show any substantial effects on diabetic polyneuropathy. Studies on animal models of diabetes indicate that IGF I enhances regeneration, and nerve growth factor (NGF) has been shown to have a beneficial effect in other experimental neuropathies. The neurotrophic effect of NGF is mainly on small myelinated and unmyelinated fibers. Evidence from clinical trials with diabetic patients are needed. If autoimmunity is of pathogenetic importance in diabetic subjects, an immunosuppressive regimen would be expected to be useful, but unfortunaly little information is available on this subject. Combined pancreas and kidney transplantation and renal transplantation in patients with longstanding type I diabetes improved peripheral nerve function. An initial improvement is probably due to the elimination of uremia. Subsequent further improvement is probably due to nerve repair and regeneration under euglycemia. Autonomic function improves also, even if to a slight lesser degree. Several methods of varying complexity can be used to counteract the postural hypotension, but all are in various ways unsatisfactory in severe cases. Obviously, if the volume into which the pooling of blood takes place on standing is reduced, this will help the patient, and if the volume of blood is increased, this will also reduce the severity of the symptoms. However, because of lack of baroreflexes all patients have a very labile blood pressure, high when lying and low when standing, and relatively low in the morning and rising towards evening. Patients can sometimes tolerate a standing systolic blood pressure as low as 80 mmHg without dizziness or syncope, probably because their capacity for cerebral autoregulation is better than normal subjects. Almost all patients with postural hypotension can be helped by tilting up the head at night. Once patients have experienced the benefit, they are usually ready to tolerate the degree of discomfort this entails. Other nonpharmacological measures include eating smaller and more frequent meals and using countermaneuvers such as standing with legs crossed. Wearing elastic support garments is of some help, but it reduces intrinsic myogenic tone and is rarely of much long-term benefit if not associated with proper daily muscle tone exercise. Plasma volume expanders (e.g., 9-fludrohydrocortisone, Florinef) are important and have been shown to have an effect on blood pressure. However, this drug at high doses is potentially dangerous, as cardiac dysfunction resulting from diabetic angiopathy may be present. Plasma expansion by an increase in salt intake or the use of prostaglandin inhibitors, such as ibuprofen or indomethacin, may sometimes be effective. The treatment of symptomatic upper gastrointestinal symptoms in diabetic autonomic neuropathy is unsatisfactory. General measures include control of nausea and vomiting and fluid and electrolyte loss. Antiemetics may provide tem-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

porary relief. Small meals are encouraged. Prokinetic drugs for hypomobility are suggested. Domperidone may relieve heartburn and dysphagia. Peripheral cholinergic agonists such as metoclopramide, bethanechol, and cisapride and 2-adrenergic agonists such as clonidine have been used to treat diabetic gut neuropathy. All available therapeutic options have resulted in only transient relief. Pancreas transplantation is reported to restore normal gastric emptying in patients with diabetic gastropheresis (46). The neurogenic bladder requires careful treatment. Patients with initial symptoms should be instructed to void every 3 hours during the day to compensate for the lack of desire to void. Straining may be required, and the Credé maneuver may be needed to increase the efficacy of bladder emptying. Sometimes the use of small-diameter urethral catheters may be necessary. Patients should be instructed to do self-catheterization; this has been found to be a more efficient way of reducing the residual urinary volume than pharmacological treatment. With more severe symptoms, cholinergic drugs are used to increase intravesical pressure and maintain a small bladder capacity. Bethanecol is the best available drug. Sometimes surgery is needed. In more severe cases transurethral resection of the prostate may be required to reduce resistance to flow at the bladder neck, lowering the propulsive force needed to empty the bladder. The choice of treatment of diabetic impotence depends upon proper identification of the cause. If psychological factors dominate the picture, careful counseling of the patient and the partner may be helpful. In selected cases the implant of a penile prosthesis has been of value. Self-injection with papaverine or prostaglandin E in the corpora cavernosa has also been shown to be effective. Use of silderefil or derivatives is under investigation and seems to be promising if contraindications are absent.

REFERENCES 1. 2. 3.

4. 5. 6.

Kopin I. Catecholamine Metabolism (and the Biochemical Assessment of Sympathetic Activity). London: W.B. Saunders, 1977. Schwartz TW, et al. Vagal cholinergic regulation of pancreatic polypeptide secretion. J Clin Invest 1978; 781–789. Rasmussen MH, et al. Impaired pancreatic polypeptide response to a meal in type I diabetic patients: vagal neuropathy or islet cell dysfunction? J Cardiovac Pharmacol 1993; 21:863–868. Palkovits M. The anatomy of central cardiovascular neurones. In Fuxe K, et al., eds. Central Adrenaline Neurons. Oxford: Pergamon Press, 1980:3–17. Thomas PK. Diabetic neuropathy: mechanisms and future treatment options. J Neurol Neurosurg Psychiatry 1999; 67:277–281. Diabetic Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993; 329:977–986.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

7. 8. 9.

10.

11. 12. 13.

14. 15. 16. 17.

18. 19.

20. 21. 22. 23. 24. 25.

Said G, Slama G, Selva J. Progressive centripetal degeneration of axons in small fibre diabetic neuropathy. Brain 1983; 106:791–807. Sugimura K, Dyck P.J. Multifocal fibre loss in proximal sciatic nerve in symmetric diabetic neuropathy. J Neurol Sci 1982; 53:501–509. Tomlinson DR. Role of aldose reductase inhibitors in the treatment of diabetic polyneuropathy. In: Dyck PJ, Thomas PK, eds. Diabetic Neuropathy. 2d ed. Philadelphia: W.B. Saunders, 1999:330–340. Bradley JL, Thomas PK, King RHM, et al. Myelinated nerve fibre regeneration in diabetic sensory polyneuropathy: correlation with type of diabetes. Acta Neuropathol 1995; 90:403–410. Eisenbarth GS. Type I diabetes mellitus. A chronic autoimmune disease. N Engl J Med 1986; 314:1360–1368. Brown FM, et al. Inheritance of anti-autonomic nervous system antibodies in type I diabetes (abstr). Diabetes 1990; 39(suppl 1):497. Rabinowe SL, et al. Complement fixing autoantibodies to the sympathetic nervous system and sciatic nerve in type I diabetes mellitus (XII Congress of the International Diabetes Federation). Diabetes Res Clin Pract 1988; 5:55–76. Low PA. Clinical Autonomic Disorders: Evaluation and Management. Philadelphia: Lippincott-Raven, 1997:509–527. Rundles RW Diabetic neuropathy. General review with report of 125 cases. Medicine 1945; 24:11–160. Ewing DJ, et al. Vascular reflexes in diabetic autonomic neuropathy. Lancet 1973; 2:1354–1356. Canal N, et al. The relationship between peripheral and autonomic neuropathy in insulin dependent diabetes: a clinical and instrumental evaluation. In: Canal N, Pozza G, eds. Peripheral Neuropathies. Amsterdam: Elsevier North Holland, 1978:247–255. Low PA, et al. Comparison of distal sympathetic with vagal function in diabetic neuropathy. Muscle Nerve 1986; 9:592–596. Dyck PJ, et al. The prevalence by stage severity of various types of diabetic neuropathy, retinopathy and nephropathy in a population based cohort: The Rochester Diabetic Neuropathy Study. Neurology 1993; 43:817–824. Ewing DJ, et al. Cardiac autonomic neuropathy in diabetes: comparison of measures of R-R interval variations. Diabetologia 1981; 21:18–24. Archer AG, et al. Blood flow patterns in painful diabetic neuropathy. Diabetologia 1984; 27:563–567. Low PA, et al. Sudomotor neuropathy. In: Dick PJ, et al., eds. Diabetic Neuropathy. Philadelphia: W.B. Saunders, 1987:140–145. Guy RJ, et al. Evaluation of thermal and vibration sensation in diabetic neuropathy. Diabetologia 1985; 28:131–137. Low PA, et al. The sympathetic nervous system in diabetic neuropathy. a clinical and pathological study. Brain 1975; 98:341–356. Veglio M, et al. Autonomic neuropathy in non insulin-dependent diabetic patients: correlation with age, sex, duration and metabolic control of diabetes. Diabetes Metab 1990; 16:200–206.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

26. 27.

28. 29.

30. 31. 32. 33. 34. 35. 36. 37.

38. 39. 40. 41. 42. 43. 44. 45. 46.

Young RJ, et al. Nerve function and metabolic control in teenage diabetics. Diabetes 1983; 32:142–147. Ewing DJ, et al. Autonomic neuropathy: its diagnosis and prognosis. In: Watkins PJ, ed. Clinics in Endocrinology and Metabolism. London: W.B. Saunders, 1986:855–888. Monserrat JM, et al. Ventilatory control in diabetes mellitus. Eur J Respir 1985; 67:112–117. Bellavere F, et al. Evidences of early impairment of parasympathetic reflexes in insulin-dependent diabetics without autonomic symptoms. Diabetes Metab 1985; 11:152–156. Bennet T, et al. Assessment of methods for estimating autonomic nervous control of the heart in patients with diabetes mellitus. Diabetes 1978; 27:1167–1174. Ewing DJ, et al. Immediate heart rate response to standing simple test of autonomic neuropathy in diabetes. Br Med J 1978; 1:145–147. Bennet T, et al. Cardiovascular reflex responses to apnoeic face immersion and mental stress in diabetic subjects. Cardiovasc Res 1976; 10:192–199. Angelone A, Coulter NA. Respiratory sinus arrthythmia: a frequency dependent phenomenon. J Appl Physiol 1964; 19:479–482. Appel PL, et al. Comparison of measurements of cardiac output by bioimpedence and thermodilution in severely ill surgical patients. Crit Care Med 1986; 14:933–935. Low PA. Laboratory Evaluation of Autonomic Function. Clinical Autonomic Disorders Evaluation and Management. Philadelphia: Lippincot-Raven 1997; 15:181–193. Akselrod S, et al. Hemodynamic regulation: investigation by spectral analysis. Am J Physiol 1985; 249:H867–H875. Pagani M, et al. Power spectral analysis of heart rate and arterial pressure variabilities as marker of sympatho-vagal interaction in man and conscious dog. Circ Res 1986; 59:178–193. Pomeranz B, et al. Assessment of autonomic function in humans by heart rate spectral analysis. Am J Physiol 1985; 248:H151–H153. Saul JP, et al. Transfer function analysis of the circulation: unique insights into cardiovascular regulation. Am J Physiol 1991; 261:H1231–H1245. Comi, et al. Spectral analysis of short-term heart rate variability in diabetic patients. J Auton Nerv Syst 1990; 30(suppl):S45–S49. Minami H, et al. The physiology and pathophysiology of gastric emptying in humans. Gastroenterology 1984; 86:1592–1610. Pfeifer M, et al. Autonomic neural dysfunction in recently diagnosed diabetic subjects. Diabetes 1984; 7:447–453. Smith SA, et al. Reduced pupillary light reflexes in diabetic autonomic neuropathy. Diabetologia 1983; 24:330–332. Smith SA, et al. Pupillary signs in diabetic autonomic neuropathy. Br Med J 1978; 2:924–927. Kennedy WR, et al. The sweating deficiency in diabetes mellitus: methods for quantitation and clinical correlation. Neurology (Cleveland) 1984; 34:758–763. Murat A, et al. Amélioration dela neuropathie périphérique et de la vidance gastrique après transplantation simultanée rénale et pancréatique. Diabetes Metab 1990; 16:419–420.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

18 Sympathetic Innervation of the Kidney in Health and Disease Lars Christian Rump Ruhr-University Bochum, Bochum, Germany

Kerstin Amann University of Erlangen, Erlangen, Germany

Eberhard Ritz Ruperto Carola University of Heidelberg, Heidelberg, Germany

The autonomic innervation of the kidneys shows large interspecies variations. In general, postganglionic efferent nerves originate from T5 to L3 with inputs from the splanchnic nerves, celiac and intermesenteric plexus. Preganglionic renal neurons receive projections from various centers in the brain. Renal sympathetic nerves release noradrenaline, ATP, and NPY as neurotransmitters with specific postsynaptic receptors located on vascular, glomerular, and tubular structures. No conclusive evidence exists for cholinergic or dopaminergic innervation. The renal sympathetic nerves regulate renin release (1 receptor), sodium and water reabsorption (1 2 receptor), and renal resistance (1 and 2 receptors) in a frequency-dependent manner. The afferent renal innervation with cell bodies in dorsal root ganglia of T6-L3 comprise calcitonin gene–related peptide and substance P–containing nerves. They are activated by mechanoreceptors that sense variations in hydrostatic pressure and chemoreceptors that respond to changes in the chemical environment of the interstitium. There is compelling evidence that efferent and afferent nerves are involved in the genesis of various forms of hypertension in experimental animals and humans. Furthermore, congestive heart failure, liver cirrhosis, and the nephrotic syndrome are associated with increased activity of renal sympathetic nerves. Only recently experimental and clinical studies demonstrated that sympathetic overactivity is a hallmark of chronic renal fail-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ure. Afferent signals arising from diseased kidneys project to the brain to increase renal neurotransmitter release, which induces proliferative processes and contributes to the development of hypertension. On the other hand, inhibition of sympathetic nerve activity slows progression of renal disease even independently from blood pressure reductions in an animal model of chronic renal failure. Thus, it is possible that interventions to interfere with renal sympathetic overactivity will provide new therapeutic approaches.

I. ANATOMY AND PHYSIOLOGY OF RENAL AUTONOMIC INNERVATION The innervation of the kidney by the autonomic nervous system is complex and shows large interspecies differences with respect to the nerves that enter (efferent) or leave (afferent) the kidney (1,2). For reasons of clarity the afferent innervation will be discussed later in some detail (intrarenal receptors and afferent innervation). The postganglionic nerves that enter the kidney originate at levels T5 to L3 (1) and receive inputs from there major sources: the celiac plexus (aorticorenal ganglion, celiac ganglion, and major splanchnic nerves), the thoracic and lumbar sympathetic trunk (thoracic and lumbar splanchnic nerves), and the intermesenteric plexus. There are some inputs from the vagus nerve to the celiac ganglion, but no direct evidence exists for parasympathetic fibers entering the kidney (2,3). The pathways from the central nervous system (CNS) to the kidney have been identified. At least in the rat, the preganglionic renal neurons in the intermediolateral column between T5 and T13 receive projections from the medullary raphe nuclei, the rostral ventral medulla, A5 cell group, and the paraventricular hypothalamic nucleus (4). Although dopamine is present in sympathetic nerves and prolonged stimulation of renal nerves increases the concentration of dopamine in the renal vein, there is no convincing structural or functional evidence for dopaminergic renal nerves (5,6). Most if not all efferent renal nerve fibers enter the kidney along with the renal blood vessels and supply the renal cortex and the outer medulla. They have been shown to innervate interlobar, interlobular, arcuate, and vasa recta as well as afferent and efferent arterioles (2). The greatest density of neuroeffector junctions is seen in the juxtaglomerular region of the inner cortex (7), whereas the inner medulla and the papillae apparently lack sympathetic innervation (8). In addition, there is evidence of direct renal tubular innervation. Most of the sympathetic neuroeffector junctions are located in the proximal tubule, thick ascending loop of Henle, distal convoluted tubule, proximal tubule, and collecting duct (9). Thus, sympathetic neurotransmitters released within the kidney are able to modulate renal function at various levels by activating specific receptor systems on renal target cells.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Noradrenaline is the major but not the only renal sympathetic neurotransmitter. At least in kidneys of rats, pigs, and humans, ATP (10–14) and neuropeptide Y (NPY) (14–16) are released from sympathetic nerves as cotransmitters of noradrenaline. Receptors for these neurotransmitter are either located on the sympathetic nerve ending itself (presynaptic receptors) or on the innervated renal structures (postsynaptic receptors). The most important presynaptic receptor in the kidney are those activated by endogenous substances. Thus, noradrenaline activates presynaptic 2 adrenoceptors to inhibit its own release (autoinhibition); 2 adrenoceptors are subdivided in four subtypes (2A, 2B, 2C, and 2D) (17). In the rat the presynaptic receptor seems to be of the 2D subtype (18), whereas in the human kidney functional evidence suggests the coexistence of 2A and 2C subtypes (19,20). This finding is supported by recent evidence from knockout mice suggesting the presence of more than one functional presynaptic 2 subtype present on one sympathetic nerve ending (21). Enhanced renal neurotransmitter release is observed in a genetic model of hypertension (spontaneously hypertensive rat), but no evidence for altered 2D autoreceptor modulation of renal noradrenaline release was observed (18). Other inhibitory renal presynaptic receptors include prostaglandin E2, dopamine D2, NPY-Y2, adenosine, and ATP-P2 receptors (6,11,22–25). On the other hand, activation of angiotensin II, bradykinin, and 2 receptors has been shown to enhance noradrenaline release in various species, including the human kidney (26). These facilitatory receptor systems are activated by endogenous substances. In renal diseases they are of special importance with respect to pathophysiological effects of noradrenaline and its cotransmitters, as discussed below. What are the effects of sympathetic nerve stimulation? Postsynaptic receptors for noradrenaline, NPY, and ATP regulate various renal functions. The effect of sympathetic nerve stimulation depends largely on the distribution of the specific receptor subtypes within the kidney. In general, direct stimulation of renal sympathetic nerves induces -adrenergic receptor–mediated constriction of afferent and efferent arterioles and the increase in resistance seems is slightly more pronounced in afferent than efferent arterioles (1). There are, however, considerable species differences with respect to -adrenergic receptor distribution (27). Briefly, in rats the  adrenoceptor regulating renal blood flow is mainly of the 1 subtype (28), whereas in rabbits and dogs both 1 and 2 subtypes are involved (29,30). The involvement of 2 adrenoceceptor–mediated renal vasoconstriction is underestimated from studies in isolated kidneys, since circulating angiotensin may be necessary to unmask 2 adrenoceptor–mediated effects on renal blood flow (31). They may be more important in hypertensive animals as shown in SHR kidneys (31). In contrast to other species, studies in humans suggest that 2 adrenoceptors dominate the regulation of renal blood flow (32), although evidence for 1 adrenoceptor–mediated vasoconstriction has been found in human renal blood vessels in vitro (33). Sympathetically mediated renal vasodilation can pos-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

sibly be achieved by activation of renovascular 2 adrenoceptors (34), but this effect is of uncertain physiological importance, since noradrenaline—in contrast to adrenaline—is a weak agonist at the 2 adrenoceptor. Stimulation of renal nerves in rats causes sodium, water, and bicarbonate reabsorption (1). The antidiuretic and antinatriuretic response is mediated by tubular 1 adrenoceptors of the 1A or 1B subtype (1). Other effects mediated by renal 1 adrenoceptors are stimulation of gluconeogenesis (35) and release of prostaglandins (23,36). In addition to noradrenaline, neuronally released ATP and NPY are both able to modulate renal blood flow. At least in the rat, NPY seems to act as a master hormone to modulate the vasoconstrictor effects of its cotransmitters, especially in a situation of elevated sympathetic tone (14), by activating the Y1-receptor subtype located on smooth muscle cells. The vascular effects of NPY are possibly of clinical relevance because NPY plasma levels are increased in patients with diabetic nephropathy (37), end-stage renal disease (38), and after renal transplantation (39). ATP is released into the extracellular space upon renal nerve stimulation. It increases renal vascular resistance through activation of purinoceptors of the P2X subtype (14,40). In contrast, ATP released from endothelial cells or other nonneuronal sources vasodilates the renal vasculature by activating P2Y receptors in a nitric oxide–dependent manner (41). In addition, P2Y receptor activation has also been implicated in renin release from juxtaglomerular cells (42,43) and renal gluconeogenesis (44). Besides the classical responses to renal nerve stimulation, sympathetic neurotransmitters may induce proliferative effects by activating specific postsynaptic receptors. Thus, it has been shown that adrenaline and the nonselective  adrenoceptor agonist isoprenaline stimulates DNA replication and mitogen-activated protein kinase (MAPK) in cultured proximal tubular cell lines (45,46). Similar observations have been made for NPY in vascular smooth muscle cells (47) and cardiomyocytes (48), in which NPY also potentiates 1 adrenoceptor–mediated MAPK activation. There is preliminary evidence that synergistic proliferative effect may also occur in human renal cells in culture (49). A growth-promoting role for extracellular ATP has been demonstrated in vascular smooth muscle (50) as well as mesangial and glomerular epithelial cells (49,51,52). Taken together, the efferent renal nerves release at least three different neurotransmitters. They not only influence renal function but also modulate renal proliferative processes through activation of specific receptor systems (Fig. 1). These long-term effects are probably of special importance with respect to end-organ damage in hypertension and progression of chronic renal failure. Based on a priori consideration, drugs such as vasodilators that activate the baroreceptor reflex leading to increased sympathetic activity and release of sympathetic cotransmitters may be inferior to sympatholytic drugs (53) or drugs that impair facilitatory presynaptic mechanisms of neurotransmitter release such as inhibitors of the renin-angiotensin system (54).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 1 Short- and long-term effects of sympathetic neurotransmitters (for explanation see text)

II. INTRARENAL RECEPTORS AND AFFERENT INNERVATION The kidneys are sensory organs with a dense afferent innervation (1). The course of the afferent renal nerves has been investigated by retrograde transport of horseradish peroxidase (55). Their cell bodies are located in the dorsal root ganglia of T6-L3 (56). The afferent nerves project directly to many brain areas, including the hypothalamus, the supraoptic and paraventricular nuclei, and the nucleus tractus solitarii (57,58). The intrarenal localization of afferent nerve fibers is further demonstrated by staining for the sensory neurotransmitters substance P and calcitonin gene–related peptide (CGRP). With this technique a vast number of sensory fibers were identified around the ureter and the renal pelvis, the venous as well as the arterial blood vessels, including afferent and efferent arterioles and peritubular capillaries (59,60). A smaller number of substance P–containing nerve fibers was found among the proximal and distal tubules. There are two types of renal sensory receptors—renal mechanoreceptors and renal chemoreceptors— which can be activated by substance P, CGRP, bradykinin, and prostaglandin E2, which seems to facilitate substance P release (1). The mechanoreceptors sense variations in hydrostatic pressure within the kidney, whereas the chemoreceptors respond to changes in the chemical environment of the renal interstitium that may be brought about by renal hypoxia/ischemia or uremia. Stimulation of afferent renal nerves has been shown to either inhibit or enhance efferent renal sympathetic

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

nerve activity leading to the hypothesis of excitatory and inhibitory afferent nerve fibers. An increased activity of mechanoreceptors by obstruction of urine flow in one kidney results in an inhibition of the contralateral efferent sympathetic nerve activity, thereby increasing urine flow and sodium excretion in the contralateral kidney (61). These renorenal reflexes may serve as a self-regulating system to balance excretory functions of the kidney (1). On the other hand, activation of mechanoreceptors and chemoreceptors by various experimental interventions increases efferent sympathetic nerve activity and blood pressure. Thus, intrarenal injections of the irritant phenol into the lower pole of the kidney of rats causes a permanent form of neurogenic hypertension (62), which could be prevented by cutting the renal nerves around the renal artery. Similar observations have been made in an experimental model of chronic renal failure (63), which will be discussed in more detail below. Renal chemoreceptors may also be activated by adenosine (64). Recordings of renal nerve activity have shown that intrarenal injections of adenosine induce an increase in ipsilateral and contralateral renal sympathetic nerve activity. This is important since in acute renal failure or ischemic nephropathy, marked intrarenal release of adenosine is likely. The stimulatory effect of adenosine on nerve traffic is accompanied by an increase in blood pressure, which is abrogated by ipsilateral renal denervation. Recently, it has also been shown that cyclosporin A causes hypertension in rats by activating afferent renal nerves (65). Apparently this involves synapsin-containing sensory microvesicles, since in knockout mice lacking the phosphoproteins synapsin I and II, the blood pressure increase by cyclosporin A is not observed.

III. EFFECT OF SYMPATHETIC ACTIVATION ON RENIN ANGIOTENSIN SYSTEM, RENAL HEMODYNAMICS, AND ELECTROLYTE EXCRETION In all species investigated renin release is brought about by 1 adrenoceptors, and intact renal nerves are essential for increasing renal angiotensin formation, as shown in rats with renal artery stenosis (66). Early clinical evidence for the important role of renal nerves for renin secretion was provided by study that showed that after renal transplantation the angiotensin II receptor blocker saralasin increased renin activity and lowered blood pressure only in patients with one or more innervated kidneys (67). The most clear-cut evidence for the direct effects of sympathetic activation and their respective dose-response relationship was shown in experiments evaluating the relation between the frequency of stimulation of the peripheral stump of renal nerves and the changes in renin release, sodium excretion, and vasoconstriction, respectively (68–72). Figure 2 shows that as the frequency of renal nerve stimulation increases, the first effect to be seen is increased renin release, followed by increased renal sodium retention and finally

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Relationship between the frequency of electrical stimulation of renal nerves and changes in renin release, sodium excretion, and vasoconstriction. (From Ref. 70.)

vasoconstriction with reduced renal cortical blood flow. These results in anesthetized animals had been confirmed by measurements of sympathetic activation using the carotid occlusion technique in conscious chronically instrumented dogs. Unloading the carotid sinus baroreceptors by this technique increases efferent renal sympathetic nerve activity by about 60% but produces little if any change in renal blood flow (72,73). The differential response to a graded activation of the renal nerves is of great importance because it explains several features of the pathophysiology of hypertension, congestive heart failure, liver cirrhosis, and hypovolemic conditions (see Secs. IV and V). The sympathetic control of renin release is quantitatively important as nonhypotensive doses of -blockers eliminate about 33% of the variability of plasma renin activity (74). The effect is directly related to receptor blockade and can be clearly dissociated from changes in renal blood flow and systemic blood pressure. At stimulation frequencies of the renal nerves of 1 Hz, when there are no effects on renal blood flow, selective concentrations of the 1 adrenoceptor blocker metoprolol prevent the rise in renin release (74,75). Higher stimulation frequencies induce  adrenoceptor–mediated renal vasconstriction leading to activation of baroreceptor and macula densa mechanisms of renin release. Still in this situation more than 30% of the renin release is under control of 1 adrenoceptors (76). The antinatriuretic action of sympathetic activation is due to increased tubular reabsorption and can be clearly dissociated from

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3 Autoregulation of renal blood flow (RBF) and glomerular filtration rate (GFR) and renin release in resting conscious dogs. (From Ref. 70.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

changes in glomerular filtration and filtered load of sodium. Decreases in sodium excretion occur at frequencies of renal nerve activity of 1–2.5 Hz that are subthreshold for decreases in renal blood flow (Fig. 2). The antinatriuretic effect is not surprising in view of the fact that 1 adrenoceptors have been demonstrated throughout the nephron with the exception of the cortical and outer medullary collecting ducts (77). The magnitude of the antidiuretic and antinatriuretic effects of renal sympathetic nerves can estimated by denervation experiments. It is proportional to the density of innervation of the nephron segments (1): thick ascending limb of the loop of Henle proximal tubule distal convoluted tubule. The 1 adrenoceptor–mediated stimulation of Na-K ATPase is enhanced by NPY in rat proximal tubular cells (78). Although 2 adrenoceptors are also widely distributed on the basolateral sites of proximal tubules and outnumber 1 adrenoceptors (3:1) as judged from binding studies (79), their functional role is uncertain (1). With respect to a functional role of renal postsynaptic 2 adrenoceptors, at least in the rat, they seem to play a role for sodium and water transport in the cortical collecting duct (80). As mentioned above, sympathetic activity affects renal blood flow and particularly autoregulation of renal blood flow. As shown in Figure 3, renin release, glomerular filtration rate, and renal blood flow are autoregulated, i.e., the three indices do not change over a wide range of blood pressure, but decrease once blood pressure is below a certain threshold. Figure 2 shows that sympathetic activation causes a frequency-dependent consecutive activation of renin release, sodium retention, and vasoconstriction. Similarly, when systemic pressure is lowered progressively, renin release increases first and renal blood flow last. This is modulated by sympathetic activity. Sympathetic activation shifts the threshold to the right. This effect is obliterated by  blockade, i.e., administration of prazosin.

IV. INVOLVEMENT OF RENAL NERVES IN GENESIS OF DIFFERENT FORMS OF HYPERTENSION The importance of the kidney in the genesis of primary hypertension has been known for a long time. Abnormal renal function seems to be a crucial step in the development and maintenance of hypertension. Experimental and clinical transplantation studies (81,82) indicate that hypertension goes with the (denervated) kidney transplant (83). Many experimental and clinical data (1,84), however, support the notion that alterations of afferent sensory or efferent sympathetic nerve activity play an important role in various forms of hypertension. Theoretically, there are many ways by which the renal nerves can contribute to hypertension. An increased firing rate of the efferent renal sympathetic nerves stimulates renin release in juxtaglomerular cells with increased local and systemic angiotensin II concentrations, increased tubular sodium and water reabsorption, as well as ele-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

vated renal vascular resistance. As a result the expansion of intravascular volume and the increase of blood pressure promote pressure natriuresis and thus reverse the increase in blood pressure (pressure-natriuresis relationship). It is well documented that high levels of efferent sympathetic nerve activity attenuate natriuresis in response to an increase of blood pressure. In anesthetized rats after renal denervation, fractional excretion of water and sodium is higher at every level of renal perfusion pressure than in rats with intact renal nerves (85). A similar shift of the pressure natriuresis relationship has been demonstrated in conscious dogs with bilateral carotid artery occlusion by using the 1 adrenoreceptor antagonist prazosin (86). There is now a substantial body of evidence to suggest that efferent sympathetic nerve activity is enhanced in patients with hypertension. Accordingly, renal noradrenaline spillover is more pronounced in patients with essential (87) and renovascular hypertension (88) than in normotensive controls. Administration of the nonselective 1, 2 adrenoceptor blocker phentolamine increased renal blood flow to a greater extent in hypertensive than in normotensive subjects (89), indicating a higher sympathetic tone of the renal vasculature of hypertensive kidneys. Further evidence for a contribution of increased renal nerve activity in the regulation of renal vascular resistance comes from another study in patients who received splanchnic blockade by bupivacaine (90). The injection of the anesthetic into the spinal canal increased renal blood flow only in the hypertensive and not in the normotensive subjects. The most compelling evidence for the involvement of renal nerves, however, comes from studies in experimental animals in which the renal nerves have been cut to prevent or attenuate the development of hypertension (1,84). These include established models of experimental hypertension such as the spontaneously hypertensive rat (SHR) (91–93), the spontaneously hypertensive stroke–prone rat (SHR-SP) (94), Goldblatt hypertension (95), the DOCA salt hypertensive rat (96), the rat renal wrap model of hypertension (97), and obesity-induced hypertension (98,99). In most of the studies a complete interruption of both afferent and efferent renal nerves was performed, whereas in others only the afferent renal nerves were cut selectively by dorsal rhizotomy. In the SHR the activity of the renal nerves is increased, as demonstrated by increased efferent nerve-firing rates (100) and a significantly greater exocytotic release of noradrenaline upon renal nerve stimulation compared to normotensive controls (18,101,102). The enhanced renal neurotransmitter release within in the kidney of the SHR is age dependent and not due to a defective  autoreceptor modulation since SHR and WKY kidneys have identical pharmacological properties (18). It is more likely that a diminished 1 adrenoceptor–mediated transjunctional adenosine modulation is involved (12). Interestingly, only complete renal denervation (93) delays the increase in blood pressure, which is then associated with an higher sodium output only in young (7 weeks) and not in old (103) SHR. Interrupting afferent signals by dorsal rhi-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

zotomy has no effect on the course of hypertension in SHR (104). This observation supports the notion of an altered efferent control of renal nerve activity in the SHR. In the rat complete renal denervation also prevents hypertension induced by hyperinsulinemia (105). Thus, it appears that as a rule renal denervation attenuates the development of hypertension. But this is clearly not the case. As a case in point renal denervation has no effect on blood pressure in the Dahl sensitive rat (106) and in hypertension-induced chronic inhibition of nitric oxide synthase (107).

V. AUTONOMIC RENAL NERVE ACTIVITY IN CONGESTIVE HEART FAILURE AND OTHER CONDITIONS Many edema-forming states are associated with sodium and water retention that can be explained by increased activity of the efferent renal sympathetic nerves. These states include congestive heart failure, liver cirrhosis, and nephrotic syndrome (108). In congestive heart failure, the sympathetic nervous system and the renin-angiotensin-aldosterone system are activated. It has been documented that the prognosis of the patients correlates not only with the degree of cardiac functional impairment but also with the degree of sympathetic activation (109). In very early stages of heart failure the high dynamic sensitivity of the arterial baroreceptors senses even a minor decrease in stroke volume and/or pulse pressure and responds by activating the renal sympathetic nerves (110). Thus, overt congestive heart failure with decreased blood pressure is not a prerequisite for an activation of renal sympathetic nerve activity. Antinatriuresis and antidiuresis in heart failure are clearly a consequence of increased efferent renal sympathetic nerve activity. This is seen even before alterations of systemic or renal perfusion pressure are present. Accordingly, enhanced renal noradrenaline spillover has been found in patients with congestive heart failure (111,112). It is important to remember that local angiotensin formation within the kidney and the heart may amplify and aggravate sympathetic overactivity by stimulating the release of noradrenaline via activation of presynaptic angiotensin (AT1) receptors representing a vicious circle (26,54). Increased spillover of noradrenaline from the kidneys has been shown also in patients with hepatic cirrhosis (113). The magnitude of spillover was not correlated with renal blood flow but with impaired salt and water excretion. In patients with the nephrotic syndrome, elevated plasma and urinary noradrenaline levels have been found (114,115). A detailed study of nephrotic patients with normal glomerular filtration rate documented that noradrenaline clearance rates were similar but renal noradrenaline spillover was increased as compared to controls (116). Formation of generalized edema in patients with liver cirrhosis and some patients with nephrotic syndrome is associated with underfilling of the circulation. This is reflected by the concentrations of volume-regulating hormones such as

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

high renin and ADH, but normal or low ANP plasma concentrations. The contribution of renal sympathetic nerves to retention of sodium and water in these states can be appreciated best from the responses to interventions that decrease sympathetic activity. In patients with hepatorenal syndrome (i.e., with ascites, impaired renal function, and absent response to volume expansion) bilateral lumbar sympathetic blockade increased renal blood flow and GFR resulting in natriuresis and diuresis (117). Similar results were obtained in rats with congestive heart failure and hepatic cirrhosis, in which renal denervation reduced sodium retention by about 35–45% (118). In addition to hypovolemia, specific hepatorenal baroreflexes and chemosensitive reflexes have been postulated to explain sympathetic overactivity in the hepatorenal syndrome (119). Other conditions that are probably associated with increased renal sympathetic nerve activity include type 2 diabetes (120), preeclampsia (121), and acute renal failure due to acute blood losses and hypoxia. Acute renal failure following cardiovascular surgery may have various causes: is the high sympathetic drive to the kidneys, which is elicited by manipulation of the infra- or suparenal aorta. One has to consider that noradrenalineinduced renal ischemia is an established experimental model of acute renal failure (122,123). In this context it is remarkable that preoperative treatment with clonidine (4
g/kg) prevents the deterioration of renal function after cardiac surgery (124). Further studies are needed to assess the role of renal sympathetic activity in patients at risk for acute renal failure. VI. EVIDENCE FOR SYMPATHETIC OVERACTIVITY IN RENAL FAILURE A. Hypertension in Renal Disease The issue of sympathetic overactivity in renal failure had not attracted much attention because conventional measurements of sympathetic activity, particularly noradrenaline concentrations, are not easily interpretable in renal disease since neuronal uptake of noradrenaline is reduced. It was only with the introduction of microneurographic techniques that Converse et al. (125) was able to document intense sympathetic overactivity in dialyzed patients. What was striking was the observation that after bilateral nephrectomy, sympathetic overactivity was completely abrogated and blood pressure was lower than in the group without nephectomy. The authors interpreted this finding as evidence that the kidney was the origin of stimulatory signals provoking sympathetic overactivity. This hypothesis finds strong support in a series of experiments of Campese (62,63,126,127). As shown in Figure 4, section of the dorsal roots (rhizotomy) abrogated to a considerable extent the progressive increase in systolic blood pressures seen in subtotally nephrectomized rats. This led to the hypothesis that in the diseased kidney mechanoreceptors or chemoreceptors are activated and afferent Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 4 Blood pressure in subtotally nephrectomized rats with chronic failure (CRF) is attenuated by cutting the afferent renal nerves (rhizotomy). (From Ref. 63.)

signals then travel via the dorsal roots into the CNS. Increased catecholamine turnover was documented in hypothalamic nuclei and brown fat of animals with renal damage (126–128). Sympathetic overactivity after subtotal nephrectomy was abrogated by renal denervation (126). It could also be shown that massive renal damage was not required to provoke sympathetic overactivity. One week after subtotal nephrectomy, rats show a progressive increase in systolic blood pressure (Fig. 4). Rats in which the dorsal roots on the level T10-L2 were cut (rhizotomy) on both sides had only slightly increased blood pressure. Noradrenaline turnover in posterior and lateral hypothalamic nuclei and the locus ceruleus was lower, indicating reduced central adrenergic activity (63). Application of as little as of 50
L of 10% phenol to the lower pole of one kidney caused persisting hypertension and sympathetic overactivity, which was reversed by renal denervation (62). The regulation of the hypothalamic activity involves stimulatory and inhibitory signals. Interleukin-1 was shown to stimulate (129) and activation of NO synthase (NOS) (130) to inhibit catecholamine turnover. Increased activity of NOS was interpreted as a compensatory mechanism, which was not completely successful in preventing hypothalamic overactivity. Accordingly, administration of an inhibitor of nitric oxide synthesis caused an increase in sympaCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

thetic outflow to the kidney, supporting the validity of this pathophysiological concept in the rat (131). In contrast, in healthy volunteers muscle sympathetic nerve activity does not increase during intravenous infusion of an inhibitor of NOS (132). The original observation of Converse concerned end-stage renal failure, but by now there is ample evidence of sympathetic overactivity in proteinuric and nonproteinuric primary renal disease (133,134). It has also been reported that surgical removal of the recipients’ contracted kidneys reduces sympathetic overactivity in renal allograft recipients (135). The stimulation of the sympathetic system is apparently more complex than was originally thought, when it was explained exclusively by stimulation of intrarenal chemo- or mechanoreceptors. There are strong interactions between the sympathetic system and the renin angiotensin system. As a case in point, Ligtenberg et al. (133) showed sympathetic overactivity in patients with preterminal renal failure. Long-term administration of enalapril significantly lowered sympathetic overactivity and more specifically shifted the dose-response relationship between blood pressure and microneurographic activity in the nervus suralis. Enalapril does not readily penetrate into the central nervous system so one could speculate that a peripheral interaction of the renin-angiotensin system with afferent sensory nerves might be involved. Interestingly long-term administration of a subpressor dose of angiotensin II to rats leads to pressor hyperresponsiveness and slow development of hypertension with an upregulation of mRNA expression for calcitonin gene–related peptide in dorsal root ganglia (136). In this context it is of interest that the renin system is variably activated in different renal diseases. In polycystic kidney disease (ADPKD), overexpression of renin was shown not only in the juxtaglomerular apparatus, but also in arterioles (137) as well as in tubular epithelial cells (138). In proteinuric renal disease, protein-loaded proximal tubular epithelial cells express all components of the renin system. Recent experimental evidence indicates that such overexpression is downregulated by administration of angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers, while renin and angiotensin II expression in the juxtaglomerular apparatus is upregulated secondary to interruption of the short feedback loop (139). It is even conceivable that further mechanisms stimulate the sympathetic nerve system. For instance, leptin concentrations are increased in renal disease (140), and leptin has been shown to stimulate the sympathetic nerve system (141). B. Involvement in Renal Progression While very good experimental and clinical evidence had been provided for a deleterious role of the activation of the renin angiotensin system in progression of renal disease, until recently no such information had been available with respect to the sympathetic nerve system. Amann et al. (142) had reasoned that sympathetic

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

overactivity, either directly or indirectly (e.g., via activating the renin angiotensin system), might have a deleterious effect on progression of renal disease. This hypothesis was subjected to an experimental test in an established model of progressive renal damage, i.e., the subtotally nephrectomized rat. Sympathetic overactivity was reduced by administering moxonidine, a sympathoplegic agent that diminishes sympathetic activity (143). It was used at a dose that failed to affect blood pressure as measured by telemetric monitoring. After subtotal nephrectomy, nephrosclerosis and albuminuria were seen. Both indices of renal damage were significantly reduced by administration of nonhypotensive doses of moxonidine. The course of proteinuria and representative histological samples of treated and untreated rats are shown in Figures 5 and 6. This is not a pharmacological effect of moxonidine on the kidney, since the comparable results could be obtained by using low nonhypotensive doses of -blockers (144). The beneficial effect of nonhypotensive doses of -blockers on renal morphology and albuminuria in the renal ablation model argues for a blood pressure–independent role of sympathetic overactivity in the genesis of progression. In addition, the effect of  adrenoceptor blockade indicates that a substantial part is not mediated by the sympathetic cotransmitters such as ATP and NPY. Definite proof for a role of sympathetic overactivity in progression has been provided by the observation that surgival denervation is similarly effective in ameliorating progression in renal damage models (145). The question arises whether the sympathetic nerve system is relevant for progression of renal disease in humans as well. At least in one disease, i.e., incipient nephropathy of type 1 di-

Figure 5 Urinary albumin excretion of subtotally nephrectomized rats is significantly attenuated by moxonidine treatment. (Data from Ref. 142.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

A

B Figure 6 (A) Glomerulus of a moxonidine (1.5 mg/kg body weight/day) treated subtotally nephrectomized rat with minimal mesangial matrix expansion. (B) Glomerulus of untreated subtotally nephrectomized rat with moderate glomerulosclerosis, i.e., mesangial matrix expansion and minimal segmental hypercellularity

abetes, Strojek et al. (146) observed that moxonidine in doses that failed to lower blood pressure by ambulatory blood pressure monitoring caused a significant reduction in urinary albumin excretion, as shown in Figure 7. Several important issues remain unresolved. Is the effect of increased sympathetic activity (and its reversal by pharmacological or surgical manipulation) a direct one, or is it indirectly mediated via the known effects of adrenergic innerCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 7 Albumin excretion rate in nighttime urine collections at the end of a 3-week period of placebo and 3-week period of moxonidine. Open squares: patients who received placebo first; filled triangles: patients who received moxonidine first. (From Ref. 146.)

vation on the juxtaglomerular apparatus? Catecholamines are known to affect several processes that are operative in progression of renal disease. For instance, they have proproliferative action (147), they affect the function of podocytes, a key cell involved in mediating glomerular damage and provoking proteinuria (49,148), and they may indirectly promote progression by vasoconstriction of cortical vesCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

sels and promoting cortical hypoxia, which has been shown to increase renal scarring (149). This is of more than academic interest because one would anticipate that pharmacological blockade of the sympathetic system would have additive benefit, superimposed on ACE inhibition, at least if the deleterious effect of sympathetic activity is not completely mediated via the RAS. REFERENCES 1. DiBona GF, Kopp UC. Neural control of renal function. Physiol Rev 1997; 77:75–197. 2. Barajas L, Liu L, Powers K. Anatomy of the renal innervation: intrarenal aspects and ganglia of origin. Can J Physiol Pharmacol 1992; 70:735–749. 3. Gattone VH, Marfurt CF, Dallie S. Extrinsic innervation of the rat kidney: a retrograde tracing study. Am J Physiol 1986; 250(2 Pt 2):F189–196. 4. Schramm LP, Strack AM, Platt KB, Loewy AD. Peripheral and central pathways regulating the kidney: a study using pseudorabies virus. Brain Res 1993; 616:251–262. 5. Dibona GF. Renal dopamine containing nerves. What is their functional significance? Am J Hypertens 1990; 3:64S–67S. 6. Rump LC, Schwertfeger E, Schuster MJ, Schaible U, Frankenschmidt A, Schollmeyer PJ. Dopamine DA2-receptor activation inhibits norepinephrine release in human kidney slices. Kidney Int 1993; 43:197–204. 7. McKenna OC, Angelakos ET. Adrenergic innervation of the canine kidney. Circ Res 1968; 22:345–354. 8. Dieterich HJ. Electron microscopic studies of the innervation of the rat kidney. Z Anat Entwicklungsgesch 1974; 145:169–186. 9. Barajas L, Powers K, Wang P. Innervation of the renal cortical tubules: a quantitative study. Am J Physiol 1984; 247(1 Pt 2):F50–60. 10. Schwartz DD, Malik KU. Renal periarterial nerve stimulation-induced vasoconstriction at low frequencies is primarily due to release of a purinergic transmitter in the rat. J Pharmacol Exp Ther 1989; 250:764–771. 11. Rump LC, Wilde K, Schollmeyer P. Prostaglandin E2 inhibits noradrenaline release and purinergic pressor responses to renal nerve stimulation at 1 Hz in isolated kidneys of young spontaneously hypertensive rats. J Hypertens 1990; 8:897–908. 12. Bohmann C, Rump LC, Schaible U, von Kügelgen. -Adrenoceptor modulation of norepinephrine and ATP release in isolated kidneys of spontaneously hypertensive rats. Hypertension 1995; 25:1224–1231. 13. Rump LC, Bohmann C, Schwertfeger E, Krumme B, Von Kügelgen I, Schollmeyer P. Extracellular ATP in the human kidney: Mode of release and vascular effects. J Auton Pharmacol 1996; 16:371–376. 14. Oberhauser V, Vonend O, Rump LC. Neuropeptide Y and ATP interact to control renovascular resistance in the rat. J Am Soc Nephrol 1999; 10:1179–1185. 15. Pernow J, Lundberg JM. Modulation of norepinephrine and neuropeptide Y (NPY) release in the pig kidney in vivo: involvement of 2, NPY and angiotensin II receptors. Naunyn-Schmiedebergs Arch Pharmacol 1989; 340:379–385.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

16.

17.

18.

19.

20.

21.

22.

23.

24. 25.

26.

27. 28. 29. 30. 31.

Oellerich FW, Malik KU. Neuropeptide Y modulates the vascular responses to periarterial nerve stimulation primarily by a postjunctional action in the isolated perfused rat kidney. J Pharmacol Exp Ther 1993; 266:1321–1329. Bylund DB, Eikenberg DC, Hieble JP, Langer SZ, Lefkowitz RJ, Minneman KP, Molinoff PB, Ruffolo RR Jr, Trendelenburg U. International Union of Pharmacology nomenclature of adrenoceptors. Pharmacol Rev 1994; 46:121–136. Bohmann C, Schaible U, Schollmeyer P, Rump LC. 2D-Adrenoceptors modulate renal norepinephrine release in normotensive and spontaneously hypertensive rats. Eur J Pharmacol 1995; 217:283–292. Trendelenburg AU, Limberger N, Rump LC. Alpha2C-adrenergic receptors mediate inhibition of norepinephrine release in human kidney cortex. Mol Pharmacol 1994; 45:1168–1176. Trendelenburg U, Sutej I, Wahl CA, Molderings GJ, Rump LC, Starke K. A reinvestigation of questionable presynaptic 2-autoreceptors: rat vena cava, rat atria, human kidney and guinea-pig urethra. Naunyn-Schmiedeberg’s Arch Pharmacol 1997; 356:721–737. Trendelenburg AU, Hein L, Gaiser EG, Starke K. Occurrence, pharmacology and function of presynaptic alpha2-autoreceptors in alpha2A/D-adrenoceptor-deficient mice. Naunyn Schmiedebergs Arch Pharmacol 1999; 360:540–515. Rump LC, Schollmeyer P. Effects of endogenous and synthetic prostanoids, the thromboxane A2 receptor agonist U-46619 and arachidonic acid on [3H]-noradrenaline release and vascular tone in rat isolated kidney. Br J Pharmacol 1989; 97:819–828. Bohmann C, Schollmeyer P, Rump LC. Methoxamine inhibits noradrenaline release through activation of 1- and 2-adrenoceptors in rat isolated kidney: involvement of purines and prostaglandins. Naunyn-Schmiedeberg’s Arch Pharmacol 1993; 347:273–279. Bohmann C, Von Kügelgen I, Rump LC. P2-Purinoceptor modulation of rat renal neurotransmission. Br J Pharmacol 1997; 121:1255–1262. Rump LC, Riess M, Schwertfeger E, Michel MC, Bohmann C, Schollmeyer P. Prejunctional neuropeptid Y receptors in human kidney and atrium. J Cardiovasc Pharmacol 1997; 29:656–661. Rump LC, Bohmann C, Schaible U, Schultze-Seemann W, Schollmeyer PJ. Adrenergic, angiotensin II, and bradykinin receptors enhance neurotransmission in human kidney. Hypertension 1995; 26:445–451. Michel MC, Rump LC. Alpha-adrenergic regulation of human renal function. Fundam Clin Pharmacol 1996; 10:493–503. Wolff DW, Gesek FA, Strandhoy JW. In vivo assessment of rat renal alpha adrenoceptors. J Pharmacol Exp Ther 1987; 241:472–476. Strandhoy JW, Wolff DW, Buckalew VM Jr. Renal alpha 1- and alpha 2-adrenoceptor mediated vasoconstriction in dogs. J Hypertens Suppl 1984; 2:S151–153. Hesse IFA, Johns EJ. An in vivo study of the alpha-adrenoreceptor subtypes on the renal vasculature of the anaesthetized rabbit. J Auton Pharmacol 1984; 4:145–152. Bohmann C, Rist W, Schollmeyer P, Rump LC. Low concentrations of angiotensin II unmask vasoconstrictory 2-adrenoceptors in isolated perfused kidneys of spontaneously hypertensive rats. Cardiovasc Res 1995; 30:857–865.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

32.

33.

34.

35. 36.

37.

38.

39.

40.

41.

42. 43.

44. 45. 46.

47. 48.

De Leeuw PW, van Es PN, de Bos R, Birkenhager WH. Role of alpha 1- and alpha 2-adrenergic receptors in the human hypertensive kidney. Hypertension 1987; 9(6 Pt 2):210–212. Rump LC, Ruff G, Wolk V, Schollmeyer P. 2-Adrenoceptor activation inhibits noradrenaline release in human and rabbit renal arteries. Eur J Pharmacol 1991; 196:277–283. Nakamura KT, Alden BM, Matheme GP, Jose PA, Robillard JE. Ontogeny of renal hemodynamic response to terbutaline and forskolin in sheep. J Pharmacol Exp Ther 1988; 247:453–459. Baines AD, Drangova R, Ho P. Neural stimulation of gluconeogenesis in isolated pyruvate-perfused rat kidneys. Can J Physiol Pharmacol. 1988; 66:106–111. Cooper CL, Malik KU. Prostaglandin synthesis and renal vasoconstriction elicited by adrenergic stimuli are linked toactivation of alpha-1 adrenergic receptors in the isolated rat kidney. J Pharmacol Exp Ther 1985; 233:24–31. Satoh C, Satoh F, Takahashi K, Murakami O, Sone M, Totsune K, Yabe T, Ohneda M, Fukuda M, Sugimura K, Ogawa S, Nagakubo H, Sato T, Mouri T. Elevated plasma immunoreactive neuropeptide Y concentrations and its increased urinary excretion in patients with advanced diabetic nephropathy. Endocr J 1999; 46:139–146. Bald M, Gerigk M, Rascher W. Elevated plasma concentrations of neuropeptide Y in children and adults with chronic and terminal renal failure. Am J Kidney Dis 1997; 30:23–27. Kokot F, Adamczak M, Wiecedilcek A, Spiechowicz U, Mesjasz J. Plasma immunoreactive leptin and neuropeptide Y levels in kidney transplant patients. Am J Nephrol 1999; 19:28–33. Von Kügelgen I, Krumme B, Schaible U, Rump LC. Vasoconstrictor responses to the P2X-agonist ,-methylene ATP in human cutaneous and renal blood vessels. Br J Pharmacol 1995; 116:1932–1936. Rump LC, Oberhauser V, von Kügelgen I. Purinoceptors mediate renal vasodilation by nitric oxide dependent and independent mechanisms. Kidney Int 1998; 54:473–481. Churchill PC, Ellis VR. Purinergic P2y receptors stimulate renin secretion by rat renal cortical slices. J Pharmacol Exp Ther 1993; 266:160–163. Van Der Weyden L, Adams DJ, Morris BJ. Capacity for purinergic control of renin promoter via P2Y(11) receptor and cAMP pathways. Hypertension 2000; 36:1093–1098. Cha SH, Jung KY, Endou H. Effect of P2Y-purinoceptor stimulation on renal gluconeogenesis in rats. Biochem Biophys Res Commun. 1995; 211:454–461. Wolf G, Helmchen U, Stahl RA. Isoproterenol stimulates tubular DNA replication in mice. Nephrol Dial Transplant 1996; 11:2288–2292. Kribben A, Herget-Rosenthal S, Lange B, Michel MC, Philipp T. Stimulation of mitogen activated protein kinase and cellular proliferation in renal proximal tubular cells. Ren Fail 1998; 20:229–234. Nie M, Selbie LA. Neuropeptide Y Y1 and Y2 receptor-mediated stimulation of mitogen-activated protein kinase activity. Reg Pept 1998; 75–76:207–213. Pellieux C, Sauthier T, Domenighetti A, Marsh DJ, Palmiter RD, Brunner HR, Pedrazzini T. Neuropeptide Y (NPY) potentiates phenylephrine-induced mitogen-ac-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

49.

50. 51. 52.

53.

54.

55.

56.

57.

58. 59. 60. 61.

62.

63. 64.

tivated protein kinase activation in primary cardiomyocytes via NPY Y5 receptors. Proc Natl Acad Sci 2000; 97:1595–1600. Rump LC, Le Talec T, Oberhauser V. Sympathetic neurotransmitters ATP, neuropeptide and norepinephrine induce proliferation of human visceral glomerular epithelial cells. Hypertension 1999; 34:711. Erlinge D. Extracellular ATP: a growth factor for vascular smooth muscle cells. Gen Pharmacol 1998; 31:1–8. Schulze-Lohoff E, Ogilvie A, Sterzel RB. Extracellular nucleotides as signalling molecules for renal mesangial cells. J Auton Pharmacol 1996; 16:381–384. Harada H, Chan CM, Loesch A, Unwin R, Burnstock G. Induction of proliferation and apoptotic cell death via P2Y and P2X receptors, respectively, in rat glomerular mesangial cells. Kidney Int 2000; 57:949–958. Rump LC, Amann K, Orth S, Ritz E. Sympathetic overactivity in renal disease: a window to understand progression and cardiovascular complications of uraemia? Nephrol Dial Transplant 2000; 11:1735–1738. Rump LC. Invited comment: advantages of Ang II receptor blockade over ACE inhibition with respect to suppression of sympathetic activity: heartening news for the kidney? Nephrol Dial Transplant 1999; 14:556–559. Marfurt CF, Echtenkamp SF. Sensory innervation of the rat kidney and ureter as revealed by the anterograde transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) from dorsal root ganglia. J Comp Neurol 1991; 311:389–404. Su HC, Wharton J, Polak JM, Mulderry PK, Ghatei MA, Gibson SJ, Terenghi G, Morrison JF, Ballesta J, Bloom SR. Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: combined retrograde tracing and immunohistochemistry. Neuroscience 1986; 18:727–747. Ciriello J, Calaresu FR. Central projections of afferent renal fibers in the rat: an anterograde transport study of horseradish peroxidase. J Auton Nerv Syst 1983; 8:273–285. Ciriello J, Calaresu FR. Hypothalamic projections of renal afferent nerves in the cat. Can J Physiol Pharmacol 1980; 58:574–576. Barajas L, Liu L, Nishiyama C. Prenatal and postnatal development of the CGRPimmunoreactive innervation in the rat kidney. Neurosci Lett 1991; 133:219–224. Knight DS, Cicero S, Beal JA. Calcitonin gene-related peptide-immunoreactive nerves in the rat kidney. Am J Anat 1991; 190:31–40. Colindres RE, Spielman WS, Moss NG, Harrington WW, Gottschalk CW. Functional evidence for renorenal reflexes in the rat. Am J Physiol 1980; 239(3):F265–270. Ye S, Gamburd M, Mozayeni P, Koss M, Campese VM. A limited renal injury may cause a permanent form of neurogenic hypertension. Am J Hypertens 1998; 11(6 Pt 1):723–728. Campese VM, Kogosov E. Renal afferent denervation prevents hypertension in rats with chronic renal failure. Hypertension 1995; 25:878–882. Katholi RE, Whitlow PL, Hageman GR. Intrarenal adenosine produces hypertension by activating the sympathetic nervous system via the renal nerves in the dog. J Hypertens 1984; 2:349–359.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

65.

66.

67.

68. 69.

70. 71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

Zhang W, Li JL, Hosaka M, Janz R, Shelton JM, Albright GM, Richardson JA, Sudhof TC, Victor RG. Cyclosporine A-induced hypertension involves synapsin in renal sensory nerve endings. Proc Natl Acad Sci 2000; 97:9765–9770. Wagner C, Hinder M, Kramer BK, Kurtz A. Role of renal nerves in the stimulation of the renin system by reduced renal arterial pressure. Hypertension 1999; 34:1101–1105. Linas SL, Miller PD, McDonald KM, Stables DP, Katz F, Weil R, Schrier RW. Role of the renin-angiotensin system in post-transplantation hypertension in patients with multiple kidneys. N Engl J Med 1978; 298:1440–1444. DiBona GF. The functions of the renal nerves. Rev Physiol Pharmacol Biochem Pharmacol 1982; 94:76–181. Kirchheim H. Regulation of renal hemodynamics in congestive heart failure. In: Just H, Bussmann WD, eds. Vasodilators in Chronic Heart Failure. New York: SpringerVerlag, 1983, 36–46. Kirchheim H, Ehmke H, Persson P. Sympathetic modulation of renal hemodynamics, renin release and sodium excretion. Klin Wochenschr 1989; 67:858–864. Ehmke H, Persson P, Fischer S, Hackenthal E, Kirchheim H. Resetting of pressuredependent renin release by intrarenal alpha 1-adrenoceptors in conscious dogs. Pflugers Arch 1989; 413:261–266. Gross R, Hackenberg HM, Hackenthal E, Kirchheim H. Interaction between perfusion pressure and sympathetic nerves in renin release by carotid baroreflex in conscious dogs. J Physiol 1981; 313:237–250. Gross R, Kirchheim H. Effects of bilateral carotid and auditory stimulation on renal blood flow and sympathetic nerve activity in the conscious dog. Pflugers Arch 1980; 383:233–239. Osborn JL, DiBona GF, Thames MD. Beta-1 receptor mediation of renin secretion elicited by low-frequency renal nerve stimulation. J Pharmacol Exp Ther 1981; 216:265–269. Kopp U, Aurell M, Nilsson IM, Ablad B. The role of beta-1-adrenoceptors in the renin release response to graded renal sympathetic nerve stimulation. Pflugers Arch 1980; 387:107–113. Kopp U, Aurell M, Sjolander M, Ablad B. The role of prostaglandins in the alphaand beta-adrenoceptor mediated renin release response to graded renal nerve stimulation. Pflugers Arch 1981; 391:1–8. Cohen HT, Takemoto F, Satoh T, Katz Al. Renal adrenergic receptors: localization of [125]prazosin binding sites along the microdissected rat nephron. Can J Physiol Pharmacol 1992; 70:1016–1020. Holtback U, Ohtomo Y, Forberg P, Sahlgren B, Aperia A. Neuropeptide Y shifts equilibrium between alpha- and beta-adrenergic tonus in proximal tubule cells. Am J Physiol 1998; 275(1 Pt 2):F1–7. Schmitz JM, Graham RM, Sagalowsky A, Pettinger WA. Renal alpha-1 and alpha2 adrenergic receptors: biochemical and pharmacological correlations. J Pharmacol Exp Ther 1981; 219:400–406. Wiborn TW, Sun D, Schafer JA. Expression of multiple alpha-adrenoceptor isoforms in rat CCD. Am J Physiol 1998; 275(1 Pt 2):F111–118.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

81.

82.

83.

84. 85. 86. 87. 88.

89.

90.

91. 92.

93.

94.

95.

96.

97.

Rettig R, Bandelow N, Patschan O, Kuttler B, Frey B, Uber A. The importance of the kidney in primary hypertension: insights from cross-transplantation. J Hum Hypertens 1996; 10:641–644. Curtis JJ, Luke RG, Dustan HP, Kashgarian M, Whelchel JD, Jones P, Diethelm AG. Remission of essential hypertension after renal transplantation. N Engl J Med 1983; 309:1009–1015. Grisk O, Rose HJ, Rettig R. The development of post-transplantation hypertension in recipients of an SHR kidney is independent of reinnvervation of the graft. Pflugers Arch 1999; 438:502–507. Katholi RE. Renal nerves in the pathogenesis of hypertension in experimental animals and humans. Am J Physiol 1983; 245:F1–14. Roman RJ, Cowley AW Jr. Characterization of a new model for the study of pressure-natriuresis in the rat. Am J Physiol 1985; 248(2 Pt 2):F190-198. Ehmke H, Persson PB, Seyfarth M, Kirchheim HR. Neurogenic control of pressure natriuresis in conscious dogs. Am J Physiol 1990; 259(3 Pt 2):F466–473. Esler M, Jennings G, Lambert G. Noradrenaline release and the pathophysiology of primary human hypertension. Am J Hypertens 1989; 2(3 Pt 2):140S–146S. Johansson M, Elam M, Rundqvist B, Eisenhofer G, Herlitz H, Lambert G, Friberg P. Increased sympathetic nerve activity in renovascular hypertension. Circulation 1999; 99:2537–2542. Hollenberg NK, Adams DF, Solomon H, Chenitz WR, Burger BM, Abrams HL, Merrill JP. Renal vascular tone in essential and secondary hypertension: hemodynamic and responses to vasodilators. Medicine (Baltimore) 1975; 54:29–44. Lorelius LE, Lofroth PO, Morlin C, Wiklund L, Aberg H. Renal haemodynamics before and after splanchnic block in patients with hypertension. Scand J Clin Lab Invest 1978; 38:233–240. Liard JF. Renal denervation delays blood pressure increase in the spontaneously hypertensive rat. Experientia 1977; 33:339–340. Saynavalammi P, Vaalasti A, Pyykonen ML, Ylitalo P, Vapaatalo H. The effect of renal sympathectomy on blood pressure and plasma renin activity in spontaneously hypertensive and normotensive rats. Acta Physiol Scand 1982; 115:289–293. Winternitz SR, Katholi RE, Oparil S. Role of the renal sympathetic nerves in the development and maintenance of hypertension in the spontaneously hypertensive rat. J Clin Invest 1980; 66:971–978. Nagaoka A, Kakihana M. Effects of renal sympathectomy on sodium and water excretion in stroke-prone spontaneously hypertensive rats. Jpn J Pharmacol 1982; 32:591–597. Katholi RE, Winternitz SR, Oparil S. Decrease in peripheral sympathetic nervous system activity following renal denervation or unclipping in the one-kidney one-clip Goldblatt hypertensive rat. J Clin Invest 1982; 69:55–62. Takahashi H, Iyoda I, Yamasaki H, Takeda K, Okajima H, Sasaki S, Yoshimura M, Nakagawa M, Ijichi H. Retardation of the development of hypertension in DOCAsalt rats by renal denervation. Jpn Circ J 1984; 48:567–574. Kline RL, Denton KM, Anderson WP. Effect of renal denervation on the development of cellophane-wrap hypertension in rabbits. Clin Exp Hypertens A 1986; 3(8):1327–1342.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

98.

99.

100.

101. 102.

103. 104.

105. 106.

107.

108. 109. 110. 111.

112.

113. 114.

Kassab S, Kato T, Wilkins FC, Chen R, Hall JE, Granger JP. Renal denervation attenuates the sodium retention and hypertension associated with obesity. Hypertension 1995; 25:893–897. Rumantir MS, Vaz M, Jennings GL, Collier G, Kaye DM, Seals DR, Wiesner GH, Brunner-La Rocca HP, Esler M. Neural mechanisms in human obesity-related hypertension. J Hypertens 1999; 17:1125–1133. Lundin S, Ricksten SE, Thoren P. Renal sympathetic activity in spontaneously hypertensive rats and normotensive controls, as studied by three different methods. Acta Physiol Scand 1984; 120:265–272. Collis MG, DeMey C, Vanhoutte PM. Renal vascular reactivity in the young spontaneously hypertensive rat. Hypertension 1980; 2:45–52. Ekas RD Jr, Steenberg ML, Lokhandwala MF. Increased norepinephrine release during sympathetic nerve stimulation and its inhibition by adenosine in the isolated perfused kidney of spontaneously hypertensive rats. Clin Exp Hypertens A 1983; 5:41–48. Yoshida M, Satoh S. Role of renal nerves on pressure natriuresis in spontaneously hypertensive rats. Am J Physiol 1991; 260(1 Pt 2):F81–85. Janssen BJ, van Essen H, Vervoort-Peters LH, Struyker-Boudier HA, Smits JF. Role of afferent renal nerves in spontaneous hypertension in rats. Hypertension 1989; 13:327–333. Huang WC, Fang TC, Cheng JT. Renal denervation prevents and reverses hyperinsulinemia-induced hypertension in rats. Hypertension 1998; 32:249–254. Wyss JM, Sripairojthikoon W, Oparil S. Failure of renal denervation to attenuate hypertension in Dahl NaCl-sensitive rats. Can J Physiol Pharmacol 1987; 65:2428–2432. Granger J, Novak J, Schnackenberg C, Williams S, Reinhart GA. Role of renal nerves in mediating the hypertensive effects of nitric oxide synthesis inhibition. Hypertension 1996; 27(3 Pt 2):613–618. DiBona GF, Herman PJ, Sawin LL. Neural control of renal function in edemaforming states. Am J Physiol 1988; 254(6 Pt 2):R1017–1024. Cohn JN. Plasma norepinephrine and mortality. Clin Cardiol 1995; 18(suppl 1):19–12. Kirchheim HR. Systemic arterial baroreceptor reflexes. Physiol Rev 1976; 56:100–177. Esler M, Jennings G, Lambert G, Meredith I, Home M, Eisenhofer G. Overflow of catecholamine neurotransmitters to the circulation: source, fate, and functions. Physiol Rev 1990; 70:963–985. Hasking GJ, Esler MD, Jennings GL, Burton D, Johns JA, Korner PI. Norepinephrine spillover to plasma in patients with congestive heart failure: evidence of increased overall and cardiorenal sympathetic nervous activity. Circulation 1986; 73:615–621. Willett I, Esler M, Burke F, Leonard P, Dudley F. Total and renal sympathetic nervous system activity in alcoholic cirrhosis. J Hepatol 1985; 1:639–648. Tulassay T, Rascher W, Lang RE, Seyberth HW, Scharer K. Atrial natriuretic peptide and other vasoactive hormones in nephrotic syndrome. Kidney Int 1987; 31:1391–1395.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

115. 116.

117.

118. 119. 120. 121. 122.

123.

124.

125.

126. 127. 128. 129.

130.

131.

Kelsch RC, Light GS, Oliver WJ. The effect of albumin infusion upon plasma norepinephrine concentration in nephrotic children. J Lab Clin Med 1972; 79:516–525. Rahman SN, Abraham WT, Van Putten VJ, Hasbargen JA, Schrier RW. Increased norepinephrine secretion in patients with the nephrotic syndrome and normal glomerular filtration rates: evidence for primary sympathetic activation. Am J Nephrol 1993; 13:266–270. Solis-Herruzo JA, Duran A, Favela V, Castellano G, Madrid JL, Munoz-Yague MT, Morillas JD, Estenoz J. Effects of lumbar sympathetic block on kidney function in cirrhotic patients with hepatorenal syndrome. J Hepatol 1987; 5:167–173. DiBona GF, Sawin LL. Role of renal nerves in sodium retention of cirrhosis and congestive heart failure. Am J Physiol 1991; 260(2 Pt 2):R298–305. Morita H, Matsuda T, Tanaka K, Hosomi H. Role of hepatic receptors in controlling body fluid homeostasis. Jpn J Physiol 1995; 45:355–368. Perin PC, Maule S, Quadri R. Sympathetic nervous system, diabetes, and hypertension. Clin Exp Hypertens 2001; 23:45–55. Schobel HP, Fischer T, Heuszer K, Geiger H, Schmieder RE. Preeclampsia—a state of sympathetic overactivity. N Engl J Med 1996; 14:1480–1485. Baehler RW, Williams RH, Work J, Gottschall J, Chuang V. Studies on the natural history of the norepinephrine model of acute renal failure in the dog. Nephron 1980; 26(6):266–273. Schafferhans K, Heidbreder E, Grimm D, Heidland A. Norepinephrine-induced acute renal failure: beneficial effects of atrial natriuretic factor. Nephron 1986; 44:240–244. Kulka PJ, Tryba M, Zenz M. Preoperative alpha2-adrenergic receptor agonists prevent the deterioration of renal function after cardiac surgery: results of a randomized, controlled trial. Crit Care Med 1996; 24:947–952. Converse RL Jr, Jacobsen TN, Toto RD, Jost CM, Cosentino F, Fouad-Tarazi F, Victor RG. Sympathetic overactivity in patients with chronic renal failure. N Engl J Med 1992; 327:1912–1918. Ye S, Ozgur B, Campese VM. Renal afferent impulses, the posterior hypothalamus and hypertension in rats with chronic renal failure. Kidney Int 1997; 51:722–727. Bigazzi R, Kogosov E, Campese VM. Altered norepinephrine turnover in the brain of rats with chronic renal failure. J Am Soc Nephrol 1994; 4:1901–1907. Bigazzi R, Kogosov E, Campese VM. Altered norepinephrine turnover in the brown fat of rats with chronic renal failure. J Am Soc Nephrol 1994; 4:1896–1900. Ye S, Mozayeni P, Gamburd M, Zhong H, Campese VM. Interleukin-1 beta and neurogenic control of blood pressure in normal rats and rats with chronic renal failure. Am J Physiol Heart Circ Physiol 2000; 279:H2786–2796. Ye S, Nosrati S, Campese VM. Nitric oxide (NO) modulates the neurogenic control of blood pressure in rats with chronic renal failure (CRF). J Clin Invest 1997; 99:540–548. Sakuma I, Togashi H, Yoshioka M, Saito H, Yanagida M, Tamura M, Kobayashi T, Yasuda H, Gross SS, Levi R. NG-methyl-L-arginine, an inhibitor of L-arginine-derived nitric oxide synthesis, stimulates renal sympathetic nerve activity in vivo. A role for nitric oxide in the central regulation of sympathetic tone? Circ Res 1992; 70:607–611.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

132. 133.

134.

135.

136.

137.

138.

139.

140. 141.

142.

143.

144.

145.

Hansen J, Jacobsen TN, Victor RG. Is nitric oxide involved in the tonic inhibition of central sympathetic outflow in humans? Hypertension. 1994; 24:439–444. Ligtenberg G, Blankestijn PJ, Oey PL, Klein IH, Dijkhorst-Oei LT, Boomsma F, Wieneke GH, van Huffelen AC, Koomans HA. Reduction of sympathetic hyperactivity by enalapril in patients with chronic renal failure. N Engl J Med 1999; 340:1321–1328. Cerasola G, Vecchi M, Mule G, Cottone S, Mangano MT, Andronico G, Contorno A, Parrino I, Renda F, Pavone G. Sympathetic activity and blood pressure pattern in autosomal dominant polycystic kidney disease hypertensives. Am J Nephrol 1998; 18:391–398. Hausberg M, Kosch M, Barenbrock M, Hohage H, Kisters K, Rahn KH. Influence of remnant kidneys on muscle sympathetic nerve activity in renal allograft recipients. Kidney Blood Press Res 2000; 23:298–299. Wu W, Zhang Y, Ballew JR, Fink G, Wang DH. Development of hypertension induced by subpressor infusion of angiotensin II: role of sensory nerves. Hypertension 2000; 36:549–552. Zeier M, Fehrenbach P, Geberth S, Mohring K, Waldherr R, Ritz E. Renal histology in polycystic kidney disease with incipient and advanced renal failure. Kidney Int 1992; 42:1259–1265. Torres VE, Donovan KA, Scicli G, Holley KE, Thibodeau SN, Carretero OA, Inagami T, McAteer JA, Johnson CM. Synthesis of renin by tubulocystic epithelium in autosomal-dominant polycystic kidney disease. Kidney Int 1992; 42:364–373. Gilbert RE, Wu LL, Kelly DJ, Cox A, Wilkinson-Berka JL, Johnston CI, Cooper ME. Pathological expression of renin and angiotensin II in the renal tubule after subtotal nephrectomy. Implications for the pathogenesis of tubulointerstitial fibrosis. Am J Pathol 1999; 155:429–440. Stenvinkel P. Leptin and its clinical implications in chronic renal failure. Miner Electrolyte Metab 1999; 25:298–302. Takekoshi K, Motooka M, Isobe K, Nomura F, Manmoku T, Ishii K, Nakai T. Leptin directly stimulates catecholamine secretion and synthesis in cultured porcine adrenal medullary chromaffin cells. Biochem Biophys Res Commun 1999; 261:426–431. Amann K, Rump LC, Simonaviciene A, Oberhauser V, Wessels S, Orth SR, Gross ML, Koch A, Bielenberg GW, Van Kats JP, Ehmke H, Mall G, Ritz E. Effects of low dose sympathetic inhibition on glomerulosclerosis and albuminuria in subtotally nephrectomized rats. J Am Soc Nephrol 2000; 11:1469–1478. Bohmann C, Schollmeyer P, Rump LC. Effects of imidazolines on noradrenaline release in rat isolated kidney. Naunyn Schmiedebergs Arch Pharmacol 1994; 349:118–124. Amann K, Koch A, Hofstetter J, Gross ML, Haas C, Orth SR, Ehmke H, Rump LC, Ritz E. Glomerulosclerosis and progression—the effect of subantihypertensive doses of - and -blockers. Kidney Int 2001; 60:1309–1323. Odoni G, Ogata H, Oberhauser V, Amann K, Rump LC, Ritz E, Orth SR Renal denervation in subtotally nephrectomized (SNX)-rats attenuates structural renal damage. Hypertension 2000; 36:670 (PG.02).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

146.

Strojek K, Grzeszczak W, Gorska J, Leschinger MI, Ritz E. Lowering of microalbuminuria in diabetic patients by a sympathicoplegic agent: novel approach to prevent progression of diabetic nephropathy? J Am Soc Nephrol 2001; 12:602–605. 147. Garcia-Sainz JA, Vazquez-Prado J, Villalobos-Molina R. Alpha 1-adrenoceptors: subtypes, signaling, and roles in health and disease. Arch Med Res 1999; 30:449–458. 148. Huber TB, Gloy J, Henger A, Schollmeyer P, Greger R, Mundel P, Pavenstädt H. Catecholamines modulate podocyte function. J Am Soc Nephrol 1998; 9:335–345. 149. Norman JT, Clark IM, Garcia PL. Hypoxia promotes fibrogenesis in human renal fibroblasts. Kidney Int 2000; 58:2351–2366.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

19 Nerve Control of Bladder Function Giorgio Gabella University College London, London, United Kingdom

The nerve apparatus controlling the bladder is very complex and belies the apparent simplicity of the bladder function—storage and expulsion of urine. The autonomy of this organ from the central nervous system is minimal, and the control exerted by motor and sensory nerves is paramount. This chapter examines the structure of various components of this apparatus: it is a hierarchical system of nerves that form several specific pathways extending between the wall of the bladder and the brainstem. Twin centers in the pons preside over the separate and antagonistic functions of urine storage and urine voiding. They discharge excitatory and inhibitory impulses to the lower lumbar and sacral spinal cord. In the spinal cord the bladder autonomic motor neurons, grouped bilaterally into nuclei, issue fibers that project out of the central nervous system (CNS) and towards the bladder, through a lumbar (sympathetic) and a sacral (parasympathetic) outflow. In addition, somatic motor neurons from the sacral spinal cord project directly to the striated musculature of the urethral sphincter. The autonomic nuclei in the spinal cord receive impulses both from the brainstem pathways and from bladder afferent nerve fibers and regional interneurons. There are also influences from pathways to and from other pelvic organs. The main efferent pathways for the bladder are interrupted in the pelvic ganglion or other small ganglia of the pelvic plexus; synapses between pre- and postganglionic neurons are cholinergic and mainly with muscarinic receptors. Some evidence points to marked differences between species in the degree of filtering and integration in the pelvic ganglia. The nerves of the plexus are all mixed nerves, and axons of different origin type and function share the support of the same Schwann cells. A capsule insulates the nerves from the surrounding tissues down to the small branches. Nerve branching within the bladder wall is extensive; motor axons find their way to the musculaCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ture and, after repeated branching, end in long varicose portions. The varicosities are packed with vesicles, lose part of their Schwann cell wrapping, and lie close to the surface of a muscle cell, forming neuromuscular junctions. There are no postjunctional specializations in the muscle cells, but there is clustering of some of the receptors in the muscle cell membrane beneath the varicosities. The sensory innervation is provided by dorsal root ganglion neurons and is highly developed, most of the fibers forming a plexus just beneath the urothelium (and some twigs penetrating between the epithelial cells); the density is highest in the region of the trigone and is progressively less further away. Some sensory fibers are found in the muscle and the serosa. The number of afferent neurons projecting into the bladder is probably as high as the number of motor neurons. Afferent axons have long terminal branches within the bladder mucosa, displaying varicosities. They may respond to mechanical and chemical stimuli (including chemicals released by the urothelium) and may themselves release substances that affect the surrounding tissues, as in neurogenic inflammation. The afferent pathways from the bladder lead to dorsal root ganglia and to the dorsal horn of the lumbar and sacral spinal cord. Central terminals of sensory fibres feed on local interneurons, which either project onto the motor preganglionic neurons or form long pathways to brainstem nuclei that are connected to the bladder motor centers located there.

I. INNERVATION OF BLADDER The urinary tract assists the elimination of urine outside the body. The urine is produced continuously, although at a variable rate, in the kidneys, it is stored in the bladder, and it is expelled intermittently. The functioning of the bladder is under the paramount control of nerves, and these form an elaborate apparatus, which includes one of the largest sections of the autonomic nervous system. A. Pelvic Plexus The system under consideration—in the simplest possible structural sketch—consists of the lumbo-sacral segment of the spinal cord on the one side and the bladder on the other, namely, the main control center in the CNS and the effector organ. Between these two structures, which are placed about the midline, there are two sets of nerves, bilaterally symmetrical, rather laminar and meshlike, rooted in the spinal cord and spinal nerves centrally, and penetrating into the wall of the bladder and other pelvic organs peripherally. This mesh of nerves—one on the right and one on the left side—the largest part of which is the pelvic plexus, contains the pathways for impulses from the spinal cord to the muscular effectors in the bladder and the pathways for afferent (sensory) impulses from the bladder to the spinal cord (Fig. 1). The structure is Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 1 Pelvic plexus of an adult male rat stained in situ and in toto for acetylcholinesterase, seen from the right. The right major pelvic ganglion (the dark triangular mass immediately left of center, with a perforation near its top, which in vivo gives passage to a large artery for the bladder) is connected to the pelvic nerves (to the left), to the genital nerve (downward and to the left), to the hypogastric nerve (upwards) and to the numerous nerves for the pelvic organs (to the right). Part of the bladder and its neck are visible near the right edge and in the right bottom corner. The right seminal vesicle, with a bulbous profile, appears vertically through the middle of the field; the left seminal vesicle is next to it, and the vas deferens (dark and cylindrical) is further to the right; all three are crossed by the right ureter (light, cylindrical, and widening near the point of entry into the bladder). To the far left is part of the rectum, displaying its myenteric plexus. Accessory ganglia are seen along the nerve to the vas deferens and the urethra and along the hypogastric nerve. (6.) Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

made up of nerve fibers and ganglion nerve cells, the latter being located in dorsal root ganglia, in the pelvic ganglia, in accessory ganglia, and within the wall of the bladder itself. The nerve plexus provides also the innervation to the genital organs and contributes to the innervation of the rectum. The plexus is bilateral and symmetrical, but its layout varies considerably between species, a matter of biological variation that would deserve a more systematic study (1). In the cat and rabbit, as illustrated by Langley and Anderson (2), the plexus has many ganglia, which are scattered over a wide area and are interconnected by nerve trunks. Similarly, in the dog, the plexus comprises 70–100 ganglia of varying size and a complex network of connected nerve bundles, somewhat variable between individuals and even between the left and the right side of an individual (3). In the guinea pig, two main ganglia, or aggregates of ganglia, are located near the vas deferens (anterior pelvic ganglion) and near the neck of the bladder (posterior pelvic ganglion) (4,5). In the mouse there is a pelvic and a hypogastric ganglion, which are approximately the same size (6). In the male rat, Purinton et al. (7) illustrate a single large ganglion, diamond-shaped, with nerves issuing at each of its four corners; in the female, the ganglion is triangular and lies parasagittally by the junction of uterus and vagina (8). Both male and female rats have a single prominent pelvic ganglion (the major pelvic ganglion) in addition to a minute hypogastric ganglion and a few small accessory ganglia and innumerable nerves of all sizes. Although even within this species there are structural differences between different strains (Sprague-Dawley vs. Wistar, for example), the plexus in the rat has been taken—for the time being—as the paradigm on which to base a description of the components of the plexus in mammals (Fig. 1). The nomenclature used in the literature is not uniform, which is not really a problem in the sense that inaccurate anatomical description is. The position of the ganglia and their nerves is perforce different between males and females. Some authors refer to the major pelvic ganglion of the female as the Frankenhäuser’s or paracervical or uterine ganglion. The right and left pelvic ganglia of the female rat are in fact located immediately lateral to the uterine cervix, being firmly anchored to the wall of the uppermost part of the vagina; identification of the ganglion in the living animal and its surgical excision are more difficult than in the male counterpart. The major pelvic ganglion of the male lies on a lobe of the prostate. The ganglion is flattened laterally (although to a lesser extent than in the female) and has a major perforation for an artery (Fig. 1). Dorso-cranially the ganglion is connected to the pelvic nerve, often divided into two main trunks, which have an apparent origin from the spinal nerves (and the spinal cord). The pelvic nerve contains the parasympathetic preganglionic fibers (efferent) for the pelvic neurons; the pelvic nerve, like all the other nerves mentioned here, also has a large component of afferent (sensory) fibers. Cranially the ganglion is connected to the hypogastric nerve, a long nerve with an apparent origin from the inferior mesenteric ganglion (a sympathetic prevertebral ganglion). One or two swellings along the caudal part of the hypogastric nerve are the hypogastric ganglia, which are mainly Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

part of the sympathetic pathway. Dorso-caudally the genital nerve, the largest of the postganglionic nerves, issues from the ganglion, running parallel to the rectum and urethra and descending to the pelvic floor; the origin of this nerve is expanded by a large mass of nerve cells, a caudal appendix of the pelvic ganglion itself. The medial surface of the ganglion has several nerves, mainly for the prostate (or the uterus and vagina), while ventrally, where the limit of the ganglion is ill defined, there are many postganglionic nerves, including the urinary nerves and nerves for seminal vesicles and vas deferens; the latter nerves are associated with small accessory ganglia. B. Main Nerves of Plexus All nerves in the plexus are mixed nerves; that is, they comprise sensory and motor fibers and often a mixture of sympathetic and parasympathetic fibers. Their areas of distribution overlap. Hence, many surgical sections do not have strictly selective effects. The pelvic ganglion is both a site of origin of nerve fibers (the majority of post-ganglionic fibers) and a place of transit of fibers (including all the sensory fibers); extirpation of the ganglion has wide consequences. The pelvic nerve carries mainly the parasympathetic preganglionic fibers from the sacral or lumbo-sacral spinal cord. It also contains some sympathetic preganglionic fibers (9), many afferent fibers, and, at least in the rat (where the total number of fibers is about 5000) a large component of sympathetic postganglionic fibers (10). The hypogastric nerve carries sensory fibers and sympathetic fibers, including preganglionic fibers abutting on neurons of the hypogastric and the pelvic ganglia. The total number of fibers in the rat is about 1600 (10). The postganglionic nerves are numerous, variable in position, of different sizes, and of mixed composition; they link the ganglion to the urinary and genital organs of the pelvic cavity. The pudendal nerves carry somatic motor fibers directly to the striated musculature of urethra and bladder neck (external urethral sphincter) and many sensory fibers for these areas and for the external genitalia. In the rat, the total number of fibers is about 5000, of which 84% are sensory (10). Some nerve trunks connect the right and the left plexus across the midline, passing over the prostate or over the ventral and dorsal surface of the vagina. As in other parts of the autonomic nervous system, microscopic aggregates of neurons can be found within any of the nerve trunks of the plexus and they are functionally equivalent to the neurons in the main ganglia. C. Intramural Ganglia Some of the neurons innervating the bladder are located not within the pelvic or accessory ganglia, but as small aggregates along the postganglionic nerves within Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

the bladder itself; hence they are termed intramural ganglia. These neurons are similar in structure and function to those located in extrinsic ganglia, and they fit in the schematic plan of the innervation presented except for being located even more peripherally; however, they are exposed to trophic and mechanical conditions different from those found in the large extrinsic ganglia. Also, there is a problem in that intramural ganglion neurons are virtually absent in some species (such as the rat), while they are quite numerous in others (such as the guinea pig) and are present in small but variable numbers in others (such as humans). At least 500 neurons are found in the human bladder, predominantly within the muscle coat but also in the adventitia and serosa (11; see also Ref. 12). In the guinea pig there are over 2500 urinary intramural neurons (providing all or most of the parasympathetic innervation to the organ) (13,14), which have been well characterized histochemically and electrophysiologically (15,16). In contrast, the rat bladder has very few or no intramural neurons (14,17,18), although it has been noted that up to 200 neurons are present in neonates (19) or after unilateral extirpation of the pelvic ganglion (20). There is no explanation as to why the autonomic neurons for the bladder, all of which originate from the neural crest, should have such major differences in the extent of their peripheral migration in different species. In the rat it has been possible to excise the pelvic ganglion from its natural location and transplant it in the middle of the ventral wall of the bladder (21); many neurons survive beyond four months, preganglionic fibers reconnect with the ganglion, new synapses are formed, and there is extensive reinnervation of the musculature.

II. PELVIC GANGLION A. Components of the Ganglion The ganglia of the pelvic plexus-irrespective of whether there is a single ganglion or a set of smaller ganglia- are enclosed by a capsule, and are made of ganglion neurons with their satellite cells (Fig. 2). The ganglia also contain SIF cells, endoneural cells, fibroblasts, mast cells and blood vessels. There is also abundant connective tissue, mainly collagen, and thousands of incoming and outgoing nerve fibres (axons and their Schwann cells).

B. Capsule The capsule is a continuous layer of connective tissue at the outer surface of the ganglion. A capsule is found around all ganglia, being proportionally more conspicuous around larger ganglia, and is physically continuous with the epineurium Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Section through a pelvic ganglion. Several ganglion neurons are visible, most of which display the nucleus. No major cell process is seen emerging from a neuronal cell body. Between the neurons is the neuropil, which comprises myelinated and unmyelinated fibers, blood vessels, Schwann cells, and endoneural cell. (370.) (Modified from Ref. 82.)

of the nerves connected to the ganglion. The capsule provides a complete insulation of the ganglion cells and fibers and a strong protection from mechanical stresses, which must be considerable within the pelvic cavity. (To the electrophysiologist the capsule is also a physical obstacle to the penetration of the ganglion with a microelectrode.) The capsule is made of fibroblast-like cells, thin and wide, grouped tile-like into one to five single cell layers, which are covered by basal lamina and are separated by few collagen fibrils. The cells are joined to each other by tight junctions (occludens junctions), and this provides an impermeable barrier between the interstitial space of the ganglion (or nerve) and the spaces around the ganglion. C. Ganglion Neurons The ganglion neurons are nerve cells whose cell body resides in the pelvic ganglion—or an accessory ganglion—and whose axon (postganglionic fiber) projects Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

into the bladder wall and other target organs. The neurons are synaptically connected with axons (preganglionic fibers) originating from spinal cord neurons. The majority of neurons are parasympathetic and receive a synaptic input of preganglionic fibers from the lowermost lumbar and the sacral levels of the spinal cord via the pelvic nerves. A smaller number of neurons are sympathetic, and their preganglionic input is from preganglionic fibers originating in the lumbar spinal cord and traveling in the hypogastric nerve. Along both pathways, transmission from preganglionic to postganglionic elements is synaptic, cholinergic, and via muscarinic receptors (see Sec. II.L). D. Viscerotopic Distribution of Neurons Ganglion neurons tend to be positioned in the ganglia nearest, or in the portion of a ganglion nearest, to the target organ they innervate. This viscerotopic organization, however, is not strong and has little predictive value. In the major pelvic ganglion of the rat, the neurons innervating the colon, vas deferens, and penis are predominantly located near the exit of the nerves for those organs; those for the bladder appear scattered throughout the entire ganglion (22). E. Number of Neurons In the rat, the pelvic ganglion of the male has 14,000–15,000 ganglion neurons (23); the female has some 6000 (23) or 5000 (24). Although the method of neuronal counting used in these studies (counts of nucleoli on serial section of waxembedded ganglia) would not be regarded as acceptable today, there is certainly a large numerical difference between male and female rats, as already noted by Langworthy (25). The total number of pelvic neurons should be markedly higher than these figures suggest, because it should include neurons in accessory ganglia [about 400 according to Hondeau et al. (24)] and in the hypogastric ganglion [some 250 neurons according to Hondeau et al. (24)], and intramural ganglia when present; the figure should then be doubled, since there is a plexus on each side of the body. The sexual difference in neuron number is mainly accounted for by the fact that the genital organs of the male are more densely innervated than those of the female, hence they are served by a greater number of ganglion neurons. Whether there are also structural differences between males and females in the pelvic neurons that innervate bladder and rectum is unknown. Interestingly, there is evidence, mainly from adult male rats, that testosterone has marked effects on morphology, transmitter synthesis, and receptor expression not only in neurons involved in reproductive functions but also in motor and sensory neurons for the bladder (26). The pelvic ganglion of the male mouse contains some 3400 neurons (27). In the female guinea pig, the paracervical ganglion, a paired chain or aggregate of ganglia, with some bilateral asymmetry, providing the innervation to the uterus, Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

comprises some 2200 neurons on each side (28), while the motor innervation of the bladder is provided mainly by the intramural neurons. The size of the neuronal population (total number of neurons) in the adult is arrived at by cell division of neuronal precursors and neuroblasts, a process that, even in an altricial species such as the rat, takes place entirely prenatally; after birth, mitoses in a pelvic ganglion are only found among satellite cells, Schwann cells, fibroblasts, and endothelial cells, but not among neurons. The numerical difference between pelvic ganglia of male and female rats is much less substantial at birth than in the adult, suggesting the possibility of a developmental loss of neurons in the ganglia of the female (unpublished observations). Postnatal loss of neurons can occur after birth, notably in the male mouse after castration at birth: less than half the neurons survive into the adult, the effect being reversed by a prompt treatment with exogenous testosterone (27). In the mouse, a gender difference in pelvic ganglion size is already present at birth, the male ganglion being markedly larger; in addition, in the male, the left ganglion is significantly larger than the right one (29). When the bladder hypertrophies in the course of an outlet obstruction, there is a marked enlargement of the pelvic ganglion and hypertrophy of its neurons, but there is no generation of new neurons. The possibility that some loss of neurons takes place in the pelvic ganglion in old age (unrelated to the occurrence of neuropathies or physical damage) has been considered, but there are only limited data, and no indication of major changes. However, an increased spatial separation between sympathetic neurons, or a decrease in their spatial packing density, in aged rats as opposed to young adults, is interpreted by Warbuton and Santer (30) as evidence of loss of pelvic sympathetic neurons. The parasympathetic population shows changes in the same direction, but they are not statistically significant. F. Fine Structure of Ganglion Neurons The neurons measure 15–40
m in diameter. In rodents and in the guinea pig, they have few or no dendrites, hence the incoming synapses are mainly axosomatic. After intracellular injection of Lucifer yellow in rat pelvic neurons, Tabatabai et al. (31) observed one to four cell processes (one of which was the axon) per cell, in sharp contrast with an average of more than six processes per cell in the sympathetic neurons of the superior cervical ganglion of the same animals. The few dendrites observed with this technique in pelvic neurons are up to 50
m long and unbranched (31). In the mouse, ganglion neurons have no dendrites (6). There are few data on other species, but observations on the cat have shown pelvic neurons with an average of six to seven dendrites, which are 120–150
m long (32). The ultrastructure of pelvic neurons, although not studied in detail, is similar to that of other autonomic neurons. Rosettes of ribosome and cisternae of rough endoplasmic reticulm are abundant; the latter are dispersed through the cytoplasm and not grouped into distinct Nissl bodies. Mitochondria, Golgi complexes, and Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

thin bundles of microtubules are conspicuous in the cytoplasm. The location of the axon hillock and the initial tract of the emerging axon have not been characterized. G. Satellite Cells Satellite cells are glial cells wrapped around the cell body (or perikaryon) of ganglion neurons, for which they provide insulation and metabolic support. The cellular sheet (neuronal capsule) is continuous except where nerve processes issue from or abut upon the perikaryon. The neuronal capsule is made of a thin cytoplasmic lamina of tightly assembled cells, and it is thicker where the nucleus is housed. A tight barrier to direct diffusion of fluids from the extracellular space proper to the surface of the perikaryon is thus formed. The sheath of satellite cells is continuous with other glial cells, which are wrapped around dendrites, incoming axons, and outgoing axons. Glial cells of pelvic neurons are linked by gap junctions, which support metabolic and ionic coupling between all the cells that are wrapped around a neuron. Intracellular recording and intracellular injection of satellite cells in intramural ganglia of the bladder show the occurrence of die coupling among some (about 40%) of the satellite cells; the dye spreads to one to four other satellite cells around the same or an adjacent neuron (33). Satellite cells are positive for S100, but not for glial fibrillary acidic protein (33). H. Histochemistry of Ganglion Neurons In the rat pelvic ganglion many neurons are neuropeptide Y (NPY)–positive and so are many fibers in the detrusor muscle and around peripheral blood vessels. NPY labels nonadrenergic parasympathetic neurons and motor fibers in the detrusor as well as also adrenergic sympathetic perivascular fibers and many terminal nerve fibers or presynaptic endings (34). VIP-positive neurons are common but not as frequent as NPY-positive neurons (34,35). Substance P (SP) immunoreactivity is found in nerve fibers but not in ganglion neurons (34, male rat; 36, female rat); SP-positive fibers impinge upon more than one third of the neurons (36). Other neuropeptides founds in neurons and nerve endings in pelvic ganglia include enkephalin, somatostatin, colecystokinin, and dynorphin. It is possible to distinguish subpopulations of neurons on the basis of their neuropeptide content. However, the existence of a precise chemical coding for the neurons with different target and function remains a matter for speculation. Even with the detailed data of recent studies, for example, in the dog (3), the matter is unresolved. A minority of pelvic neurons are adrenergic (tyrosine-hydroxylase-immunoreactive). In the female rat, they are 9% of the total in the major pelvic ganglion, 13% in the hypogastric and the accessory ganglia (24). Of the intramural neurons of the human bladder, 14% are adrenergic (11); in the guinea pig the figure is 10% (37). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

I. Types of Neurons Many observations suggest that there are separate groups, or subpopulations, of neurons in the pelvic ganglion. Neurons can be subdivided on the basis of their peripheral projection to one of the pelvic organs: although diffusely distributed, the neurons tend to be located near the nerve they project into (3,22). Neurons can be subdivided into sympathetic and parasympathetic on the basis of the level of origin of their spinal cord input and the nature of their transmitters. Sympathetic neurons are in a minority, and they lie in parallel with the more numerous sympathetic neurons in the paravertebral and prevertebral ganglia. Pelvic ganglion sympathetic neurons are called “short adrenergic neurons” (38) because their axon travels only a short distance to reach their target. Further classifications of ganglion neurons based on the synaptic pattern, degree of convergence and divergence of the preganglionic input, distribution of receptors, and functional properties are likely to emerge. J. SIF Cells The pelvic ganglion contains a population of small intensely fluorescent (SIF) cells, which are also observed in sympathetic ganglia. The cells are small (5–10
m in diameter), are often clustered into groups of 3–10, and lie close to capillaries (38–40). The nucleus is spherical and has condensed chromatin; the cytoplasm and thick processes emanating from it are packed with large electron dense vesicles (or granules). These store noradrenaline or dopamine or serotonin, which give these cells an intense formaldehyde-induced fluorescence. Ultrastructurally, two subpopulations of SIF cells are distinguished on the basis of the size of the vesicles: one type has relatively small vesicles, 80–140 nm in diameter, while the other type has vesicles 200–300 nm in diameter. SIF cells are common in the pelvic ganglion and in sympathetic ganglia. Their number and distribution appear very variable, and their significance is not well clear. K. Synapses Synapses on ganglion neurons are predominantly, when not exclusively, on the perikaryon or on small intracapsular projections of both the sympathetic and parasympathetic neurons (Fig. 3). Some synapsing nerve endings are found in a deep tunneling invagination within the neuron (39). The synapsing nerve endings are relatively large (by comparison with those in sympathetic ganglia) and are packed with small clear vesicles 40–60 nm in diameter. They also contain mitochondria, occasional large granular vesicles, and some minute sacs of smooth endoplasmic reticulum. The ultrastructural features are consistent with cholinergic synapses. The vast majority of synapses disappear after preganglionic nerve section, confirming that they derive from preganglionic neurons in the spinal cord. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 3 Pelvic ganglion of a rat. A frozen section was stained with an antibody against synaptophysin (a protein located in synaptic vesicles) and examined in a fluorescence microscope. The bright spots are nerve endings packed with vesicles; they are mainly distributed around ganglion neurons and correspond to axon-somatic synapses from preganglionic nerve fibres. The cytoplasm of the neurons is faintly stained, while the nucleus is unstained. (360.)

In addition, the pelvic ganglion may contain a small number of adrenergic synapses. For example, in the guinea pig, 1% of all synapses in the pelvic ganglion are adrenergic (the nerve endings are packed with small granular vesicles) (41), and they are probably postganglionic sympathetic fibers abutting on parasympathetic cholinergic neurons. Histochemically, adrenergic endings are seen close to some nonadrenergic ganglion neurons (11,42). There are also small nerve endings synapsing onto SIF cells; they too are of preganglionic origin and cholinergic. Whether, or in what circumstances, SIF cells issue synapses onto ganglion neurons (as they do in the superior cervical ganglion, at least in the rat) is not certain; a study of the pelvic ganglion of male rats suggests that this may be the case (43). Processes of SIF cells, making some specialized contacts in the form of symmetrical membrane thickenings with principal neurons, are also interpreted as synapses (44). L. Ganglionic Transmission Excitatory synaptic transmission from the preganglionic fibers, both sympathetic and parasympathetic, to ganglion neurons is cholinergic, mediated by nicotinic Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

cholinergic receptors; transmission via these receptors is blocked by hexamethonium. There are also some muscarinic cholinergic receptors, which are inhibitory and are blocked by atropine (45), together with some muscarinic cholinergic receptors that are excitatory, in some species (46). These muscarinic receptors have a modulatory role in ganglion transmission. In the cat, for example, they may have a slow inhibitory effect on the ganglionic transmission mediated via nicotinic receptors; their slow inhibitory postsynaptic potentials are more evident with repetitive stimulation, are suppressed by atropine, and are mimicked by acetylcholine when the nicotinic receptors are blocked with hexamethonium (47). Neuropeptides colocalized with acetylcholine in nerve endings may act as cotransmitters and have an inhibitory or a facilitatory action, acting presynaptically or postsynaptically. Ganglion neurons also have adrenoceptors, with an inhibitory (alpha adrenoceptors) or excitatory function (beta adrenoceptors) (46). Every ganglion neuron is innervated and driven by preganglionic fibers in either the pelvic or the hypogastric nerve. Electrical stimulation of either nerve is followed by synaptically mediated impulses in the postganglionic nerves to the bladder (in addition to axonal volleys, which are due to fibers simply passing through the ganglion) (48–50). In the rat, the number of preganglionic inputs impinging onto a ganglion neuron is only one or two (each of which can have several synapsing terminals on the same neuron) (31). In contrast, in the cat, ganglion neurons have a synaptic input from seven preganglionic fibers, on average (31,51). In the guinea pig there can be up to 10 preganglionic fibers converging onto a ganglion neuron (52). There are no data on the extent of the divergence of preganglionic fibers, namely, the number of neurons that one fiber innervates with its terminal arborization. Both with divergence and convergence, the average figures may mask a variation between different functional groups of ganglion neurons. In the cat and the guinea pig, but not in the rat, the electrophysiological studies quoted above show that a small percentage of neurons receive a dual input from both sympathetic and parasympathetic fibers. On the basis of comparative electrophysiological studies of pelvic neurons in the cat and rat, de Groat and Booth (46) describe ganglia, such as those of the rat, where transmission occurs with a high safety factor: each neuron receives a synaptic input from only one or two preganglionic fibers, and the EPSPs they generate are supratheshold for initiating an action potential; similar findings are obtained in the mouse (6). In species such as the cat the safety factor is low, there is synaptic input from an average of seven preganglionic fibers, low-frequency stimulation produces EPSPs, which are usually subthreshold for initiating an action potential, and repetitive stimulation has a facilitatory effect, thus producing a high temporal facilitation. Based on this evidence, the pelvic ganglion of species such as the rat appears to function as a simple relay station, whereas in species such as the cat it has a more complex function, which includes filtering and temporal facilitation of the efferent impulses. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

III. NERVES AND NERVE FIBERS A. Preganglionic Sympathetic Fibers The sympathetic preganglionic neurons for the bladder are located in the lumbar spinal cord; in the rat, L1 and L2 are involved (53). They are part of a large population of neurons providing the sympathetic input to lumbar and pelvic organs and are located in the intermediomedial nucleus, the intercalated nucleus, the central nucleus, and the nucleus of the lateral funiculus of the spinal cord at levels L2–L5 (54,55). The axons of these neurons project ventrally, exit the spinal cord inside a ventral root, pass through a white ramus communicans and into the paravertebral sympathetic chain, where they predominantly acquire a descending course. Some axons terminate in the sympathetic chain, synapsing on ganglion neurons. Other axons continue into the splanchnic nerve and reach the inferior mesenteric ganglion (a prevertebral sympathetic ganglion) and synapse on ganglion neurons therein. Some preganglionic axon travel further away from the spinal cord and from the inferior mesenteric ganglion enter the hypogastric nerve; they terminate synapsing onto sympathetic neurons in the hypogastric or the pelvic ganglion. B. Preganglionic Parasympathetic Fibers Parasympathetic preganglionic neurons are located in the intermediolateral region of the lower lumbar and upper sacral spinal cord (55–57). In the rat, the levels involved are L6 and S1 (56). Their axons exit the spinal cord via a ventral root, then gather to form the pelvic nerves. In the cat, these axons are myelinated, with a conduction velocity of 2–15 m/sec (58); in the rat and mouse, they are unmyelinated and have a much lower conduction velocity (6,50). The fibers terminate synapsing on ganglion neurons in the pelvic ganglion or in an accessory ganglion. When the bladder contains intramural ganglion neurons, preganglionic parasympathetic fibers travel all the way from the sacral spinal cord to the wall of the bladder. C. Postganglionic Nerve Fibers The postganglionic sympathetic fibers for the bladder, the largest part of which are vasomotor fibers, arise at three different levels: paravertebral, prevertebral, and hypogastric or pelvic ganglia. The ratio of the length of preganglionic fibers over the corresponding postganglionic fibers is markedly different in the three groups of axons (the neurons in the pelvic ganglion are short adrenergic neurons innervated by a very long preganglionic fiber). Furthermore, a nerve such as the hypogastric nerve contains both preganglionic and postganglionic fibres. The conduction velocity is slower in the postganglionic fibres than in the corresponding preganglionic ones. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The postganglionic parasympathetic fibers are issued by ganglion neurons—in the pelvic ganglion, or in accessory ganglia, or in the bladder wall. They join other postganglionic fibers and sensory fibers and enter the bladder and spread over the entire wall. D. Nerves in Bladder Wall Postganglionic nerves radiate from the pelvic ganglion, predominantly in a parasagittal plane, and reach various pelvic organs. The nerves for the bladder emerge from the ventral aspect of the pelvic ganglion (Fig. 1). There are usually 6–12 nerves, which run to the bladder and penetrate its wall at various points situated around the site of entry of the ureters; blood vessels too enter at this level. The nerves then branch and spread over the bladder; although it is common to find branches that merge with each other, the predominant pattern of these nerves is tree-like, and the nerves branch and grow progressively smaller as they move away from the point of entry. The large nerve trunks lie predominantly in the serosa (Fig. 4), but as they branch the nerves penetrate further into the wall, first

Figure 4 Large nerve in transverse section within the bladder wall, photographed in a light microscope. Smooth muscle cells are seen at the top (obliquely sectioned) and to the right (transversely sectioned); the serosal side is at the bottom. Blood vessels, emptied of blood by the perfusion procedure, surround the nerve. The bulk of the nerve is made of unmyelinated axons, the outline of which can just be recognized in this micrograph; blurred dots within the axons are mitochondria. Four myelinated axons are visible. (1650.) (From Ref. 59.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

between the bundles of musculature, then in the mucosa. The nerves vary in size from trunks made of a thousand or more axons, at the point of entry into the bladder, to progressively smaller trunks, down to trunks of only a few axons and eventually to single axons. The nerve trunks contain both motor and sensory axons, which are thoroughly mixed and cannot be distinguished structurally except when seen near termination. However, the smaller nerve trunks within the musculature contain predominantly or exclusively motor axons, and those in the mucosa contain sensory axons. Only the axons that are myelinated, a small percentage of the total (less than 1% in the rat, more in the cat) can be identified as being afferent. Similarly, it is impossible to tell apart sympathetic (adrenergic) and parasympathetic (cholinergic) axons except by knowing their site of origin or by seeing their terminal portions or the cell body of origin. Sensory and motor axons, and axons of any type, seem to travel together within intramural nerves and to share the same Schwann cells.

Figure 5 Electron micrograph of an intramural nerve of the bladder. In this microscopic field several axon-glial bundles with about 85 unmyelinated axons and 3 glial nuclei are visible. Mainly longitudinally oriented collagen fibrils (fine dots of uniform size) occupy the spaces between axon bundles. (5200.) (Modified from Ref. 59.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 6 For these drawings, electron micrographs at 20,000 of a nerve of the bladder were traced by hand on tracing paper. The drawings are from the same nerve and illustrate different forms of display. (a) The lines represent the axolemma of every axon in this nerve, the parts of glial cell membrane at the surface of the axon-glial bundles beneath the basal lamina, and the inner surface of the capsule (outer line). (b) The axon-glial bundles are filled in. The white spaces are extracellular and would be occupied by collagen fibrils. (c) The axonal profiles have been filled in to highlight spatial density, size, and shape of the axons. (d) All the glial cell processes are filled in grey. (e) The extracellular space is filled in black. The major axis of this nerve is 19 mm.

The nerves have a capsule (the perineurium), which is continuous with the capsule of the pelvic ganglion, is made of thin laminar cells tightly joined, forms a continuous and impermeable barrier around the axons, and extends down to nerve trunks made of only 8–15 axons; the perineurium ends abruptly and is absent in the smallest nerve trunks. In consequence, in any part of the bladder wall the nerves are insulated from the extracellular space at large, except close to their termination. The metabolic supply to the nerves is separated from that of the bladder; it is provided by arterial vessels, usually one per trunk, which originate in the pelvic ganglion, travel inside the nerve, then turn into capillaries running between the axons within trunks down to the size of about 500 axons, and eventually they exit the nerve trunk. These data, from the rat, show that there is no blood return from the peripheral nerves into the ganglion (59). The unmyelinated axons, which amount to more than 90% of the total are fully surrounded by Schwann cells (Fig. 5). Ten to 20 axons are bundled together by a Schwann cell, each individually, so that there are never two axons in direct contact with each other, and they are not exposed to the extracellular space within the nerve except through the narrow laminar space provided by the mesoaxon (Fig. 6). The axon profiles range in diameter from 0.25 to 1.25
m (in the rat), and they are not circular (this would put constraints on their ability to withstand bendCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ing and compression) but oval or more irregular. Although they are not varicose, these axons oscillate considerably in size along their length, an ultrastructural detail that can only be seen with serial sections. Neurofilaments and small mitochondria are their main cellular components. There are also microtubules, sacs of endoplasmic reticulum, isolated large granular vesicles; clusters of small agranular vesicles (similar to those found in the varicosities of the terminal portions) are found along the axons (59). In contrast with the observations on rats reported above, Elbadawi (60) observes axo-axonal contacts between cholinergic and adrenergic axons in intramural nerves and terminal nerves of the bladder of cats and rats; these contacts, which are common in the musculature of the trigone, are described as being synaptic. The axons in the smallest nerve trunks and those traveling singly turn into terminal axons, which provide the motor and sensory contacts with the peripheral tissues (See Secs. IV.G and VI.C, respectively).

IV. NEUROMUSCULAR JUNCTIONS A. Nerve Terminal Branching After an extensive branching within the wall, down to very small bundles and single axons, the nerve fibers become terminal axons. While the nerve trunks branch and become progressively smaller and more numerous, the individual axons in them hardly branch at all. The terminal axons are those parts of the nerve fiber where sensory transduction or transmission to muscle cells takes place; they can be hundreds of
m long, present some branching, and lie very close to the innervated structures. B. Structure of Bladder Wall The effector of all motility in the bladder is the musculature. This is the smooth muscle in the bladder body (detrusor muscle), in the base of the bladder (trigonal muscle), in the neck (internal sphincter), and in intramural blood vessels. A ring of striated musculature is located outside the internal sphincter; in humans, is under voluntary control. There is no boundary between detrusor and trigonal muscle; the latter tends to be more compact and layered, and it continues into the musculature of the neck of the bladder without a distinct boundary. The muscle of the trigone has different functional properties from the detrusor (61) and probably has a specialized role related to its position between the ureteric and urethral openings. In addition, in large species, such as humans, there are conspicuous bundles of musculature in the lamina propria of the mucosa; these do not form a continuous layer but are amply connected to each other and represent a kind of muscularis mucosae, whose role in the bladder has not yet been clarified. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The detrusor muscle is a layer of tissue spread around the entire bladder, except the trigone; its thickness is relatively uniform around the bladder, but it varies by at least a factor of six between full and empty bladder. In the cat, the detrusor presents distinct layers of circular and longitudinal musculature (longitudinal here means in approximate continuation of the urethra in the direction of the cranial end of the bladder); longitudinal muscle bundles are outermost in the ventral and lateral walls but lie beneath a layer of circular musculature in the dorsal wall; elsewhere the circular muscle bundles lie deep to the longitudinal ones, and oblique bundles are always present between the two components; the longitudinal bundles converge towards the neck and arch around the dome (62). In the rat and other rodents, there is a broad sheet of longitudinal musculature in the ventral wall; elsewhere, but especially in the lateral walls, the bundles run in all directions, although always parallel to the serosa: they do not have points of termination but continuously split or merge with each other. When fully distended, the muscle is 50–100
m thick in the rat (63). The musculature is also associated with endothelial cells of capillaries, with Schwann cells and axons, and with occasional mast cells. There are no interstitial cells of Cajal. By comparison with the musculature of the intestine, the bladder detrusor is less uniform in thickness and apparently much less regular in its architecture. Mechanically, it is essential that there is continuity of the wall, so that transmural pressure and wall stress are spread uniformly, as evidenced by the slightly elongated shape of the bladder and by the smoothness of its outer surface. The mechanical continuity is provided partly by the fibrous connective tissue of the mucosa and partly by the almost flat meshwork of muscle bundles. The active mechanical function is an isotonic contraction: a shortening of muscle bundles, which are all linked and which discharge the tension they generate on the interstitial collagen and on the conspicuous amount of collagen in the lamina propria. If the outlet of the bladder does not open, then the contraction of the detrusor is isometric: the muscle develops tension and hardens, without apparent shortening but with an increase in intraluminal pressure. C. Smooth Muscle Cells While the detrusor muscle can be considered a unit, capable of being stimulated and of responding synchronously, its component units are the muscle bundles— bundled together into a lamina or into a mesh—sharply outlined from the nonmuscle tissue. The bundles are made of smooth muscle cells; these have the standard appearance of visceral muscle cells (muscles working at comparatively low pressure), as opposed to vascular muscle cells. The cells are spindle shaped, are up to 1 mm in length when fully distended (and down to 100–150
m when fully shortCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ened), and are linked to each other by specialized junctions that provide the mechanical coupling (Fig. 7) (63). The cell volume varies little with the body size of the species and is always minute: the average cell volume of 3000–4000
m3 gives a packing density of about 180,000 muscle cells per mm3 or per mg of tissue. Each muscle cell has some 5000
m2 of cell membrane (plasmalemma), which amounts to almost 1 m2 of cell membrane per mg of pure muscle tissue (64).

Figure 7 Electron micrograph of detrusor the muscle of a rat bladder. The muscle cells are in transverse section, and between them are two small nerve trunks, each with two axons. One of the two axons in each bundle is partly denuded of its Schwann cell cover (a window appears), and it is closely apposed to a muscle cell (neuromuscular junction). (23,000.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

D. Contractile Apparatus and Mechanical Coupling Smooth muscle cells—and the bundles they form—are capable of shortening to less than half their length at rest. The shortening is accompanied by a lateral expansion of the cell, or the bundle, so that the volume of either does not change. The contractile apparatus consists chiefly of filaments of actin and myosin and of many types of molecules associated with them. Force is generated by transient interactions between actin and myosin at cross-bridges; cross-bridges are short-lived and cycle rapidly if energy is available. In certain circumstances longlived stable cross-bridges (latch-bridges) can lock filaments together and maintain tension with minimal expenditure of energy. It can be calculated that a single muscle cell from the rabbit detrusor muscle generates in situ a force of 5–6
N (64). Tension or shortening is produced at the cross-bridges, and it is transmitted within the units of the contractile apparatus along the cell. The actin filaments are bundled into fascicles, are very long, have a longitudinal polarity, run almost parallel to the long axis of the cell, and are inserted to dense material (dense bands) encrusting the internal side of the cell membrane. Other fascicles of actin filaments are inserted to dense bodies in the cytoplasm, but these too eventually discharge their tension to the cell membrane (Fig. 7). At the surface of the muscle cells, tension is transmitted to other muscle cells within the bundle via cell-to-cell junctions of the adherens type (intermediate junctions), where molecules of the cadherin type provide a firm mechanical link across a narrow intercellular gap. The cell membrane of muscle cells also firmly links the contractile apparatus to fibrillar elements of the stroma; via these cell-to-stroma junctions, the stroma transmits passively the tension or the shortening generated by the muscle cells (65). E. Stroma or Matrix Some collagen fibrils lie within the muscle bundles. More abundant collagen and associated extracellular materials such as elastin fibers and microfibrils are present between the muscle bundles, together with fibroblasts, and lie continuous with the collagen of the serosa and tunica propria. Contracting muscle cells transmit tension and shortening along their length (and there is also a conspicuous effect in an orthogonal direction: lateral expansion and compression), then across cell-to-cell junctions, and eventually onto the connective tissue of the stroma, which is the load-bearing element of the wall and a major contributor to the passive mechanical properties of the bladder. The stroma is particularly abundant in the tunica propria of the mucosa, where it has a complex arrangement. In the deepest parts of the mucosa the collagen forms large dense fibers constituting a fibrous meshwork close to the musculature; in contrast, thinner fibers and a looser meshwork are found near the urothelium (66). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The function of the stroma is to distribute the stress, generated by muscle contraction or by the increased lumenal content, uniformly over all parts of the bladder wall. While upon distension of the bladder the mucosa is thinned and the fibers of the stroma are stretched in a direction parallel to the surface of the organ, upon contraction and maximal reduction of the lumen the fibers of the stroma are compressed, and they may also be stretched in a direction across the wall thickness. F. Ionic Coupling Between Muscle Cells In several tissues, including some smooth muscles, gap junctions provide channels for intercellular diffusion of ions and small molecules, supporting electrical and metabolic coupling. Gap junctions have not been observed in the detrusor of the rat (62), whereas they are seen (by connexin 43 immunofluorescence) in the guinea pig detrusor (but rarely observed by electron microscopy) (67). Absence of gap junctions has been noted also in the human bladder (68). Even in the guinea pig, however, the electrical coupling between muscle cells is limited (69): the length constant for injected currents is 425
m in the longitudinal direction and 12
m in the transverse direction (67). There is, once again, much variation between species (even before considering developmental changes and pathological conditions). However, the ionic coupling between muscle cells is either absent or, when present, is limited and not comparable to that found in the musculature of the small intestine or the heart. G. Junctions Between Nerve Endings and Muscle Cells Junctions between motor nerve endings and muscle cells are very numerous in the bladder, far more abundant than in the musculature of the intestine and airways. These specialized junctions are intimate and should be regarded as true autonomic neuromuscular junctions, the site of transmission from nerve endings to muscle cells (70). Each terminal branch of an axon, which in the case of the bladder corresponds to that part of the axon that runs inside of a muscle bundle, develops a beaded shape with varicosities and intervaricose segments. The varicosities contain axonal vesicles (which are mainly of the small agranular type), microtubules (which are usually grouped into small bundles and have little connection with axolemma or vesicles), some neurofilaments (which are rare or absent in the more fully developed varicosities), mitochondria (which are markedly smaller than those in the corresponding neuronal perikarya in the pelvic ganglion), and a minute amount of endoplasmic reticulum. The beaded pattern appears gradually along the length of the terminal axon. Initially the varicosities are only modest swellings of the axon, separated by intervaricose portions that are only moderately Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

constricted. Expansions and constrictions become more marked distally, and in the more peripheral portions of an axon the varicosities are largest (1.5
m in diameter) and the intervaricose portions smallest (down to a minimum of about 0.05
m in diameter). The intervaricose distance varied between 2 and 4
m (Fig. 8). The Schwann cells of intramural nerves group the axons together and at the same time intervene between them so that there are no direct contacts between axons. Even between axons that are nearest to each other, there is always an intervening Schwann cell process, however thin. The Schwann cells continue around single axons, but the sheath they form becomes progressively thinner, then becomes discontinuous (and portions of the axolemma are not covered by it) and eventually disappears. The termination of the Schwann cell occurs before that of the axon, so that the latter is devoid of sheath in its most peripheral part, however briefly. The contacts between axons and bladder muscle cells are morphologically very variable, a situation not different from that found in other smooth muscles. The very notion of neuromuscular junction is not as clear-cut as in skeletal muscles and has been controversial; it is possible that in some muscles transmission from nerves to muscle takes place without the occurrence of specialized junctions. In the bladder, however, there is usually a close relation between axons and muscle cells, and neuromuscular junctions are identified by the occurrence of four structural characteristics: 1. 2.

The axon is a varicosity, packed with vesicles. The axon is devoid of, or only partly sheathed by, a Schwann cell, so that at least part of the axolemma is in contact with the basal lamina; the partly wrapped varicosities have a “window” in which the axolemma, together with the vesicles behind it, are directly exposed and face the membrane of a muscle cell across a short distance. 3. The distance between axolemma and muscle cell membrane ranges between 10 and 100 nm, mostly 30–50 nm; hence, there is a very close apposition between nerve endings and cell membrane. 4. The intercellular gap is occupied by a single basal lamina, and it excludes extracellular materials such as collagen fibrils; occasionally even the basal lamina is excluded from the gap (or junctional cleft) (70). Neuromuscular junctions fulfilling these criteria are abundant in the bladder; each axon forms junctions with many muscle cells, and there are muscle cells that receive junctions from more than one axon (Fig. 9). However, some varicosities remain fully wrapped by their Schwann cell (this, again, can only be established by examining serial sections in the electron microscope); in others the window does not correspond to the part of the axon profile closest to a muscle cell; and in yet others the separation between muscle cell membrane and axolemma exceeds 100 nm and collagen may occur in the cleft. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

The two striking features described above, namely the structural variability of neuromuscular junctions and the existence of a morphological gradient (and possibly a maturation) in varicosities along the axonal length, are probably related; however, it remains to be discovered how they affect neuromuscular transmission. It should be kept in mind that the structural features of a neuromuscular junction have evolved and do develop in individual organisms, not only so as to achieve neuromuscular transmission; other functions of a neuromuscular junction, such as allowing trophic influences between muscle cell and neuron to occur or just maintaining a given configuration of the junction, may account for certain structural features. Some of these features may also correlate with dynamic properties, such as the axons’ ability to regenerate, to grow, and to sprout, which are present in the bladder of mature organisms. No structural specialization is apparent in the muscle cell membrane lying beneath a varicosity, where neuromuscular transmission occurs. Muscarinic cholinergic receptors and purinergic receptors operate in the detrusor muscle (see next section), and they can be detected by autoradiographic labeling (71,72). In the case of purinergic receptors, which are of the P2X type, their localization had been detected at high resolution in the rat detrusor muscle (73). These receptors are not distributed over the entire muscle cell surface, but they are clustered into elliptical patches, about 1.2  0.9
m, located immediately adjacent to a varicosity. There are also smaller patches of P2X receptors, about 0.4
m in diameter, which are not associated with varicosities. The autonomic neuromuscular junctions are different in structure from those of skeletal muscles in several respects. The volume of each autonomic ending or varicosity is at least an order of magnitude smaller than in the somatic endings, and so is, approximately, the number of synaptic vesicles. In the somatic junctions, the axon is longer, minimally branched, and larger, and the bulbous terminal expansions face a prominent postjunctional specialization. In the autonomic junctions, the arrangement in a long terminal axon with a string of varicosities allows many nerve endings (i.e., separate sites of transmitter release) to be formed without a great increase in total axonal length and number of branching points; the number of true anatomical terminations of axons remains very small, in spite of the large number of sites of neuromuscular transmission. The significance of the

Figure 8 Serial sections of a motor axons and muscle cells of the detrusor muscle of a rat. The selected pictures (originally at 27,000 and about 0.1 mm thick) cover a distance of 10 mm along the length of the axon. The sequence of 20 micrographs (i–xx) illustrates the blossoming of a varicose nerve fiber (if an arrangement along an axis in space is present as a temporal event) or the formation of an autonomic neuromuscular junction. In the top left micrograph (i) an intervaricose portion of the axon is fully wrapped by a Schwann cell process. The Schwann cell wrapping then retracts (iii onwards) and a “window” ap-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

pears where the axonal membrane is in contact with the basal lamina. The axon grows, increasing over 35-fold in cross-sectional area, the bundle of microtubules is displaced to one side, and many axonal vesicles appear and pack the varicosity. The “window” expands, and the distance between axolemma and smooth muscle cell membrane is reduced to around 20 nm over a wide area. The postjunctional cell membrane shows no specific structural specializations. (15,500.) (From Ref. 70.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

varicose axons is not entirely clear. Key data such as the conduction velocity along varicose axons, percentage of varicosities activated by a single action potential, and morphogenetic process leading to the beaded structure are still awaiting clarification. H. Neuromuscular Transmission and Transmitters The motor nerve fibers exert their influence on the detrusor muscle by chemical transmission at sites identified as neuromuscular junctions. Stimulation of nerves triggers release of neurotransmitters, and these induce muscle contraction. The excitatory input to the muscle is mediated by cholinergic receptors of the muscarinic type (which are metabotropic receptors) and is abolished by atropine. In addition, an atropine-resistant excitatory input is present in the rat (where there is also a high density of purinergic receptors) and is due to ATP (or a related purine) coreleased with acetylcholine from the same motor nerve endings and acting on purinergic receptors (which are ionotropic receptors) (71); this atropine-resistant excitatory input is weak or absent in other species, such as humans, rabbit, and guinea pig (71). When cholinergic and purinergic transmission coexist, the latter is responsible for the initial transient excitation, which is atropine-resistant, while the cholinergic transmission is responsible for a secondary sustained excitation (74–76). The striated musculature of the external urethral sphincter is innervated by cholinergic axons acting on nicotinic receptors. The endings of the somatic motor axons to this muscle form neuromuscular junctions that resemble to some extent those in skeletal muscles. I. Density of Innervation—Regional Differences The density of innervation of the bladder musculature is very high. The axon of ganglion neuron has a terminal arborization that is many hundreds of
m long and bears many hundreds of minute varicosities 2–4
m apart from each other. Differences in the density of innervation of the muscle in different parts of the detrusor have not been noticed.

Figure 9 A similar preparation as in Figure 8, following two axons, at top and bottom in each panel, over a distance of 30 mm. The axon on top has a varicosity (i–iii) with a large window facing a muscle cell; the nerve-muscle apposition is close, the gap being about 30 nm over most of the extent of the window. The intervaricose portion that follows is long (iv–viii), but it is interrupted by a transient swelling (vi) of the axon. Another varicosity along this axon is formed (ix) followed by an intervaricose segment and another varicosity (xi). The last varicosity is very long and has at least two windows, each apposed to a dif-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

ferent muscle cell. The axon at bottom is at an intervaricose level (i), then forms a short varicosity which is fully wrapped by a Schwann cell. Following an intervaricose segment a large varicosity appears, which has a window apposed to the same muscle cell, which is contacted by two varicosities of the other axon. The axon then shows another intervaricose segment and another varicosity with a window apposed to the same muscle cell. (13,300.) (From Ref. 70.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

A single motor axon can make more than one neuromuscular junction with the same muscle cell (Fig. 9), and it forms junctions with hundreds of different muscle cells. A single muscle cell can have more than one neuromuscular junction from the same axon and can have junctions from more than one axon (Fig. 9). It seems likely that each muscle cell is directly innervated by a motor axon, and multiple innervation of a single cell is common. J. Adrenergic Innervation In addition to the widespread cholinergic innervation (parasympathetic), some species, such as the cat, display an adrenergic innervation (sympathetic) throughout the detrusor muscle. Detected histochemically, these fibers in the cat are less abundant in the dome than in the lower part of the body and in the base of the bladder. They are more abundant in the muscle bundles close to the serosa than in the deep ones in this species, and thyroxyne-hydroxylase-immunoreactive varicosities are seen close to muscle cells (77). The adrenergic fibers of the feline detrusor muscle are inhibitory and act on beta adrenoceptors on the muscle cells; they may also act on alpha adrenoceptors on the cholinergic nerve endings, inhibiting acetylcholine release (78). In the trigone, the adrenergic innervation is denser and the fibers have an excitatory effect on the muscle, mediated by alpha adrenoceptors (78). A less significant inhibitory effect is mediated by beta adrenoceptors. In many other species, such as humans and rat, there is hardly any adrenergic innervation in the detrusor muscle, except around the blood vessels (9). (Even in the cat, earlier studies noted a very limited adrenergic innervation of the detrusor muscle.) In contrast, an adrenergic innervation is present in the trigone muscle (9,71), where it has a predominantly excitatory role. K. Degeneration and Regeneration of Nerves Pelvic ganglionectomy in the rat eliminates almost entirely the adrenergic innervation (both the adrenergic fibers originating from and those passing through the ganglion). After unilateral ganglionectomy, the demarcation of the denervated area at the midline in the bladder is rather sharp, and few adrenergic fibers seem to cross the midline from one side to the other (79). At 6–9 weeks after unilateral pelvic ganglionectomy, the adrenergic innervation is restored in the whole bladder with the same appearance as in controls, evidence of the growth of new adrenergic fibers, presumably from fibers of the contralateral half of the bladder. While in the control rat, sympathetic nerve stimulation elicits a relaxing response mediated by beta adrenoceptors in the detrusor muscle, after long-term ganglionectomy nerve stimulation produces a prominent contractile response mediated by alpha receptors (79). Section of the hypogastric nerve at the level of the bifurcation of the aorta (i.e., cranial to the hypogastric ganglion) does not cause reduction in bladder adrenergic innervation (79). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

As to the parasympathetic innervation, unilateral excision of the pelvic ganglion reduces choline acetyltransferase activity (an indicator of cholinergic innervation): the effect is much more marked on the side of the lesion than on the contralateral side (80). After 6 weeks the enzyme level has recovered and is again at the control levels in both halves of the bladder (81), but it appears that the demarcation at the midline is not as sharp as for adrenergic fibers, and almost one third of the fibers of one side cross over to the other side. The results are explained as due to degeneration of the pelvic parasympathetic postganglionic fibers, which project predominantly but not exclusively to the same side of the bladder; the recovery is due to axonal sprouting and growth from axons in the contralateral half of the bladder. The neurons in the contralateral ganglion end up innervating twice as much muscle as they do in the control condition, and they undergo hypertrophy (82). After complete denervation of the bladder (bilateral pelvic ganglionectomy) or complete decentralization (bilateral section of pelvic and hypogastric nerves), the bladder is paralyzed, becomes overdistended, and has to be emptied by manual distension by the operator. The weight of the bladder increases fourfold by hyperplasia and hypertrophy of muscle cells and mucosal cells (83). Both denervation and decentralization are followed by sensitization to cholinergic agonists at 7 days with either procedure; 4 weeks after denervation, however, acetylcholine sensitivity is much decreased (83). Sensitization arises also in disused bladders (diverting the ureters into an artificial reservoir for 1–3 weeks) (84), when the bladder nerve connection are intact but presumably there is a loss of nerve traffic due to the fact the bladder is permanently empty. With long-term parasympathetic denervation—as in patients with lower motor neuron lesions—there is an excessive growth of sympathetic fibers in the detrusor muscle (detected histochemically) and alpha adrenoceptors appear in the detrusor (85). These observations in human subjects confirm studies on cats: sprouts (detected by immunofluorescence) from adrenergic fibres in the bladder are observed 6 weeks after parasympathetic denervation at the level of the ventral roots (85), and later a much increased number of adrenergic varicosities is observed in the detrusor muscle. Concomitantly, the effect of electric stimulation of the hypogastric nerve changes from an initial contraction followed by relaxation to a sustained contraction in the parasympathetically denervated bladder. Following a partial and chronic outlet obstruction, the musculature of the bladder undergoes a conspicuous increase in volume (muscle hypertrophy) by an increase in muscle cell size and number (86). The hypertrophic growth involves the innervation too. There is enlargement of the pelvic ganglion neurons projecting to the bladder and a growth of new axonal branches within the muscle (82,87). The neuronal growth is stimulated by upregulation in the production of NGF by hypertrophic muscle cells (88). In spite of this growth the density of motor innervation is lower than in the control bladder (82). A reduction of the innervation is observed also in human detrusor muscle in outflow obstruction (89). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

V. SENSORY INNERVATION A. Localization of Sensory Ganglion Neurons The sensory innervation to the bladder is produced by neurons in dorsal root ganglia. Their distribution is studied by injecting tracers into the bladder wall and searching for labeled neurons in dorsal root ganglia. In the rat, sensory neurons projecting to the bladder are in the ganglia T13–L2 and in L6, S1 (18,90,91); a minor labeling in T12 and L3 was found by Sharkey et al. (92). In the cat, neurons retrogradely labeled from the bladder are found in ganglia L2–L5 and S1–S4 (90). B. Afferent Pathways From the dorsal root ganglion neurons the central processes (conducting impulses centrifugally, away from the cell body) project to the dorsal horn of the spinal cord of the same nominal level, while the distal process (conducting centripetally, toward the cell body) extends uninterrupted to the peripheral organ. There are coordinated responses of the rectum and other pelvic organs following bladder distension (93), and this occurrence suggests that there exists some convergence of sensory pathways from different organs, probably in the spinal cord. The majority of bladder sensory fibers travel in the pelvic nerves and pass through the pelvic ganglion (or ganglia) before reaching the bladder wall in the postganglionic nerves. A lesser number of sensory fibers are found in the hypogastric nerve, and these too pass through the pelvic ganglion. The pudendal nerve contains many sensory fibres, few of which extend to the bladder. Jansco and Maggi (91) raise the possibility of afferent vagal fibers reaching the bladder in the rat. Each sensory neuron projects to one nerve only and to only one peripheral organ. Only a few percent of all neurons may project to two peripheral organs (see Ref. 94). In humans, sensations arising in the bladder and urethra report not only distension towards the end of the filling phase (this becomes an unpleasant sensation when the bladder is maximally distended and cannot void) but also contraction of the detrusor, fall of pressure immediately preceding voiding, the flow of urine, and the completed contraction of the bladder. Pain is reported with inflammation and tissue damage. Brain-imaging studies in humans have prompted the suggestion that the brain pathways activated in the perception of an urge to void are distinct from those activated with the appreciation of bladder fullness (95). Smet et al. (11) note that less than 5% of the total bladder innervation are sensory fibers, and these have a restricted distribution (in the subepithelium, around blood vessels, around intramural ganglion neurons). In contrast, calculations carried out in the rat nerves indicate that the bladder is served approximately by an equal number of motor and sensory neurons in the pelvic ganglia and in dorsal root ganglia, respectively (96). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

While passing through the pelvic ganglion, sensory fibres may issue small collateral branches, which are distributed among the ganglion neurons, occasionally producing a perineuronal basket (36,96,97), but apparently only on parasympathetic neurons, not on sympathetic ones (97). The extent of this occurrence is unclear. In other autonomic ganglia, such as the celiac ganglion of the guinea pig, the presence of these contacts is documented and well investigated. In the pelvic ganglion the evidence of a possible contact between afferent fibers and ganglion neurons is mainly histochemical, and its functional significance remains to be elucidated. Papka and McNeill (36) have suggested that there are synaptic contacts between sensory fibers and pelvic ganglion neurons in the rat. C. Innervation of Mucosa In the bladder wall the sensory fibers remain in mixed nerves for long distances, but they find their way predominantly into the nerve branches that extend to the mucosa. To follow the intramural course of the afferent fibers to their termination, advantage is taken of histochemical markers for the peptides substance P and CGRP (calcitonin gene–related peptide) (Fig. 10). In species such as the rat, CGRP antibodies label all the afferent fibers and no other fiber type (98). There are fewer fibers staining with SP antibody in the rat [none according to Alm et al. (99)] than with CGRP, but they are abundant in the guinea-pig (99). With immunofluorescence methods it is possible to dissect manually the mucosa of the entire bladder, stain it, and mount it flat as a single preparation for topographical studies (96). In the rat bladder the sensory fibers are found in an elaborate meshwork or plexus of small nerve bundles and long single axons, intensely immunofluorescent for CGRP and with a clear varicose structure (Fig. 11). This plexus is located in the mucosa immediately beneath (1–5
m) the epithelium, and short varicose axons emerge out of it at right angle, penetrate into the epithelium (urothelium), and expand across it almost reaching the lumenal surface. The plexus is thickest in the regions of the trigone, bladder neck, and initial segment of the urethra; it becomes progressively less well developed towards the equatorial region of the bladder, and it is absent in the region of the dome, where (at least in the rat) there is hardly any sensory innervation in the mucosa (96). The distribution of sensory fibers very close to the epithelium and the presence of some intraepithelial fibers is noted not only in the rat (96,98,100,101), but also in humans (102) and dog (103). D. Sensory Fibers Outside Mucosa There are also sensory fibers within the musculature (usually around the muscle bundles, rather then inside them), around the blood vessels, which have a muscle Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 10 Sections of a rat bladder wall examined by immunofluorescence. (A) The lumen is at the top and the epithelium is barely visible and is underlined by numerous nerve fibers stained with an antibody for CGRP (sensory fibers). At bottom is the musculature, where faintly immunostained fibers are seen. (B) A similar preparation, at higher magnification, showing a subepithelial sensory fibers, which penetrates into the epithelium. (C) Circular and longitudinal bundles of the musculature occupy this microscope field. The muscle contains innumerable small nerve fibers, which are varicose and are stained with an antibody against synaptophysin (motor axons). (A, 80; B and C, 160.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 11 Whole mount stretch preparation of the bladder mucosa, stained for CGRP. A sensory fiber seen in its terminal portion is distinctly varicose and branching; it runs beneath the epithelium and parallel to it. (350.) (Modified from Ref. 96.)

coat, and in the serosa of the bladder (Fig. 11). The spatial density of these fibers is much less than in the mucosa of the trigone; the fibers also appear smaller and less intensely immunofluorescent for CGRP. E. Types of Sensory Fibers Sensory fibers (or, more generally, afferent fibres) from the bladder are thinly myelinated (A-fibers) or unmyelinated fibers (C-fibers). Functionally, the fibers transmit impulses from mechanoreceptors—which detect stretch or, more likely, tension—and probably from chemoreceptors; there are also polymodal nociceptive fibers. Most of the receptors are slow-adapting and they discharge already at physiological intraluminar pressures when these rise. F. Origin of Afferent Impulses The site of mechanoelectrical transduction (physical deformation of an element of the tissue, giving rise to an action potential within a sensory fibers) is at the nerve ending, but the mechanism involved is unclear. There are no sensory corpuscles around the peripheral termination of sensory fibers in the bladder and no distinct structural specializations. The sensory endings (which are varicosities in a chain) Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

are partially devoid of Schwann cell wrapping; at these sites their plasma membrane is covered by a basal lamina and faces the extracellular space with fibrous elements of the stroma in it. A novel mechanism of sensory transduction is based on the observation that the urothelium (the epithelium lining the lumen of the bladder) contains ATP and releases it upon stimulation (strips of rabbit bladder electrically stimulated in vitro) (104). Since sensory nerve terminals possess on their membrane P2X receptors (105), then the ATP released from the urothelium may activate the intraepithelial and subepithelial sensory fibers: the initial transduction takes place at stretch-sensitive ionic channels in the urothelium, and the purines thus released act on the varicosities of chemosensitive sensory fibers (104). ATP is also released from injured tissues, and it has been suggested that the substance plays a role in pain mechanisms in the bladder by being released from the epithelium when it is damaged or when it is maximally distended and by acting on purinergic receptors (of the P2X3 type) in sensory axons An important aspect of the sensory innervation of the bladder is that each sensory axon branches and spreads over a large area. The branching points, at right angle, produce a tree- or bush-like pattern that overlaps extensively with similar terminal structures from other axons. The terminal portions of these axons are varicose, and each sensory axon thus produces many hundreds of varicosities spread over hundreds of
m of axonal length (96). These varicosities are smaller and more heterogeneous in size than those of the motor axons within the muscle. They contain some small agranular vesicles and some large granular vesicles. The small vesicles are much less abundant and less densely packed than in motor fiber varicosities (and they are accompanied by some large granular vesicles); nevertheless, they constitute a characteristic features of autonomic sensory axons, in contrast with somatic sensory axons associated with terminal corpuscles, which contain no vesicles. The presence of vesicle in sensory fiber varicosities suggests that upon stimulation there is release of substances, for example, neuropeptides, from these axons into the extracellular space. This occurrence may lead to stimulation of other cells in the proximity and of the varicosity itself, if it has autoreceptors—possibly a process of positive feedback. G. Axonal Reflex—Neurogenic Inflammation Nerve impulses can travel in either direction along an axon, the orthodromic one being the direction away from the physiological site of generation of the action potential. Impulses propagate peripherally along motor axons and centripetally along sensory axons, but antidromic conduction can be effected by experimental electric stimulation. At the terminal arborization of a sensory fiber, antidromic stimulation occurs when impulses traveling centripetally also invade adjacent branches and spread centrifugally along them, an axonal reflex. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

When axonal reflexes occur in the bladder of rats and guinea pigs, they lead to the release of neurochemicals such as substance P and CGRP from the varicosities of sensory fibers. Antidromic electric stimulation of bladder nerves induces inflammation of the innervated tissue: there is plasma extravasation, hyperemia, and leukocyte migration (107). This neurogenic inflammation is blocked by chronic denervation, is mimicked by administration of substance P and CGRP, and is inhibited by treatment with doses of capsaicin that deplete substance P from nerves (107–109). The effect is obtained also with intense stimulation of intramural nociceptive fibers but not of mechanoceptive fibers, and it is absent when Cfibers are selectively destroyed (107,108). The possibility of antidromic release of neurochemicals, at least in some species, extends the significance of the ummyelinated sensory fibers of the bladder, and the notion of sensorimotor fibers has been proposed for them (110). The possible chemical relevance of these phenomena in the inflammatory processes that do not involve infection, such as interstitial cystitis, has been discussed (109).

VII.

SPINAL CORD AND URINARY REFLEXES

A. Organization of Reflex Arc The key mechanism of bladder motor function is a reflex arc, with an ascending (centripetal) and a descending (centrifugal) limb, effected by a chain of four groups of neurons: sensory neurons, spinal cord interneurons, autonomic preganglionic neurons, and ganglion neurons. The arc comprises dual pathways, for bladder detrusor muscle and for urethral sphincters, providing a reciprocal relationship of activation of one of the two and inhibition of the other. The primary sensory neurons, located in dorsal root ganglia, spread their terminals in the bladder wall while sending via the dorsal root a centripetal process into the spinal cord, as described in sec V.A. Centrally, these fibers terminate at the level of the point of entrance in the spinal cord, synapsing on neurons in various regions of the dorsal horn. There is some overlap between the spinal cord projections from different pelvic organs and probably also from some somatic areas. The primary afferent fibers do not reach the preganglionic neurons directly but only via one or more interneurons; hence these reflexes are not monosynaptic but di- or polysynaptic (in addition to the synapse in the pelvi ganglion). The spinal interneurons in the dorsal horn, synaptically activated by terminals of primary afferent neurons, project either centrally, i.e., up the spinal cord onto neurons in the brainstem, or transversely (segmentally) onto other interneurons or onto preganglionic neurons at the same level of the spinal cord. They are identified by electrophysiology (111,112), by localization of injected and transported tracers (113–115), and by histochemistry of the c-fos gene protein product (116,117). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Various types of interneurons are involved in the control of bladder and urethra, supporting either inhibition or excitation, in either the peripheral reflex or in the spino-bulbo spinal reflex loop (111). Some of these interneurons allow for an input from cutaneous regions or from the terminal intestine to converge onto the urinary motor pathway. Many of the interneurons have an excitatory influence on parasympathetic preganglionic neurons, mediated by glutamic acid. In addition, there are also inhibitory interneurons, which release glycine and GABA (112,118). A maturational change in a group of interneurons in the rat around the end of the third week of life may explain the developmental transition from the urinary reflex being predominantly triggered by a cutaneous sensory input (from the skin of the perineum), as is found at birth, to a reflex triggered by a sensory input from the bladder, as is found in the adult (119). Preganglionic neurons and the pelvic ganglion neurons they drive, discussed in Secs. III.A and III.B, provide the efferent or descending limb of the reflex arc to the bladder. The reflex controlling the external sphincter of the bladder, which is a striated muscle under voluntary control, involves motor neurons in the sacral spinal cord. In humans, these neurons are located in a nucleus extending from the caudal part of S1 to the cranial part of S3, originally described by Onufrowicz (120) and known as the Onuf’s nucleus. The nucleus, which also innervated the external anal sphincter (121), has a marked sexual dimorphism in the rat (122) and dog (123), but not in humans (124). The neurons of the Onuf’s nucleus have ultrastructural and histochemical characteristics of other somatic motor neurons. However, they receive in common with autonomic preganglionic neurons a large peptidergic synaptic input (125,126) and have synaptic input from midbrain nuclei that project also to the preganglionic neurons (127). B. Supraspinal Centers The main control centers of bladder motor activity are in the brainstem (128). In the cat, two areas in the pons have an overriding influence on detrusor contractility and on bladder neck contractility, the so-called M-region and the L-region (129). The M-region or pontine micturition center (PMC) or Barrington’s nucleus, located in the dorso-lateral pontine tegmentum, has an excitatory projection to the bladder parasympathetic preganglionic neurons in the sacral spinal cord, and when electrically stimulated causes bladder contraction (129,130). Ultrastructural evidence in the cat indicates that this synaptic input is specific for neurons that project towards the bladder, rather than for those that project towards the rectum or for interneurons (131). The pontine micturition center has also a projection to interneurons in the dorsal grey commissure of the sacral spinal cord (132), which in turn project onto the motoneurons of the Onuf’s nucleus (114,133); this pathCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

way from the pons has an inhibitory effect and causes relaxation of the bladder external sphincter. The L-region, located more ventrally and laterally, projects directly onto neurons in the Onuf’s nucleus, and when electrically stimulated causes a contraction of the pelvic floor, including the external urethral sphincter (129). There is a clear-cut antagonism between the two regions of the tegmentum, the M-region effecting contraction of the detrusor and relaxation of the sphincter, hence voiding, while the L-region effects contraction of the sphincter, hence retention. Afferent stimuli from the bladder via dorsal root ganglia are conveyed, in the cat and rat, by ascending fibers in the spinal cord and project mainly onto the periaqueductal grey (134,135), which in its turn project to the pontine tegmentum. The periaqueductal grey seems, therefore, to have a tonic influence on the L-region, the effect of which is that the bladder outlet is closed and continence is maintained; via a phasic effect on the M-region, the periaqueductal grey controls the onset of micturition. Two regions equivalent to those described in the cat are identified by positron emission tomography (PET) in men and women (137,138). In men, micturition is associated with activation (increased blood flow) of the dorsal pontine tegmentum, hypothalamus, right inferior frontal gyrus, and right anterior cyngulate gyrus (131). In women, micturition is associated with activation in the right dorsal pontine tegmentum (roughly equivalent to the M-region of the cat); retention (or inability to void in the experimental conditions) is associated with activation of the right ventral pontine tegmentum (roughly equivalent to the L-region of the cat). In micturition, there is also activation of the right anterior cyngulate gyrus and in the right inferior frontal gyrus. The predominant localization on the right side in human subjects observed in these experiments is not yet explained, and it has not been found in other studies on humans (95,139); furthermore, there is no counterpart of that asymmetry in animals, where a bilateral destruction of the pontine micturition center is required to cause urine retention (140).

REFERENCES 1.

Wozniak W, Skrowronska U. Comparative anatomy of pelvic plexus in cat, dog, macaque and man. Anat Anz 1967; 120:457–473. 2. Langley JN, Anderson HK. The innervation of the pelvic and adjoining viscera. Part II. The bladder. J Physiol 1895; 19:71–84. 3. Li M-Z, Masuko S. Target specific organization and neuron types of the dog pelvic ganglia: a retrograde-tracing and immunohistochemical study. Arch Histol Cytol 2001; 64:267–280. 4. Costa M, Furness GB. Observations on the anatomy and amine histochemistry of the nerves and ganglia which supply the pelvic viscera and on the associated chromaffin tissue in the guinea-pig. Z Anat Entwicklungsgesch 1973; 140:85–108.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

5.

6.

7. 8. 9. 10. 11.

12. 13. 14.

15.

16. 17. 18.

19. 20. 21. 22.

23.

Morris JL, Gibbins IL. Neuronal colocalization of peptides, catecholamines, and catecholamine-synthesizing enzymes in guinea pig paracervical ganglia. J Neurosci 1987; 7:3117–3130. Rogers H, Kennedy C, Henderson G. Characterization of the axons of the mouse hypogastric ganglion: morphology and electrophysiology. J Auton Nerv Syst 1990; 29:255–270. Purinton PT, Fletcher TF, Bradley WE. Gross and light microscopic features of the pelvic plexus in the rat. Anat Rec 1973; 175:697–706. Baljet B, Drukker J. The extrinsic innervation of the abdominal organs in the female rat. Acta Anat 1980; 104:241–267. Alm P, Elmér M. Adrenergic and cholinergic innervation of the rat urinary bladder. Acta Physiol Scand 1975; 94:36–45. Hulsebosch CE, Cogeshall RE. An analysis of the axon populations in the nerves to the pelvic viscera of the rat. J Comp Neurol 1982; 211:1–10. Smet PJ, Edyvane KA, Jovanicius J, Marshall VR. Neuropeptides and neurotransmitter-synthesizing enzymes in intrinsic neurons of the human urinary bladder. J Neurocytol 1996; 25:112–124. Gilpin CJ, Dixon JS, Gilpin SA, Gosling JF. The fine structure of autonomic neurones in the wall of the human urinary bladder. J Anat 1983; 137:705–713. Gabella G. Intramural neurons in the urinary bladder of the guinea-pig. Cell Tissue Res 1990; 261:231–237. Zhou Y, Tan CK, Ling EA. Distribution of NADPH-diaphorase and nitric oxidesynthase-containing neurons in the intramural ganglia of guinea pig urinary bladder. J Anat 1997; 190:135–145. Crowe R, Haven AJ, Burnstock G. Intramural neurones of the guinea-pig urinary bladder: histochemical localization of putative neurotransmitters in cultures and newborn animals. J Auton Nerv Syst 1986; 15:319–339. Hanani M, Maudlej N. Intracellular recordings from intramural neurons in the guinea pig urinary bladder. J Neurophysiol 1995; 74:2358–2365. Carpenter FG, Rand SA. Relation of acetylcholine release to responses of the rat urinary bladder. J Physiol 1965; 180:371–382. McNeill DL, Traugh NE, Vaidya AM, Hua HT, Papka RE. Origin and distribution of NADPH-diaphorase-positive neurons and fibers innervating the urinary bladder of the rat. Neurosci Lett 1992; 147:33–36. Alian M, Gabella G. Decrease and disappearance of intramural neurons in the rat bladder during post-natal development. Neurosci Lett 1996; 218:103–106. Uvelius B, Gabella G. Intramural neurons appear in the urinary bladder wall following excision of the pelvic ganglion in the rat. NeuroReport 1995; 6:2213–2216. Gabella G, Uvelius B. Homotransplant of pelvic ganglion into bladder wall in adult rats. Neurosci 1998; 83:645–653. Keast JR, Booth AM, de Groat WC. Distribution of neurons in the major pelvic ganglion of the rat which supply the bladder, colon or penis. Cell Tissue Res 1989; 256:105–112. Greenwood D, Coggeshall RE, Hulsebosch CE. Sexual dimorphism in the number of neurons in the pelvic ganglia of adult rats. Brain Res 1985; 340:160–162.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

24.

25. 26. 27. 28.

29. 30.

31. 32.

33.

34.

35.

36.

36.

37. 38.

39.

Hondeau E, Prud’homme M-J, Rousseau A, Rousseau J-P. Distribution of noradrenergic neurons in the female rat pelvic plexus and involvement in the genital tract innervation. J Auton Nerv Syst 1995; 54:113–125. Langworthy OR. Innervation of the pelvic porgans of the rat. Invest Urol 1965; 2:491–511. Keast JR. Effects of testosterone on pelvic autonomic pathways: progress and pitfalls. J Auton Nerv Syst 2000; 79:67–73. Melvin JE, Hamill RW. Androgen-specific critical periods for organization of the major pelvic ganglion. J Neurosci 1989; 9:736–742. Mitchell BS, Stauber VV. Morphological, histochemical and immunohistological studies of the paracervical ganglion in prepubertal, pregnant and adult, non-pregnant guinea-pigs. J Anat 1990; 172:177–189. Suzuki Y, Arai Y. Laterality associated with sexual dimorphism in the volume of the mouse hypogastric ganglion. Exp Neurol 1986; 94:241–245. Warburton AL, Santer RM. Sympathetic and sensory innervation of the urinary tract in young adult and aged rats: a semi-quantitative histochemical and immunohistochemical study. Histochemical J 1994; 26:127–133. Tabatabai M, Booth AM, de Groat WC. Morphological and electrophysiological properties of pelvic ganglion cells in the rat. Brain Res 1986; 382:61–70. Tabatabai M, Booth AM, de Groat WC. Comparison of morphology and electrophysiological properties of pelvic parasympathetic ganglion cells in the rat and cat. Fed Proc 1984; 43:998. Hanani M, Maudlej N, Härtig W. Morphology and intercellular communication in glial cells of intramural ganglia from the guinea-pig urinary bladder. J Auton Nerv Syst 1999; 76:62–67. Mattiasson A, Ekblad E, Sundler F, Uvelius B. Origin and distribution of neuropeptide Y-, vasoactive intestinal polypeptide- and substance P-containing nerve fibers in the urinary bladder of the rat. Cell Tissue Res 1985; 239:141–146. Dail WG, Moll MA, Weber K. Localization of vasoactive intestinal polypeptide in penile erectile tissue and in the major pelvic ganglion of the rat. Neurosci 1983; 10:1379–1386. Papka RE, McNeill DL. Coexistence of calcitonin gene-related peptide and galanin immunoreactivity in female rat pelvic and lumbosacral dorsal root ganglia. Peptides 1992; 13:761–767. Papka RE, McNeill DL. Light- and electron-microscopic study of synaptic connections in the paracevical ganglion of the female rat: special reference to calcitonin gene-related peptide-, galanin- and tachykinin (substance P and neurokinin A)-immunoreactive nerve fibers and terminals. Cell Tissue Res 1993; 271:417–428. Werkström V, Alm P, Persson K, Andersson K-E. Inhibitory innervation of the guinea-pig urethra; roles of CO, NO and VIP. J Auton Nerv Syst 1998; 74:33–42. Kanerva L, Lietzén R, Teräväinen H. Catecholamines and cholinesterases in the paracervical (Frankenhäuser) ganglion of normal and pregnant rats. Acta Physiol Scand 1972; 86:271–277. Watanabe H. Adrenergic nerve elements in the hypogastric ganglion of the guineapig. Am J Anat 1971; 130:305–330.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

40.

41. 42. 43.

44. 45.

46.

47. 48. 49.

50. 51. 52. 53. 54.

55.

56. 57.

Dixon JS, Gosling JA. The distribution of noradrenergic nerves and small, intensely fluorescent (SIF) cells in the cat urinary bladder. Cell Tissue Res 1974; 150:147–159. Yokota R, Burnstock G. Synaptic organization of the pelvic ganglion of the guineapig. Cell Tissue Res 1983; 232:379–397. de Groat WC, Booth AM. Inhibition and facilitation in parasympathetic ganglia of the urinary bladder. Fed Proc 1980; 39:2990–2996. Wang B-R, Senba E, Tohyama M. Met-5-enkephalin-arg6-gly7-leu8-like immunoreactivity in the pelvic ganglion of the male rat: a light and electron microscope study. J Comp Neurol 1990; 293:26–38. Dail WG, Evan AP, Eason HR. The major ganglion in the pelvic plexus of the male rat: a histochemical and ultrastructural study. Cell Tissue Res 1975; 159:49–62. Griffith WH, Gallagher JP, Shinnick-Gallagher P. Sucrose-gap recordings of nerveevoked potentials in mammalian parasympathetic ganglia. Brain Res 1981; 209:446–451. de Groat AM, Booth AM. Synaptic transmission in pelvic ganglia. In: Maggi CA, ed. Nervous Control of the Urogenital System. Chur, Switzerland: Harwood, 1993:291–347. Gallagher JP, Griffith WH, Shinnick-Gallagher P. Cholinergic transmission in cat parasympathetic ganglia. J Physiol 1982; 332:473–486. de Groat WC, Saum WR. Sympathetic inhibition of the urinary bladder and pelvic ganglionic transmission in the cat. J Physiol 1972; 220:297–314. Purinton PT, Fletcher TF, Bradley WE. Innervation of pelvic viscera in the rat: evoked potentials in nerves to bladder and penis (clitoris). Invest Urol 1976; 14:28–32. Mallory B, Steers WD, de Groat WC. Electrophysiological study of micturition reflex in rats. Am J Physiol 1989; 257:R410–R421. Booth AM, de Groat WC. A study of facilitation in vesicel parasympathetic ganglia of the cat using intracellular ecording techniques. Brain Res 1979; 169:388–392. Crowcroft PJ, Szurszewski J. A study of the inferior mesenteric and pelvic ganglia of the guinea-pigs with intracellular electrodes. J Physiol 1971; 219:421–441. Hancock MB, Peveto CA. A preganglionic autonomic nucleus in the dorsal grey commissure of the lumbar spinal cord of the rat. J Comp Neurol 1979; 183:65–72. Neuhuber W. The central projections of visceral primary afferent neurons of the inferior mesenteric plexus and hypogastric nerve and the location of the related sensory and preganglionic sympathetic cell bodies in the rat. Anat Embryol 1982; 164:413–425. Morgan C, de Groat WC, Nadelhaft I. The spatial distribution of sympathetic preganglionic and visceral primary afferent neurons which send axons in the hypogastric nerve of the cat. J Comp Neurol 1986; 243:23–40. Tanaka S, Zukeran C.. A macroscopical study of the somatic visceral nerves innervating the male urogenital organs. Acta Anat 1981; 56:413–414. Mawe GM, Brenahan JC, Beattie MS. A light and electron microscopic analysis of the sacral parasympathetic nucleus after labeling primary afferent and efferent elements with HRP. J Comp Neurol 1986; 250:33–57.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

58.

59. 60.

61.

62.

63. 64. 65. 66.

67.

68. 69. 70. 71.

72.

73.

74.

de Groat WC, Booth AM, Yoshimura N. Neurophysiology of micturition and its modification in animal models of human disease. In: Maggi CA, ed. Nervous Control of the Urogenital System. Chur, Switzerland: Harwood, 1993:227–290. Gabella G. Structure of the intramural nerves of the rat bladder. J Neurocytol 1999; 28:615–637. Elbadawi A. Ultrastructure of vesicourethral innervation. II. Postganglionic axoaxonal synapses in intrinsic innervation of the vesicourethral lissosphincter: a new structural and functional concept in micturition. J Urol 1984; 131:781–790. Speakman MJ, Walmsley D, Brading AF. An in vitro pharmacological study of the human trigone—a site of non-adrenergic non-cholinergic transmission. Br J Urol 1987; 61:304–309. Wakabayashi Y, Makiura Y, Tomoyoshi T, Kitahama K, Geffard M, Maeda T. Adrenergic innervation of the urinary bladder body in the cat with special reference to structure of the detrusor muscle: an immunohistochemical study of noradrenaline and its synthesizing enzymes. Arch Histol Cytol 1994; 57:277–289. Gabella G, Uvelius B. Urinary bladder of the rat: fine structure of normal and hypertrophic musculature. Cell Tissue Res 1980; 262:67–79. Uvelius B, Gabella G. Relation between cell length and force production in urinary bladder smooth muscle. Acta Physiol Scand 1980; 110:357–365. Gabella G. The structural apparatus for transmission of force in smooth muscles. Physiol Rev 1984; 64:455–477. Murakumo M, Ushiki T, Abe K, Matsumara K, Shinno Y, Koyanagi T. Three-dimensional arrangement of collagen and elastin fibers in human urinary bladder: scanning electron microscopy study. J Urol 1995; 154:251–256. Hashitani H, Fukuta H, Takano H, Klemm MF, Suzuki H. Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladder. J Physiol 2001; 530:273–286. Daniel EE, Cowan W, Daniel VP. Structural bases for neural and myogenic control of human detrusor muscle. Can J Physiol Pharmacol 1983; 61:1247–1273. Bramich NJ, Brading AF. Electrical properties of smooth muscle in the guinea-pig urinary bladder. J Physiol 1996; 492:185–198. Gabella G. The structural relation between nerve fibres and muscle cells in the urinary bladder of the rat. J Neurocytol 1995; 24:159–187. Bo X, Burnstock G. Species differences in characteristics and distribution of [3H] methylene ATP binding sites in urinary bladder and urethra of rat, guinea-pig and rabbit. Eur J Pharmacol 1992; 216:59–66. Bo X, Burnstock G. Characterization and autoradiograpohic localization of [3H] methylene adenosine 5-triphosphate binding sites in human urinary bladder. Br J Urol 1995; 76:297–302. Hansen MA, Balcar VJ, Barden JA, Bennett MR. The distribution of p21-receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder. J Neurocytol 1998; 27:529–539. Craggs MD, Rushton DN, Stephenson JD. A putative non-cholinergic mechanism in urinary bladder of new but not old world primates. J Urol 1986; 136:1348–1350.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

75. 76.

77.

78. 79. 80. 81.

82. 83.

84. 85.

86. 87.

88.

89. 90. 91. 92.

93.

Fujii K. Evidence for adenosine triphosphate as an excitatory transmitter in the guinea pig, rabbit and pig urinary bladder. J Physiol 1988; 404:39–52. Hoyle CHV, Burnstock G. Postganglionic efferent transmission in the bladder and urethra. In: Maggi CA, ed. Nervous Control of the Urogenital System. Chur, Switzerland: Harwood, 1993:349–381. Wakabayashi Y, Makiura Y, Tomoyoshi T, Kitahama K, Maeda T. Immunoelectron microscopic study of tyrosine hydroxylase in the cat urinary bladder and proximal urethra. J Auton Nerv Syst 1993; 44:243–252. Creed KE. The role of the hypogastric nerve in bladder and urethral activity in the dog. Br J Pharmacol 1979; 65:367–375. Alm P, Elmér M. Adrenergic reinnervation of the denervated rat urinary bladder. Experientia 1979; 35:1387–1388. Ekström J, Elmer M, Banns H. Transient supersensitivity in the partially denervated urinary bladder of the rat. Acta Pharmacol Toxicol 1978; 43:318–322. Ekström J, Henningsson A-C, Henningsson S, Malmberg L. Hyperplasia and hypertrophia in the denervated and distended rat urinary bladder. Acta Physiol Scand 1984; 122:45–48. Gabella G, Berggren T, Uvelius B. Hypertrophy and reversal of hypertrophy in rat pelvic ganglion neurons. J Neurocytol 1992; 21:649–662. Ekström J, Malmberg L. Development of supersensitivity to methacholine in the rat detrusor following either parasympathetic denervation or decentralization. Acta Physiol Scand 1984; 122:175–179. Ekström J, Malmberg L. Disuse as cause of supersensitivity in the rat urinary bladder. Acta Physiol Scand 1986; 126:429–432. Sundin T, Dahlström A, Norlen L, Svedmyr N. The sympathetic innervation and adrenoceptor function of the human lower urinary tract in the normal state and after parasympathetic denervation. Invest Urol 1997; 14:322–328. Mattiasson A, Uvelius B. Changes in contractile properties in hypertrophic urinary bladder. J Urol 1982; 128:1340–1342. Steers WD, Ciambotti J, Etzel B, Erdman S, de Groat WC. Morphological plasticity in efferent pathways to the urinary bladder of the rat following urethral obstruction. J Neurosci 1990; 10:1943–1951. Steers WD, Kolbeck S, Creedon D, Tuttle JB. Nerve growth factor in the urinary bladder of the adult regulates neuronal form and function. J Clin Invest 1991; 88:1709–1715. Gosling JA, Gilpin SA, Dixon JA, Gilpin CJ Decrease in the autonomic innervation of human detrusor muscle in outflow obstruction. J Urol 1986; 136:501–504. Applebaum AE, Vanve WH, Coggeshall RE. Segmental localization of sensory cells that innervate the baldder. J Comp Neurol 1980; 192:203–209. Jancso G, Maggi CA. Distribution of capsaicin-sensitive urinary bladder afferents in the rat spinal cord. Brain Res 1987; 418:371–376. Sharkey KA, Willaims RG, Schultzberg M, Dockray GJ. Sensory substance P innervation of the urinary bladder: possible site of action of capsaicin in causing urine retention in rats. Neurosci 1983; 10:861–868. Buntzen S, Nordgren S, Delbro D, Hultén L. Anal and rectal motility responses to distension of the urinary bladder in the cat. J Auton Nerv Syst 1994; 49:261–268.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

94. Janig W, Koltzenburg M. Pain arising from the urogenital tract. In: Maggi CA, ed. Nervous Control of the Urogenital System. Chur, Switzerland: Harwood, 1993:525–578. 95. Athwal BS, Berkley KJ, Hussain I, Brennan A, Craggs M, Sakakibara R, Frackowiak RSJ, Fowler CJ. Brain responses to changes in bladder volume and urge to void in healthy men. Brain 2001; 124:369–377. 96. Gabella G, Davis C. Distribution of afferent axons in the bladder of rats. J Neurocytol 1998; 27:141–155. 97. Keast JR, Chiam H-C. Selective association of nerve fibres immunoreactive for substance P or bombesin with putative cholinergic neurons of the male rat major pelvic ganglion. Cell Tissue Res 1994; 278:589–594. 98. Su HC, Wharton J, Polak JM, Mulderry PK, Ghatei MA, Gibson SJ, Terenghi G, Morrison JFB, Ballesta J, Bloom SR. Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: combined retrograde tracing and immunohistochemistry. Neurosci 1986; 18:727–747. 99. Alm P, Alumets J, Brodin E, Håkanson R, Nilsson G, Sjöberg N-O, Sundler GF. Peptidergic (substance P) nerves in the genito-urinary tract. Neurosci 1978; 3:419–425. 100. Yokokawa K, Toyama M, Shiosaka S, Shiotani Y, Sonoda T, Emson PC, Hill-Yard CV, Girgis S, McIntyre S. Distribution of calcitonin gene-related peptide containing fibers in the urinary bladder of the rat and their origin. Cell Tissue Res 1986; 244:271–278. 101. Iuchi H, Satoh Y, Ono K. Postnatal development of neuropeptide Y- and calcitonin gene-related peptide immunoreactive nerves in the rat urinary bladder. Anat Embryol 1994; 189:361–373. 102. Dixon JS, Gilpin CJ. Presumptive sensory axons of the human urinary bladder: a fine ultrastructural study. J Anat 1987; 151:199–207. 103. Tamaki M, Iwanaga T, Takeda M, Adachi I, Sato S, Fujita T. Calcitonin gene-related peptide (CGHRP)-immunoreactive nerve terminal in the whole mount preparations of the dog urethra. Arch Histol Japon 1992; 55:1–11. 104. Ferguson DR, Kennedy I, Burton TJ. ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changes—a possible sensory mechanism? J Physiol 1997; 505:503–511. 105. Evans RJ, Surprenant A. P2X receptors in autonomic and sensory neurons. Semin Neurosci 1996; 8:217–223. 106. Cockayne DA, Hamilton SG, Zhu Q-M, Dunn PM, Zhong Y, Novakovic S, Malmberg AB, Cain G, Berson A, Kassotakis L, Hedley L, Lachnit WG, Burnstock G, McMahon SB, Ford APSW. Urinary bladder hyporeflexia and reduced pain-related behaviour in PrX3-deficient mice. Nature 2000; 407:1011–1014. 107. Lundberg JM, Brodin E, Hua X, Saria A. Vascular permeability changes and smooth muscle contraction in relation to capsaicin-sensitive substance P afferents in the guinea-pig. Acta Physiol Scand 1984; 120:217–227. 108. Koltzenburg M, McMahon SB. Plasma extravasation in the rat urinary bladder following mechanical, electical and chemical stimuli: evidence for a new population of chemosensitive primary sensory afferents. Neurosci Lett 1986; 72:352–236. 109. Bjorling DE, Saban MR, Saban R. Neurogenic inflammation of guinea-pig bladder. Mediat Inflamm 1994; 3:189–197.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

110.

111. 112.

113.

114.

115. 116. 117. 118.

119. 120. 121.

122.

123. 124. 125.

126.

Maggi CA. The dual, sensory and “efferent” function of the capsaicin-sensitive primary sensory neurons in the urinary bladder and urethra. In: Maggi CA, ed. Nervous Control of the Urogenital System. Chur, Switzerland: Harwood, 1993:383–422. McMahon SB, Morrison JFB. Two groups of spinal interneurones that respond to stimulation of the abdominal viscera of the cat. J Physiol 1982; 322:21–34. Araki I, de Groat WC. Unitary excitatory synaptic currents in preganglionic neurons mediated by two distinct groups of interneurons in neonatal sacral parasympathetic nucleus. J Neurophysiol 1996; 76:215–226 Vizzard MA, Erickson VL, Card JP, Roppolo JR, de Groat WC. Transneuronal labeling of neurons in the adult rat brainstem and spinal cord after injection of pseudorabies virus into the urethra. J Comp Neurol 1995; 355:629–640. Nadelhaft I, Vera PL. Neurons in the rat brain and spinal cord labeled after pseudorabies virus injected into the external urethral sphincter. J Comp Neurol 1996; 375:502–517. Sugaya K, Matsuyama K, Takakusaki M, Mori S. Electrical and chemical stimulations of the pontine micturition center. Neurosci Lett 1987; 80:197–201. Birder LA, de Groat WC. Increased c-fos expression in spinal neurons after irritation of the lower urinary tract in the rat. J Neurosci 1992; 12:4878–4889. Birder LA, de Groat WC. Induction of c-fos expression in spinal neurons by nociceptive and non-nociceptive stimulation of LUT. Am J Phys 1993; 265:R326–R333. Araki I. Inhibitory postsynaptic currents and the effects of GABA on visually identified sacral parasympathetic preganglionic neurons in neonatal rats. J Neurophysiol 1994; 72:2903–2910. Araki I, de Groat WC. Developmental synaptic depression underlying reorganization of visceral reflex pathway in spinal cord. J Neurosci 1997; 17:8402–8407. Onufrowicz B. On the arrangement and function of the cell groups of the sacral region of the spinal cord in man. Arch Neurol Psychopathol 1890; 3:387–411. Kuzuhara S, Kanayawa I, Nakanishi T. Topographical localisation of the Onuf’s nuclear region innervating the rectal and vesical striated sphincter muscles: a retorgrade fluorescent double labelling in cat and dog. Neurosci Lett 1980; 16:125–130. Breedlove SM, Arnold AP. Sexually dimorphic motor nucleus in the rat lumbar spinal cord: response to adult hormone manipulation, absence in androgen insensitive rats. Brain Res 1981; 225:297–357. Forger N, Breedlove SM. Sexual dimorphism in human and canine spinal cord: role of early androgen. Proc Nat Acad Sci USA 1986; 83:7527–7531. Pullen AH, Tucker D, Martin JE. Morphological and morphometric characterisation of Onuf’s nucleus in the spinal cord of man. J Anat 1997; 191:201–213. Gibson S, Polak JM, Katagiri T, Su H, Weller RO, Brownell DB, et al. A comparison of the distribution of eight peptides in spinal cord from controls and cases of motor neurone disease with special reference to Onuf’s nucleus. Brain Res 1988; 474:255–278. Tashiro T, Sadota T, Matsishima R, Mizuno N. Convergence of serotonin-, enkephalin- and substance P-like immunoreactive afferent fibres on single pudendal motoneurones in Onuf’s nucleus of the cat: a light microscope study combining the triple immunocytochemical staining technique with the retrograde HRP-tracing method. Brain Res 1989; 481:392–398.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

127.

128. 129.

130. 131.

132.

133. 134.

135.

136.

137. 138. 139. 140.

Holstege G, Tan J. Supraspinal control of motoneurones innervating the striated muscles of the pelvic floor including urethral and anal sphincters in the cat. Brain 1987; 110:1323–1344. Barrington RJ. The effect of lesions of the hind and midbrain on micturition in the cat. Quart J Experiment Physiol 1925; 15:81–102. Holstege G, Griffith D, de Wall H, Dalm E. Anatomival and physiological observations on supraspinal control of bladder and urethral sphincter muscles in the cat. J Comp Neurol 1986; 250:449–461. Mallory BS, Roppolo JR, de Groat WC. Pharmacological modulation of the pontine micturition center. Brain Res 1979; 546:310–320. Blok BFM, Holstege G. Ultrastructural evidence for a direct pathway from the pontine micturition center to the parasympathetic preganglionic motoneurons of the bladder of the cat. Neurosci Lett 1997; 222:195–198. Blok BFM, De Weerd H, Holstege G. The pontine micturition center projects to sacral cord GABA immunoreactive neurons in the cat. Neurosci Lett 1997; 233:109–112. Konishi A, Itoh K, Sugimoto T, Yasui Y, Kaneko T, Takada M. Neurosci Lett 1985; 61:109–113. Noto H, Roppolo JR, Steers WD, de Groat WC. Excitatory end inhibitory influences on bladder activity elicited by electrical stimulation in the pontine micturition center in the rat. Brain Res 1989; 492:99–115. Vanderhorst VG, Mouton LJ, Blok BF, Holstege G. Distinct cell groups in the lumbosacral cord in the cat project to different areas in the periaqueductal grey. J Comp Neurol 1996; 376:361–385. Blok BFM, Holstege G. Direct projection from the periaqueductal grey to the pontine micturition center (M-region). An anterograde and retrograde tracing study in the cat. Neurosci Lett 1994; 166:93–96. Blok BFM, Willemsen AT, Holstege G. A PET study on brain control of micturition in humans. Brain 1997; 120:111–121. Blok DF, Sturms LM, Holstege G. Brain activation during micturition in women. Brain 1998; 121:2033–2042. Nour S, Svarer C, Kristensen JK, Paulson ON, Law I. Cerebral activation during micturition in normal men. Brain 2000; 123:781–789. Mallory BS, Roppolo JR, de Groat WC. Pharmacological modulation of the pontine micturition center. Brain Res 1991; 546:310–320.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

20 Sleep Regulation and the Autonomic Nervous System Harvey Moldofsky and Wah-Ping Luk University of Toronto, Toronto, Ontario, Canada

I. FUNCTIONAL ANATOMY A. Circadian Rhythms, Sleep, and the Autonomic Nervous System The autonomic nervous system is integral to the homeostatic regulatory functions of the sleeping/waking brain. The autonomic changes that occur over the course of the 24-hour of sleep/wake cycle in humans have rhythmic physiological characteristics. Not only do cholinergic, adrenergic, histaminergic, orexin and adenosine neurotransmitters, operating via the brain stem, facilitate the transition between wake and sleep, but they also influence the cycling between non–rapid eye movement (non-REM) and REM sleep. The autonomic system and the sleep/wake system integrate central and peripheral bodily and behavioral functions. A variety of physiological functions under autonomic system control operate within the circadian system in humans. These include the circadian changes in temperature, hormonal, cardiovascular, respiratory, gastrointestinal, and genitourinary functions. In order to understand how the autonomic functions operate as a component of the circadian rhythms of the body and how these functions are linked to the sleep/wake cycle, the underlying functional anatomy of the central nervous system will be reviewed. B. Circadian Rhythms Sleep and waking regulation involves both homeostatic and circadian influences. In the absence of periodic time cues such as the light dark cycle, humans operate Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

in about a 24-hour rhythmic schedule. The diurnal circadian sleep/wake cycle is controlled by a collection of neurons in the preoptic area of the anterior hypothalamus known as the suprachiasmatic nucleus (SCN). Inputs into this region come from two sources (for review, see Ref. 1). Photic (light) influences come directly from the retinohypothalamic tract, and nonphotic inputs are relayed via the geniculohypothalamic tract. SCN efferents project to several major regions. An important region is the preoptic area of the hypothalamus. This region is intimately involved in the control of reproduction, fluid homeostasis, sleep, and thermoregulation. Projections from the SCN to the subparaventricular zone and paraventricular nucleus are critical to maintaining diurnal rhythms of melatonin (2). These projections may also act as the gateway for circadian control of neuroendocrine and autonomic systems (3). Dorsal medial nucleus inputs from the SCN influence corticosterone rhythms (4). Ablation of the SCN does not cause the total time of sleeping and waking to change greatly. However, the schedules of sleeping and waking become fragmented (5).

II. SLEEP AND WAKE MECHANISMS A. Reticular Modulatory System Moruzzi and Magoun’s (6) seminal experiments point to the existence of a specific “wakefulness”-generating system in the brainstem. Specifically, electrical stimulation of the brainstem reticular formation, and not the somatosensory pathways, results in a state of cortical activation similar to that of wake. Additional experiments with selective lesions of the reticular formation that avoid the somatosensory pathways produce a loss of cortical activation (7). This suggests that sleep involves the inhibition of a wakefulness-activating system located in the brainstem. Since those experiments numerous studies have converged on the idea of an “activating” system located in the reticular formation, which is defined as a diffuse neuronal region running in the brainstem from the medulla to the thalamus (for a detailed review, see Ref. 8). Labeling studies show that the reticular activating system sends projections via two pathways: a dorsal route to the thalamus, which then projects to the cortex, and a ventral route to the posterior hypothalamus and basal forebrain, which then projects diffusely into the cortex. From this rudimentary view of the wake active system the convergence of these neuronal pathways with sympathetic control regions can be seen. Hess (9) first noted this overlap in the 1950s when he observed that stimulation of the posterior hypothalamus and midbrain reticular formation elicited behavioral arousal. Cortical activation, operating together with the brainstem activation, increases blood pressure and heart rate and causes pupillary dilation.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

B. Neurotransmitters of the Reticular Activating System Reticular formation is a diffuse system of neurons and fiber tracts in the brainstem running from the medulla to the thalamus. Thus, when we speak of the “reticularactivating system” within this formation, we are referring to a number of distinct neuronal groups within the reticular formation that utilize specific neurotransmitters related to arousal states. Three main activating neurotransmitter systems arise from discrete populations of neurons in the brainstem reticular formation. The cholinergic system, one of the first to be recognized, originates in the laterodorsal tegmental nuclei (LDT) and pedunculopontine tegmental nuclei (PPT) of the dorsal pons (10,11). Neurons in this region are most active during wake and REM sleep when the cortex is in an activated state (for review, see Ref. 12). Fibers from this system extensively innervate and activate the thalamus along a dorsal route. The action of acetylcholine depolarizes thalamic neurons, causing them to convert the firing mode of the neurons from burst mode to single spike mode, a state that removes the inhibition of somatosensory information transfer (13). LDT and PPT fibers that run ventrally also innervate the cholinergic basal forebrain, which can also strongly activate the cortex. Noradrengeric neurons arising from the locus coeruleus (LC) have extensive projections that cover the entire cortex (14). Neurons in the LC display their highest activity during waking and decrease their discharge in non-REM sleep, becoming relatively inactive in REM sleep (15,16). Inhibition of target neurons in the basal forebrain via pharmacological means produces sleep, as do lesions directly in the LC region (17). The dopaminergic system, arising from the substantia nigra and the ventral tegmental area of the midbrain, projects mainly to the striatum, amygdala, hippocampus, and frontal cortex. This system plays an important role in behavioral arousal (8).

C. Other Activating Systems Outside of the brainstem reticular formation there are three main activating systems: the histamine, orexin (the recently discovered hypothalamic peptides involved in appetite regulation and sleep), and cholinergic systems. 1. Histamine The ability of antihistamines to produce drowsiness was one of the first clues that histamine was involved in the maintenance of arousal. Histaminergic neurons are located in the tuberomammillary nucleus (TMN) of the posterior hypothalamus (18). These neurons display their highest activity during wake and greatly taper off during non-REM and REM sleep (19). TMN fibers project widely throughout the cortex. These fibers are thought to reciprocally inhibit temperature-sensitive

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

basal forebrain preoptic neurons that are implicated in sleep (20). Histamine injections into the preoptic area produce arousal. Selective lesions of the TMN neuronal bodies cause a decrease in arousal and an increase in both non-REM and REM sleep (21). 2. Orexins Orexin-containing neurons are found in the tuberal region of the posterior hypothalamus just rostral of the turbomammillary nucleus (22). Orexin fibers ascend in a dorsal pathway to innervate the whole cortex and ventrally throughout the hypothalamus and basal forebrain. Descending fibers of this system innervate the locus coeruleus, LDT, PPT, nucleus of solitary tract, and dorsal motor nucleus of the vagus (23). The loss of these neurons are implicated in canine and human narcolepsy. Injections of orexin A into rats at light onset cause increase in arousal and decrease in REM sleep without affecting non-REM sleep (24). It has been postulated that orexin neurons promote wakefulness by exciting wake active neurons (22). Their extensive connections suggest involvement of orexin-containing neurons in the control of various autonomic, neuroendocrine, and neuroregulatory systems that affect wakefulness functions (25). Intracerebroventricular injections of orexins can dose dependently increase heart rate, blood pressure, gastric acid secretion, drinking behavior, and renal sympathetic nerve activity in conscious unrestrained rats (26–28). Moreover, there are numerous orexin fibers in the supraoptic and paraventricular nucleus, which are involved extensively in autonomic control (23). 3. Basal Forebrain Cholinergic Neurons Apart from the cholinergic neurons in the LDT and PPT of the reticular formation, cholinergic neurons of the basal forebrain are also implicated in arousal (for review, see Ref. 29). Located in the septum, diagonal band of Broca, magnocellular preoptic nucleus, and substantia innominata extending to the globus pallidus, these neurons project extensively to the hippocampus and cortex. III. SLEEP-PROMOTING REGIONS OF THE BRAIN A. Non-REM Centers In the late 1950s Batini (30) in Moruzzi’s group utilized transsection studies to discover sleep-promoting regions of the brainstem. Batini observed insomnia in animals when the brainstem was transected behind the oral pontine tegmentum, indicating that sleep was an active phenomenon. Subsequently, it was shown that electrical stimulation of the dorsal medullary reticular formation and the nucleus of the solitary tract induce cortical synchronization indicative of non-REM sleep Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

(31). The nucleus of the solitary tract receives a great diversity of inputs, including taste afferents, chemoreceptor systems, namely detectors of carbon dioxide and oxygen tension, and inputs from the vagus nerves (32). The somnogenic actions of peripheral interleukin-1 (IL-1) are signaled via the vagus (33). Direct efferents from the solitary tract can be traced to the limbic forebrain structures, thalamus, hypothalamus, and preoptic area (34,35). This suggests that the solitary tract mediates forebrain areas that are involved in both autonomic and sleep control. Projecting serotoninergic neurons within the raphe nuclei of the brainstem are thought to be involved in sleep-promoting actions. Blocking the synthesis of serotonin, or 5-hydroxytryptamine (5HT), via PCPA causes insomnia, which is reversed by direct application of serotonin precursors (8). The discreet neuronal regionalization of 5-HT receptor subtypes also implicates 5-HT as a mediator of sleep/wake activity. Excitatory postsynaptic 5-HT receptors (36) are located preferentially on GABAergic inhibitory neurons in the cortex and forebrain. Inhibitory postsynaptic 5-HT receptors (37) are located on cholinergic basal forebrain neurons involved in cortical activation. Electrical stimulation of the anterior hypothalamus and the adjacent preoptic and basal forebrain areas induce sleep in chronically implanted animals (38). Lesions in these areas produce a significant decrease in sleep (39). Inhibitory GABAergic neurons in the anterior hypothalamus and basal forebrain descend to innervate the posterior hypothalamus (40) and ascend via long-range projections to diffusely innervate the cortex (41). Benzodiazepines, a major class of hypnotic drugs, have sedative and sleep-promoting actions that are mediated via the GABA receptor system. Cortical GABA levels are highest during non-REM sleep as compared to REM and wake (42). Although the exact mechanism remains to be elucidated, much anatomical overlap exists between sleep and parasympathetic centers in the anterior hypothalamus and adjacent preoptic regions (43). Stimulation of these regions elicits sleep in conjunction with decreases in blood pressure and heart rate and causes pupillary constriction. B. REM Centers Cortical activation during REM sleep is similar to that of arousal. However, accompanying this is widespread muscle atonia and periodic bursts of rapid eye movement. Transection studies show that the neuronal machinery that generates REM sleep is located in the pons (for detailed review, see Ref. 44). Destruction of the oral pontine reticular nucleus located just ventral to the locus coeruleus prevents the EEG and behavioral changes of REM sleep (45). Direct microinjection of acetylcholine into the oral pontine reticular nucleus produces the behavioral signs of REM sleep (46). Cholinergic neurons in the LDT/PPT are important for the expression of REM. Their fibers innervate oral pontine reticular formation. The activity of these neurons is highest during wake and REM. They become quiescent in non-REM (44). The increase in activity during REM is thought to result Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

from disinhibition. 5-HT inputs from the dorsal raphe and noradrenergic inputs from the locus coeruleus normally inhibit LDT/PPT neurons so that they become virtually silent during REM. This results from the activation of inhibitory GABAergic neurons, which synapse onto these areas (44). However, the mechanisms that activate the GABAergic neurons to date are unknown. The functional significance of these various pathways in the central nervous system provide the basis for rhythmic functions that integrate the autonomic system to the sleep/wake system. Not only do the rhythmic functions of autonomic activities occur over the 24-hour sleep/wake cycle and within the approximate 90minute cycle of non-REM and REM sleep, but there are also rhythmic changes in autonomic functions that occur within non-REM sleep. This non-REM rhythmic phenomenon is suggested to be linked to fluctuations in arousability from sleep. One of the core features of sleep is its reversibility, so that although sleep involves a behavioral disengagement from the environment, the ability to be aroused from the sleeping state is an essential characteristic. That reversibility normally operates within a cyclical system of physiological phasic changes that occur within a 60-second time interval of non-REM sleep. This cyclical electrophysiological phenomenon is known as the cyclical alternating pattern (CAP). There are two distinctive phases: phase A, which is accompanied by a transient increase in sympathetic activity and an increased tendency to be aroused from sleep, and phase B, which follows phase A, where there is a transient dominance of parasympathetic activity and a transient decrease in arousability from sleep. The CAP is identified by polysomnography in distinctive periodic changes in the EEG of non-REM sleep that are coupled to subtle fluctuations in muscle tone and vegetative functions. That is, there are periodic fluctuations, usually at intervals of 20–40 seconds, in EEG arousals, blood pressure, pulse frequency, and respiration (47). REM sleep is characterized by tonic and phasic activities. During the tonic state there is a loss of postural motor control. The episodic bursts of ocular movements are associated with variable heart rate, blood pressure, and irregular breathing. These phasic activities in REM state physiology are the result of central changes in the regulation of autonomic outflow (48). IV. THERMAL REGULATION A. Temperature and Circadian Rhythms The endogenous circadian pacemaker regulating the sleeping/waking brain is located in the suprachiasmatic nucleus. This pacemaker is highly stable and varies over an approximately 24-hour period (49). Molecular biological studies have demonstrated that a set of genes in Drosophila and mammals (Clock, Bmal1, Period, and Timeless) provide a molecular framework for the circadian mechanism. Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

These genes define a transcription-translation–based negative autoregulatory feedback loop that comprises the core elements generating circadian rhythmicity (50). Circadian rhythms are driven entrained by the suprachiasmatic nucleus (SCN) of the hypothalamus that are coupled to the light/dark cycle. Until recently the research has suggested that the SCN is the master clock of the body that entrains the phase of peripheral clocks via chemical cues, such as rhythmically secreted hormones. Such nonphotic cyclic inputs as temperature, noise, exercise, social cues, and feeding affect the timing of the circadian pacemaker (51,52). However, recent research indicates that there are peripheral circadian oscillators that operate separately from the SCN pacemaker. The liver is shown to operate under the control of times for feeding behavior and independent of the influence of the SCN (53,54). Considerable interest has focused upon the rhythm of core body temperature rhythm, which is tightly coupled to variations in melatonin and cortisol in healthy young and older individuals living in controlled lighting conditions. The temperature varies within a range of about 0.5°C, with the highest during the midday and the lowest during slow-wave sleep (SWS) early in the sleep period (55). The daily rhythm of bodily temperature operates independently of activity and posture. Moreover, the circadian variations in temperature may influence the timing and duration of sleep/wakefulness. In an environment that is free of time cues, sleep/wake times become uncoupled from temperature. In such situations, bedtime is likely to occur near the trough of the temperature curve and waking occurs during the rising phase about 8 hours later. On the other hand, if bedtime occurs at the peak of the temperature curve, awake time will still occur during the rise time about 14.5 hours later (56). B. Temperature and Sleep/Wakefulness McGinty and Szymusiak (57) hypothesize that slow-wave sleep (SWS) in mammals and birds is controlled by these thermoregulatory mechanisms and provides brain and body cooling as a primary homeostatic feedback process. Cooling of the brain and body that is induced during SWS provides for various adaptive functions. These functions regulate timing of behavior over the circadian cycle and affect a variety of fundamental processes including lower energy utilization, reduced cerebral metabolism, protection of the brain against the sustained high temperatures of wakefulness, and facilitation of immune defense processes. CNS thermoregulation resides within the preoptic/anterior hypothalamic area (POAH) where thermosensitive neurons are implicated in the regulation of both body temperature and non-REM sleep. During non-REM sleep, there is increased discharge of a majority of POAH warm-sensitive neurons (WSNs) as compared to wakefulness. On the other hand, cold-sensitive neurons (CSNs) exhibit reduced discharge in non-REM sleep as compared to wakefulness. However, the change in therCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

mosensitivity from wakefulness to non-REM sleep is related to the change in discharge rate in WSNs but not in CSNs (58). With onset of sleep and decline of core temperature, heat loss occurs from the skin in humans. In neutral environments sweat rate increases so that thermoregulation occurs at a lower body temperature. When the ambient temperature is low, shivering is preserved during non-REM sleep but is lost in REM sleep (59). The occurrence of the thermoregulatory shift from wake to sleep is in addition to the circadian changes that occur in body temperature. Brain temperature declines during non-REM sleep and increases during REM sleep. The thermoregulatory functions of the body during sleep are dependent upon the autonomic control over changes in blood pressure and the circulation of the blood. Heat that is produced by cellular metabolism in the brain is transferred to the perfusing arterial blood. The circulating blood is maintained at a lower temperature than that of the brain by systemic and selective cooling mechanisms. In mammals, the systemic mechanism involves the return of cool venous blood from the heat exchangers of the body, e.g., upper airway mucosa, ear pinna, horn, glabrous skin, and tail to the heart, where it mixes with warm venous blood returning from body tissues. The brain undergoes selective cooling via cool venous blood from the systemic heat exchangers from vasomotor control of the nasal mucosa and ear pinna traversing carotid rete so that a temperature difference exists between the vertebral artery blood system and the carotid system. Therefore, there is a difference between the pontine temperature and hypothalamic temperature. The selective cooling in these regions is impaired during REM sleep (60). Overall, thermoregulation is inhibited during REM sleep. The observations of alterations in thermal functions during sleep have spawned various theoretical functions for sleep (61). These theories include that sleep serves to promote energy conservation and that non-REM sleep is a thermally regulated adaptation to the increase in heat produced by cellular metabolism during wakefulness.

V. RESPIRATORY FUNCTIONS A. Respiration and Circadian Rhythm Other coupled rhythms to core body temperature include respiration. Ventilation, as measured by hypercapnic ventilatory response (HCVR), showed that HCVR is aligned to circadian phase core body temperature with a variation ±12.1% of 24 h and accompanied by a circadian variation in metabolism (±3.2% of 24 h mean). Although there are no detectable rhythms in tidal volume, respiratory frequency, or ventilation, cosinor analysis showed that core body temperature is linked to ventilatory control mechanisms with circadian variations in FEV1 and FEV1/FEVC. The Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

peak-to-trough ranges in circadian change in spirometric variables were 2.0–3.2% of the mesor, with the minima of all variables occurring during sleep (62,63). Pulmonary function values normally decline at night (64). Central parasympathetic outflow is an essential factor for the circadian rhythm of bronchial tone and, thus, for the increase in bronchial resistance at night. This circadian variation in airway resistance affects airway conductance and bronchial reactivity, which occur with peak times at approximately 1600 and 0440 hours, respectively (65). B. Respiration and Sleep Ventilation during sleep depends upon three processes that affect the brainstem respiratory centers: chemical information arriving from chemoreceptors that respond to PaO2, PaCO2, and pH; mechanical information from receptors in the lung and chest wall; and behavioral information from higher cortical centers. Within this context at the onset to sleep, respiration slows and is regular. In the transition from wakefulness to sleep, ventilation falls by approximately 20% (66). Ventilatory responses to hypoxia and CO2 fall during sleep, and both are substantially reduced during REM sleep. Although airway resistance is greater during non-REM sleep in humans, in cats resistance is higher during REM sleep (67). Furthermore, in REM sleep respiration is irregular in rate and amplitude. It is during REM that postural muscle tone is lost so that in the absence of intercostal muscle tone and auxiliary respiratory muscle activity, breathing is solely dependent upon the intact phrenic nerves that operate the diaphragm. VI. CARDIOVASCULAR SYSTEM A. Cardiac Functions and Circadian Rhythm Common observation reveals that heart rate slows when a person falls asleep. Indeed, over the course of sleep onset, from presleep wakefulness to stable Stage 2 non-REM sleep, heart rate declines by an average of 6.64 beats (68). Various measures of electrocardiographic activity that are presumed to reflect autonomic functions have been employed in studies of their changes between wakefulness and sleep. One measure of frequency spectrum analysis of beat-to-beat electrocardiographic activity is the respiratory sinus arrhythmia (RSA). The RSA is considered to reflect the activity of the parasympathetic nervous system (PNS). Studies of the relationship of the transitions between wakefulness and sleep and of circadian rhythms indicate that although RSA increases in the transition from wakefulness to non-REM sleep and further increases with progression of non-REM sleep, RSA is found to be more strongly influenced by the circadian system than by the sleep system (68). Another measure of autonomic function, the magnitude of the 0.1 Hz peak, is thought to indicate sympathetic nervous system (SNS) activity. Studies of Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

the magnitude of the 0.1 Hz peak show slight decreases or not at all from wakefulness to non-REM sleep. However, the 0.1 Hz peak is now generally thought to reflect both PNS and SNS afferents and efferents in the control of blood pressure. Another measure, thought to reflect SNS activity, is the preejection period (PEP). This does not change during the initial transition from wakefulness to sleep, but decreases progressively with continued non-REM sleep accompanied by a fall in the activity of peripheral sympathetic nerves to skeletal muscle blood vessels (68). The shift from sleep to awakening in the morning reverses the effect of entry into sleep on RSA and the 0.1 Hz peak. The increase in heart rate following spontaneous arousal from sleep is associated with cardiac sympathetic activation and parasympathetic withdrawal, whereas stable awakening into relaxed wakefulness is associated mainly with sympathetic activation (69). Autonomic functions change during REM sleep. Intraneural recordings of efferent sympathetic nerve activity from the peroneal nerve posterior to the fibular head during REM sleep showed that sympathetic nerve activity increases above the levels recorded during wakefulness. The measures for blood pressure and heart rate return to those recorded during wakefulness. When postural muscle tone is restored during brief muscle twitches in REM sleep, sympathetic nerve discharge ceases, and there is an increase in blood pressure. Therefore, while vasoconstriction in postural muscles occurs during REM sleep, vasodilation occurs in the mesenteric and renal vascular systems (70,71). B. Blood Pressure and Circadian Rhythm Blood pressure varies over the course of the day with highest mean values occurring in the mid-morning and the lowest values at 0300 hours. The mean arterial BP (MAP) begins to rise again during the early hours of the morning before waking (72). MAP drops on average 9 mmHg and heart rate (HR) on average 18 beats per minute during the night, which is accompanied by a substantial decrease in cardiac output, mainly through a decrease in HR. Stroke volume also decreases slightly in the night, and total peripheral resistance increases considerably during the night compared with the daytime, followed by a sharp decrease when subjects arise in the morning. The sleep-related decline in BP may be the result of recumbency and the absence of physical activity during the night. When subjects rest during the daytime instead of being ambulant, the circadian variation in BP and HR is significantly decreased. Because BP is in part controlled by blood flow, which is regulated by peripheral vasoconstriction and vasodilatation, a decreased blood flow during the nighttime suggests an increase in peripheral resistance (73). The maintenance of a normal circadian BP pattern requires both an intact sympathetic and an intact afferent parasympathetic ANS (74). Detailed analyses of the circadian and sleep-related changes in blood pressure and heart rate show that both change in a bimodal pattern with a morning Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

acrophase (around 1000 hours), a small afternoon nadir (around 1500 hours), an evening acrophase (around 2000 hours), and a profound nocturnal nadir (around 0300 hours). Therefore, the major changes in cardiovascular function occur just prior to and following morning awakening. The amplitude of the diurnal variations are largest for heart rate, intermediate for diastolic blood pressure, and smallest for systolic blood pressure (respectively, 19.9%, 14.1%, and 10.9% of the 24hour mean). The increases in blood pressure and heart rate begin before awakening and continue after awakening in the morning. Postural changes and the sleep wake transition are the major factors responsible for the 24-hour rhythm in blood pressure. Both reclining and sleep accounted for 65–75% of the nocturnal decline in blood pressure. However, the nocturnal 50% of the decline in heart rate suggests that the 24-hour rhythm of heart rate reflects an endogenous circadian rhythm, amplified by the effect of sleep (75). Plasma epinephrine levels are correlated to mean arterial blood pressure and have a circadian rhythm with the peak occurring at 12.20  0.40 hours and amplitude of 15  2 pg/mL, but plasma norepinephrine and dopamine levels do not follow a circadian rhythm and are related to arterial blood pressure (68). The relationship of sleep to autonomic control over blood pressure is evident in the observation that sleep deprivation causes an increase in blood pressure and a decrease in muscle sympathetic nerve activity but no changes in heart rate, forearm vascular resistance, and plasma catecholamines. Therefore, this increase in blood pressure is not mediated by muscle sympathetic vasoconstriction or tachycardia (76,77). C. Blood Pressure and Sleep Like heart rate, blood pressure and systemic vascular resistance are reduced during non-REM sleep. More specifically, blood pressure progressively declines from wakefulness through stages 1 to 4 non-REM or slow-wave sleep. On the other hand, blood pressure varies considerably during REM sleep and is on the ascendancy during the increased periods of REM that occur during the last part of the nighttime sleep (70). VII.

GASTROINTESTINAL FUNCTIONS

A. Gastrointestinal System and Circadian Rhythms Circadian changes in autonomic function appear to affect gastrointestinal motility. Diurnal variation in esophageal contractions are determined partly by meal intake and partly by clustering of nocturnal contractions. During the night, propagated contractions and simultaneous contractions are less frequent than during both meal and nonmeal daytimes. However, the nocturnal segmental contraction frequency of the esophagus is similar to nonmeal daytime contraction frequency Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

(78). With regard to circadian activity of the stomach, gastric emptying is 53% longer following evening meals at 2000 hours than morning meals at 0800 hours. These differences occur in the handling of solid foods rather than liquid foods (79). During sleep there appears to be a reduced level of vagally mediated intestinal motor activity compared to wakefulness (80). B. Gastrointestinal System and Sleep During nighttime sleep periods the absence of esophageal motor activity is interspersed by clusters of contractions detected 5 and 15 cm above the lower esophageal sphincter. Primary contractions progressively decrease from the lightest stages of non-REM sleep to deep (SWS) and REM sleep. Secondary contractions, which are lower in amplitude and of shorter duration than primary ones, show a similar pattern except that during REM sleep the frequency of secondary contractions increase to levels similar to awake and light stage I sleep. The increased number of secondary contractions during REM sleep is likely the result of an increase in autonomic nervous system activity during REM sleep (81). In contrast to differences noted in REM sleep in esophageal activity, no differences occur between REM and non-REM sleep in the migrating motor complex that begins in the stomach and progresses through the bowel (80). VIII. GENITAL FUNCTIONS AND SLEEP Cycles of penile erections and of clitoral erections with increased vaginal blood flow in humans are intimately linked to the cycles of REM sleep. These automatic physiological activities are not related to dream content of REM sleep, but are influenced by a complex interplay of autonomic, motor, and neuroendocrine functions. Investigators have speculated that penile tumescence occurs because REM sleep is associated with decreased sympathetic tone and increased in parasympathetic activity. But the role of the autonomic nervous system is unknown. The phasic alterations in autonomic activity during REM may have variable parasympathetic and sympathetic effects on REM and differing end organ responses. Such variability and the complexity of the influence of forebrain, brainstem, and spinal nerve projections to the genital region as well as the involvement of neurohormonal androgen influences on penile and clitoral erections remain to be unraveled (82). IX. AUTONOMIC NERVOUS SYSTEM AND PATHOPHYSIOLOGY A. Sleep Deprivation and the Autonomic Nervous System Circadian changes in temperature and cortisol continue during sleep deprivation (61), but the amplitude of change over the 24-hour period is reduced. Whereas

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

normally sleep is accompanied by reduction of sympathetic activity, total sleep deprivation and as little as 4 hours of nocturnal sleep cause an augmentation of sympathetic nervous system activity. Such changes contribute to disturbances in metabolic and neuroendocrine functions. The disturbances include a reduction in glucose tolerance and thyrotropin concentrations and increased evening cortisol concentrations. Because these changes are similar to those seen in normal aging, Spiegel et al. (83) speculated that sleep debt may increase the severity of age-related chronic disorders. B. Diagnostic and Clinical Implications 1. Effects of Autonomic Nervous System Pathophysiology on Sleep A common feature of degenerative diseases of the nervous system that result in disorders of the autonomic nervous system is the loss of circadian and sleep patterns. For example, in familial dysautonomia there is a decrease in REM sleep and increased time to onset of REM. Abnormal breathing patterns occur during sleep, and apneas are common (84). Similarly, in Shy-Drager’s disease sleep-related breathing is irregular in amplitude and regularity (85). In the autosomal dominant prion disease fatal familial insomnia, circadian autonomic and neuroendocrine rhythmicity are lost and the intrinsic organization of sleep disappears. The degeneration of the thalalmus is accompanied by unbalanced sympathergic activation with preserved parasympathetic drive with chronic secondary hypertension and loss of the nocturnal sleep–related decrease in blood pressure. Neuroendocrine studies show hypercortisolism, abnormal suppression of adrenocorticotrophic hormone, persistent elevation of catecholamines, abnormal secretory patterns of growth hormone, prolactin, and melatonin (86). Similarly, in another prion disease involving the central nervous system, Creutzfeldt-Jakob disease, the organization and characteristic features of non-REM sleep are lost (87). Diseases of the peripheral nervous system that affect autonomic functions result in REM-related loss of penile tumescence. This link of penile tumescence to REM sleep is employed in the sleep laboratory to differentiate between sexual impotence that is the result of pathophysiological changes in autonomic functions and psychogenic sexual impotence. For example, whereas REM-related erections are impaired in patients as the result of diabetic autonomic neuropathy, REM-related erections are usually preserved in psychogenic impotence. 2. Effects of Primary Sleep Disorders on Autonomic Nervous System Functions As indicated above, the cyclical alternating pattern (CAP) phenomenon, which occurs in non-REM sleep, is normally coupled to subtle fluctuations in muscle tone and vegetative functions. Where this pattern of CAP is quite dominant in the

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

sleep, there is an entrainment of periodic pathological conditions that occur in primary sleep disorders (88). In fatal familial insomnia, CAP is quite prominent and is accompanied by a disintegration of the continuity of sleep. The CAP is also integral to such periodic arousal disorders as sleep apnea and periodic limb movement disorder. a. Sleep Apnea. In sleep apnea, the cessation of breathing is triggered in phase B of the sleep EEG or the quiescent phase of CAP. During this phase there is an increase in vagal efferent parasympathetic activity with transient slowing of the heart rate, decrease in blood pressure, and decrease in arterial oxygen saturation. Carotid body or baroreceptor activation affects the degree of slowing of the heart rate. Ventricular ectopic beats are common. Phase A triggers EEG arousal with loud snoring, heavy breathing, acceleration of heart rate, increased blood pressure, and improvement in arterial oxygen saturation. This phase is thought to occur as the result of a combination of decrease in vagal tone and activation of the sympathetic arm of the autonomic nervous system. The termination of the apnea in severe hypoxia with oxygen desaturation of less than 65% may cause ventricular tachycardia and occasional sinus pauses of 2–13 seconds. Chemoreceptor activation as the result of hypoxia and carbon dioxide retention facilitates sympathetic activity. This results in peripheral vasoconstriction and elevation in blood pressure (89). Systolic and diastolic blood pressure increases by an average of 25% in those with patients with moderate to severe obstructive apneas (90). Patients with sleep apnea have high levels of sympathetic activity during both sleep and wakefulness. There are high levels of norepinephrine, faster heart rates, decreased heart variability, and increased low-frequency oscillatory component of the heart rate (89). This sympathetic activity is independent of hypertension and obesity, which occur frequently in such patients. The changes in autonomic and cardiovascular functions may predispose to hypertension, especially in those who do not have a drop in blood pressure during sleep. Other risks include a greater predisposition to myocardial infarction and stroke (90). b. Periodic Movements in Sleep and Restless Legs Syndrome. The CAP phenomenon that is evident in sleep-related periodic movement disorder is greater than the time and rate of CAP found in normal subjects. The phasic EEG arousals that coincide with leg movements during non-REM sleep are associated with an acceleration of heart rate and transient increase in blood pressure. The increased frequency of CAP acts as a phasing mechanism that entrains the motor activity during sleep (91). This increased CAP activity seen in sleep EEG is an indication of the arousal and autonomic instability that characterizes this sleep disorder. Overall, the mean increase in systolic blood pressure of 23% is similar to the rise in blood pressure seen in patients with obstructive sleep apnea. However, treatment with temazepam in order to reduce EEG arousals does not alter the swings in blood pressure. (92). Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

c. Parasomnia Disorders. Parasomnia disorders are characterized by partial arousals from sleep. During these arousals from sleep, there are sudden increases in motor behavior and autonomic activities. One of the most dramatic parasomnia disorders is sleep terrors. In sleep terrors there is a precipitous arousal from slow-wave sleep with marked increase in muscle tone, heart rate, respiration, perspiration, and dilated pupils. The physiological changes are similar to an exaggerated startle response with a tremendous increase in autonomic arousal from non-REM sleep. The individual is partially asleep but may scream, appear terrified, and behaves in an automatic fashion without clear awareness of the surroundings. Rarely in the ensuing confusional state the person might leap from the bed and behave in a violent manner. Usually the behavioral and autonomic disturbances subside in several minutes and the person returns to sleep. Often there is no recollection of the event on the following day (93). Bursts of autonomic activity with awakenings from sleep that have variable intensity also accompany other parasomnia disorders, e.g., nocturnal panic attacks or awakenings from REM-related nightmares. Transient increased autonomic activities occur in sleep-related epilepsy, where there is a violent paroxysmal stereotypical motor arousal from sleep with behavioral and brain electrophysiological features of seizure.

REFERENCES 1.

2.

3. 4.

5.

6. 7.

8.

Card JP. Anatomy of the mammalian circadian timekeeping system. In: Lydig R, Baghdoyan HA, eds. Handbook of Behavioral State Control. Boca Raton, FL: CRC Press, 1999: 13–25. Bujis RM, Hermes MH, Kalsbeek A. The suprachiasmatic nucleus-paraventricular nucleus interactions: a bridge to the neuroendocrine and autonomic nervous system, Prog Brain Res 1998; 119:365–382. Moore RY. Neural control of the pineal gland. Behav Brain Res 1996; 73:125–130. Kalsbeek A, Buijs RM, van Herrikhuize JJ, Arts M, van der Woude TP. Vasopressin containing neurons of the suprachiasmatic nuclei inhibit corticosterone release. Brain Res 1992; 580:62–67. Edgar DM, Dement WC, Fuller CA. Effect of SCN lesions on sleep in squirrel monkeys: evidence for opponent processes in sleep-wake regulation. J Neurosci 1993; 13:1065–1079. Moruzzi G, Magoun H. Brain stem reticular formation and activation of the EEG. Electroencephalogr Clin Neurophysiol 1949; 1:455–473. Lindsley DB, Schreiner LH, Knowles WB, Magoun HW. Behavioral and EEG changes following chronic brain stem lesions in the cat. Electroencephalogr Clin Neurophysiol 1950; 2:483–495. Jones BE. Basic mechanisms of sleep-wake states. In: Kryger MH, Roth T, Dement WC, eds. Principles and Practice of Sleep Medicine. 3rd ed. Philadelphia: W.M. Saunders Company, 2000:134–154.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

9. Hess WR. Diencephalon: Autonomic and Extrapyramidal Functions. New York: Grune & Stratton Inc., 1954:17–24. 10. Hallanger AE, Levy AI, Lee HJ, Rye DB, Wainer BH. The origins of cholinergic and other subcortical afferents to the thalamus in the rat. J Comp Neurol 1987; 262:105–124. 11. Shute CC, Lewis PR. The ascending cholinergic reticular system neocortical, olfactory and subcortical projections. Brain 1967; 90:497–520. 12. Siegel JM. Brainstem mechanisms generating REM sleep. In: Kryger MH, Roth T, Dement WC, eds. Principles and Practice of Sleep Medicine. 3rd ed. Philadelphia: W.B. Saunders Company, 2000:112–133. 13. Steriade M, McCormick DA, Sejnowski TJ. Thalamocortical oscillations in the sleeping and aroused brain. Science 1993; 262:679–685. 14. Jones BE, Yang TZ. The efferent projections from the reticular formation and the locus coeruleus studied by anterograde and retrograde axonal transport in the rat. J of Comparative Neurology 1985; 242:56–92. 15. Hobson JA, McCarley RW, Freedman R, Pivik RT. Time course of discharge rate changes by cat pontine brain stem neurons during sleep cycle. Neurophysiol 1974; 37:1297–1309. 16. Gervasoni D, Darracq L, Fort P, Souliere F, Chouvet G, Luppi PH. Electrophysiological evidence that noradrenergic neurons of the rat locus coeruleus are tonically inhibited by GABA during sleep. Eur J Neurosci 1998; 10:964–970. 17. Jones BE, Harper ST, Halaris AE. Effects of locus coeruleus lesions upon cerebral monoamine content, sleep-wakefulness states and the response to amphetamine. Brain Res 1977; 124:473–496. 18. Steinbusch HWM, Mulder AH. Immunohistochemical localization of histamine in neurons and mast cells in the rat brain. In: Bjorklund A, Hokfelt T, Kuhar MJ, eds. Handbook of Chemical Neuroanatomy. Vol. 3. Classical Transmitters in the CNS, Part II. Amsterdam: Elsevier, 1984:126–163. 19. Szymusiak R, McGinty D. Sleep-related neuronal discharge in the basal forebrain of cats. Brain Res 1986; 370:82–92. 20. Shiromani PJ, Scammell T, Sherin JE, Saper CB. Hypothalamic regulation of sleep. In: Lydig R, Baghdoyan HA, eds. Handbook of Behavioral State Control. Boca Raton, FL: CRC Press, 1999: 311–325. 21. Lin JS, Sakai K, Jouvet M. Hypothalamo-preoptic histaminergic projections in sleepwake control in the cat. Eur J Neurosci 1994; 6:618–625. 22. Kilduff TS, Peyron C. The hypocretin/orexin ligand-receptor system: implications for sleep and sleep disorders. Trends Neurosci 2000; 23:359–365. 23. Peyron C, Tighe DK, van den Pol AN, de Lecea L, Heller C, Sutcliffe JG, Kilduff KS. Neurons containing hypocretin (orexin) project to multiple neuronal systems. J Neurosci 1998; 18:9996–10015. 24. Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hemmati P, Munton RP, Ashmeade TE, Shah AS, Hatcher JP, Hatcher PD, Jones DNC, Smith MI, Piper DC, Hunter JA, Porter RA, Upton N. Orexin A activates locus coeruleus cell firing and increases arousal in the rat. PNAS 1999; 96:10911–10916.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

25.

26.

27.

28. 29.

30.

31.

32. 33.

34. 35.

36. 37.

38. 39. 40.

41.

Date Y, Ueta Y, Yamashita H, Yamaguchi H, Matsukura S, Kangawa K, Sakurai T, Yanagisawa M, Nakazato M. Orexins, orexigenic hypothalamic peptides, interact with autonomic, neuroendocrine and neuroregulatory systems. PNAS 1999; 96:748–753. Shirasaka T, Nakazato M, Matsukura S, Takasaki M, Kannan H. Sympathetic and cardiovascular actions of orexins in conscious rats. Am J Physiol 1999; 277(6 Pt 2):R1780–R1785. Takahashi N, Okumura T, Yamada H, Kohgo Y. Stimulation of gastric acid secretion by centrally administered orexin-A in conscious rats. Biochem Biophys Res Commun 1999; 254(3):623–627. Kunii K, Yamanaka A, Nambu T, Matsuzaki I, Goto K, Sakurai T. Orexins/hypocretins regulate drinking behaviour. Brain Res 1999; 842:256–261. Szymusiak R, Alam N, McGinty D. Discharge patterns of neurons in cholinergic regions of the basal forebrain during waking and sleep. Behav Brain Res 2000; 115(2):171–182. Batini C, Moruzzi G, Palestini M, et al. Effects of complete pontine transections of the sleep-wakefulness rhythm: the midpontine pretrigeminal preparation. Arch Ital Biol 1959; 97:12–25. Magnes J, Moruzzi G, Pompeiano O. Synchronization of the EEG produced by low frequency electrical stimulation of the region of the solitary tract. Arch Ital Biol 1961; 99:33–67. Kandel ER, Schwartz JH, Jessel TM, eds: Principles of Neural Science. 4th ed. New York: McGraw-Hill, 2000. Hansen, MK Taishi P, Chen Z, Krueger JM. Vagotomy blocks the induction of interleukin-1 (IL-1) mRNA in the brain of rats in response to systemic IL-1. J Neurosci 1998; 18:2247–2253. Norgren R. Projections from the nucleus of the solitary tract in the rat. Neuroscience 1978; 3:207–218. Ricardo JA, Koh ET. Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat. Brain Res 1978; 153:1–26. Morales M, Bloom FE The 5-HT3 receptor is present in different subpopulations of GABAergic neurons in the rat telencephalon J Neurosci 1997; 17:3157–3167. Khateb A, Fort P, Alonso A, Jones BE, Muhlethaler M. Pharmacological and immunohistochemical evidence for serotonergic modulation of cholinergic nucleus basalis neurons. Eur J Neurosci 1993; 5(5):541–547. Sterman MB, Clemente CD. Forebrain inhibitory mechanisms: sleep patterns induced by basal forebrain stimulation in the behaving cat. Exp Neurol 1962; 6:102–117. McGinty DJ, Sterman MB, Sleep suppression after basal forebrain lesions in the cat. Science 1968; 160:1253–1255. Gritti I, Mainville I, Jones BE. Projections of GABAergic and cholinergic basal forebrain and GABAergic preoptic-anterior hypothalamic neurons to the posterior lateral hypothalamus of the rat. J Comp Neurol 1994; 339:251–268. Vincent SR, Hokfelt T, Skirboll LR, Wu JY. Hypothalamic gamma-aminobutyric acid neurons project to the neocortex. Science 1983; 220(4603):1309–1311.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

42. 43. 44.

45. 46.

47. 48.

49. 50. 51.

52.

53.

54. 55.

56.

57. 58.

59.

Jasper HH, Khan RT, Elliott KAC. Amino acids released from the cerebral cortex in relation to its state of activation. Science 1965; 147:1448–1451. Hess WR. In: Diencephalon: Autonomic and Extrapyramidal Functions. New York: Grune & Stratton Inc., 1954:17–24. Siegel JM. Brainstem mechanisms generating REM sleep. In: Kryger MH, Roth T, Dement WC, eds. Principles and Practice of Sleep Medicine. 3rd ed. Philadelphia: W.B. Saunders Company, 2000:112–133. Carli G, Zanchetti A. A study of pontine lesions suppressing deep sleep in the cat. Arch Ital Biol 1965; 103:751–788. Yamamoto K, Mamelak AN, Quattrochi H, Hobson JA. A cholinoceptive desynchronized sleep induction zone in the anterodorsal pontine tegmentum: locus of sensitive region. Neuroscience 1990; 39:279–293. Terzano MG, Parrino L. Clinical applications of cyclic alternating pattern. Physiol Behav 1993; 54:807–813. Parmeggiano PL. Physiological regulation in sleep. In: Kryger MH, Roth T, Dement WC, eds. Principles and Practice of Sleep Medicine. 3rd ed. Philadelphia: WB Saunders Co., 2000:169–178. Lavie P. Sleep-wake as a biological rhythm. Ann Rev Psychol 2001; 52:277–303. King DP, Takahashi JS. Molecular genetics of circadian rhythms in mammals. Annu Rev Neurosci 2000; 23:713–742. Klerman EB, Rimmer DW, Jan-Dijk D, Kronauer RE, Rizzo JF 3rd, Czeisler CA. Nonphotic entrainment of the human circadian pacemaker. Am J Physiol 1998; 274:R991–996. Buxton OM, Frank SA, L’hermite-Baleriax M, Leproult R, Turek F, et al. Roles of intensity and duration of nocturnal exercise in causing phase delays of human circadian rhythms. Am J Physiol 1997; 36:E536–542. Damiola F, Le Minh N, Preitner N, Kornmann B, Fleury-Olela F, Schibler U. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Gene Dev 2000; 14:2950–2961. Stokkan K-A, Yamazaki S, Tei H, Sakaki Y, Menaker M. Entrainment of the circadian clock in the liver by feeding. Science 2001; 291:490–493. Czeisler CA, Duffy JF, Shanahan TL, Brown EN, Mitchell JF, Rimmer DW, Ronda JM, Silva EJ, Allan JS, Emens JS, Dijk D-J, Kronauer RE. Stability, precision, and near-24-hour period of the human circadian pacemaker. Science 1997; 284:2177–2181. Czeisler CA, Weitzman ED, Moore-Ede MC, Zimmerman JC, Knauer RS. Human sleep: its duration and organization depend on its circadian phase. Science 1980; 210:1264–1267. McGinty D, Szymusiak R. Keeping cool: a hypothesis about the mechanisms and functions of slow-wave sleep. Trends Neurosci 1990; 13:480–487. Alam MN, McGinty D, Szymusiak R. Preoptic/anterior hypothalamic neurons: thermosensitivity in wakefulness and non rapid eye movement sleep. Brain Res 1996; 718:76–82. Parmeggiani PL, Rabini C. Shivering and panting during sleep. Brain Res 1967; 6:78–791.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

60. 61.

62. 63. 64.

65.

66. 67.

68. 69.

70. 71. 72. 73. 74.

75.

76.

77.

Azzaroni A, Parmeggiani PL Changes in selective brain cooling across the behavioral states of the ultradian wake–sleep cycle. Brain Res 1999; 844:206–209. Glotzbach SF, Heller HC. Temperature Regulation. In: Kryger MH, Roth T, Dement WC, eds. Principles and Practice of Sleep Medicine. 2nd ed. London: W.B. Saunders Co., 1994:260–275. Spengler CM, Shea SA. Endogenous circadian rhythm of pulmonary function in healthy humans. Am J Respir Crit Care Med 2000; 162:1038–1046. Spengler CM, Czeisler CA, Shea SA. An endogenous circadian rhythm of respiratory control in humans. J Physiol Lond 2000; 526:683–694. Ballard RD, Irvin CG, Martin RJ, Pak J, Pandey R, White DP. Influence of sleep on lung volume in asthmatic patients and normal subjects. Appl Physiol 1990; 68:2034–2041. Dreher D, Koller EA. Circadian rhythms of specific airway conductance and bronchial reactivity to histamine: the effects of parasympathetic blockade. Eur Respir J 1990; 3:414–420. Trinder J, Whitworth F, Kay A, Wilkin P. Respiratory instability during sleep onset. J Appl Physiol 1992; 73:2462–2469. Douglas NJ. Control of ventilation during sleep. In: Kryger M H, Roth T, Dement WC eds. Principles and Practice of Sleep Medicine. 2nd ed. London: W.B. Saunders Co., 1994:204–211. Burgess HJ, Kleiman J, Trinder J. Cardiac activity during sleep onset. Psychophysiology 1999; 36:298–306. Horner RL, Brooks D, Kozar LF, Tse S, Phillipson EA. Immediate effects of arousal from sleep on cardiac autonomic outflow in the absence of breathing in dogs. J Appl Physiol 1995; 79:151–162. Mancia G. Autonomic modulation of the cardiovascular system during sleep. N Engl J Med 1993; 328:347–349. Somers VK, Dyken ME, Mark AL, Abboud FM. Sympathetic-nerve activity during sleep in normal subjects. N Engl J Med 1993; 328:303–307. Millar-Craig MW, Bishop CN, Raftery EB. Circadian variation of blood pressure. Lancet 1978; 1:795–797. Veerman DP, Imholz BPN, Wieling W, Wesseling KH, van Montfrans GA. Circadian profile of systemic hemodynamics. Hypertension 1995; 26:55–59. Carvalho MJ, van den Meiracker AH, Boomsma F, Lima M, Freitas J, Veld AJM, Falcao de Freitas A. Diurnal blood pressure variation in progressive autonomic failure. Hypertension 2000; 35:892–897. Degaute JP, van de Borne P, Linkowski P, Van Cauter E. Quantitative analysis of the 24-hour blood pressure and heart rate patterns in young men. Hypertension 1991; 18:199–210. Stern N, Beahm E, McGinty D, Eggena P, Littner M, Nyby M, Catania R, Sowers JR. Dissociation of 24-hour catecholamine levels from blood pressure in older men. Hypertension 1985; 7:1023–1029. Kato M, Phillips BG, Sigurdsson G, Narkiewicz K, Pesek CA, Somers VK. Effects of sleep deprivation on neural circulatory control. Hypertension 2000; 35:1173–1175.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

78. Armstrong D, Emde C, Bumm R, Castiglione F, Cilluffo T, Blum AL. Twenty-fourhour pattern of esophageal motility in asymptomatic volunteers. Digest Dis Sci 1990; 35:1190–1197. 79. Goo RH, Moore JG, Greenberg E, Alazraki NP. Circadian variation in gastric emptying of meals in humans. Gastroenterology 1987; 93:515–518. 80. Orr WC. Gastrointestinal physiology. In: Kryger M H, Roth T, Dement WC, eds. Principles and Practice of Sleep Medicine. 3rd ed. Philadelphia: WB Saunders Co., 2000:279–288. 81. Castiglione F, Emde C, Armstrong D, Schneider C, Bauerfeind P, Stacher G, Blum AL. Nocturnal oesophageal motor activity is dependent on sleep stage. Gut 1993; 34:1653–1659. 82. Schmidt MH. Sleep-related penile erections. In: Kryger M H, Roth T, Dement WC, eds. Principles and Practice of Sleep Medicine. 3rd ed. Philadelphia: W.B. Saunders Co., 2000:305–318. 83. Spiegel K, Leproult R, Van Cauter E. Impact of sleep debt on metabolic and endocrine function. Lancet 1999; 354:1435–1439. 84. Gadoth N, Sokol J, Lavie P. Sleep structure and nocturnal disordered breathing in familial dysautonomia. J Neurol Sci 1983; 60:117–125. 85. McNicholas WT, Rutherford R, Grossman R, Moldofsky H, Zamel N, Phillipson EA. Abnormal respiratory pattern generation during sleep in patients with autonomic dysfunction. Am Rev Respir Dis 1983; 128:429–433. 86. Montagna P, Cortelli P, Gambetti P, Lugaresi E. Fatal familial insomnia: sleep, neuroendocrine and vegetative alterations. Adv Neuroimmunol 1995; 5:13–21. 87. Terzano MG, Parrino L, Pietrini V, Mancia D, Spaggiari MC, Rossi G, Tagliavini F. Precocious loss of physiological sleep in a case of Creutzfeldt Jakob disease: a serial polygraphic study. Sleep 1995; 18:849–858. 88. Terzano MG, Parrino L. Origin and significance of the cyclical alternating pattern. Sleep Med Rev 2000; 4:101–123. 89. Somers VK. Mechanisms regulating sympathetic nerve traffic—a focus on normal sleep and obstructive sleep apnea. In: Bolis CL, Licinio J, eds. The Autonomic Nervous System. Geneva: World Health Organization, 1999:81–85. 90. Shepard JW. Hypertension, cardiac arrhythmias, myocardial infarction, and stroke in relation to obstructive sleep apnea. Clin Chest Med 1992; 13:437–458. 91. Parrino L, Boselli M, Buccino GP, Spaggiari MC, Di Giovanni G, Terzano MG. The cyclic alternating pattern plays a gate-control on periodic limb movements during non-rapid eye movement sleep. J Clin Neurophysiol 1996; 13:314–323. 92. Ali NJ, Davies RJ, Fleetham JA, Stradling JR. Periodic movements of the legs during sleep associated with rises in systemic blood pressure. Sleep 1991; 14:163–165. 93. Moldofsky H, Gilbert R, Lue FA, MacLean AW. Sleep-related violence. Sleep 1995; 18:731–739.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

21 Major Depression and the Autonomic Nervous System Gina Rinetti and Ma-Li Wong David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A.

The symptomatic characterization of major depression includes low or depressed mood, loss of interest in most activities (anhedonia), and a decrease in energy level. There are several subtypes of major depression, each of which is distinguished by a specific pattern of symptoms. The melancholic subtype consists of qualitatively distinct symptoms that are comparable with those that occur during a state of overarousal. Though still controversial, accumulating evidence suggests that those with major depression with melancholic features have increased sympathetic nervous system and hypothalamo-pituitary-adrenal (HPA) activation. This hyperarousal state has recently been confirmed by indices of sustained activation of the HPA axis and norepinephrine (NE) system in a serial cerebrospinal fluid (CSF) sampling study. That study supports a dysregulation of the HPA axis and NE system in major depression with melancholic features because 1) hypercortisolemia detected in these patients was inefficacious in providing the appropriate feedback to the HPA axis and therefore unable to properly downregulate corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) secretion and 2) sustained activation of NE in the CSF of these patients persisted throughout a 30-hour period, leading to the assumption that NE and CRH activate the central nervous system of melancholic patients regardless of their state of consciousness (asleep or awake). These findings are compatible with previous data on NE spillover in plasma and the apparent correlation between urinary-free cortisol and urinary NE outputs in major depression. They support the notion that central and peripheral NE systems are overactive, while the data caution against the generalization of central nervous system (CNS) deficiency of

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

NE postulated in the monoamine hypothesis for major depression. Thus, several lines of evidence suggest that major depression affects central as well as systemic systems.

I. INTRODUCTION Disorders of mood are classified by symptom presentation, severity of symptoms, and longitudinal course. They include major depression, dysthymia (chronic mild depression), bipolar disorder (manic depressive disorder), and cyclothymia. In this chapter, we will focus on recent findings that have better characterized major depression, particularly of the melancholic subtype. The current understanding of this disorder has been based on a descriptive cluster of symptoms, such as low mood, lack of interest or pleasure in most activities (anhedonia), decreased energy or fatigue, low self-esteem, and dysregulation of the following functions: sleep, psychomotor, appetite, body weight, and self-image. The difference between major depression and a healthy physiological response to an event or memory that evokes sad feelings is the severe, disabling, and unremitting nature of the disorder. It is predicted that by the year 2020, major depression will be the second leading cause of disability around the world (1,2). Current estimation of the incidence of this disorder indicates that about 17.1% of the American population has had at least one episode of major depression (3), making it the most common mood disorder. The World Health Organization has assessed that the grade of disability caused by major depression is greater than that caused by chronic diseases, such as diabetes mellitus, hypertension, and back pain (4). In addition, the percentage of severely depressed patients with suicidal behavior who commit suicide is 15% (5). The cost of depression to the United States is estimated to be around $44 billion per year (6). Major depression constitutes a public health problem, which is reaching alarming proportions. Political, social, economical, and scientific efforts to lessen the impact of this disorder in our society should be high on our priority list. According to the DSM-IV (5), major depression can be divided into four subtypes: melancholic, atypical, catatonic, and postpartum onset depression. Another accepted classification system is to categorize major depression into two subgroups: melancholic and nonmelancholic (atypical, catatonic, and postpartum onset) depression (7,8). In this chapter, we will focus our discussion on evidence that the stress system is implicated in melancholia, which is characterized by anhedonia, worse symptoms in the morning, increased anxiety, insomnia, early morning awakening, psychomotor retardation or agitation, significant appetite and weight loss, and excessive or inappropriate guilt (5).

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

II. MAJOR DEPRESSION—DISEASE MECHANISMS Two main hypotheses for the pathophysiological mechanisms for major depression have been postulated and extensively investigated. Alternative hypotheses have been developed recently. A. The Monoamine Hypothesis The conception of the monoamine hypothesis for major depression occurred in the early 1960s. The hypothesis is based on reports that various antidepressant drugs increase synaptic concentrations of norepinephrine or serotonin and that the catecholamine-depleting drug reserpine causes depression-like states. In this hypothesis, major depression is postulated to reflect a deficiency in NE or serotonin in the central nervous system. This hypothesis has fostered a surge of scientific research in the past 30 years that has revealed limitations to this theory. A major gap in this theory is the lack of an appropriate explanation for the fact that while antidepressants have immediate action in the NE system, their antidepressant effects happen after several weeks of daily treatment. Also, multiple studies have shown that NE levels can increase in major depression, such as recent studies showing NE spillover in plasma and an increase in NE output in the cerebrospinal fluid in patients with melancholic depression (9). B. Neurohormonal Hypothesis A role for neuropeptides in the pathophysiology of major depression has been suggested by studies showing increased cortisol production in depressed patients (10). Despite the fact that anxiety disorders (panic disorder and phobias) are classified separately from major depression, rating scales used to assess depression and anxiety reflect a considerable overlap in symptomatology of these two classes of disorders. Activation of the hypothalamo-pituitary-adrenal (HPA) axis and NE system that occurs during acute stress could be perpetuated by the persistence of the stress and result in inability to maintain homeostasis. The cloning and characterization of corticotropin-releasing hormone has been pivotal in determining that the increased levels of plasma cortisol do not adequately suppress CRH levels in major depression. Indeed, clinical research data have supported the hypothesis that increased bioactivity of CRH in the CNS causes depression (11–14). C. Alternative Hypotheses Based on recent findings, investigators have proposed that abnormalities in circadian regulation in systems such as sleep, temperature, hormonal secretion, and activity cycles may cause depression. The roles of infectious agents (especially Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Borna disease virus) and inflammatory mediators have also been implicated in some forms of major depression. Genetic transmission is implicated in cases of familial aggregation of this common complex disorder.

III. STRESS AND GENERALIZED ADAPTATION SYNDROME Selye has described the generalized adaptation syndrome as the reaction to adverse circumstances or stress (15). This reaction to a physically or emotionally threatening situation disturbs a previous state of equilibrium and initiates a cascade of reactions that begins when the CNS becomes aware of a potentially alarming situation. If the threatening stimulus is determined to be significant, several CNS pathways will be activated in response. The cortex will stimulate subcortical brain circuitries, which activate many of the symptoms that are automatically felt when experiencing stress. These symptoms include increase in respiration rate, heart rate, heart contraction strength, paleness of skin, sweating, dilation of pupils, and slowdown of the gastrointestinal tract. Two main systems act as effectors of the stress response: the sympathetic system and the HPA axis. A. Norepinephrine NE is a major neurotransmitter that is involved in the stress response. It inhibits feeding, grooming, and sleeping behaviors (9). Moreover, NE modulates the encoding of emotion to the stress reaction through its role in the amygdala (16–19). NE acts in the frontal cortex to inhibit novel over well-versed behaviors, activates the HPA axis, and can mediate direct sympathetic nervous system inputs to the adrenal gland. Peripherally, the secretion of epinephrine and NE by the adrenal glands propels the immediate sympathetic response to a frightening stimulus. Independent groups of neurons located in the hypothalamic paraventricular nucleus (PVN) seem to be involved in the control of neuroendocrine mechanisms regulating the posterior and anterior pituitary lobes and in the sympathetic and parasympathetic autonomic mechanisms (20). The activation of the sympathetic nervous system is influenced by signals coming from the hypothalamic and brainstem (A6, A7, A5, and A1) sympathetic centers (21). These major cell groups have descending projections to the spinal cord as well as extensive connections to preganglionic neurons, which in turn innervate sympathetic ganglia and adrenal medulla (22). Descending neuronal pathways arise from the same cell groups, which terminate in the hypothalamus and other CNS regions (22). Therefore, the stress response sets into motion a sequence of reactions that leads to immediate sympathetic responses, stimulation of anabolic pathways in somatic tissue, changes in immunological functions (which can be sustained by hypercorti-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

solemia), neuroendocrine changes, as well as the stereotyped behaviors and affects that are associated with stress.

B. Corticotropin-Releasing Hormone CRH is a 41-amino-acid peptide that has been characterized as the main hypothalamic-releasing factor of the HPA axis. Centrally administrated, CRH initiates a set of behavioral, physiological, and neuroendocrine events that resemble those in the stress response and major depression. In the amygdala, CRH is involved in the acquisition and storage of conditioned fear responses. CRH pathways descending from the hypothalamus to the brainstem can modulate the activation of the NE system.

IV. MELANCHOLIA AND STRESS Besides being associated with an intense sense of pessimism and hopelessness, melancholic depression is characterized by a particular state of fear or hyperarousal, similar to what occurs in stressful situations (9). Patients with major depression experience increased sympathetic activity such as increased heart and respiration rates, more frequent eye blinking, and abnormal skin responses (23). Furthermore, elevated levels of plasma and urinary catecholamines (CA) and/or their metabolites [vanillylmandelic acid (VMA), 3-methoxy-4-hydroxyphenylglycol (MHPG), and normethinephrine] have been correlated with the presence of symptoms such as agitation, anxiety, guilt, diurnal variation, psychomotor retardation, loss of energy, decreased appetite, and weight loss (24). The notable increase of sympathetic symptoms found in depressed patients could be regarded as a sign of a hyperaroused state or a protective inhibition (25) in which an alert state is maintained during critical circumstances in life (23). However, the stress response and melancholic depression do differ in terms of severity, intensity, and duration. In depression, some aspects of the normal stress response seem to reach a pathological status due to dysregulation in the mechanisms of downregulation, resulting in a perpetuated alarm state (26). Hormonal changes related to the stress response have been found in patients with melancholic depression. Significantly greater output of urinary-free cortisol has been detected, and urinary-free cortisol has been correlated to urinary outputs of NE and its metabolite VMA (27,28). Increased plasma cortisol levels has been one of the most reproducible findings in such patients. Moreover, increased sympathetic activity has also been detected in these patients during resting, orthostasis, and moderate exercise (29). Furthermore, the increased heart rates and de-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

creased heart rate variability of depressed patients with melancholic features suggest an alteration of sympatho-vagal activities in the heart. Clearly, the most persuasive indications of the pathophysiological role of stress systems in depression are provided by three sets of findings. First, attenuation of the neuroendocrine changes in response to stress can be achieved by antagonism of CRH actions. CRH antagonists also attenuate autonomic and behavioral responses to stress in primates (30). A limited pilot study has claimed that CRH antagonists are effective in the treatment of depression. Second, antidepressants downregulate the HPA axis in rodents and humans (31–33). Third, increased CSF NE concentrations in medication-free patients with depression are sustained throughout the day and night (9). Elevated CSF NE even during sleep suggests that this is not a reaction to depressed mood but rather a dysregulation of the stress system. A. HPA and NE Systems in Melancholic Depression Though still controversial, several studies have indicated an association between the activation of the HPA axis and the activation of the sympathoadrenal system in major depression (9,16–19,23–25,27,28,34–36). In a recent report, Wong et al. (9) simultaneously evaluated the HPA axis and NE system in order to address the following questions: Are there alterations in CSF NE and CRH levels in patients with melancholic depression? What is the relationship between CSF NE and CRH? Does hypercortisolism influence these systems? Are the alterations in the CSF related to the sleep-wake cycle? Continuous serial CSF and plasma sampling were performed for 30 consecutive hours. CSF NE and CRH levels and plasma adrenocorticotropic hormone (ACTH) and cortisol levels were measured and circadian curves were subsequently generated (Fig. 1). In order to minimize the effects of hormone or neurotransmitter changes related to the acute stress during the initiation and during the termination of CSF collection, data from the first and the last 2 hours were excluded from those analyses. Mean CSF NE levels were significantly elevated, but mean CSF CRH levels were not different in depressed patients when compared to control subjects. These findings indicate that NE is abnormally elevated in patients with melancholic depression and that the CRH component of the HPA axis is not effectively suppressed by the elevated cortisol levels. Hypercortisolism of noncentral origin produces a remarkable suppression of CSF CRH levels, as seen in Cushing’s disease (37). In contrast, high cortisol levels do not elicit the appropriate signals to decrease CRH secretion in depressed patients with melancholia subtype. Plasma cortisol, plasma ACTH, and CSF CRH levels have been previously reported to exhibit circadian rhythmicity. CSF NE levels were found to have significant diurnal variations, and its pattern was analogous to that of plasma cortisol. In fact, the circadian pattern of CSF NE had characteristics that suggest that its rhythm could be as tightly regulated by the circadian clock as that of plasma Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

cortisol (Fig. 2). The pattern of circadian variation of CSF CRH has been previously described as opposite to that of plasma cortisol in normal subjects (38). Cross-correlation, calculated after lagging the time series of the level of the first hormone relative to the time series of the level of the second hormone, revealed that CSF CRH and plasma cortisol levels have a negative correlation in control subjects that is lost in patient with melancholic depression. This loss of association between CSF CRH and plasma cortisol levels in these patients could be interpreted as a disruption or bypass of the negative feedback mechanisms for glucocorticoids, possibly by another activating system (9). However, it could also reflect an increase in variability of hormonal levels in patients. This latter explanation is appealing because when the effects of the baseline shifts and of the circadian component were diminished by detrending the data, the cross-correlation analysis between plasma ACTH and plasma cortisol was decreased in patients (unpublished observations). A positive correlation was found between CSF NE and plasma cortisol. Elevated levels of CSF NE and plasma cortisol persisted throughout a 30-hour period in melancholic patients, suggesting that the activation of the HPA and NE stress-responsive systems is sustained regardless of state of consciousness (awake or asleep) (Fig. 1A, C). That correlation persisted even when the series of measurements were detrended. Therefore, the measurements become more independent of the effects of baseline shifts and of the circadian component. Cross-correlation analysis has shown that the relationship between CSF NE and cortisol seems to be as robust as the well-characterized relationship between plasma ACTH and plasma cortisol. But those analyses are not suited or designed to ascertain which of these two substances is triggering the response of the other. One could be tempted to speculate that plasma cortisol is contributing to central NE release since the average peak time of plasma cortisol secretion in this study seems to be 3 hours earlier than the peak time for CSF NE secretion (Fig. 2). However, the relationship between CSF NE and plasma cortisol can only be clarified by studies specifically designed to dissect this issue. Although the anatomical substrates describing the relationship between the HPA axis and peripheral NE are well known (see Sec. III), the circuitries or pathways that cause this positive crosscorrelation need to be determined. The existence of a blood-brain barrier to peripheral levels of NE (39) and the decrement content in the CSF and brain after bilateral lesioning of the locus coeruleus (40,41) support the notion that the NE content in the CSF reflects mostly CNS release. B. Other Neurotransmission Systems Involved in Depression in Relation to the Autonomous Nervous System It is beyond the scope of this book to discuss in detail the other neurotransmitter systems including the serotoninergic, dopaminergic, and cholinergic ones that are thought to be dysregulated in major depression. However, a brief overview of reCopyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 1 Diurnal curves of plasma cortisol (A), plasma ACTH (B), CSF NE (C), and CSF CRH (D) levels (mean  SE) in 14 healthy volunteers and 10 patients with major depression, melancholic subtype. Curves resulted from the averaged measurement per time point across a group of subjects by using the cropped hormonal series. The shaded area rep-

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

resents data recorded with the lights off (23:00–7:00 h). In the right corner insets under each pair of curves, the bar graphs represent the average of the mean value for each series of hormonal measurements (mean  SE). *, p  0.02. (From Ref. 9.)

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Figure 2 Polar representations of the biological rhythm’s % amplitude/MESOR (radius) and acrophase (angle) relations obtained by single cosinor analysis of CSF NE and plasma cortisol 24-hour circadian rhythm. In these graphs a statistical summary is shown with a bivariate statistical confidence region computed for a 24-hour period to detect a rhythm by a confidence region not overlapping the pole using the cropped hormonal series. Note that in both polar graphs all the acrophases are within a 12-hour time span, and most of them are within a 6-hour time span. These characteristics are indicative that the rhythm of CSF NE seems to be as tightly regulated as plasma cortisol is by the circadian clock. The circadian representation for plasma cortisol is shown as a reference of our analysis and population. (From Ref. 9.) Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

cent advances revealed the following data that are pertinent to our chapter: Enhanced serotonin-induced platelet calcium mobilization has been recently proposed to be a biological marker for depression (42). Previous reports had already shown that plasma serotonin levels were increased in depressed patients, while platelet serotonin reuptake was decreased (29). Furthermore, the clinical and anatomical data have consistently pointed to a possible parasympathetic dysregulation in patients with major depression (43–45). This observation has recently initiated a clinical trial with a device developed to supply vagus nerve stimulation. In this device, a small electrical generator implanted in the patient’s left upper chest provides vagal stimulation that can be regulated by the patient. A noncontrolled clinical study has shown that this experimental procedure decreased depression in 40% of severely affected patients (46).

V. CONCLUSIONS Recently, a study on serial CSF measurements of NE, CRH and concomitant measurements of plasma cortisol and ACTH in patients has shown that the central NE system is activated in melancholic depression. CSF NE levels provide evidence for a state of sustained activation, which is independent of the state of consciousness. These findings have cast serious doubt on the universality of the monoamine hypothesis for major depression, which claims that depression is a state of deficiency of NE. This study has also provided strong evidence for the dysregulation of the HPA axis and the NE systems in melancholia. The sustained hypercortisolemia evidenced in this particular disorder does not allow an efficient downregulation of the HPA axis. Evidence for the involvement of several neurotransmitter systems, particularly the central and peripheral components of the HPA axis and NE system as well as the parasympathetic and neuroimmune mediators, and for the systemic consequences of depression, such as loss of bone mineral density (33), increased risk of myocardial infarction, and the increased morbidity and mortality for coronary disease (47), strongly suggests that major depression involves multiple central and peripheral systems.

REFERENCES 1.

Murray CJ, Lopez AD. Alternative projections of mortality and disability by cause 1990–2020: Global Burden of Disease Study. Lancet 1997; 349:1498–1504. 2. Murray CJL, et al. The Global Burden of Disease: A Comprehensive Assessment of Mortality and Disability from Diseases, Injuries, and Risk Factors in 1990 and Projected to 2020. Boston, 1996.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

3.

4. 5. 6. 7.

8.

9.

10.

11.

12.

13.

14.

15. 16.

17.

18.

Kessler RC, McGonagle KA, Zhao S, Nelson CB, Hughes M, Eshleman S, et al. Lifetime and 12-month prevalence of DSM-III-R psychiatric disorders in the United States. Results from the National Comorbidity Survey. Arch Gen Psychiatry 1994; 51:8–19. Ustun TB SN, et al. Mental Illness in General Health Care: An International Study. London, 1995. APA. Diagnostic and Statistical Manual of Mental Disorders. 4th ed. Washington, DC: American Psychiatric Association, 1994. Greenberg PE, Stiglin LE, Finkelstein SN, Berndt ER. The economic burden of depression in 1990. J Clin Psychiatry 1993; 54:405–418. Parker G, Roussos J, Austin MP, Hadzi-Pavlovic D, Wilhelm K, Mitchell P. Disordered personality style: higher rates in non-melancholic compared to melancholic depression. J Affect Disord 1998; 47:131–140. Parker G, Roy K, Wilhelm K, Mitchell P, Austin MP, Hadzi-Pavlovic D, et al. Subgrouping non-melancholic depression from manifest clinical features. J Affect Disord 1999; 53:1–13. Wong ML, Kling MA, Munson PJ, Listwak S, Licinio J, Prolo P, et al. Pronounced and sustained central hypernoradrenergic function in major depression with melancholic features: relation to hypercortisolism and corticotropin-releasing hormone. Proc Natl Acad Sci USA 2000; 97:325–330. Sachar EJ, Hellman L, Fukushima DK, Gallagher TF. Cortisol production in depressive illness. A clinical and biochemical clarification. Arch Gen Psychiatry 1970; 23:289–298. Gold PW, Loriaux DL, Roy A, Kling MA, Calabrese JR, Kellner CH, et al. Responses to corticotropin-releasing hormone in the hypercortisolism of depression and Cushing’s disease. Pathophysiologic and diagnostic implications. N Engl J Med 1986; 314:1329–1335. Gold PW, Chrousos G, Kellner C, Post R, Roy A, Augerinos P, et al. Psychiatric implications of basic and clinical studies with corticotropin-releasing factor. Am J Psychiatry 1984; 141:619–627. Nemeroff CB, Widerlov E, Bissette G, Walleus H, Karlsson I, Eklund K, et al. Elevated concentrations of CSF corticotropin-releasing factor-like immunoreactivity in depressed patients. Science 1984; 226:1342–1344. Holsboer F, Von Bardeleben U, Gerken A, Stalla GK, Muller OA. Blunted corticotropin and normal cortisol response to human corticotropin-releasing factor in depression. N Engl J Med 1984; 311:1127. Selye H. A syndrome produced by diverse nocuous agents. Nature 1936; 138:32. Brown MR, Fisher LA, Spiess J, Rivier C, Rivier J, Vale W. Corticotropin-releasing factor: actions on the sympathetic nervous system and metabolism. Endocrinology 1982; 111:928–931. Gold PW, Chrousos GP. The endocrinology of melancholic and atypical depression: relation to neurocircuitry and somatic consequences. Proc Assoc Am Physicians 1999; 111:22–34. Gold PW, Goodwin FK, Chrousos GP. Clinical and biochemical manifestations of depression. Relation to the neurobiology of stress (2) [published erratum appears in N Engl J Med 1988; 319:1428]. N Engl J Med 1988; 319:413–420.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

19. 20.

21. 22.

23.

24.

25. 26.

27.

28.

29.

30.

31.

32.

33.

Sutton RE, Koob GF, Le Moal M, Rivier J, Vale W. Corticotropin releasing factor produces behavioural activation in rats. Nature 1982; 297:331–333. Swanson LW, Sawchenko PE. Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology 1980; 31:410–417. Sourkes TL. Pathways of stress in the CNS. Prog Neuropsychopharmacol Biol Psychiatry 1983; 7:389–411. Westlund KN, Bowker RM, Ziegler MG, Coulter JD. Origins and terminations of descending noradrenergic projections to the spinal cord of monkey. Brain Res 1984; 292:1–16. Guinjoan SM, Bernabo JL, Cardinali DP. Cardiovascular tests of autonomic function and sympathetic skin responses in patients with major depression. J Neurol Neurosurg Psychiatry 1995; 59:299–302. Wyatt RJ, Portnoy B, Kupfer DJ, Snyder F, Engelman K. Resting plasma catecholamine concentrations in patients with depression and anxiety. Arch Gen Psychiatry 1971; 24:65–70. Giedke H, Heimann H. Psychophysiological aspects of depressive syndromes. Pharmacopsychiatry 1987; 20:177–180. Musselman DL, Evans DL, Nemeroff CB. The relationship of depression to cardiovascular disease: epidemiology, biology, and treatment. Arch Gen Psychiatry 1998; 55:580–592. Roy A, Linnoila M, Karoum F, Pickar D. Urinary-free cortisol in depressed patients and controls: relationship to urinary indices of noradrenergic function. Psychol Med 1988; 18:93–98. Maes M, Vandewoude M, Schotte C, Martin M, Blockx P. Positive relationship between the catecholaminergic turnover and the DST results in depression. Psychol Med 1990; 20:493–499. Lechin F, van der Dijs B, Orozco B, Lechin ME, Baez S, Lechin AE, et al. Plasma neurotransmitters, blood pressure, and heart rate during supine-resting, orthostasis, and moderate exercise conditions in major depressed patients. Biol Psychiatry 1995; 38:166–173. Habib KE, Weld KP, Rice KC, Pushkas J, Champoux M, Listwak S, et al. Oral administration of a corticotropin-releasing hormone receptor antagonist significantly attenuates behavioral neuroendocrine, and autonomic responses to stress in primates. Proc Natl Acad Sci USA 2000; 97:6079–6084. Brady LS, Gold PW, Herkenham M, Lynn AB, Whitfield HJ, Jr. The antidepressants fluoxetine, idazoxan and phenelzine alter corticotropin-releasing hormone and tyrosine hydroxylase mRNA levels in rat brain: therapeutic implications. Brain Res 1992; 572:117–125. Brady LS, Whitfield HJ, Jr., Fox RJ, Gold PW, Herkenham M. Long-term antidepressant administration alters corticotropin-releasing hormone, tyrosine hydroxylase, and mineralocorticoid receptor gene expression in rat brain. Therapeutic implications. J Clin Invest 1991; 87:831–837. Michelson D, Galliven E, Hill L, Demitrack M, Chrousos G, Gold P. Chronic imipramine is associated with diminished hypothalamic-pituitary-adrenal axis responsivity in healthy humans. J Clin Endocrinol Metab 1997; 82:2601–2606.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

34. Veith RC, Lewis N, Linares OA, Barnes RF, Raskind MA, Villacres EC, et al. Sympathetic nervous system activity in major depression. Basal and desipramine-induced alterations in plasma norepinephrine kinetics. Arch Gen Psychiatry 1994; 51:411–422. 35. Arnsten AF, Mathew R, Ubriani R, Taylor JR, Li BM. Alpha-1 noradrenergic receptor stimulation impairs prefrontal cortical cognitive function. Biol Psychiatry 1999; 45:26–31. 36. Bornstein SR, Chrousos GP. Clinical review 104: Adrenocorticotropin (ACTH)- and non-ACTH-mediated regulation of the adrenal cortex: neural and immune inputs. J Clin Endocrinol Metab 1999; 84:1729–1736. 37. Kling MA, Roy A, Doran AR, Calabrese JR, Rubinow DR, Whitfield HJ, et al. Cerebrospinal fluid immunoreactive corticotropin-releasing hormone and adrenocorticotropin secretion in Cushing’s disease and major depression: potential clinical implications [see comments]. J Clin Endocrinol Metab 1991; 72:260–271. 38. Kling MA, DeBellis MD, O’Rourke DK, Listwak SJ, Geracioti TJ, McCutcheon IE, et al. Diurnal variation of cerebrospinal fluid immunoreactive corticotropin-releasing hormone levels in healthy volunteers [published erratum appears in J Clin Endocrinol Metab 1994 Dec;79(6):1762]. J Clin Endocrinol Metab 1994; 79:233–239. 39. Ziegler MG, Wood JH, Lake R, Kopin IJ. Norepinephrine and 3-methoxy-4-hydroxyphenyl glycol gradients in human cerebrospinal fluid. Am J Psychiatry 1977; 134:565–568. 40. Anselmo-Franci JA, Franci CR, Krulich L, Antunes-Rodrigues J, McCann SM. Locus coeruleus lesions decrease norepinephrine input into the medial preoptic area and medial basal hypothalamus and block the LH, FSH and prolactin preovulatory surge. Brain Res 1997; 767:289–296. 41. Fornai F, Alessandri MG, Torracca MT, Bassi L, Corsini GU. Effects of noradrenergic lesions on MPTP/MPP kinetics and MPTP-induced nigrostriatal dopamine depletions. J Pharmacol Exp Ther 1997; 283:100–107. 42. Uchitomi Y, Kugaya A, Akechi T, Nakano T, Wenner M, Okamura H, et al. Three sets of diagnostic criteria for major depression and correlations with serotonin-induced platelet calcium mobilization in cancer patients. Psychopharmacology (Berl) 2001; 153:244–248. 43. Dilsaver SC, Coffman JA. Cholinergic hypothesis of depression: a reappraisal. J Clin Psychopharmacol 1989; 9:173–179. 44. Janowsky DS, el-Yousef MK, Davis JM, Sekerke HJ. A cholinergic-adrenergic hypothesis of mania and depression. Lancet 1972; 2:632–635. 45. Janowsky DS, Risch SC, Huey LY, Kennedy B, Ziegler M. Effects of physostigmine on pulse, blood pressure, and serum epinephrine levels. Am J Psychiatry 1985; 142:738–740. 46. George MS, Sackeim HA, Marangell LB, Husain MM, Nahas Z, Lisanby SH, et al. Vagus nerve stimulation. A potential therapy for resistant depression? Psychiatr Clin North Am 2000; 23:757–783. 47. Glassman AH, Shapiro PA. Depression and the course of coronary artery disease. Am J Psychiatry 1998; 155:4–11.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.