Functional Neuroanatomy of the Nitric Oxide System (Handbook of Chemical Neuroanatomy)

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Author: H.W.M. Steinbusch | J. de Vente | S.R. Vincent

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FUNCTIONAL NEUROANATOMY OF THE NITRIC OXIDE SYSTEM

FUNCTIONAL NEUROANATOMY OF THE NITRIC OXIDE SYSTEM

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H A N D B O O K OF CHEMICAL NEUROANATOMY Series Editors: A. Bj6rklund and T. H6kfelt

Volume 17

FUNCTIONAL NEUROANATOMY OF THE NITRIC OXIDE SYSTEM Editors:

H.W.M. STEINBUSCH and J. DE VENTE European Graduate School of Neuroscience (EURON), Department of Psychiatry and Neuropsychology, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands

S.R. VINCENT Graduate Program in Neuroscience, University of British Columbia, Kinsman Laboratory of Neurological Research, Department of Psychiatry, Vancouver, BC V6T 1Z3, Canada

2000

ELSEVIER Amsterdam-

Lausanne - New York-

Oxford-

Shannon - Singapore - Tokyo

ELSEVIER SCIENCE B.V. Sara Burgerhartstraat 25 P.O. Box 211, 1000 AE Amsterdam, The Netherlands

© 2000 Elsevier Science B.V. All rights reserved.

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I S B N 0-444-50285-8 (volume) ISBN: 0-444-90340-2 (series) The paper used in this publication meets the requirements of A N S I / N I S O Z39.48-1992 (Permanence of Paper). Printed in The Netherlands

List of Contributors J. AIJON Dpto. Biologfa Celular y Patologfa Universidad de Salamanca Avda. Alfonso X el Sabio 1 37007 Salamanca Spain

T.EC. BATTEN Institute of Cardiovascular Research University of Leeds The Worsley Building Leeds LS2 9JT UK

J.R. ALONSO Dpto. Biologfa Celular y Patologfa Universidad de Salamanca Avda. Alfonso X el Sabio 1 37007 Salamanca Spain

J.G. BRINON Dpto. Biolog/a Celular y Patologfa Universidad de Salamanca Avda. Alfonso X el Sabio 1 37007 Salamanca Spain

S. AMIR Center for Studies in Behavioral Neurobiology Concordia University 1455 de Maisonneuve Boulevard West Montreal, PQ H3G 1M8 Canada

M.S. DAVIDOFF Institute of Anatomy University of Hamburg Martinistr. 52 D-20246 Hamburg Germany

C.R. ANDERSON Department of Anatomy and Cell Biology University of Melbourne Parkville, VIC 3052 Australia R. ARI~VALO Dpto. Biologfa Celular y Patologfa Universidad de Salamanca Avda. Alfonso X el Sabio 1 37007 Salamanca Spain L. ATKINSON School of Biomedical Sciences University of Leeds The Worsley Building Leeds LS 2 9NQ UK

J. DEUCHARS School of Biomedical Sciences University of Leeds The Worsley Building Leeds LS 2 9 NQ UK J. DE VENTE European Graduate School of Neuroscience (EURON) Department of Psychiatry and Neuropsychology Maastricht University PO Box 616 6200 MD Maastricht The Netherlands

W.D. ELDRED Department of Biology Laboratory of Visual Neurobiology Boston University 5 Cummington Street Boston, MA 02215 USA J.B. FURNESS Department of Anatomy and Cell Biology University of Melbourne Parkville, VIC 3052 Australia L.J. IGNARRO Department of Molecular and Medical Pharmacology UCLA School of Medicine CHS 23-120, Box 951785 Los Angeles, CA 90095-1735 USA A. JACOBS Department of Molecular and Medical Pharmacology UCLA School of Medicine CHS 23-120, Box 951785 Los Angeles, CA 90095-1735 USA R. MIDDENDORFF Institute of Anatomy University of Hamburg Martinistr. 52 D-20246 Hamburg Germany A. PORTEROS Dpto. Biologfa Celular y Patologfa Universidad de Salamanca Avda. Alfonso X el Sabio 1 37007 Salamanca Spain

vi

N.L. SCHOLZ Northwest Fisheries Science Center Seattle, WA 98112 USA H.W.M. STEINBUSCH European Graduate School of Neuroscience (EURON) Department of Psychiatry and Neuropsychology Maastricht University PO Box 616 6200 MD Maastricht The Netherlands J.W. TRUMAN Department of Zoology University of Washington Box 351800 Seattle, WA 98195-1800 USA S.R. VINCENT Graduate Program in Neuroscience Kinsmen Laboratory of Neurological Research Department of Psychiatry University of British Columbia Vancouver, BC V6T 1Z3 Canada E. WERUAGA Dpto. Biolog/a Celular y Patologfa Universidad de Salamanca Avda. Alfonso X el Sabio 1 37007 Salamanca Spain B. WOODSIDE Center for Studies in BehaVioral Neurobiology Concordia University 1455 de Maisonneuve Boulevard West Montreal, PQ H3G 1M8 Canada

W. WU Department of Anatomy University of Hong Kong Faculty of Medicine 5 Sassoon Road Hong Kong People's Republic of China

H.M. YOUNG Department of Anatomy and Cell Biology University of Melbourne Parkville, VIC 3052 Australia

vii


Preface This volume concerns the neuronal isoform of nitric oxide synthase (nNOS, bNOS or NOS I). Only in the first chapter, the endothelial isoform and the inducible form are briefly mentioned in an attempt to provide a more complete picture. In agreement with the goal of the Handbook Series, the focus of this volume is on the localization of NOS in the nervous system where today extensive information is present from studies on the brain, spinal cord and peripheral nervous system. This is to a considerable extent a consequence of the important discovery that NADPH-diaphorase activity is a part of NOS, which made it possible to use an earlier established histochemical staining method to visualize NOS systems. With this method brilliant staining patterns can be achieved in an easy and relatively cheap way. There are, however, certain caveats in the use of this NADPH-diaphorase method, which are discussed in more or less detail in several chapters of this volume. Nevertheless, this enzyme histochemical staining method remains a valuable and preferred tool for many researchers involved in NOS neuroanatomy. We have tried to bring together a number of reviews on different aspects of NOS, ranging from detailed descriptions of the nNOS distribution to analysis of NO signaling with the aim to provide a first major reference guide to the localization of NOS in different species. In addition we wanted to present an overview of the co-localization of other (co)transmitters with NOS. A more functional approach to NO can be achieved by the combined visualization of the localization of NOS and activated soluble guanylyl cyclase as a target for NO. Although many more direct effects of NO on a number of molecular targets, e.g. ion channels and NMDA receptors, have been reported in the literature, it remains to be demonstrated that these effects are relevant to the physiological function of NO in the nervous system. NOS is present in virtually every area throughout the central and peripheral nervous system. Over the last decade NO has emerged as an important regulator of many brain processes, and its physico-chemical properties and synthetic and release characteristics make NO an unconventional messenger molecule. It is synthesized upon demand and can diffuse over considerable distances in spite of its short half-life. The fact that NO is a free radical makes it potentially harmful to the living organism. Therefore, the expression of the genes for the different NOS isoforms as well as the metabolism of NO in the nervous system have to be rigidly controlled. This potential neurotoxic role may have been accentuated in the first decade of NO research at the expense of its physiological signaling function. It is our hope that this volume will help to further stimulate research on NO as a messenger molecule in the nervous system, which is one of the aims with the publication of this volume of the Handbook Series. We would like to express our gratitude to the authors of this volume who have taken the time and made the effort to produce such excellent chapters, as well as to the highly competent and cooperative staff at Elsevier. Maastricht, Vancouver, Lund and Stockholm, January 2000 HARRY W.M. STEINBUSCH ANDERS BJORKLUND

JAN DE VENTE

STEVEN R. VINCENT TOMAS HOKFELT ix


Contents List of Contributors Preface

v

ix

NITRIC OXIDE SYNTHASE AND THE PRODUCTION OF NITRIC O X I D E L.J. IGNARRO AND A. JACOBS 1. 2. 3. 4. 5. II.

1 3 6 10 11

HISTOCHEMISTRY OF NITRIC OXIDE SYNTHASE IN THE CENTRAL NERVOUS SYSTEM- S.R. VINCENT 1. 2.

3. 4. 5. 6. III.

Introduction and historical perspectives NO synthase isoforms Protein structure of NO synthase isoforms Neuronal NO synthase References

Introduction Localization of nitric oxide synthase in the brain 2.1. The olfactory bulb 2.2. The cerebral cortex 2.3. The hippocampal formation 2.4. The basal forebrain 2.5. The basal ganglia 2.6. The thalamus 2.7. The superior colliculus 2.8. The auditory system 2.9. The hypothalamus 2.10. Circumventricular organs 2.11. The mesopontine tegmentum 2.12. The cerebellum 2.13. The medulla 2.14. The spinal cord Arginine metabolism in the brain Heme oxygenase Conclusions References

19 21 21 22 24 26 27 28 29 31 31 33 33 36 36 37 38 39 39 39

COMPARATIVE AND DEVELOPMENTAL NEUROANATOMICAL ASPECTS OF THE NO SYSTEM- J.R. ALONSO, g. ARI~VALO, E. WERUAGA, A. PORTEROS, J.G. BgIlqON AND J. AIJON 1. 2.

Introduction Methods to localize nitric oxide and nitric oxide synthases in brain tissue 2.1. Histological detection of NADPH-diaphorase/nitric oxide synthase

51 52 52 xi

3.

4.

5. 6. IV.

53 53 54 54 54 55 55 55 59 63 67 71 76 87 87 89 91 92 94 101

NITRIC OXIDE IN THE RETINA - W.D. ELDRED 1. 2.

3. 4.

5.

xii

2.1.1. NADPH-diaphorase histochemistry 2.1.2. Immunohistochemistry 2.1.3. In situ hybridization 2.2. Biochemical detection of nitric oxide synthase, nitric oxide production, and nitric oxide proper Results 3.1. Fish 3.1.1. Cyclostomes 3.1.2. Teleosts 3.2. Amphibians 3.3. Reptiles 3.4. Birds 3.5. Mammals 3.6. General pattern of NADPH-diaphorase expression during rat brain development Discussion 4.1. Methodological aspects 4.2. Interspecies differences in the NADPH-diaphorase distribution pattern 4.3. Implications of the NADPH-diaphorase expression during the developmental processes 4.4. NADPH-diaphorase/nitric oxide synthase distribution pattern and nitric oxide functional implications Abbreviations References

Introduction Localization of nitric oxide synthase in the retina 2.1. Methodological considerations 2.1.1. NADPH-diaphorase histochemistry 2.1.2. NOS immunocytochemistry 2.2. Isoforms of NOS in the retina 2.3. Anatomical localizations of NOS 2.3.1. Mammals 2.3.2. Lower vertebrates 2.4. Regional distribution of NOS 2.5. Efferents Development of NOS in the retina and central visual targets Biochemistry and molecular biology of NOS in the retina 4.1. NOS 4.2. Photoreceptors Function of NO in specific retinal cell types 5.1. Photoreceptors 5.2. Horizontal cells 5.3. Bipolar cells 5.4. Amacrine cells 5.5. Ganglion cells

111 111 112 112 113 113 114 114 117 120 122 122 125 125 126 128 128 129 131 131 132

6. Release of NO in the retina 7. Modulation of transmitter release by NO 8. The cGMP signal transduction pathway in retina and its modulation by NO 9. Future areas of investigation 10. Abbreviations 11. Acknowledgements 12. References V.

I32 133 133 138

I39 140 I40

NITRIC OXIDE SIGNALING IN THE HYPOTHALAMUS - B. WOODSIDE AND S, AMIR Introduction 147 NOS in the hypothalamus 147 Co-localization 149 Regulation 151 Functional considerations 157 5.1. Stress axis 157 5.2. Magnocellular neurosecretory system 159 5.2.1. Modulation of vasopressin and oxytocin release 159 5.2.2. Drinking 161 5.2.3. Reproductive behavior 161 5.3. The reproductive axis 161 5.3. I . Modulation of luteinizing hormone-releasing hormone secre161 tion 5.3.2. Modulation of prolactin release 163 5.3.3. Sexual behavior 163 5.4. Somatostatin release 163 5.5. Circadian regulation 1 64 5.6. Autonomic regulation 164 5.7. Plasticity 165 6. Conclusions 166 7. Acknowledgements 167 8. References 167

1. 2. 3. 4. 5.

VI.

NITRIC OXIDE SYSTEMS IN THE MEDULLA OBLONGATA AND THEIR INVOLVEMENT IN AUTONOMIC CONTROL - T.F.C. BATTEN, L. ATKINSON AND J. DEUCHARS 1. Introduction 2. Methods 3. Results and discussion 3.1. Anatomy of NOS in the medulla oblongata 3. I . 1. Description of the location and morphological characteristics of NOS-IR neurones in the rat medulla oblongata 3.1.2. Relative distribution of NOS-IR and NADPH-diaphorase activity 3.1.3. Co-localisation of NOS-IR or NADPH-diaphorase activity with other neuronal phenotypes in the medulla oblongata

177 177 180 180 180 187 188 ...

Xlll

3.2.

,

5. 6.

Role of nitric oxide in medullary pathways involved in autonomic functions 3.2.1. NO in the control of the cardiovascular system 3.2.2. NO in neuronal circuitry underlying control of the oesophagus 3.2.3. NO in CNS control of the stomach and large intestine 3.2.4. NO in central respiratory control 3.2.5. NO in pathways coordinating autonomic and nociceptive responses Abbreviations Acknowledgements References

199 199 204 204 205 206 207 208 208

VII. NITRIC OXIDE IN THE PERIPHERAL AUTONOMIC NERVOUS SYSTEMH.M. YOUNG, C.R. ANDERSON AND J.B. FURNESS Introduction 1.1. Brief history of the identification of NO as a peripheral neurotransmitter 1.2. General properties of NO-mediated neurotransmission 1.3. Scope of this review 2. NO in autonomic ganglia 2.1. Nitric oxide and sympathetic pre- and postganglionic neurones 2.1.1. Presence of NOS in sympathetic pre- and postganglionic neurones 2.1.2. Functionally identified subclasses of sympathetic preganglionic NOS neurones 2.1.3. Presence of NOS in parasympathetic pre- and postganglionic neurones 2.2. Nitric oxide and ganglionic transmission 3. Role of NO in the neural control of the vasculature and the heart 3.1. Role of neurally derived NO in the control of the vasculature 3.1.1. Autonomic vasodilator neurones 3.1.2. Sensory vasodilation 3.1.3. Role of neurally released NO in regulation of blood vessels summary 3.2. Role of neurally derived NO in the control of the heart 4. Role of NO in the neural control of the gastrointestinal tract 4.1. Introduction 4.2. NO is a neurotransmitter of enteric inhibitory motor neurones 4.3. Role of NO in co-transmission from enteric inhibitory motor neurones 4.4. Axo-axonal interactions involving NO 4.5. NOS interneurones within the intestine 4.6. NOS in nerve fibres innervating oesophageal motor endplates 4.7. NOS innervation of the mucosa in the stomach 4.8. NOS in intestinofugal neurones 4.9. NOS in the biliary system and pancreas 5. Role of NO in the neural control of the trachea and lower airways 5.1. Types of neurones innervating the trachea and lower airways xiv

215 215 216 216 217 217 217 219 221 223 224 224 224 228 228 229 232 232 232 233 237 237 238 238 238 239 240 240

5.2.

Pharmacology of transmission and source of NOS neurones in the trachea Role of NO in the neural control of salivary glands and other secretory tissues 6.1. Salivary glands 6.1.1. Types of neurones innervating the salivary glands 6.1.2. Role of NO in vasodilation and secretion in the salivary glands 6.2. Sweat glands 6.3. The nasal mucosa 7. Role of NO in the innervation of the adrenal medulla 7.1. Presence of NOS in neurones in the adrenal medulla 7.2. Role of neurally derived NO in the adrenal medulla 8. Overview of peripheral autonomic NO neurones 9. Acknowledgements 10. References .

241 242 242 242 242 244 244 245 245 246 248 249 249

VIII. THE NITRIC OXIDE SYSTEM IN THE UROGENITAL TRACTM.S. DAVIDOFF AND R. MIDDENDORFF 1.

2.

3. 4. IX.

The urinary tract 1.1. The upper urinary tract 1.1.1. The kidney 1.1.2. The renal pelvis 1.1.3. The ureter 1.2. The lower urinary tract 1.2.1. The urinary bladder 1.2.2. The urethra The genital tract 2.1. The male reproductive organs 2.1.1. The testis 2.1.2. The spermatozoa 2.1.3. The epididymis 2.1.4. The vas deferens 2.1.5. The seminal vesicle 2.1.6. The prostate 2.1.7. The penis 2.2. The female genital tract 2.2.1. The ovary 2.2.2. The Fallopian tube 2.2.3. The uterus 2.2.4. The vagina 2.2.5. The placenta and umbilical artery Acknowledgements References

267 267 267 277 277 279 279 281 282 282 282 286 288 290 290 291 291 292 292 294 294 297 297 301 301

RESPONSE OF NITRIC OXIDE SYNTHASE TO NEURONAL INJURY - W. WU 1. 2.

Introduction Injury-induced expression of NOS

315 316 XV

2.1.

Up-regulation of NOS expression in injured neurons 317 2.1.1. Up-regulation of NOS expression in the neurohypophyseal systern 317 2.1.2. Up-regulation of NOS expression in the nuclei of cranial nerves 317 2.1.3. Up-regulation of NOS expression in the spinal cord 317 2.1.4. Up-regulation of NOS expression in the peripheral nerve and ganglia 319 2.2. De novo expression of NOS in injured neurons 321 2.2.1. De novo expression of NOS in the cerebral cortex 321 2.2.2. De novo expression of NOS in the cerebellar cortex 321 2.2.3. De novo expression of NOS in the nuclei of cranial nerves 321 2.2.4. De novo expression of NOS in spinal motoneurons 324 2.2.5. De novo expression of NOS in nuclei associated with the long descending and ascending pathways 324 325 2.3. Time course of NOS expression in injured neurons 2.3.1. Time course of NOS expression is different in different populations of neurons 325 2.3.2. Time course of NOS expression can be different in the same population of neurons following different types of injuries 326 3. Co-expression of NOS with other injury-relevant components 326 4. Age-related expression of NOS in injured neurons 328 5. Species-related expression of NOS in injured neurons 329 6. Ultrastructure of injury-induced NOS-positive neurons 330 7. Regulation of NOS expression in injured neurons 334 7.1. Regulation of injury-induced NOS by the length of the remaining proximal axons following axotomy 334 7.2. Regulation of injury-induced NOS by peripheral nerve (PN) graft transplantation 336 7.3. Regulation of injury-induced NOS by neurotrophic factors 338 8. Mechanisms of NOS expression in injured neurons 338 8.1. Expression of injury-induced NOS in neurons is a general response to injury 339 8.2. Expression of injury-induced NOS results from the interruption of axonal transport 340 8.3. Expression of injury-induced NOS results from stimulation of NMDA receptors 340 8.4. Expression of injury-induced NOS results from deprivation of neurotrophic factors 341 Potential roles of NOS expression in neuronal degeneration and regeneration 341 342 9.1. Potential role of NOS in neuronal degeneration 342 9.1.1. Involvement of NOS in neural degeneration 343 9.1.2. Potential mechanisms of NO/NOS-mediated neurotoxicity 9.1.3. Evidence suggesting that NO/NOS is not involved in degener344 ative processes after neuronal injury 345 9.2. Potential role of NOS in neuronal regeneration 346 10. Summary 346 11. Acknowledgements 347 12. References ,

xvi

Xo

NITRIC OXIDE-cGMP SIGNALING IN THE RAT BRAIN - J. DE VENTE AND H.W.M. STEINBUSCH 1. 2. 3. 4. 5.

Introduction 355 Soluble guanylyl cyclase 356 Phosphodiesterase activity and the termination of the NO-cGMP signal 357 cGMP immunocytochemistry 358 Immunocytochemical localization of NOS and NO-mediated cGMP synthesis 359 5.1. A note on the use of brain slices 359 5.2. A note on the use of phosphodiesterase inhibitors in in vitro studies 360 5.3. NOS activity in brain slices 361 5.4. Localization of NO-mediated cGMP accumulation in brain slices 362 5.4.1. Telencephalon 362 5.4.2. Diencephalon 365 5.4.3. Mesencephalon 367 5.4.4. Cerebellum 367 5.4.5. Pons and medulla oblongata 372 5.5. Colocalization of cGMP and NOS 378 6. Localization of cGMP in the cerebellum 384 7. NO-cGMP signaling in other vertebrates 389 8. NO-cGMP signaling in the rat brain during development 390 9. Colocalization of cGMP with neurotransmitter systems in the rat brain 395 10. Abbreviations 405 11. Acknowledgements 405 12. References 406

XI.

INVERTEBRATE MODELS FOR STUDYING NO-MEDIATED SIGNALINGN. SCHOLZ AND J.W. TRUMAN 1. 2. 3.

4. 5.

6. 7. 8. 9.

Introduction Components of the NO signaling pathway in invertebrates Developmental roles of NO/cGMP signaling for invertebrates 3.1. NO signaling and proliferation control 3.2. NO sensitivity during neuronal development 3.3. Functional significance of NO/cGMP signaling The role of NO in feeding behavior The role of NO in regulating rhythmic motor networks 5.1. The gastropod feeding circuit 5.2. The crustacean stomatogastric ganglion 5.3. The crustacean cardiac network NO and nociception NO as a potential blood-borne neurohormone Acknowledgements References

Subject Index

417 418 419 419 420 427 428 429 429 430 433 435 436 438 438 443

xvii

CHAPTER I

Nitric oxide synthase and the production of nitric oxide L.J. IGNARRO AND A. JACOBS

1. INTRODUCTION AND HISTORICAL PERSPECTIVES Not too long ago, nitric oxide (NO) was viewed by many as a noxious component of polluted air over industrial cities or as a gas that could be purchased in gas cylinders for the purpose of conducting chemical experiments. The possible biological importance of NO emerged in the 1970s, when a variety of studies from different laboratories revealed that nitroso compounds and related chemical species that were suspected of decomposition or conversion to NO could stimulate the production of cyclic GMP in mammalian tissues by activating the cytosolic isoform of guanylate cyclase (DeRubertis and Craven, 1976; Arnold et al., 1977; Katsuki and Murad, 1977; Katsuki et al., 1977; Miki et al., 1977: Schultz et al., 1977; B6hme et al., 1978; Craven and DeRubertis, 1978; Murad et al., 1978; Axelsson et al., 1979; Craven et al., 1979; Kukovetz et al., 1979; Ignarro, 1989). The well known chemical effects of some of these nitro compounds, such as nitroprusside and nitroglycerin, led us to suspect that their conversion to NO might account for the mechanism of vasodilation elicited by nitroprusside, and organic nitrate esters and organic nitrite esters. Accordingly, NO was tested and found to be a potent vascular smooth muscle relaxant which caused vasorelaxation via the second messenger actions of cyclic GMP (Gruetter et al., 1979, 1980, 1981; Napoli et al., 1980). These observations were confirmed and extended by several laboratories (Ignarro et al., 1981, 1984; Axelsson et al., 1982; Keith et al., 1982; Axelsson and Andersson, 1983; Galvas and DiSalvo, 1983; Horowitz et al., 1983; Ignarro and Kadowitz, 1985; Ignarro, 1989). The discovery that NO itself is a potent relaxant helped to explain the many earlier observations that diverse nitro and nitroso compounds caused both vasorelaxation and tissue accumulation of cyclic GME The clinical awareness that certain nitrovasodilators also interfered with platelet aggregation led us to ascertain whether NO and cyclic GMP were responsible for such anti-platelet actions. NO and a series of S-nitrosothiols, which are used as NO donor agents, were found to inhibit human platelet aggregation and by mechanisms involving the second messenger actions of cyclic GMP (Mellion et al., 1981, 1983). Inhibition of platelet aggregation occurred regardless of the agent used to promote aggregation, including ADP, collagen, thrombin, or thromboxane analogs. It is of historic interest, perhaps, that the first two pharmacological actions described for NO were vasorelaxation and inhibition of platelet aggregation, now appreciated to be the two most important actions of endothelium-derived relaxing factor (EDRF). In the process of elucidating the mechanism by which nitroglycerin causes vascular smooth muscle relaxation, we found that sulfhydryl (-SH) containing compounds or thiols Handbook of Chemical Neuroanatom~; Vol. 17: Functional Neuroanatomv of the Nitric Oxide System H.W.M. Steinbusch, J. De Vente and S.R. Vincent, editors (~ 2000 Elsevier Science B.V. All rights reserved.

Ch. I

L.J. Ignarro and A. Jacobs

(cysteine, dithiothreitol) were required for the activation of cytosolic guanylate cyclase by nitroglycerin, other organic nitrate esters and some organic nitrite esters (Ignarro and Gruetter, 1980; Ignarro et al., 1980a,b). The reason for this thiol requirement was the liberation of NO from intermediate S-nitrosothiols formed, as a result of a chemical reaction between the nitrovasodilator and thiol (Ignarro et al., 1981). These studies constituted the first demonstration of the pharmacological actions of S-nitrosothiols and their characteristic property as NO donor agents. Two of these S-nitrosothiols have become widely used NO donor agents, namely S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO). The extraordinary high potency of nitroglycerin as a vasodilator and the finding that NO is responsible for its pharmacological effects suggested that mammals might possess an endogenous nitroglycerin, another NO donor species, or NO itself. This hypothesis was confirmed when the EDRF, discovered in 1980 (Furchgott and Zawadzki, 1980), was identified as NO in 1986-1987 (Ignarro et al., 1986a, 1987a,b, Palmer et al., 1987). This observation provided explanations for previous findings that EDRF activates guanylate cyclase (Frrstermann et al., 1986; Ignarro et al., 1986b) and inhibits platelet aggregation (Azuma et al., 1986). Subsequent experiments revealed the mechanism by which EDRF inhibits platelet aggregation and adhesion compared with the anti-platelet effect of prostacyclin (Radomski et al., 1987). Most of the early biological research on NO focused on its effects on vascular smooth muscle, platelets and cytosolic guanylate cyclase, whereas little or no attention was given to the possible influence of NO on neuronal cell function or as a neurotransmitter. Early studies revealed that certain regions of the brain were rich in cyclic GMP and cytosolic guanylate cyclase (Garthwaite, 1990). Not only NO (Miki et al., 1977) but also an unidentified low molecular weight factor from rat forebrain (Deguchi, 1977) were shown to activate guanylate cyclase, and enzyme activation was inhibited by hemoglobin. This soluble endogenous activator of guanylate cyclase was later identified as arginine (Deguchi and Yoshioka, 1982), although we had observed that arginine alone could not directly activate purified preparations of guanylate cyclase (unpublished observations). In retrospect, these original observations made by the Deguchi laboratory were well ahead of their time. We now understand that in crude soluble fractions from certain brain regions, arginine serves as the substrate for neuronal NO synthase to generate NO, which was likely responsible for the activation of guanylate cyclase in the experiments of Deguchi. This interpretation also explains the earlier finding by Ferrendelli (Ferrendelli et al., 1974) that glutamate caused a calcium-dependent stimulation of cyclic GMP accumulation in cerebellar slices. Now the stage was set for the pioneer studies of Garthwaite in 1988 that NMDA stimulated cyclic GMP accumulation in rat cerebellar cells (Garthwaite et al., 1988). This response to NMDA was associated with the release of an EDRF-like factor, which in turn acted on distinct target cells to elevate cyclic GMP levels. The EDRF-like factor was quickly shown to be NO (Moncada et al., 1991 ). Following the important observation that vascular endothelial cells could synthesize NO and citrulline from arginine by a process requiring NADPH (see Moncada et al., 1991), similar observations were made in brain tissue and the arginine-to-NO pathway was identified and elucidated as the NO synthase enzymatic pathway (Bredt and Snyder, 1989, 1990; Bredt et al., 1990). These pioneering studies showing the calcium- and calmodulin-dependent catalytic activity of NO synthase were performed with enzyme purified from rat cerebellum. The enzyme was termed neuronal NO synthase (nNOS). The discovery and characterization of the other two isoforms of NOS came later. Soon after the identification of nNOS in the brain~ many investigators began to elucidate the possible physiological and pathophysiological roles of endogenous NO in the brain. Since cellular and subcellular localizations of neurotransmitters

Neuronal NO synthase

Ch. I

often help to suggest and clarify biological function, antibodies to nNOS were developed and the tissue distribution and localization of nNOS in brain neurons as well as in peripheral neurons were demonstrated (Bredt and Snyder, 1994). Unfortunately, specific definitive roles for nNOS or NO in the brain are not well established and this is an area of extensive investigation (Bredt and Snyder, 1994). NO has been implicated in long-term potentiation in the hippocampus where it may function as a retrograde messenger in memory and learning processes. Direct evidence for specific neurotransmitter actions of NO derives from research conducted in the peripheral nervous system. In the central nervous system, although NO appears to function in synaptic transmission, the neuronal release of excess quantities of NO is associated with neurotoxicity. For example, excess glutamate release stimulates NMDA receptors and consequent NO production, which mediates the focal schemia of vascular stroke (Choi, 1988) and perhaps other CNS disorders (Bredt and Snyder, 1994). In the peripheral nervous system, NO appears to function as a neurotransmitter mediating vascular and nonvascular smooth muscle relaxation. These tissues are innervated by nonadrenergic-noncholinergic (NANC) neurons, nNOS containing neurons exist in the myenteric (Auerbach's) plexus of the gastrointestinal tract (Bredt et al., 1990; Dawson et al., 1991) and neuronal stimulation causes NO release and nonvascular smooth muscle relaxation (Bult et al., 1990; Boeckxstaens et al., 1991; Desai et al., 1991 ; Tottrup et al., 1991) associated with inhibition of peristalsis. NANC neurons exist also in the outer adventitial layers of large blood vessels, and in arteries within erectile tissue (Burnett et al., 1992). Functional, constitutive nNOS was recovered from rabbit corpus cavernosum (Bush et al., 1992a) and later localized to neurons innervating the erectile tissue (Burnett et al., 1992). In the erectile tissue (corpus cavernosum) of the penis, NO is the NANC neurotransmitter that mediates penile erection by provoking both vascular and nonvascular smooth muscle relaxation, thereby allowing the trabecular and sinusoidal vascular beds to become engorged with blood (Ignarro et al., 1990; Bush et al., 1992a,b,c; Rajfer et al., 1992; Trigo-Rocha et al., 1993).

2. NO SYNTHASE ISOFORMS After the discovery of the nNOS isoform, the inducible isoform of NOS (iNOS) was found. Early studies by Hibbs and colleagues revealed that activated rodent macrophages killed target tumor cells by arginine-dependent mechanisms, and could be inhibited by chemical analogs of arginine such as NG-methylarginine (Hibbs et al., 1987a). Other studies showed that murine macrophages activated by lipopolysaccharide or other agents could produce nitrite and nitrate, and that these latter products might be responsible for the resulting target cell cytotoxicity (Stuehr and Marietta, 1985, 1987a,b; Iyengar et al., 1987; Miwa et al., 1987). Also, arginine was identified as the precursor for nitrite and nitrate in these latter studies (Iyengar et al., 1987). None of these investigators suggested that NO was actually generated first, as the intermediate product of arginine conversion, and subsequently oxidized to nitrite and nitrate, as is now known to occur. Indeed, the first proposed enzymatic pathway for this conversion of arginine to nitrite was via arginine deiminase, which produces citrulline plus ammonia, followed by oxidation of ammonia to nitrite (Hibbs et al., 1987b). The first clue that the conversion of arginine to nitrite and nitrate might involve the intermediate formation of NO came from experiments conducted with activated rat neutrophils, which generated a vascular smooth muscle relaxing factor with the properties of NO (Rimele et al., 1988; Sturm et al., 1988). The definitive observations that arginine can be converted to NO plus

Ch. I


citrulline came from experiments utilizing vascular endothelial cells (Palmer et al., 1988). It turns out, however, that the endothelial cells contain a constitutive isoform of NOS, whereas the activated macrophages and neutrophils contain a distinct inducible isoform of NOS. Research conducted in other laboratories in the early 1980s revealed that the production of nitrate in mammals could occur by pathways that were independent of intestinal microbial metabolism, and present endogenously in the animal (Green et al., 1981). Administration of lipopolysaccharide to rats led to increased urinary output of nitrate (Wagner et al., 1983). The mechanism of this effect was not understood until Stuehr and Marletta (1985) showed that lipopolysaccharide stimulated nitrate production in mice, and that this effect was probably mediated by macrophages, as lipopolysaccharide-activated macrophages were capable of generating nitrite and nitrate. Additional experiments showed that both nitrite and nitrate were derived from a common precursor, arginine (Iyengar et al., 1987). Therefore, research from several independent laboratories showed that lipopolysaccharide-treated animals and lipopolysaccharide-activated macrophages and other cell types could synthesize nitrite and nitrate from arginine. Following the demonstration that NO could be synthesized from arginine by vascular endothelial cells (Palmer et al., 1988), it became apparent that NO was likely an intermediate in the production of nitrite and nitrate from arginine by activated macrophages. These findings led to subsequent studies confirming that activated macrophages synthesized NO from arginine and that the NO was subsequently oxidized to nitrite and nitrate (Hibbs et al., 1988; Marletta et al., 1988; Stuehr et al., 1989). After the initial discovery and purification of nNOS (Bredt and Snyder, 1994), the inducible iNOS and endothelial NOS (eNOS) isoforms were characterized (F6rstermann et al., 1994; Griffith and Stuehr, 1995). Two distinct nomenclatures have been used to designate the three isoforms of NOS. One nomenclature refers to the NOS isoforms according to principal tissue distribution and the inducibility of one isoform, and these isoforms are designated as nNOS, eNOS and iNOS. Some of the cited problems with this nomenclature are that the so-called constitutive isoforms (nNOS and eNOS) can be expressionally regulated (upregulation and downregulation), and that certain isoforms can be found in locations other than those of their principal designation. This led some to propose the use of type I (nNOS), type II (iNOS) and type III (eNOS), according to the chronological order of their isolation and purification. The main problem with this latter nomenclature is the non-descriptive nature of the terms. In general, the NOS isoforms catalyze the oxidation or oxygenation of arginine to NO plus citrulline (Fig. 1). One atom of dioxygen (molecular oxygen) is incorporated into NO and a second atom of dioxygen is incorporated into citrulline (Kwon et al., 1990; Leone et al., 1991). One mole of L-arginine yields one mole of NO plus one mole of L-citrulline (Taych and Marietta, 1989; Bush et al., 1992d). This is a cytochrome P450-1ike oxidation reaction and requires NADPH as the principal electron donor. Like cytochrome P450, NOS contains heine as a prosthetic group for oxygen binding and subsequent incorporation into substrate to yield the products of the reaction (Griffith and Stuehr, 1995). An important intermediate in this enzymatic reaction is N~-hydroxyarginine (Stuehr et al., 1991; Wallace and Fukuto, 1991), which elicits pharmacological and perhaps physiological effects of its own (Buga et al., 1996). In addition to being a hemoprotein, NOS is a flavoprotein and requires both FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide) for the full expression of catalytic activity (Stuehr et al., 1991; Griffith and Stuehr, 1995). The enzyme-bound flavins function to transfer the electrons from the NADPH to the heine prosthetic group, thereby keeping the heme iron in the reduced state (ferrous: Fe 2+) to facilitate oxygen binding. Thus, NOS is a flavo-hemoprotein, one of the very few known to occur in mammals. Calmodulin, a calcium-binding protein, is also required for NOS catalytic activity (Abu-Soud


Ch. I

NG-hydroxy-L-arginine

L-arginine

+H3N--CH~CO 2I

(~H2)3 NH [

H2N/C~NH

02

~-~

H20

/z~ "NADPH NADP +

+H3N~CH----CO2" 02 H20 ( )H2)3 NH

+H3N~CH~CO 2I

0.5 NADPH 0.5 NADP§

I

H2N/ c %

L-citrulline

N

I

OH

(~H2)3 NH

I

H2N/C~o +NO

nitric oxide Fig. 1. The conversion of L-arginine to L-citrulline plus nitric oxide (NO) is catalyzed by the enzyme NO synthase. This conversion involves an initial 2-electron oxidation of one of the two equivalent guanidino nitrogen atoms of L-arginine to form NG-hydroxy-L-arginine, which can dissociate from the enzyme. A second, 3-electron oxidation of NG-hydroxy-L-arginine forms L-citrulline plus NO. Two molecules of dioxygen and 1.5 equivalents of NADPH are consumed in the overall enzymatic reaction.

and Stuehr, 1993; Griffith and Stuehr, 1995). Calmodulin binds free calcium to form a complex which binds to. nNOS and eNOS and functions to facilitate the transfer of electrons from enzyme-bound FAD to the heine iron. Since this transfer of electrons is essential for the expression of catalytic activity, calmodulin, and therefore calcium, acts as an on/off switch for NO biosynthesis from arginine. The iNOS isoform contains calmodulin already bound tightly as a subunit and, therefore, does not require calcium for enzyme activation (Cho et al., 1992). An additional cofactor that appears to be required for full NOS catalytic activity is tetrahydrobiopterin (Gross and Levi, 1992; Stuehr and Griffith, 1992; Griffith and Stuehr, 1995). It was speculated initially by some that the pterin may participate in the redox chemistry of the catalytic cycle. It is now believed that tetrahydrobiopterin functions in stabilizing the NOS protein rather than participating in catalysis (Giovanelli et al., 1991), and later reports added to this view based on the findings that tetrahydrobiopterin prevented the direct negative feedback effect of NO on NOS catalytic activity (Rogers and Ignarro, 1992; Griscavage et al., 1993). A summary of some of the properties of the NOS isoforms is given in Table 1. The NOS isoforms appear to catalyze an atypical, odd-numbered electron (5-electron) oxidation of L-arginine to NO plus L-citrulline (Griffith and Stuehr, 1995). In the first step of the reaction, one of the two basic guanidino nitrogen atoms of arginine undergoes a 2electron oxidation to yield NG-hydroxyarginine (typical of cytochrome P450 monooxygenase chemistry), and in the second step of the reaction, NG-hydroxyarginine undergoes a 3-electron oxidation to yield NO and citrulline (atypical of cytochrome P450). The stoichiometry of the NOS catalytic cycle has been studied extensively and often debated, but the conclusion has been reached that if NO is indeed the immediate product of the NADPH-dependent oxidation of NG-hydroxyarginine, then a 5-electron oxidation of arginine must occur. A more typical, even-numbered, 4-electron oxidation of arginine would necessarily yield HNO (nitroxyl in the protonated state) instead of NO (Fukuto et al., 1992, 1993a). Interestingly, HNO is chemically unstable and is readily oxidized to NO by numerous physiologically important oxidants (Fukuto et al., 1993b). Indeed, the pharmacological effects of HNO are virtually identical to those for NO (Fukuto et al., 1993a), and this may be due to the rapid oxidation of HNO to NO. These observations prompted additional studies aimed at elucidating the chemical nature

Ch. I


TABLE 1. Properties of representative NO svnthase isoforms Property

nNOS (I)

eNOS (III)

iNOS (II)

Principal source Subunit Mr (kDa) Subcellular localization Native structure Constitutively expressed Inducible Regulation ECso Ca2+ (nM) Km arginine (IxM) Vmax (gmol min-1 mg-1) Km NG-hydroxyarginine(gM) IC5o NG-methylarginine(gM) IC5o N~-nitroarginine (IxM)

Neuronal 160 Cytosolic Dimer Yes No Ca2.`./calmodulin 300 1-4 1-3 25 1.6 0.9

Endothelial 133 Membrane-bound Dimer Yes No Ca2§ 300 1-5 0.8-1 0.9 0.2

Macrophages 130 Cytosolic Dimer Yes Yes Protein synthesis Ca2+ not required 2-20 1-2 25 7.5 200

See Griffith and Stuehr (1995) and Hobbs and Ignarro (1997) for more details.

of the NOS products. Using a sensitive chemiluminescence technique that is selective for NO (relative to HNO and other oxides of nitrogen), evidence was obtained that HNO is an intermediate in the oxidation of arginine to NO by iNOS and nNOS (Hobbs et al., 1994; Hobbs and Ignarro, 1997). These studies suggested that NOS might catalyze a 4-electron oxidation of arginine to HNO, and the HNO would be readily and rapidly oxidized under physiological conditions to NO. These observations are more consistent with the chemistry of cytochrome P450-type monooxygenases and would solve the potential problem of having to deal with atypical 5-electron oxidation reactions of arginine to yield NO directly. These observations and conclusions have been confirmed by others (Schmidt et al., 1996).

3. PROTEIN STRUCTURE OF NO SYNTHASE ISOFORMS In 1991, Bredt and co-workers reported the cDNA sequence that codes rat cerebellum nNOS, which has a molecular mass of approximately 160 kDa (Bredt et al., 1991). There is good homology of the C-terminal portion of nNOS to the C-terminal portion of iNOS and eNOS, and all NOS isoforms show close homology to NADPH cytochrome P450 reductase (Bredt et al., 1991; Sessa, 1994; Xie et al., 1994; F6rstermann and Kleinert, 1995). NOS isoforms and NADPH cytochrome P450 reductase possess very similar characteristics including consensus sequences for NADPH, FMN and FAD binding sites. Many investigators have shown high homology among the individual NOS isoforms across species (Bredt and Snyder, 1994; Griffith and Stuehr, 1995). Table 2 gives information on the cDNAs encoding the three NOS isoforms (McMillan and Masters, 1993). Fig. 2 illustrates schematically the relationships among the cofactor sequences and other sequences for NOS isoforms and cytochrome P450 reductase. The observations of sequence homology between the C-terminal portion of NOS isoforms and cytochrome P450 reductase, together with the findings that NOS possesses heme that is required for catalysis, suggested that there must be a binding site for heine. In view of the fact that the heme for cytochrome P450 reductase actually resides within a distinct and separate protein (cytochrome P450), and that the NOS protein itself is capable of catalysis,

Neuronal N O synthase

Ch. I

TABLE 2. cDNAs encoding ttm three NO svnthase isq[olvns Isoform

cDNA source

nNOS (I) eNOS (III) iNOS (II)

mRNA (kb)

Predicted protein

Brain Endothelium Macrophage Smooth muscle Hepatocyte

Amino acids

Mr (kDa)

1429-1433 1203-1205 1144 1147 1147

160-161 133 130-131 131 131

10-10.5 4.05-4.8 4 4 4.5

See F6rstermann and Kleinert (1995) for more details.

the hypothesis developed that the heme may bind at the N-terminal portion of NOS (Griffith and Stuehr, 1995). This would mean that NOS is composed of a distinct reductase domain (C-terminal) and a distinct oxygenase domain (N-terminal). Initial studies revealed this to be the case for nNOS, as limited trypsin proteolysis generated an N-terminal heme-containing fragment that binds L-arginine, and a C-terminal FMN- and FAD-containing fragment that catalyzes the NADPH-dependent reduction of cytochrome c (Sheta et al., 1994). These studies suggested that nNOS is a bi-domain enzyme in which the oxygenase and reductase domains can fold and function independently of one another. Similar observations were subsequently made with the iNOS isoform (Ghosh and Stuehr, I995). In a relatively short period of time since the initial purification of nNOS from cerebellum (Bredt and Snyder, 1990; Mayer et al., 1990), a great deal has been learned about structurefunction relationships for all three NOS isoforms. As discussed above, NOS binds both arginine and NADPH, and contains heme, tetrahydrobiopterin and flavins. The two constitutive isoforms (nNOS, eNOS) bind calmodulin, whereas the inducible isoform (iNOS) contains

Oxygenase Domain Heme I NH2H NH2HI

Heme I Heme I

Reductase Domain

II

CaM FMN

m

~

FAD NADPH

I

[~

neuronal NOS

I I! IIF ~176176161kDa

I

I

I

I ~ [l~ cOOH

1inducibleNOS31 kDa

I 11 ]]1-C~176

1end~kDa

I ]] 'Ub COOH

NADPH-cytochromeP450Reductase 76 kDa

H 1O0 a.a. Fig. 2. Primary sequence map of the three isoforms of human nitric oxide synthase (NOS) and comparison to human NADPH-cytochrome P450 reductase. All NOS isoforms are flavo-hemoproteins which utilize NADPH as a substrate for reducing equivalents. Illustrated in the diagram are binding sites for the cofactors: NADPH, FAD, FMN, and Ca2+/calmodulin. Calmodulin is constitutively bound to the enzyme in the inducible NOS isoform. Not illustrated are the residues which bind tetrahydrobiopterin. The oxygenase domain of NOS contains a conserved cysteine residue which acts as an axial ligand for the heme iron. This contrasts with the cytochrome P450 isoforms, which contain a histidine ligand.

Ch. I


tightly bound calmodulin. The heine is the site of oxygen activation for both sequential monooxygenase reactions catalyzed by NOS. The heine iron must be in the reduced or ferrous (Fe 2+) state to bind oxygen, and the electrons required to achieve iron reduction are derived from NADPH. The nNOS isoform is a bi-functional enzyme comprised of an N-terminal oxygenase domain and a C-terminal reductase domain. The N-terminal oxygenase domain contains binding sites for heme, tetrahydrobiopterin and L-arginine, whereas the C-terminal reductase domain contains binding sites for FMN, FAD and NADPH (Bredt et al., 1990; McMillan and Masters, 1993, 1995: Klatt et al.. 1994: Sheta et al., 1994; Nishimura et al., 1995). The binding of the flavins to NOS is structurally analogous to the binding of flavins to NADPH cytochrome P450 reductase and related proteins (Bredt and Snyder, 1994; Griffith and Stuehr, 1995). The flavins function to transfer NADPH-derived electrons either to an external heme protein acceptor (cytochrome P450) or an internal heme prosthetic group (NOS) (Gachhui et al., 1996). Calmodulin binding to nNOS increases the rate of electron transfer from NADPH to the flavins and triggers the interdomain transfer of electrons from the flavins to the heine iron, providing a basis by which the calcium-calmodulin complex activates nNOS and eNOS (Abu-Soud and Stuehr, 1993: Gachhui et al., 1996). This explains the requirement by nNOS and eNOS of calcium-facilitated calmodulin binding in order to stimulate NOS catalytic activity. Thus, nNOS and eNOS can participate in signal transduction pathways by generating NO only in response to increases in intracellular free calcium. Similarly, prolonged calcium influx, which is characteristic of reperfused or reoxygenated ischemic tissues, can lead to neuropathological conditions attributed to excess localized NO production (Garthwaite and Boulton, 1995). The high-output production of NO by iNOS is attributed to the sustained generation of relatively large quantities of NO by cells for many hours or days. This is explained by the capacity of iNOS to bind calmodulin as a subunit at ambient levels of intracellular free calcium (Cho et al., 1992: Stevens-Truss and Marietta, 1995; Ruan et al., 1996). The nNOS and eNOS isoforms contain a unique polypeptide insert in their FMN binding domains that is not present in the iNOS isoform (Salerno et al., 1997a). This polypeptide insert may function as an autoinhibitory domain by binding to endogenous peptides that could compete with calmodulin for binding sites on nNOS and eNOS but not iNOS. Further advances in this field could lead to the development of potent peptide inhibitors of nNOS or eNOS. The eNOS isoform resembles the other NOS isoforms in possessing similar heme ligation geometry (Salerno et al., 1997b). In each isoform, the heine is 5-coordinate and retains the axial thiolate ligand. Utilization of optical difference spectroscopy (McMillan and Masters, 1993) and EPR spectroscopy (Salerno et al., 1995) on all three NOS isoforms expressed in, and purified from, stably transfected human kidney embryonic cells or from Escherichia coli has allowed a comprehensive analysis of the interactions between isoforms and probes such as substrate, intermediate and substrate analog inhibitors (Salerno et al., 1997b). This work makes it possible to map the active sites of the three NOS isoforms via analysis of various substrate analogs and. therefore, to design more isoform-specific NOS inhibitors. The eNOS isoform is distinct from nNOS and iNOS in having a particulate or membrane-bound subcellular distribution (F6rstermann et al., 1991; Pollock et al., 1991; Busconi and Michel, 1993; Feron et al., 1996). The eNOS isoform appears to undergo a complex series of covalent modifications that influence its subcellular targeting. In endothelial cells, N-myristoylation and thiopalmitoylation of eNOS represent important determinants for its subcellular localization (Busconi and Michel, 1993: Liu and Sessa, 1994; Sase and Michel, 1997). Moreover, eNOS is specifically targeted to plasmalemmal caveolae as a consequence


Ch. I

of palmitoylation of eNOS (Shaul et al., 1996). The subcellular translocation of eNOS suggests that the targeting of eNOS to endothelial cell caveolae may be dynamically regulated (Robinson and Michel, 1995; Robinson et al., 1995). Plasmalemmal caveolae are signal-transducing membrane microdomains consisting of cholesterol- and glycosphingolipid-enriched domains that may function to sequester diverse membrane-targeted signaling proteins (Anderson, 1993). Caveolin is an oligomeric integral membrane protein that appears to serve as a structural scaffold within caveolae, and can interact with and inhibit numerous structurally diverse signaling proteins (Li et al., 1995, 1996; Couet et al., 1997). It appears that the caveolin-scaffolding domain can specifically and potently inhibit eNOS catalytic activity and may function as a competitive inhibitor of the allosteric activation of eNOS by calmodulin (Garcia-Cardena et al., 1996; Michel et al., 1997a). More recent work using myristoylationand palmitoylation-deficient eNOS mutants suggests that the association between eNOS and caveolin is independent of the state of acylation of eNOS and that agonist-evoked, calmodulin-dependent disruption of the caveolin-NOS complex is what attenuates caveolin-mediated tonic inhibition of eNOS catalytic activity (Feron et al., 1998). Therefore, caveolin may play a role as an eNOS chaperone by regulating NO production independently of the presence of eNOS within caveolae or its state of acylation. Information on the crystal structure of the three NOS isoforms has recently become available (Crane et al., 1997, 1998; Raman et al., 1998). Crane et al. (1997) determined the crystal structure for iNOS, which revealed an unusual fold and heme environment for stabilization of activated oxygen intermediates that are key for enzyme catalysis. An active center created by an interface allows substrate and effector molecules such as tetrahydrobiopterin and calmodulin to modulate catalysis by altering the association between domains and subunits. NOS appears to be highly conducive to multiple regulatory pathways, and the authors believe that NO may have evolved to be a very effective signaling molecule in higher organisms. Additional studies by this group (Crane et al., 1998) revealed the dynamic structure of the iNOS dimer containing binding sites for arginine substrate and tetrahydrobiopterin cofactor at the catalytic center of the enzyme protein. The authors interpreted their findings to suggest that pterin binding causes a conformational change at the central interface region to expose the heine prosthetic group for interactions with the reductase domain. Pterin binding may also have electronic influences on heine-bound oxygen. L-Arginine appears to bind to glutamic acid-371 via hydrogen bonding and interacts with heine in a hydrophobic pocket to facilitate activation of heine-bound oxygen. This interaction may help to explain the two steps of oxidation of arginine, first to NG-hydroxyarginine and second to NO plus citrulline. The crystal structure of the constitutive isoform eNOS has been recently reported (Raman et al., 1998) and the conclusions drawn by these authors are considerably different from those drawn by Crane et al. for the inducible iNOS isoform (Crane et al., 1997, 1998). Raman et al. (1998) have addressed pterin function in NOS by determining the high-resolution crystal structure of the dimeric eNOS home domain. The protein structures reveal that pterin binding is not required for dimerization, as suggested earlier (Crane et al., 1997, 1998), but rather may be critical for enzyme catalysis. Moreover, Raman et al. (1998) argue that pterin binding fails to cause any conformational changes in the enzyme protein either at the pterin binding site or anywhere else in the protein. Another difference between these two groups is the finding by Raman et al. (1998) that the bottom region of the interface between monomers contains two conserved cysteine residues, rather than a disulfide bridge (Crane et al., 1997), that are involved in coordinating a zinc atom. The function of zinc may be stabilization of the pterin binding site and facilitation of stereospecific pterin recognition. The electropositive surface around the zinc metal center may serve as a docking site for the reductase domain of NOS.

Ch. I

L.J. lgnarro and A. Jacobs

These observations may have direct implications for understanding the molecular basis of vascular dysfunction in pterin-deficient disorders.

4. NEURONAL NO SYNTHASE The biochemical properties of constitutive neuronal NOS (nNOS) are strikingly similar to those for constitutive eNOS and inducible iNOS. The crystal structure of the nNOS dimer should prove to be closely similar to the crystal structures of eNOS and iNOS. Like the other isoforms of NOS, nNOS is a modular enzyme which consists of a flavin-containing reductase domain and a heine-containing oxygenase domain linked by a chain of amino acids containing a calmodulin binding site. Calmodulin binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, thereby resulting in the oxygenation of arginine to yield NO plus citrulline. Like the other NOS isoforms, nNOS is capable of generating superoxide anion (O_;-) from oxygen when arginine availability is limited (Miller et al., 1997). It is not only interesting but also important to recognize that nearly all organs in the mammal are innervated by neurons that release NO as a neurotransmitter, which elicit both physiological and pathophysiological actions. The nNOS is distributed to both the cytosolic and particulate or membrane-bound fractions in most peripheral neurons. The membrane-bound nNOS may be attributed to the PDZ/GLGF motif found in the NH2-terminal sequence of the nNOS protein (Brenman et al., 1996a,b). nNOS is also distributed in skeletal muscle mainly to the particulate fraction, where it is attached to the sarcolemma-dystrophin complex via the PDZ/GLGF motif and interacts with (L-syntrophin (Brenman et al., 1995). The significance of these findings is that a selective loss of sarcolemmal nNOS is associated with muscular dystrophy where the dystrophin gene is mutated (Brenman et al., 1995" Chao et al., 1996; Grozdanovic et al., 1996). Although nNOS is generally considered to be present constitutively in most neurons and other tissues, there is increasing evidence that nNOS can undergo expressional regulation. Upregulation of nNOS mRNA seems to represent a general response of neuronal cells to stress induced by a widespread diversity of physical, chemical and biological agents (F6rstermann et al., 1998). This upregulation of nNOS may represent a part of a more global expression of numerous genes, in response to cellular stress, ultimately resulting in cellular injury and apoptosis. Downregulation of nNOS may occur in response to endotoxin and cytokines that cause induction of iNOS. Since expressional changes in constitutive nNOS may lead to changes in the quantity of NO generated and released from neurons, it is important to develop a better understanding of genetic control of nNOS expression. The human nNOS gene has been mapped to the q l4-qter position of chromosome 12 (Kishimoto et al., 1992), and the genomic structure of nNOS is well documented from human brain (Hall et al., 1994; Marsden et al., 1994). The catalytic activity of nNOS is turned on in neuronal cells by an increase in the intracellular concentration of free calcium as a result of the action potential or sodium current. Calcium binding to calmodulin allows the calcium-calmodulin complex to bind to the appropriate amino acid sequence in nNOS. As discussed above, the binding of calmodulin triggers the electron transport cycle in nNOS, which results in activation of heine iron-bound molecular oxygen and consequent oxygenation of arginine to yield NO plus citrulline. Although depriving cells of flavins, heine, tetrahydrobiopterin or arginine can each lead to diminished catalytic activity, the principal physiological mode of regulation of nNOS activity 10


Ch. 1

is a change in the intracellular concentration of free calcium. This m e c h a n i s m affords rapid changes in the catalytic activity of nNOS, whereas expressional changes in nNOS protein levels in response to cellular stress represents a much slower and delayed or long-term regulation of nNOS activity and N O production. Therefore, rapid increases in N O production by n N O S result primarily from the firing of action potentials in the neuron. The only known m e c h a n i s m for rapid decreases in N O production by nNOS is negative feedback control of n N O S catalytic activity by N O itself (Rogers and Ignarro, 1992; Buga et al., 1993; Griscavage et al., 1993, 1994; A b u - S o u d et al., 1995; Hyun et al., 1995; Cohen et al., 1996). Evidence from experiments using e n z y m e s , isolated tissues, cell culture and in vivo experiments indicate that p h y s i o l o g i c a l l y relevant concentrations of NO directly inhibit the catalytic activity of all three NOS isoforms, and that NO plays an important autoregulatory role.

5. REFERENCES Abu-Soud HM, Stuehr DJ (1993): Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer. Proc Natl Acad Sci USA 90:10769-10772. Abu-Soud HM, Wang J, Rousseau DL, Fukuto JM, Ignarro LJ, Stuehr DJ (1995): Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis. J Biol Chem 270:22997-23006. Anderson RG (1993): Caveolae: where incoming and outgoing messengers meet. Ptvc Natl Acad Sci USA 90:10909-10913.

Arnold WE Mittal CK, Katsuki S, Murad F (1977): Nitric oxide activates guanylate cyclase and increases guanosine 3':5'-cyclic monophosphate levels in various tissue preparations. Proc Natl Acad Sci USA 74:3203-3207. Axelsson KL, Andersson KG (1983): Tolerance towards nitroglycerin, induced in vivo, is correlated to a reduced cGMP response and an alteration in cGMP turnover. EurJ Pharmacol 88:71-79. Axelsson KL, Wikberg JES, Andersson RGG (1979): Relationship between nitroglycerin, cyclic GMP and relaxation of vascular smooth muscle. Life Sci 24:1779-1786. Axelsson KL, Andersson RGG, Wikberg JES (1982): Vascular smooth muscle relaxation by nitro compounds: reduced relaxation and cGMP elevation in tolerant vessels and reversal of tolerance by dithiothreitol. Acta Phapvnacol Toxicol 50:350-357. Azuma H, Ishikawa M, Sekizaki S (1986): Endothelium-dependent inhibition of platelet aggregation. Br J Pharmacol 88:411-415.

Boeckxstaens GE, Pelckmans PA, Ruytjens IF, Bult H, De Man JG, Herman AG, Van Maercke YM (1991): Bioassay of nitric oxide released upon stimulation of non-adrenergic non-cholinergic nerves in the canine ileocolonic junction. Br J Pharmacol 103:1085-1091. B6hme E, Graf H, Schultz G (1978): Effects of sodium nitroprusside and other smooth muscle relaxants on cyclic GMP formation in smooth muscle and platelets. Adv Cyclic Nucleotide Res 9:131-143. Bredt DS, Snyder SH (1989): Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci USA 86:9030-9033. Bredt DS, Snyder SH (1990): Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 87:632-685. Bredt DS, Snyder SH (1994): Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem 63:175-195. Bredt DS, Hwang PM, Snyder SH (1990): Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347:768-770. Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, Snyder SH (1991): Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 351:713-714. Brenman JE, Chart DS, Xia H, Aldape K, Bredt DS (1995): Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy. Cell 82:743-752. Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, Santillano DR, Wu Z, Huang F, Xia H, Peters ME Froehner SC, Bredt DS (1996a): Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alphal-syntrophin mediated by PDZ domains. Cell 84:757-767. Brenman JE, Christopherson KS, Craven SE, McGee AW, Bredt DS (1996b): Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein. J Neutvsci 16:7407-7415. Buga GM, Griscavage JM, Rogers NE, Ignarro LJ (1993): Negative feedback regulation of endothelial cell function by nitric oxide. Circ Res 73:808-812. 11

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L.J. I g n a r r o a n d A. J a c o b s

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Stuehr DJ, Marietta MA (1985): Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc Natl Acad Sci USA 82"7738-7742. Stuehr DJ, Marletta MA (1987a): Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma. J Immunol 139:518-525. Stuehr PJ, Marletta MA (1987b): Synthesis of nitrite and nitrate in routine macrophage cell lines. Cancer Res 47:5590-5594. Stuehr DJ, Gross SS, Sakuma I, Levi R, Nathan CF (1989)" Activated routine macrophages secrete a metabolite of arginine with the bioactivity of endothelium-derived relaxing factor and the chemical reactivity of nitric oxide. J Exp Med 169:1011 - 1020. Stuehr DJ, Kwon NS, Nathan CE Griffith OW. Feldman PL. Wiseman J (1991): N(-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine. J Biol Chem 2666259-6263. Sturm RJ, Adams LM, Holloway DA, Weichman BM, Rimele TJ (1988)" Functional comparison of a rat polymorphonuclear leukocyte (PMN)-derived relaxing factor with nitric oxide (NO), sodium nitrite (NaNO~), sodium nitrate (NaNO3), and sodium nitroprusside (NP) (abstract). FASEB J 2:A519. Taych MA, Marletta MA (1989): Macrophage oxidation of L-arginine to nitric oxide, nitrite, and nitrate. Tetrahydrobiopterin is required as a cofactor. J Biol Chent 264:19654-19658. Tottrup A, Svane D, Forman A (1991): Nitric oxide-mediating NANC inhibition in opossum lower esophageal sphincter. Am J Phvsiol 260:G385-G389. Trigo-Rocha F, Aronson WJ, Hohenfellner M, Ignarro LJ, Rajfer J, Lue T (1993): Nitric oxide and cGMP: mediators of pelvic nerve-stimulated erection in dogs. Am J Phvsiol 264:H419-H422. Wagner DA, Young VR, Tannenbaum SR (1983): Mammalian nitrate biosynthesis" incorporation of 15NH3 into nitrate is enhanced by endotoxin treatment. Proc Natl Acad Sci USA 80:4518-4521. Wallace GC, Fukuto JM (1991)" Synthesis and bioactivity of N'"-hydroxyarginine: a possible intermediate in the biosynthesis of nitric oxide from arginine. J Med Chem 34:1746-1748. Xie QW, Cho H, Kashiwabara Y, Baum M, Weidner JR, Elliston K. Mumford R, Nathan C (1994): Carboxyl terminus of inducible nitric oxide synthase contribution to NADPH binding and enzymatic activity. J Biol Chem 269:28500-28505.

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CHAPTER II

Histochemistry of nitric oxide synthase in the central nervous system S.R. VINCENT

1. INTRODUCTION Our current understanding of the distribution of the neurons that can produce nitric oxide has been built on a number of complementary technical developments. The introduction of NADPH diaphorase histochemistry in formaldehyde-fixed material led to the frequent use of this method for neuroanatomical studies even before the nature of the enzyme responsible was discovered (Scherer-Singler et al., 1983). The demonstration that the NADPH diaphorase histochemical staining is due to the activity of nitric oxide synthase (NOS) (Hope et al., 1991) quickly enabled the detailed anatomical analysis of NO-producing neurons throughout the nervous system (Vincent and Hope, 1992; Vincent and Kimura, 1992). The direct relationship between NADPH diaphorase staining and NOS expression has been well documented (Bredt et al., 1991; Dawson et al., 1991 ). The absence of neuronal expression of NOS immunoreactivity and NADPH diaphorase activity in knockout mice lacking nNOS provided definitive evidence for the specificity of this simple histochemical procedure (Huang et al., 1993). The distribution of NADPH diaphorase-positive neurons in the rat brain has been reviewed previously, together with discussions on the specificity of this method (Leigh et al., 1990; Vincent and Hope, 1992; Vincent and Kimura, 1992; Vincent, 1994; Blottner et al., 1995; Norris et al., 1995). Ultrastructural examination of NADPH diaphorase has also been described (Hope and Vincent, 1989; Rothe et al., 1998). The relationship of NADPH diaphorase staining and NOS immunohistochemistry to soluble guanylyl cyclase (Schmidt et al., 1992a) and to NO-induced cGMP-immunoreactivity has been reviewed (Southam and Garthwaite, 1993; De Vente et al., 1998). More recently, the development of a World Wide Web accessible digital atlas of NADPH diaphorase staining in the mouse brain [http://nadph.anatomy.lsumc.edu] presents an exciting new tool for the neuroscience community. The results obtained with NADPH diaphorase histochemistry have been confirmed and extended using antibodies against the various NOS isoforms (Bredt et al., 1990) as well as in situ hybridization (Bredt et al., 1991). Of particular irnportance has been the description of alternatively spliced forms of nNOS expressed in certain brain regions (Brenman et al., 1997; Eliasson et al., 1997). Another approach for localizing NOS that has been use~t is the autoradiographic localization of [3H]L-NG-nitro-arginine binding to the enzyme (Burazin and Gundlach, 1995; Kidd et al., 1995; Hara et al., 1996; Rao and Butterworth, 1996). Certain caveats must of course be kept in mind when using any of these techniques. For example, the NADPH diaphorase staining observed in cell groups lacking NOS expression Hamlhook ,{f CIwmical Neuroanatomx. Vol. 17: Functional Nettroanatomy ,!t the Nitric Oxide System H.W.M. Steinbusch, J. De Vente and S.R. Vincent. editors @ 2000 Elsevier Science B.V. All rights reserved.

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often appears to be due to technical artifacts, including fixation difficulties (Hope and Vincent, 1989; Matsumoto et al., 1993; Spessert and Layes, 1994; Buwalda et al., 1995; Gonzalez-Hernandez et al., 1996). Indeed, some have suggested running the NADPH diaphorase reaction in the presence of formaldehyde to overcome this problem (Nakos and Gossrau, 1994; Grozdanovic et al., 1995). Poor fixation has been reported to result in the NADPH diaphorase staining of astrocytes as well (Gabbott and Bacon, 1996: Kugler and Drenckhahn, 1996). Alkaline phosphatase activity can sometimes hydrolyze NADPH to NADH which can then be used as a substrate for other diaphorase reactions leading to false-positive formazan formation (Song et al., 1994; Grozdanovic and Gossrau, 1995b). Cytochrome-P450 reductase has also been suggested as a possible source for NADPH diaphorase activity, however, although some neuronal populations do express both NOS and cytochrome-P450 reductase-immunoreactivity, this enzyme does not appear to contribute to NADPH diaphorase staining (Norris et al., 1994; Young et al., 1997). Poor fixation may also contribute to false-positive staining with NOS antibodies (Wendland et al., 1994; Kugler and Drenckhahn, 1996). Finally, some neurons deep in the rat cortex have been reported to be diaphorase-positive but nNOS-immunonegative (Kharazia et al., 1994). It may be that these cells contain an isozyme of nNOS which is detected histochemically but not with the particular antibody used. There have been attempts to develop pharmacological methods to help ensure the specificity of NADPH diaphorase staining for NOS. Biochemical studies on the unfixed enzyme indicate that the NADPH diaphorase activity of nNOS is Ca 2+/calmodulin-independent (Schmidt et al., 1992b). This is consistent with observations that calcium chelators are without effect on NADPH diaphorase histochemistry (Hope and Vincent, 1989; Spessert et al., 1994). However, one group has reported that EDTA can inhibit neuronal NADPH diaphorase staining (Sancesario et al., 1993). Furthermore, studies by Morris et al. (1997) indicate that neuronal NADPH diaphorase staining is dependent on Ca2+/calmodulin and suggest that the intensity of the NADPH diaphorase staining may be related to the level of enzyme activation at the moment of tissue fixation. A number of groups have reported inhibition of NADPH diaphorase with the non-selective flavoprotein inhibitor, diphenyleneiodonium (Blottner and Baumgarten, 1992, 1995; Spessert and Claassen, 1998). NADPH diaphorase staining of formaldehyde-fixed intermediolateral spinal neurons was blocked by NG-nitro-L-arginine (L-NNA), but was still observed in the presence of NG-monomethyl-L-arginine (NMMA) and 7-nitroindazole (Blottner and Baumgarten, 1992, 1995). The NADPH diaphorase staining in the olfactory epithelium was stimulated by addition of the NOS substrate L-arginine, and was inhibited by the NOS inhibitor L-NG-nitro arginine (Dellacorte et al., 1995). Methylene blue, which is a very non-specific inhibitor of NOS, can also block NADPH diaphorase staining (Luo et al., 1995). ~-NADPH can be substituted for the physiological [3-NADPH, and this may result in a diaphorase staining more specific for NOS (Hope and Vincent, 1989; Grozdanovic and Gossrau, 1995a), although one group found that neuronal NADPH diaphorase in the olfactory bulb could not utilize ~-NADPH (Spessert et al., 1994; Spessert and Claassen, 1998). Over the past decade, the use of NADPH diaphorase histochemistry, complemented by these other methods, has allowed NO-producing neurons to be described in a wide variety of species. The comparative anatomy of the NO system in various vertebrates is reviewed in another chapter in this volume (Alonso et al. this volume). In this chapter, the distribution and characteristics of such cells in the mammalian central nervous system will be reviewed. Unless otherwise mentioned, this description is based on work in the rat brain.

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2. LOCALIZATION OF NITRIC OXIDE SYNTHASE IN THE BRAIN

2.1. THE OLFACTORY BULB Within the nasal mucosa, a dense innervation of NOS-positive fibers derived from the sphenopalatine ganglion is present (Hanazawa et al., 1993; Kulkarni et al., 1994; Lee et al., 1995; Jeon et al., 1997; Kim et al., 1997). NADPH diaphorase staining has also been reported in cells with morphology reminiscent of microvillar olfactory cells, but not in the respiratory epithelium and the sustentacular cells (Dellacorte et al., 1995). nNOS is expressed in the presumptive nervous layer of the olfactory placode and the cells that differentiate into embryonic olfactory receptor neurons (Roskams et al., 1994). However, no evidence for NOS expression was found either in the mature main olfactory epithelium nor in the vomeronasal organ, in spite of the strong diaphorase staining of the surface of the main olfactory epithelium (Kishimoto et al., 1993; Kulkarni et al., 1994; Roskams et al., 1994; Lee et al., 1995). There is intense NADPH diaphorase staining of the glomeruli of the olfactory bulb (Vincent and Kimura, 1992). In the accessory olfactory bulb the entire vomeronasal nerve and all vomeronasal glomeruli were strongly labeled, contrary to the main olfactory bulb, where only dorsomedial olfactory glomeruli displayed NADPH diaphorase activity. This glomerular NADPH diaphorase reaction is pharmacologically different from that due to NOS, and does not co-localize with NOS immunohistochemistry (Spessert et al., 1994). Thus NOS does not appear to be present in mature primary olfactory neurons. Interneurons containing NOS protein and mRNA, and exhibiting NADPH diaphorase activity have been well described in the plexiform layer of the main olfactory bulb and the granule cell layer of main and accessory olfactory bulbs (Fig. 1A,C) (Kishimoto et al., 1993). Periglomerular cells (Fig. 1B) and granule cells in the main olfactory bulb were also NOS-positive (Kishimoto et al., 1993). NADPH diaphorase-positive neurons were identified as periglomerular cells in the glomerular layer and external plexiform layer, horizontal cells in the internal plexiform layer, and granule cells and deep short-axon cells in the granule cell layer (Porteros et al., 1994). Some of these cells appear to correspond to the superficial short-axon cell described in Golgi and electron microscopic studies, and the dendrites of these cells lie within the periglomerular region and in the superficial external plexiform layer (Scott et al., 1987). In addition to NOS, these cells also express somatostatin as well as NPY and the C-terminal flanking peptide of NPY, C-PON (Scott et al., 1987; Villalba et al., 1989). Positive periglomerular cells are more frequently associated with typical than atypical glomeruli (Crespo et al., 1996). The NADPH diaphorase-positive periglomerular cells appear to form a distinct population, and do not express tyrosine hydroxylase (Samama and Boehm, 1996), calbindin Dzgk (Alonso et al., 1993), calretinin or parvalbumin (Brifirn et al., 1997). A type of short-axon cell which is also stained for NADPH diaphorase, NPY, C-PON and somatostatin, lies deep in the granule cell layer, frequently near the ventricular layer and its dendrites lie parallel to that layer (Scott et al., 1987; Villalba et al., 1989). Many NADPH-stained neurons are also found in regions known to provide centrifugal inputs to the olfactory bulb, including all subdivisions of the anterior olfactory nucleus (Fig. 1D), the anterior hippocampal rudiment, anterior and posterior levels of the piriform cortex, and the vertical and horizontal limbs of the diagonal band of Broca (Davis, 1991; Garcia-Ojeda et al., 1994).

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2 5 t.tm d i a m e t e r as "very large'.

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Figs. 1-4. Atlas of NOS-IR neurones in the rat medulla oblongata visualised with the recombinant nNOS antibody and Cy3 Iabelling. Four coronal levels of sectioning are shown corresponding to bregma -14.60, -13.80, -13.30 and -12.80 of the Paxinos and Watson (1997) rat brain atlas. The boxed areas marked on the low power photomontage indicate areas containing NOS-IR neurones that are shown at higher magnification in panels A-J.

Fig. 1. Caudal level of the medulla (-14.60). Nuclei illustrated in boxed areas: (A) Gr, (B) Cu, (C) dorsal part of MdD (or DRt), (D) dorsomedial part of Sp5C, (E) neurones close to the cc, encapsulating the XII, in the DVN and coNTS, (F) dorsal part of MdV, (G) IRt in the region of the NA, (H) superficial Sp5C.

S p 5 C and the r e t i c u l a r f o r m a t i o n ; i n t e r p o l a r division ( S p 5 C I ) - - m a n y f e w e r cells and fibres, r e s t r i c t e d to the m o s t superficial layers; d o r s o m e d i a l division ( D M S p 5 ) - - a p r o m i n e n t c l u s t e r o f t r i a n g u l a r and ovoid n e u r o n e s , with a high d e n s i t y o f i m m u n o r e a c t i v e fibres. 181

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Fig. 2. Intermediate level of the medulla (-13.80). Nuclei illustrated in boxed areas: (A) AR coNTS and meNTS, (B) ts, dNTS and Gr, (C) ventral part of Cu, dorsal part of MdD (or DRt) and dorsomedial pole of Sp5C, (D) dorsal part of IRt, (E) ventral part of Sp5I, (F) dorsal MdV (nucleus of Probst's bundle) and Ro, (G) ROb and caudal part of PMn, (H) ventromedial part of MdV, (I) ventral part of MdV between LRt and IO, (J) ventral part of IRt surrounding the NA.

Paratrigeminal nucleus (Pa5) very dense network of varicose fibres and small ovoid intensely immunoreactive cells. Vestibular/auditoi3'. Medial vestibular nucleus (MVe) rather evenly distributed, well labelled, small triangular and ovoid neurones, with many dendritic and fibre profiles. Spinal vestibular nucleus (SpVe) very few isolated, weakly labelled, medium-sized neurones and few fibres. Nucleus prepositus hypoglossi (Pr) and nucleus intercalatus (In) clusters of small ovoid 182

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Fig. 3. Central level of the medulla (-13.30). Nuclei illustrated in boxed areas: (A) Ro, (B) dNTS, sgNTS and ceNTS, (C) vlNTS, (D) IRt, (E) dorsal part of PCRt (probably forming the caudal pole of DMSp5), (F) PMn, (G) ROb, (H) Gi, overlying the LRt, (I) C1 cell area, (J) central part of PCRt. neurones and varicose fibres, most numerous towards the ventricular surface. Visceromotor areas. Dorsal vagal nucleus (DVN) - - some weakly positive, medium-sized ovoid neurones and a few more intense elongated neurones, mostly situated in the lateral part. Nucleus ambiguus ( N A ) - isolated bipolar neurones in this region, but only very weak cellular labelling and varicose fibres occur within the compact zone. Somatomotor areas. Hypoglossal motor nucleus (XII) - - contains fibres and varicosities, but very few isolated neurones occur within the confines of the nucleus itself. A few large intensely labelled multipolar cells located medially to the XII bordering the central canal (cc) at caudal levels, and at the lateral margin of the nucleus more rostrally, with dendrites and 183

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Fig. 4. Rostral level of the medulla (-12.80). Nuclei illustrated in boxed areas: (A) Ro, In and medial tip of MVe, (B) ventral part of NTS and dorsal part of PCRt, (C) MVe and dorsal part of meNTS, (D) ts and vlNTS, (E) DMSp5 and dorsal part of Sp5I, (F) ROb, (G) GiV between the component nuclei of the IO, (H) LPGi, (I) IRt, (J) compact formation of NA.

varicose fibres crossing the XII towards the midline (but see also Ro and Prb below). Facial motor nucleus (VII) - - no labelled neurones, very few fibres or varicosities. Reticular formation. Nucleus of Roller (Ro), nucleus of Probst's bundle (Prb), and nucleus intermedius ( I n M ) several prominent groups of medium-sized, mainly ovoid, bipolar or triangular neurones occur in the most dorsomedial part of the reticular formation near the intersection of XII and DVN. Dorsal reticular area (MdD) - - many large mostly triangular multipolar neurones with prominent branching dendrites in the dorsal part, stretching down below vlNTS and DVN (but 184


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Fig. 5. Photomontages of the NTS at central level taken from adjacent 30 lxm sections stained by NADPH-d histochemistry and NOS immunohistochemistry (ABC-VIP method). Note that the relative numbers and distributions of the labelled cell bodies and fibres in the various subnuclei of the NTS are almost identical with both methods, with densely packed aggregations of small neurones and fibres in the central subnucleus (ceNTS), and more loosely packed groups of larger neurones in the medial (raNTS). ventral I vNTS) and ventrolateral (vlNTS) subnuclei.

most of these lie within the IRt). Similar, isolated neurones in the lateral part adjoining the Sp5C, but only a few isolated cells in the more ventrolateral parts. Intermediate reticular area (IRt) - - an almost continuous band of large triangular and ovoid neurones with prominent dendrites radiating dorsomedially and ventrolaterally into the reticular formation. Neurones appear to avoid the NA and A1 cell areas, but dendrites and fibres extend into these areas. Cells most numerous in this area at intermediate and rostral levels of the medulla. 185

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Parvocellular reticular field (PCRt) - - dense networks of fibres and varicosities, with widely scattered ovoid and triangular multipolar neurones, many rather weakly labelled. Some larger clusters occur in the dorsal part bordering the IRt, DMSp5 and Sp51. Lateral reticular nucleus (LRt) - - very few neurones within this nucleus, although many are scattered along the medial and dorsal aspects in the reticular formation, with dendrites penetrating into the LRt. Scattered fibres and varicosities. Ventral reticular area (MdV) - - large triangular and ovoid neurones with 2-5 main dendrites radiating in all directions are scattered throughout this area. At caudal levels these are aggregated towards the dorsal edge of the pyramidal tract (py) and medial to the NA and A1 cell areas but mainly aggregated in the ventral zone over the inferior olive, between the ROb and the LRt. Paramedian reticular area (PMn) - - small clusters of mainly bipolar and triangular neurones, some within the medial longitudinal fasciculus (mlf). Gigantocellular field (Gi) - - rather evenly scattered medium-sized neurones, mostly triangular with branching dendrites. Ventral gigantocellular field (GiV) - - as Gi, but a higher density of labelled neurones, many bipolar with long prominent dendrites, extending over and between the IO nuclei. Lateral paragigantocellular field (LPGi) - - similar large neurones to those of the Gi, but forming large clusters. Those towards the ventral surface of the medulla are very large in size with extensive dendritic arborisations. Raphe obscurus (ROb) ~ varicose fibres and isolated bipolar and multipolar neurones throughout, extending into the mlf. Inferior olive (IO) ~ scattered beaded fibres, but extremely few NOS-IR neurones within olivary complex, although cells similar to those seen in ventral reticular field do penetrate between the olivary subnuclei.

3.1.2. Relative distribution of NOS-IR and NADPH-diaphorase activity Two different methods have been used to map the distribution of nitric oxide-producing neurones in the central nervous system: the NADPH-diaphorase histochemical method (Scherer-Singler et al., 1983; Bredt et al., 1990: Leigh et al., 1990; Vincent and Kimura, 1992), and the later immunohistochemical method using antisera raised against nitric oxide synthase (NOS) (Dun et al., 1994; Egberongbe et al., 1994; Rodrigo et al., 1994). Evidence has been presented for several areas of the nervous system that in all likelihood the two techniques result in the visualisation of the same neuronal population (Bredt et al., 1991a; Dawson et al., 1991; Hope et al., 1991; Schmidt et al., 1992). The distributions of NOS-IR and NADPH-diaphorase staining throughout the medulla

Fig. 6. NOS-IR neurones are present at various levels of the NTS of the rat medulla./A. B) At caudal levels, NOS immunoreactivity can be identified by the presence of gold particles in cells in the dorsal part of the commissural NTS (A). The neurones in the boxed area can be seen at higher magnification in B. (C, D) At a caudal level similar to that shown in A and B, a discrete group of NOS-IR neurones can be observed ventral to the central canal. D is a higher magnification of the boxed area in C and illustrates that the labelling is clearly in neuronal somata and dendrites. (E, F) In areas of the NTS below the rostral pole of the area postrema a densely packed group of immunoreactive neurones can be observed in the central subnucleus (encircled in E). At higher magnification it is clear that the labelled neurones are small, with rounded somata IF). (G. H) In the rostral pole of the NTS, NOS-IR neurones are numerous (G), loosely packed (H) and vary in size and shape (H). 187

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of the rat and the cat have been compared by double-labelling sections and by examining series of adjacent sections. In dual-labelled 50 ~m vibratome sections, in which the NADPH reaction was performed prior to NOS immunolabelling, virtually all NADPH-positive cells displayed blue and brown reaction product within the cytoplasm of their soma and proximal dendrites. Approximately 80% of NADPH-positive cells in the rat medulla showed NOS-IR, and approximately 95% of NOS-IR cells showed NADPH staining. In adjacent 30 ~tm sections at all transverse levels, there was a close correspondence between the positions of NOS-IR cells or fibres and NADPH-diaphorase-positive cells or fibres in most anatomical divisions of both cat and rat medulla (see Fig. 5). Groups of cells intensely or weakly stained by one method showed a similar staining intensity with the other method. A few regions showed slight discrepancies, i.e. external cuneate nucleus, hypoglossal nucleus, inferior olive, nucleus ambiguus, where a large number of weakly NADPH-diaphorase-positive cells were sometimes found, but few NOS-IR cells were seen. It can be concluded, therefore, that identical sets of neurones in the medulla are labelled by the two different techniques. The small discrepancies noticed in the above studies are most likely explained by technical reasons inherent in the two methods. Firstly, additional neurones might be stained by the NADPH method, due to its relative lesser specificity for the nNOS enzyme than the well-characterised NOS antisera used. It is possible that structurally related enzymes such as endothelial NOS and cytochrome P450 oxyreductase (Bredt et al., 1991b; Norris et al., 1994) could be responsible for additional weakly reactive neurones not labelled by the NOS antisera. Secondly, discrepancies could be due to differential penetration of reagents into vibratome sections, with the smaller molecular size of the reagents for the NADPH reaction better able to penetrate into the interior of the sections than the larger antibodies used in the immunolabelling. Finally, and probably most important, are the different sensitivities of detection of the methods for neurones with low levels of NOS expression, allied with the effects of fixation on the reactive sites in the enzyme. This is demonstrated by the numbers of cells detected on adjacent sections using different antisera to NOS, and by the fact that adding higher concentrations of glutaraldehyde (>0.2%) to the fixative dramatically reduces the numbers of neurones detected by the NADPH method, whereas immunostaining with some NOS antibodies can tolerate glutaraldehyde concentrations up to 2% without any noticeable reduction in labelling.

3.1.3. Co-localisation of NOS-IR or NADPH-diaphorase activity with other neuronal phenotypes in the medulla oblongata Many authors have described NADPH-d activity co-localised with neuroactive substances within specific regions of the central nervous system. The presence of the enzyme has been reported in serotonergic cells in the mesopontine areas of the brain (Johnson and Ma, 1993). Other studies have demonstrated co-localisation with calcitonin gene-related peptide (CGRP)-IR and substance P-IR in neurones within cranial and dorsal root sensory neurones (Aimi et al., 1991; Ichikawa and Helke, 1996), and in cholinergic, glycinergic and GABAergic neurones of the spinal dorsal horn (Spike et al., 1993). NOS-IR has been found in a subpopulation of GABAergic local circuit neurones in the ventrobasal complex of the cat (Meng et al., 1996), and of GABAergic amacrine cells in the retina (Oh et al., 1998). Both GABA and/or the calcium-binding protein calbindin D28K have been detected in NOS-IR cortical neurones of several species (Bertini et al., 1996; Yan and Garey, 1997), while calbindin has also been detected in nitrergic neurones of the intermediolateral spinal cord (Grkovic and Anderson, 1997), lateral septum (Doutrelant-Viltart and Poulain, 1996) and in the petrosal and 188

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nodose ganglia (Ichikawa and Helke, 1996). A further calcium-binding protein, calretinin, was reported in significant proportions of NADPH-d-reactive neurones of the hippocampus and dentate gyrus (Megias et al., 1997). In hypothalamic neurosecretory neurones, co-localisation of NADPH-d with somatostatin, vasopressin, angiotensin and calretinin has been reported (Alonso et al., 1992; Ar6valo et al., 1993; Calka and Block, 1993a,b), and in other areas of the hypothalamus immunoreactivities for enkephalin and substance-P were frequently found in NOS-IR neurones (Yamada et al., 1996), with other peptides such as galanin, cholecystokinin (CCK) and somatostatin (SS) also occurring in minor populations of nitrergic cells in some areas. SS and avian pancreatic polypeptide-like (probably NPY) IR were also found to coexist with NADPH-d activity in neurones of the striatum and neocortex (Vincent et al., 1983a). NADPH-d activity has also been found to co-exist with choline acetyl-transferase (CHAT) in a number of regions including the NTS, the compact zone of the NA, raphe nuclei, PGLi, lateral tegmental field, dorsal horn and sympathetic columns of the spinal cord (Ruggiero et al., 1993; Spike et al., 1993), and in neurones of the pontine reticular formation (Vincent et al., 1983b). Both galanin and 5-HT immunoreactivities were found to coexist with NOS-IR in dorsal raphe neurones (Xu and H6kfelt, 1997). Neurones of the areas of the medulla that control autonomic functions contain a wide range of neuropeptides and neurotransmitters (Leibstein et al., 1985; Ruggiero et al., 1989; Batten, 1995). Two of the neuronal groups that have been most extensively studied are the vagal parasympathetic preganglionic neurones, and the vasomotor neurones of the RVLM that project onto the sympathetic preganglionic motorneurones located in the thoracic spinal cord. The latter group of neurones have been identified as the C 1 group of adrenergic neurones (Ross et al., 1984), and these occur in a very similar area to a large group of NADPH-d-reactive neurones (Iadecola et al., 1993). It has also been suggested that the sympathoexcitatory vasomotor neurones of the RVLM may be glutamatergic rather than catecholaminergic (Sun et al., 1988a,b). Therefore, it was of great interest to examine the possible co-existence of NADPH-d or NOS activities with immunoreactivities for catecholamine synthesising enzymes and glutamate in this region, and furthermore with immunoreactivities for other neuropeptides and neurotransmitters (e.g. SS, NPY, CCK) which have been localised to RVLM neurones projecting to the area containing the sympathetic preganglionic neurones of the intermediolateral columns of the spinal cord (Mantyh and Hunt, 1984; Blessing et al., 1986, 1987; Millhorn et al., 1987). The following sections therefore discuss the co-localisation of NOS with vagal afferent and efferent structures, bioamines, neuropeptides, amino acids and calcium-binding proteins. (a) Presence of NOS in vagal afferent and efferent structures. One possible source of NO in the NTS is the vagal afferent fibres. Studies to support this include: (i) neurones in the nodose ganglia stain positive for NADPH-d (Aimi et al., 1991; Morris et al., 1993; Ruggerio et al., 1996); (ii) in situ hybridisation indicates that cell bodies in the nodose ganglia express mRNA encoding NOS (Lawrence et al., 1996). NOS could therefore be transported from these cell ~odies to vagal afferent terminals in the NTS. The presence of NOS in central vagal terminals has also recently been inferred by a decrease in NOS-IR in a restricted portion of the ipsilateral medial NTS following nodose ganglionectomy (Lawrence et al., 1998), and this was illustrated by the presence of NOS immunoreactivity in 67% of degenerating terminals in the NTS following a nodose ganglionectomy (Lin et al., 1998). However, we have been unable to confirm the presence of NOS in vagal afferents despite combination of anterograde tracing from the nodose ganglion with NOS immunohistochemistry (Fig. 7). There are many other possible sources of NOS-IR fibres and terminals in the NTS as shown by studies injecting tracers into this nucleus. These include the dorsal horn of the spinal cord (Esteves 189

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Fig. 7. Relationship between NOS-IR neurones (B, D, F) with vagal preganglionic efferent neurones (A) and vagal afferent fibres (C, E) shown by double labelling. (A) Cell bodies of vagal efferent neurones in the DVN labelled by CTb injection into the vagus nerve, visualised with Cv2. (B) Corresponding area, showing NOS-IR neurones and fibres labelled with Cy3. Some cells are strongly labelled for both CTb and NOS (longer arrows in A and B), some CTb-containing cells are weakly immunoreactive for NOS (open-headed arrows), and some cells labelled lot CTb are not NOS-IR (double-headed arrow in A). A strongly NOS-IR neurone that is not labelled for CTb is indicated by a larger arrowhead in B. (C) Vagal afferent fibres in the TS and central NTS at an intermediate level, labelled by injection of BDA into the nodose ganglion, and visualised by the TSA-fluorescein method. (D) Same area, showing NOS-IR neurones and fibres (Cy3 labelling). Although NOS-IR fibres are concentrated in the same areas as the vagal afferents, there is no clear evidence of dual-labelled fibres or terminals. (E) Vagal afferent fibres in the TS and central NTS at a more rostral level (TSA-fluorescein). (F) Same area showing NOS immunoreactivity (Cy3). Again, there is little evidence for dual labelling. 190


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Fig. 8. NOS-IR neurones are present in the dorsal vagal nucleus (DVN) and the nucleus ambiguus (NA). (A) Photomontage of the dorsomedial area of a coronal section of the rat medulla showing both dorsal vagal nuclei. NOS-IR neurones are indicated by the arrows. The DVN on the right-hand side contains more labelled neurones than the left-hand side, and the labelling of the cells is noticeably more intense. This increased NOS expression may be a result of damage to the axons of the vagal neurones on the right-hand side. due to injections of tracer into the vagus nerve. (B) NOS immunoreactivity in the compact region of the NA can be detected in a dense network of fibres as well as some somata. (C) A NOS-IR neurone in the compact NA is apposed by two NOS-IR terminals (arrows).

et al., 1999), paratrigeminal nucleus (Armstrong and Hopkins, 1998) and raphe obscurus (Fig. 14). NOS and NADPH-d have also been detected in vagal efferent neurones (Mizukawa et al., 1989; Krowicki et al., 1997; Zheng et al., 1999: Figs. 7 and 8). Several anatomical studies have localised NOS in DVN neurones (e.g. cat: Mizukawa et al., 1989; Figs. 7 and 8). These nitrergic neurones are likely to be vagal neurones, since intra-peritoneal injection of Fluorogold, which labels all preganglionic neurones, combined with NADPH-d histochemistry revealed double-labelled neurones in the caudal and rostral, but not intermediate, zones of the DVN (Krowicki et al., 1997; see also Figs. 7 and 8). Retrograde tracing from the gastric fundus labelled a discrete population of NOS-immunoreactive neurones in the medial portion of the DVN (Zheng et al., 1999). However, it can be difficult to interpret this kind of double labelling since neurones in the DVN may upregulate NOS expression in response to 191

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axonal damage, such as that which might occur during a retrograde tracing experiment as a result of nerve section or merely the injection of a tracer (Fig. 8; Jia et al., 1994; Yu, 1994). While it is therefore difficult to determine if NOS immunoreactivity would have been detectable in the double-labelled cells without the experimental intervention, it is clear that weak staining, probably representing low levels of NOS, occurs within the DVN in a few neurones with the morphology of parasympathetic efferent neurones even in intact animals (Figs. 8 and 9). A few moderately stained NOS-immunoreactive neurones have been localised within or near to the nucleus ambiguus (Dun et al., 1994; Fig. 8). However, these neurones are not immunoreactive for cholinergic markers (Dun et al., 1994) and are therefore unlikely to be vagal efferent neurones. This is consistent with lack of double-labelled neurones in the NA following combination of retrograde tracing from the cervical vagus or the heart with NOS immunohistochemistry (E. Corbett, unpubl, observations). (b) Bioamines. Our double-labelling studies using either enzymatic or fluorescence methods on cat and rat brain stem sections demonstrated that very few NOS-containing neurones in the medulla were immunoreactive for the catecholamine synthesising enzymes tyrosine hydroxylase (TH), dopamine-[3-hydroxylase (DBH) or phenylethanolamine N-methyl transferase (PNMT). In both the caudal and rostral ventrolateral medulla the NOS cells were generally located more dorsal and more medial than the TH-IR cells (of the A1 and C1 cell groups, respectively). This distinction was most striking at the more rostral levels of the brain stem, and we have found no dual-labelled cells in either area (Fig. 10). In the dorsomedial medulla, the two groups of neurones were more intimately mingled, but catecholamine cells of the DMV and NTS (A2 and C2 groups) were only rarely (~ 10% of cases) found to be NOS positive (Fig. 10). NOS-IR terminals were found apposing catecholamine neurones in both the NTS and ventrolateral medulla, and TH-IR or DBH-IR terminals were observed on NOS/NADPH-reactive neurones in both these areas. These results are in general agreement with the earlier findings of Iadecola et al. (1993), Ohta et al. (1993), Dun et al. (1994) and Simonian and Herbison (1996), although the latter authors reported a small degree of TH-NOS coexistence in both A 1 and C 1 regions of the ventrolateral medulla. NOS-IR neurones in the nucleus raphe obscurus and ventral gigantocellular reticular nucleus have a similar morphology and distribution to the 5-HT-IR neurones in these nuclei. Nevertheless, in agreement with Dun et al. (1994) dual-labelling studies in the rat have demonstrated that the two immunoreactivities are generally localised to separate neurones, with only occasional examples of dual-labelled cells being found (Fig. 11). These studies revealed, however, evidence for the existence of an intimate reciprocal innervation of NOS-IR cells by 5-HT-IR fibre varicosities and vice versa. (c) Neuropeptides. Neurones staining for both NOS and SS immunoreactivity were found scattered within the paramedian and lateral tegmental field of the cat medulla. These dual-labelled cells accounted for 6% of the total NOS cells in both these areas and comprised 15% of the paragigantocellular NOS cells (Maqbool et al., 1995). In the most rostral medulla oblongata a few double-labelled cells were observed in the NTS (12%) and in the Pr (9%). In the rat medulla, dual-labelled neurones could be found scattered throughout several areas of the reticular formation, particularly in the dorsal (Fig. 12) and intermediate medullary reticular nuclei, in the InM, Ro and Prb. SS-IR was co-localised to very few NOS-IR cells throughout the NTS (Fig. 12) and spinal trigeminal nuclei, and to a small percentage of neurones in the DMSp5. Similar double-labelling studies demonstrated that NOS-reactive neurones were not immunoreactive for CCK although they were co-distributed with CCK-IR cells within the 192


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Fig. 9. Vagal afferent fibres labelled from the nodose ganglion closely appose NOS-immunoreactive neurones in the dorsal vagal complex. (A) A low power view of the dorsal vagal nucleus tDVN). The tractus solitarius (TS) can be seen at the top of the micrograph. The boxed region indicates the area shown at higher magnification in B. (B) The DVN contains many neurones immunoreactive for NOS. The neurone enclosed by the box is magnified in C. (C) A NOS-IR neurone in the DVN, identified by the presence of silver intensified gold particles, is closely apposed by a vagal afferent terminal (arrow). (D) Another NOS-IR neurone in the DVN is also closely apposed by two vagal afferent fibres (arrows).

lateral reticular field, within the paramedian field and dorsal to the inferior olive. Neither was NOS-IR found to be present in neurones immunoreactive for NPY, even though the two types of cells were often co-distributed within the same brain stem section. However, numerous NPY-IR terminals were found making close appositions with NOS neurones in the ventrolateral medulla. (d) Amino acids and acetylcholine. Neurones double-labelled for both NOS activity and glutamate-immunoreactivity were observed in the ventrolateral region throughout the extent of the cat medulla oblongata (Maqbool et al., 1995). These cells were scattered in the lateral tegmental field (9% of glutamate neurones), nucleus ambiguus (7%), lateral reticular nucleus (49%) and gigantocellular tegmental field (10%). Double-labelled cells were also seen in the paramedian (23%), the external cuneate nucleus (62%) and within the infratrigeminal nucleus (30%). At the most rostral levels examined double-labelled cells were also found in the nucleus prepositus hypoglossi (12%) and occasionally in the NTS (1%). A slightly 193

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Fig. 10. Relationship between NOS-IR neurones and catecholaminergic (TH-IR) neurones in the dorsomedial medulla (A2 cell group) and RVLM (C1 group) shown by double labelling. (A) NOS-IR neurones in the medial NTS at an intermediate level of the nucleus (Cy3 labelling). (B) Same area. showing TH-IR neurones labelled by Cy2, several of which (longer arrows) are dual-labelled. Other neurones in this cell group are single-labelled for either TH or NOS (larger arrowhead). (C. D) As A and B. but at a central level of the NTS. (C) Clearly dual-labelled neurones (arrows) are far less numerous here. (D) NOS-IR neurones in the RVLM (Cy3). (E) Corresponding area, showing TH-IR neurones of the C I group labelled with Cy2. These are clearly separate from the NOS-IR neurones, which lie in a more ventromedial position than the TH-IR neurones.

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Fig. 11. Relationship between NOS-IR neurones and 5-HT-IR neurones shown by double labelling. (A) Group of 5-HT-IR neurones and terminals in the ROb (Cy3 labelling), tB) Same area labelled for NOS-IR with (~y2. One neurone (longer arrow) is clearly dual-labelled, while others are only immunopositive for 5-HT or NOS (larger arrowheads). (C) Group of 5-HT-IR neurones in the superticial ventral medulla, immediately medial to a rootlet of the XII nerve (Cy3). (D) Corresponding area labelled for NOS-IR with Cy2. Large multipolar cells with extensive dendritic arborisations are located both medially and laterally to the nerve rootlet. Note that the two groups of labelled cells shown in panels C and D are clearly separate populations.

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different pattern of co-existence was observed in the rat (Fig. 12). In the NTS, particularly in the commissural, medial, ventral and ventrolateral subnuclei (Fig. 12), NOS-IR cells were also found to be moderately glutamate-IR. In certain reticular areas, for example the PMn, IRt, dorsal PCRt, Prb and Ro (Fig. 12), clearly dual-labelled cells were quite numerous. Occasional examples of coexistence were also seen in the DMV, DMSp5, MeV, Pr and Sp5C. Despite the evidence for the coexistence of GABA in nitrergic neurones of the dorsal horn and forebrain, and for the coexistence of acetylcholine in nitrergic cells of the spinal cord and pontine reticular nuclei (see above), we have not been able to gather any conclusive proof from dual-labelling studies of such a coexistence in any nuclei of the medulla. (e) Calcium-binding proteins. Neurones immunoreactive for NOS and for calbindin were found to have a broadly similar distribution throughout the rat medulla, and were also similar in their morphology (Fig. 13). However, coexistence of these two markers was found to be restricted to certain areas (Fig. 13). In the sensory nuclei of the medulla calbindin-IR neurones far outnumbered NOS-IR neurones, and coexistence was either absent or rarely observed. The two populations of neurones were entirely separate in the dorsal column nuclei (Gr, Cu and ECu) and in the AP. Few cells were dual-labelled in the NTS, even in the central and rostral zones, where numerous small ovoid or triangular cells immunoreactive for the two markers were coextensive and intermingled (Fig. 13). In the spinal trigeminal nuclei, coexistence was restricted to a few cells in the dorsal superficial zone of the pars caudalis, and to a larger population of cells in the DMSp5 at rostral levels. In the reticular formation no coexistence was seen in the ventral, parvocellular, paramedian or lateral paragigantocellular fields, or in the NA or the LRt. Only a few scattered neurones displayed coexistence in the MdD or Gi. However, many of the large neurones forming the prominent group in the IRt were immunoreactive for both NOS and calbindin at levels both caudal and rostral to obex. Labelling for calretinin or parvalbumin immunoreactivities was found overlapping populations of small, densely packed ovoid neurones throughout the NTS, and were particularly numerous at rostral levels (Fig. 13). Dual labelling demonstrated that the cells containing these calcium-binding proteins were usually different from the nitrergic neurones, with which they were intimately intermingled within the NTS. A few examples of weakly dual-labelled calretinin-NOS neurones were, however, observed in some brains (Fig. 13). From the above evidence, it appears, therefore, that nitric oxide production in the medulla is not confined exclusively to neurones of one particular transmitter phenotype or function. Although high levels of coexistence of NOS-IR occur in certain neurone populations that are presumed excitatory by virtue of their labelling for glutamate, groups of nitrergic neurones in other areas label for SS and calbindin, more usually associated with inhibitory neurones.

Fig. 12. Relationships between NOS-IR neurones and neurones containing somatostatin (SS) and glutamate (Glu) immunoreactivities in different regions of the medulla shown by double labelling. (A) Somatostatin-IR neurones in the dorsomedial area of the reticular formation (Cy2 labelling). (B) Same area showing NOS-IR neurones (Cy3). Two neurones are clearly dual-labelled (arrows), while at least one other cell is labelled for NOS but not somatostatin (larger arrowhead). (C) Somatostatin-IR neurones and fibres in the medial NTS (Cy2). (D) Same area, showing that there is no coexistence of somatostatin-IR and NOS-IR in this area of the NTS. The larger, multipolar NOS-IR neurones are more ventrally located than the smaller, ovoid somatostatin-IR cells. (E) Glutamate-IR neurones lying immediately ventral to the XII motor nucleus, within the nucleus of Roller (Ro), labelled with Cy2. (F) Same area, showing that these neurones also contain NOS immunoreactivity (arrows), visualised by Cy3 labelling. However, other neurones in this nucleus are NOS-IR (larger arrowhead), but have undetectable levels of glutamate-IR. (G) Clearly glutamate-IR neurones (arrows) in the ventral INTS at intermediate level of this nucleus (Cy2). (H) The same neurones (arrow) also show intense NOS-IR (Cy3). 197

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Fig. 13. Relationship between NOS-IR neurones and neurones containing calcium-binding proteins shown by double labelling. (A) Group of NOS-IR neurones in the dorsal NTS at an intermediate level (Cy3 labelling). (B) Same area labelled for calbindin-IR with Cy2. Only a few dual-labelled neurones can be recognised. (C) NOS-IR neurones and fibres in the DMSp5 (Cy3). (DI Same area showing labelling for calbindin-IR with Cy2. Many neurones are clearly dual-labelled (longer arrow), but others are only labelled for calbindin (larger arrowhead). (E) Large group of weakly NOS-IR neurones in the medial NTS at a rostral level (AMCA labelling). (F) Same area labelled for calretinin-IR with Cy3. Only a few neurones are clearly identified as being dual-labelled for both markers (arrow).

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It is clear, however, that the primary neurotransmitters co-existing with NOS in many of the nitrergic cell groups of the medulla (most notably the NTS) remain to be established. These neurones may contain peptides such as enkephalins that are difficult to visualise in somata and dendrites by immunohistochemistry, and it may be that methods aimed at defining the transmitters present in the terminals of these neurones after intracellular filling with markers such as biocytin may prove to be a more fruitful approach to resolving this question. 3.2. ROLE OF NITRIC OXIDE IN MEDULLARY PATHWAYS INVOLVED IN AUTONOMIC FUNCTIONS Considerable evidence exists implicating NO as a modulator of various central control pathways in the medulla, including those regulating the cardiovascular, oesophageal, gastrointestinal, respiratory and (anti)nociceptive systems. 3.2.1. NO in the control of the cardiovascular system The centrally mediated baroreceptor reflex provides a rapid negative feedback mechanism that dampens fluctuations in cardiovascular parameters. A rise in blood pressure increases the firing of baroreceptors in the carotid sinus and aortic arch. Sensory information from these receptors is carried to the NTS via the aortic and carotid sinus nerves (components of the vagus and glossopharyngeal nerves, respectively). This rise in baroreceptor firing causes an increase in the discharge of first-order neurones that receive synaptic inputs from the baroreceptor nerve fibres. Outputs from the NTS, via neuronal pathways that are still only partly resolved, modulate the activity of neurones in other medullary nuclei that results in compensatory adjustments of parasympathetic and sympathetic outflow, from efferent neurones located in the vagal motor nuclei and the RVLM, respectively. Ultimately, an increase in blood pressure results in an increased vagal tone and a decrease in sympathetic outflow to the heart and blood vessels, resulting in a decrease in blood pressure. Anatomical studies, using either NOS immunohistochemistry or NADPH-d histochemistry, have demonstrated nitrergic neurones in regions of the medulla implicated in central cardiovascular control, e.g. the NTS (Figs. 1-5), the RVLM (see Figs. 1-4); the raphe nuclei (Fig. 14), the DVN (Figs. 7 and 8) and the nucleus ambiguus (Fig. 8), which contains the majority of the vagal preganglionic neurones projecting to the heart. Of these regions, the NTS and the RVLM are those most likely to contain nitrergic neurones involved in cardiovascular control, since we have been unable to demonstrate the presence of NOS within cardiac vagal preganglionic neurones retrogradely labelled by application of tracers to the heart (E. Corbett et al., unpubl, results). (a) The nucleus tractus solitarius. Nitrergic neurones have been localised in several subnuclei of the NTS which may have roles in central cardiovascular control NTS (Ruggerio et al., 1996; Lawrence et al., 1998; Fig. 6). At least some of these neurones may be baroreceptive, since a substantial proportion of neurones in the dorsal NTS that expressed c-fos in response to phenylephrine-induced hypertension also stained positive for NADPH-d and NOS mRNA (Chan and Sawchenko, 1998). This region of the NTS is known to contain some terminals from aortic depressor nerve afferent fibres (Wallach and Loewy, 1980; Ciriello and Calaresu, 1981; Youfsi-Malki and Puizillout, 1995). However, since it is impossible to determine if these c-fos immunoreactive cells are mono- or polysynaptically activated by baroreceptor stimulation, their exact role in cardiovascular control remains uncertain. Nevertheless, we 199

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provide some support for this notion by illustrating that NOS-immunoreactive neurones in the NTS receive synaptic input from vagal afferents (Figs. 6, 15 and 16). Experiments in vitro indicate clearly that NO can have cellular actions in the NTS. Single-unit extracellular recordings from rat brain stem slices have shown that perfusion of the slices with L-arginine increases the neuronal activity of NTS neurones (Tagawa et al., 1994). Perfusion of the same slices with L-NMMA (a NOS inhibitor) or haemoglobin (a NO trapper) blocks the increase in neuronal activity evoked by L-arginine, as does the guanylate cyclase inhibitor methylene blue (Tagawa et al., 1994). Since haemoglobin does not penetrate into neurones (Stewart et al., 1987; Ignarro, 1991), its action suggests that NO diffuses out of the neurones in which it is formed and penetrates the adjacent neurones to increase their activity through an elevation in cGMP levels. This is consistent with good correlation between cGMP and NOS immunostaining in many areas of the brain including the NTS (De Vente et al., 1998). Evidence for a physiological function of NO in the baroreceptor reflex pathway has come from administering NO-like substances and antagonists to the NTS in vivo. Microinjection of S-nitrocysteine (a NO-like substance) into the NTS of anaesthetised (Lewis et al., 1991; Tseng et al., 1996) and conscious (Machado and Bongama, 1992) rats resulted in an immediate bradycardia and hypotension. Conversely, L-NMMA (a NOS inhibitor) microinjected into the NTS of rats has been shown to attenuate the depressor effect evoked with glutamate microinjection into the NTS (Paola et al., 1991), and to elicit an increase in arterial pressure and heart rate (Harada et al., 1993). The bradycardia and hypotension caused by injection of NO-like substances into the NTS can be blocked by inhibition of soluble guanylate cyclase (methylene blue), suggesting that NO mediates the control of blood pressure and heart rate via its actions on guanylate cyclase (Lewis et al., 1991). (b) The ventrolateral medulla. The rostral portion of the ventrolateral medulla (RVLM) contains sympathoexcitatory neurones (Barman and Gebber, 1985; Dampney et al., 1985; Ciriello et al., 1986) which provide a tonic sympathetic drive to blood vessels. Injection of L-arginine (a precursor of NO) or NO donors into the RVLM of anaesthetised rats (Tseng et al., 1996; Kagiyama et al., 1997), cats (Shapoval et al., 1991; Zanzinger et al., 1995) and rabbits (Kagiyama et al., 1998) elicits a depressor effect. This action is blocked by inhibiting soluble guanylate cyclase with methylene blue (Shapoval et al., 1991; Kagiyama et al., 1998). Similar injections of NOS inhibitors (L-NMMA; L-NAME) into the RVLM elicit a pressor response and an increase in blood pressure (in rat: Tseng et al., 1996; Kagiyama et al., 1997; in cat: Shapoval et al., 1991; in rabbit: Kagiyama et al., 1998). Injection of the NOS inhibitor L-NMMA into the RVLM prior to injection of L-arginine also attenuates the cardiovascular effects of L-arginine (Tseng et al., 1996). In contrast, other studies have shown that microinjection of NO donors into the pressor region of the RVLM of anaesthetised rabbits (Hirooka et al., 1996) or the RVLM of freely moving rats (Martins-Pinge et al., 1997) elicits a pressor effect. In both of these studies this effect was attenuated by prior injection of methylene blue (a guanylate cyclase inhibitor) into the RVLM (Hirooka et al., 1996; Martins-Pinge et al., 1997). Similar injections of the NOS inhibitor (L-NAME) in anaesthetised rabbits caused a depressor effect (Hirooka et al., 1996). These studies suggest that NO has a pressor and sympathoexcitatory action in the RVLM. These contrasting results may be due to variations in the methods used. All of the studies used different volumes and doses of NO donors and three different animal species were used (cat: Shapoval et al., 1991; Zanzinger et al., 1995; rat: Liu et al., 1996; Tseng et al., 1996; Kagiyama et al., 1997; Martins-Pinge et al., 1997; rabbit: Hirooka et al., 1996). Studies were 200


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Fig. 14. Raphe obscurus nucleus (ROb) at an intermediate level of the medulla showing the presence of NOS immunoreactivity in neurones projecting to NTS. (A) Scattered cell bodies in the ROb labelled by injection of CTb into the commissural NTS (Cy2 labelling). (B) Same area showing labelling for NOS-IR with Cy3. Some neurones are clearly dual-labelled (longer arrow), whereas other cells containing CTb are not NOS-IR (larger arrowhead). (C, D) Higher magnifications of areas boxed in A and B, respectively. Again, two cells are clearly dual-labelled (longer arrows), whereas other cells are only single-labelled for CTb or NOS-IR (larger arrowheads).

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Fig. 15. Vagal afferent fibres closely appose NOS-IR neurones in the NTS. (A) Low power view of the NTS on one side of the medulla oblongata. A dense network of vagal afferent fibres, labelled by the injection of BDA into the nodose ganglion, can be detected in the NTS. Some retrogradely labelled neurones are visible in the dorsal vagal nucleus (DVN). The boxed area lateral to the capillary (Cap) is illustrated at higher magnification in B. (B) The capillary (Cap) is the same as that indicated in A. The boxed area contains a NOS-IR neurone and is shown at higher magnification in C. ((7) The NOS-IR neurone, identified by silver intensified gold particles, is closely apposed by DAB-labelled vagal afferent tei-minals.

p e r f o r m e d on both a n a e s t h e t i s e d and c o n s c i o u s a n i m a l s , and differences m a y be due to the level and type of a n a e s t h e s i a . T h e r e is also the possibility that the injections w e r e m a d e into different areas o f the R V L M . O n l y one study injected N O d o n o r s into a region f u n c t i o n a l l y identified as the p r e s s o r region of the R V L M ( H i r o o k a et al., 1996); no others m a d e this distinction. For d i s c u s s i o n see H i r o o k a et al. (1996). 202


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Fig. 16. Electron microscopic verification that vagal afferent synaptic terminals form synaptic contacts with somata and dendrites of NOS-IR neurones in the NTS. IA) A NOS-IR neurone in the NTS identified by the silver intensified gold particles (broken arrows) is apposed by a vagal afferent terminal containing electron dense DAB reaction product (boxed area). (B) Higher magnification of the boxed area in A. A synaptic specialisation (two arrows) occurs between the labelled vagal afferent bouton and the NOS-IR cell (silver indicated by the broken arrow). (C) A large-calibre dendrite of a silver containing NOS-IR neurone is apposed by two electron dense vagal afferent terminals (arrows), illustrated at higher magnification in D and E. (D) The labelled vagal afferent terminal forms a synaptic contact (arrows) with the silver (broken arrow) containing NOS-IR dendrite. (E) The second terminal also forms a synaptic contact (arrows) with the NOS-IR dendrite (broken arrowl.

N O released in the R V L M m a y arise from nitrergic cells located in this region (Figs. 1 - 4 and 10), or from the axon terminals (or possibly dendrites) of neurones located in other areas such as the raphe nuclei or NTS. One possible source is the group of N O S m R N A and N A D P H - d - p o s i t i v e neurones in the d N T S , which were shown to express c-fos in response to p h e n y l e p h r i n e - i n d u c e d hypertension, and which were also retrogradely labelled by tracers injected into the R V L M (Chart and S a w c h e n k o , 1998). This suggests that there is a direct route involving N O neurones, by which the b a r o r e c e p t o r inhibition of s y m p a t h e t i c outflow m a y be affected. 203

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3.2.2. NO in neuronal circuitry underlying control of the oesophagus Reflex central control of the oesophagus may be conducted via a disynaptic pathway. Sensory vagal neurones carry information from the oesophagus to a discrete part of the NTS, the central subnucleus (ceNTS; Fryscak et al., 1984; Altschuler et al., 1989). Vagal afferents form synaptic contacts with NOS-immunoreactive NTS neurones near this region (Figs. 15 and 16). Neurones in the ceNTS emit a dense and topographically discrete projection to the compact formation of the nucleus ambiguus (NAc), where terminals surround the oesophageal motorneurones that project back to the oesophagus (Cunningham and Sawchenko, 1989). NO may be involved at every step in this pathway. The ceNTS is also rich in nitrergic neurones. Histochemical localisation of NADPH-d has revealed a cluster of positive neurones in the central subnucleus in a number of species (rat, dog, guinea pig, ferret and cynomolgus monkey: Bieger and Sharkey, 1993; humans: Gai and Blessing, 1996; rat: Vincent and Kimura, 1992: cat: Maqbool et al., 1995). NOS-IR has also been found in the neurones of the ceNTS/see Figs. 5 and 6: Gai et al., 1995; Maqbool et al., 1995). These cells appear to receive information from oesophageal afferents since nitrergic cells in the ceNTS are innervated by afferents labelled from the oesophagus (rat: Wiedner et al., 1995; rabbit: Gai et al., 1995). The NOS neurones in the ceNTS act as oesophageal premotor neurones and project to oesophageal motor neurones in the NAc (Cunningham and Sawchenko, 1989). NOS terminals in the NAc were found to be derived from ceNTS neurones as NTS lesions caused a decrease in the density of the NOS terminals (Gai et al., 1995). Furthermore, terminals in the NAc labelled anterogradely from the ceNTS surround motorneurones and show NADPH and NOS-IR (rat: Wiedner et al., 1995; rabbit: Gai et al., 1995). The nitrergic neurones in the ceNTS therefore act as interneurones in a disynaptic pathway connecting afferent and efferent neurones controlling oesophageal peristaltic activity. By releasing NO they are thought to excite the oesophageal motorneurones of the NAc, the axons of which terminate in nicotinic cholinergic neuromuscular junctions on the oesophageal muscle fibres, and may thus potentiate nicotinic synaptic transmission (O'Sullivan and Burgoyne, 1990; Anderson et al., 1993). This prevalence of NOS in the neuronal circuitry involved with control of the oesophagus is consistent with a prominent role for NO as a neurotransmitter involved in oesophageal peristalsis.

3.2.3. NO in CNS control of the stomach and large intestine It is well known that NO is a nonadrenergic noncholinergic (NANC) neurotransmitter in the vagal nerve fibres that mediates the relaxation of the GI tract (Desai et al., 1991: Lefebvre et al., 1992). However, the role of NO in the central control of this system is less well described, although the location of NOS and NADPH-d-positive cells suggests a physiological role for NO in the central regulation of gastrointestinal activity. Vagal afferent fibres from the stomach enter the NTS at intermediate levels and project sparsely to the medial and commissural nuclei, and densely to the subnucleus gelatinosus (sgNTS; Shapiro and Miselis, 1985). Afferents terminating in the sgNTS have been shown to synapse directly onto the dendrites of vagal gastric efferent neurones situated in the nearby DVN and projecting back to the stomach (Rinaman et al., 1989). NO may play a role here, as NADPH-d histochemistry has visualised a relatively dense network of fibres and terminal in the sgNTS (e.g. Krowicki et al., 1997). In addition, vagal afferents closely appose the somata of NOS-immunoreactive neurones in the dorsal vagal nucleus (Fig. 9). 204


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As discussed earlier (Section 3.1.3), NOS has been localised in vagal preganglionic neurones in the dorsal vagal nucleus (see also Figs. 7 and 8). Retrograde labelling studies have shown that the DVN provides the major source of efferent innervation to the stomach (Norman et al., 1985; Shapiro and Miselis, 1985), and it has been estimated that 95% of preganglionic DVN neurones innervate the stomach specifically (Leslie et al., 1982). There is also a lesser innervation by neurones in the DVN of the small intestine (Shapiro and Miselis, 1985; Zhang et al., 1991), large intestine (Berthoud et al., 1991) and pancreas (Rinaman and Miselis, 1987). Recently, Zheng et al. (1999) studied specific areas of the DVN thought to mediate particular reflexes such as the adaptive response of the stomach after ingesting a meal (i.e. the capacity of the stomach to receive large volumes with only a slight increase in pressure). They combined injection of retrograde tracers into the gastric fundus of rats and labelled a discrete neuronal population of NOS-immunoreactive neurones in the medial portion. At a cellular level it is clear that NO can excite DVN neurones (Travagli and Gillis, 1994). Perfusion of the rat DVN in vitro with L-arginine (the NO precursor) or S-nitrosoN-acetylpenicillamine (SNAP, a NO donor) has been shown to increase the spontaneous firing rate of DVN motoneurones, an excitatory effect that can be counteracted by the NOS antagonist N-nitro-L-arginine (L-NNA). Perfusion of the cGMP analogue dibutyryl cGMP onto the DVN also increased the spontaneous firing rate of the DVN neurones, and perfusion with LY88583 (an inhibitor of guanylate cyclase) counteracted the excitatory effect of L-arginine. These results suggest that the excitatory effect of NO on DVN neurones is due to the accumulation of cGMP (Travagli and Gillis, 1994). As DVN cells are known to mediate gastrointestinal activity and be excited by NO, much research has been carried out on the effect of NO agonists and antagonists on the GI system. In vivo there is a rostrocaudal separation of function in the DVN. The excitation of rostral DVN neurones by L-glutamate increased gastric motility and lower oesophageal pressure, whereas a similar excitation of caudal DVN neurones resulted in gastric relaxation and a decrease in lower oesophageal pressure (Rossiter et al., 1990). In keeping with an excitatory effect of NO on DVN neurones, in vivo microinjections of L-arginine into the rostral DVN of cats increased gastric motility, and injection of SNAP increased antral activity (Panico et al., 1995). This effect was mediated by NOS since the NOS inhibitor N-nitro-L-arginine-methyl-ester (L-NAME) prevented L-arginine from exerting an effect (Panico et al., 1995). In contrast, microinjection of NO-liberating compounds into the region of the sgNTS decreased intragastric pressure and NOS inhibitors increased intragastric pressure (Krowicki et al., 1997). These latter authors attribute the discrepancy between the effect of NTS and DVN injections to the different locations of the injection sites. They suggested that the functionally more relevant changes are as a result of microinjections into the sgNTS since this is the area where vagal afferents terminate on the dendrites of DVN neurones. Since the NOS antagonist L-NAME inhibits NTS neuronal excitability (Ma et al., 1995), this suggests that the NTS neurones activated by NOS may either be inhibitory neurones, or may project to the rostral DVN, where stimulation evokes relaxation effects. However, since vagal afferents closely appose the soma of neurones in the DVN (Fig. 9), this raises some questions about where the functionally most relevant sites of action might be, as synapses onto somata may have a greater influence on neurones than those on distal dendrites.

3.2.4. NO in central respiratory control To date the most likely region of the medulla oblongata in which NO appears to be involved in central control of respiration is the NTS. The caudal NTS is involved in the control 205

Ch. VI


of respiration since it receives input from the carotid sinus nerve (Housley et al., 1987), which carries afferent information from peripheral chemoreceptors (Donoghue et al., 1984; Housley and Sinclair, 1988). Stimulation of the peripheral chemoreceptors by hypoxia releases glutamate from the central synaptic terminals of the carotid sinus nerve and a reflex increase in ventilation occurs to correct the hypoxia and maintain body homeostasis (Neubauer et al., 1990). Injection of a NO donor into the NTS enhances the ventilatory response to hypoxia by increasing glutamate release in the NTS, probably from the presynaptic terminals of the chemoreceptor afferents (Ogawa et al., 1995). The formation of NO in response to hypoxia is blocked by an NMDA antagonist, suggesting that NO is produced in NTS neurones. However, it cannot be excluded that NO is released from the chemoreceptor terminals themselves since vagal afferent fibres have recently been shown to contain NMDA receptor immunoreactivity (Aicher et al., 1999), although it is uncertain that NOS is in vagal afferent fibres (see earlier). A role for the nitrergic neurones of the ventral medulla in chemoreception and in the ventilatory responses to cerebral hypoxia has been proposed (Iadecola et al., 1993). The NOS-IR neurones in the RVLM are completely separate from the catecholaminergic C1 cell group (Iadecola et al., 1993; Ohta et al., 1993; Fig. 10 of Maqbool et al., 1995), being located more medially and ventrally, approaching the surface of the medulla. Thus they are ideally positioned to act as central chemoreceptors, although firm evidence in support of this hypothesis is still lacking.

3.2.5. NO in pathways coordinating autonomic and nociceptive responses It has been known for many years that there are mutual interactions between the central mechanisms controlling autonomic, particularly cardiovascular responses and those promoting nociception or anti-nociception, which may involve reciprocal neural connections between the spinal cord and the medulla (Randich and Maixner, 1986; Randich and Gebhart, 1992; Wilson and Hand, 1997). Thus, peripheral somatosensory and viscerosensory inputs can modify autonomic reflex responses, and vagally mediated afferent inputs can disengage nociceptive responses. These interactions might occur both at the level of the spinal cord and within the brain, either in the medulla or in higher centres, as the effects of stimulating several brain sites, including the RVLM and raphe nuclei, suggest that they have a role in coordinating cardiovascular and nociceptive responses (Lovick, 1991). Inhibitory responses elicited in RVLM neurones appeared to be mediated through a GABAergic input, and might involve a more indirect spinal projection via another area of the medulla such as the NTS (Sun and Spyer, 1991). Electrical stimulation of vagal afferent fibres, particularly those of the cardiopulmonary branches, inhibited nociceptive responses, and the prime relay site in the medulla oblongata concerned with this inhibition was the NTS. Application of local anaesthetics to the NTS abolished the anti-nociceptive effects of vagal stimulation, whereas glutamate injection or electrical stimulation in the NTS strongly inhibited spinal nociceptive responses (Randich and Maixner, 1986; Randich and Gebhart, 1992), although other brain stem areas, including the locus coeruleus, raphe magnus, RVLM and adjacent reticular formation were also shown to be involved. NTS neurones mediating nociceptive inhibition may do so through indirect descending connections to the spinal cord via one or all of these three noradrenergic or serotoninergic brain stem nuclei which are also known to contain opiate neurones (Basbaum and Fields, 1984). However, direct connections from the NTS to the dorsal horn have been reported (Kuypers and Maisky, 1975; Loewy and Burton, 1978; Basbaum and Fields, 1979), and the participation of such projections in vagal afferent modulation of nociception warrants further investigation. It is interesting that nitrergic neurones are present 206


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in all the areas of the medulla that have been shown to participate in these responses (Sun and Spyer, 1991), and it has been proposed that NO plays an important role in central nociceptive function (Haley et al., 1992; Schuman and Madison, 1994; Anderson, 1998). In this regard, it has recently been demonstrated that visceral hyperalgaesia is associated with an increase in the numbers of cells in the RVLM labelled tbr NOS or NADPH-d, and furthermore, that injection of L-NAME into this region attenuated the hyperalgaesic response (Coutinho et al., 1998). Although the exact nature of the pathways within the medulla involved in these responses are still poorly understood, there is now evidence that at least two areas of the CNS known to integrate and relay peripheral inputs, i.e. the paratrigeminal nucleus (Armstrong and Hopkins, 1998) and lamina I of the dorsal horn (Esteves et al., 1999), contain NOS-IR neurones projecting to NTS.

4. Abbreviations AP A1 CC Cu DVN DMSp5 ECu Gi GiV Gr IO In InM IRt k-NAME L-NMMA LPGi LRt MdD MdV mlf MVe NA NADPH-d NAc NANC nNOs NO NOS NTS

Pa5

area postrema catecholamine cell area of the caudal ventrolateral medulla central canal cuneate nucleus dorsal motor vagal nucleus spinal trigeminal nucleus, dorsomedial division external cuneate nucleus gigantocellular field medial gigantocellular field gracile nucleus inferior olive nucleus intercalatus nucleus intermedius intermediate reticular nucleus NW-nitro-L-arginine methyl ester (a NOS inhibitor) N~-monomethyl-L-arginine (a NOS inhibitor) lateral paragigantocellular field lateral reticular nucleus dorsal medullary reticular nucleus ventral medullary reticular nucleus medial longitudinal fasciculus medial vestibular nucleus nucleus ambiguus nicotinamide adenine dinucleotide phosphate (reduced) diaphorase compact region of the nucleus ambiguus nonadrenergic nonchloinergic neuronal nitric oxide synthase nitric oxide nitric oxide synthase nucleus tractus solitarii (subnuclei: ce = central; co = commissural; d = dorsal; i = interstitial; me = medial; ts = tractus solitarius; v = ventral; vl --- ventrolateral) paratrigeminal nucleus 207

Ch. VI

PCRt PMn Pr Prb PY Ro ROb RVLM SNAP Sp5C Sp5CI SpVe TS VII XII


parvocellular reticular field paramedian reticular area nucleus prepositus hypoglossi nucleus of Probst's bundle pyramidal tract nucleus of Roller raphe obscurus nucleus rostral ventrolateral medulla S-nitroso-N-acetylpenicillamine (a NO donor) spinal trigeminal nucleus, caudal division spinal trigeminal nucleus, interpolar division spinal vestibular nucleus tractus solitarius facial motor nucleus hypoglossal motor nucleus

5. ACKNOWLEDGEMENTS We are especially grateful to Dr. Piers Emson (Molecular Neuroscience Group, University of Cambridge) for providing the antiserum to recombinant rat nNOS. We wish to thank Brenda Frater and Jean Kaye for their invaluable technical assistance, and Drs. Azhar Maqbool, Filomena Esteves and Eric Corbett who contributed greatly to many of the experiments we report here. We are also grateful to the Medical Research Council for Project Grant and JREI Equipment Award funding to TFCB, and to the Wellcome Trust for a project grant to JD, as well as an Equipment Grant awarded to JD, TFCB and others.

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Paola EDD, Vidal MJ, Nistico G (1991): k-glutamate evokes the release of an endothelium derived relaxing factor like substance from the rat nucleus tractus solitarius. J Catdiovasc Pharmacol 17:$268-$272. Paxinos G, Watson C (1997): The Rat Brain in Stereotaxic Co-oldinates. San Diego, CA: Academic Press. Randich A, Gebhart GF (1992): Vagal afferent modulation of nociception. Brain Res Rev 77:99. Randich A, Maixner W (1986): The role of sinoaortic and cardiopulmonary baroreceptor reflex arcs in nociception and stress-induced analgesia. Ann NY Acad Sci 467:385-401. Rinaman L, Miselis RR (1987): Organisation of the vagal innervation of rat pancreas using cholera-toxin horseradish peroxidase conjugate. J Auton Neta' Svst 21:109-125. Rinaman L, Card PJ, Schwaber JS, Miselis RR (1989): Ultrastructural demonstration of a gastric monosynaptic vagal circuit in the nucleus of the solitary tract in rat. J Neurosci 9:1985-1996. Rodrigo J, Springall DR, Uttenthal O. Bentura ML, Abadia-Molina E Riveros-Moreno V, Martinez-Murillo R, Polak JM, Moncada S (1994): Localization of nitric oxide synthase in the adult rat brain. Philos Trans R Soc Lond (B) 345:175-221. Ross CA, Ruggiero DA, Park DH, Job TH, Sved AF, Fernandez-Pardal J, Saavedra JM, Reis DJ (1984): Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C I adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin. J Neurosci 4:474-494. Rossiter CD, Norman WE Jain M, Hornby PG, Benjamin S, Gillis RA (1990): Control of lower esophageal sphincter pressure by two sites in the dorsal motor nucleus of the vagus in the cat. Am J Phvsiol 259:G899G906. Ruggiero DA, Cravo S, Arango V, Reis DJ (1989): Central control of the circulation by the rostral ventrolateral reticular nucleus: anatomical substrates. Prog Brain Res 81:47-79. Ruggiero DA, Mtui EE Anwar M, Otake K, Reis DJ t 1993): Structural basis for a role of nitric oxide in cholinergic autonomic control. Soc Neurosci Abstr 19:184.3. Ruggerio DA, Mtui EE Otake K, Anwar M (1996): Central and primary visceral afferents to nucleus tractus solitarii may generate nitric oxide as a membrane-permeant neuronal messenger. J Comp Neurol 361:51-67. Scherer-Singler U, Vincent SR, Kimura H, McGeer EG 11983): Demonstration of a unique population of neurons with NADPH-diaphorase histochemistry. J Neurosci Methods 9:229-234. Schmidt HHW, Gagne CD, Nakane M. Pollock JS, Miller ME Murad F (1992): Mapping of neuronal nitric oxide synthase in the rat suggests frequent colocalization with NADPH-diaphorase but not soluble guanylyl cyclase and novel paraneural functions for nitrergic signal transduction. J Histol Cvtochem 40:1439-1456. Schuman EM, Madison DV (1994): Nitric oxide and synaptic function. Am~u Rex' Neutvsci 17:153-183. Shapiro RE, Miselis RR (1985): The central organization of the vagus nerve innervating the stomach of the rat. J Comp Neurol 238:437-488. Shapoval LN, Sagach VF, Pobegaiio LS (1991): Nitric oxide influences ventrolateral medullary mechanisms of vasomotor control in the cat. Neurosci Lett 132:47-50. Simonian SX, Herbison AE (1996): Localization of neuronal nitric oxide synthase-immunoreactivity within sub-populations of noradrenergic A I and A2 neurons in the rat. Brain Res 732:247-252. Spike RC, Todd AJ, Johnston M (1993 I: Coexistence of NADPH diaphorase with GABA, glycine and acetylcholine in rat spinal cord. J Comp Neurol 335:320-333. Stewart DJ, Munzel T, Bassenge E (1987): Reversal of acetylcholine induced coronary resistance vessel dilation by hemoglobin. Eur J Pharmacol 136:239-242. Sun MK, Spyer KM (1991): Nociceptive inputs into rostral ventrolateral medulla-spinal vasomotor neurones in rats. J Phvsio1436:685-700. Sun MK, Hackett JT, Guyenet PG (1988a): Sympathoexcitatory neurons of rostral ventrolateral medulla exhibit pacemaker properties in the presence of a glutamate receptor antagonist. Brain Res 438:23-40. Sun MK, Young BS, Hackett JT, Guyenet PG (1988b): Rostral ventrolateral medullary neurons with intrinsic pacemaker properties are not catecholaminergic. Brain Res 451:345-349. Tagawa T, Imaizumi T, Harada S, Endo T, Shiramoto M. Hirooka Y, Takeshita A (1994): Nitric oxide influences neuronal activity in the nucleus tractus solitarius of rat brainstem slices. Circ Res 75:70-76. Thomas E, Pearse AGE (1961): The fine localization of dehydrogenases in the nervous system. Histochimie 2:266-282. Travagli RA, Gillis RA (1994): Nitric oxide mediated excitatory effect on neurons of dorsal motor nucleus of the vagus. Am J Phvsiol 266:G 154-G 160. Tseng CJ, Liu HY, Lin HC, Ger LE Tung CS, Yen MH (1996): Cardiovascular effects of nitric oxide in the brain stem nuclei of rats. Hypertension 27:36-42. 212

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Vincent SR, Kimura H (1992): Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46:755-784. Vincent SR, Johansson O, H6kfelt T, Skirboll L, Elde RE Terenius L, Kimmel J, Goldstein M (1983a): NADPH-diaphorase: a selective histochemical marker for striatal neurones containing both somatostatin- and avian pancreatic polypeptide (AAP)-like immunoreactivities. J Comp Neuro! 217:252-263. Vincent SR, Satoh K, Armstrong DM, Fibiger HC (1983b): NADPH-diaphorase: a selective histochemical marker for the cholinergic neurons of the pontine reticular formation. Neurosci Lett 43:31-36. Wallach JH, Loewy AD (1980): Projections of the aortic nerve to the nucleus tractus solitarius in the rabbit. Brain Res I88:247-251. Wiedner EB, Bao X, Altschuler SM (1995): Localization of nitric oxide synthase in the brain stem neural circuit controlling esophageal peristalsis in rats. Gastroenterology 108:367-375. Wilson LB, Hand GA (1997): The pressor reflex evoked by static contraction: neurochemistry at the site of the first synapse. Brain Res Rev 23:196-209. Xu ZQ, H6kfelt T (1997): Expression of galanin and nitric oxide synthase in subpopulations of serotonin neurons of the rat dorsal raphe nucleus. J Chem Neuroanat 13:169-187. Yamada K, Emson P, H0kfelt T (1996): Immunohistochemical mapping of nitric oxide synthase in the rat hypothalamus and colocalization with neuropeptides. J Chem Neuroanat 10:295-316. Yan XX, Garey LJ (1997): Morphological diversity of nitric oxide synthesising neurons in mammalian cerebral cortex. Hirnforschung 38:165-172. Youfsi-Malki M, Puizillout JJ (1995): Study of brainstem projections of aortic baroreceptor afferents in the rabbit using transganglionic anterograde transport of choleragenoid-HRP. Prim Sens Neuron 1:143-155. Yu WH (1994): Nitric oxide synthase in motor neurons after axotomy. J Histochem Cvtochem 42:451-457. Zanzinger J, Czachurski J, Seller H (1995): Inhibition of basal and reflex-mediated sympathetic activity in the RVLM by nitric oxide. Am.J Phvsiol 268:R958-R962. Zhang X, Fogel R, Simpson P, Renehan WE (1991): The target specificity of the extrinsic innervation of the rat small intestine. J Auton Nerv Svst 32:53-62. Zheng ZL, Rogers RC, Travagli RA (1999): Selective gastric projections of nitric oxide synthase-containing vagal brainstem neurons. Neuroscience 90:685-694.

213


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Nitric oxide in the peripheral autonomic nervous system H.M. YOUNG, C.R. ANDERSON AND J.B. FURNESS

1. INTRODUCTION 1.1. BRIEF HISTORY OF THE IDENTIFICATION OF NO AS A PERIPHERAL NEUROT RA N S M ITTER In the mid-1960s, following the availability of specific drugs to block adrenergic receptors or to block noradrenaline release, it was realised that some responses in peripheral tissues were not mediated by either of the then-known neurotransmitters of peripheral neurones, noradrenaline or acetylcholine (Burnstock et al., 1966). Shortly afterwards, Burnstock et al. (1970) proposed that ATP is an inhibitory neurotransmitter to gastrointestinal smooth muscle. Subsequently, the discovery of peptides with potent pharmacological activities in many peripheral nerves led to the demonstration that substances such as vasoactive intestinal peptide (VIP), neuropeptide Y and substance P can also act as neurotransmitters (see H6kfelt et al., 1980; Lundberg and H6kfelt, 1986: Fumess et al., 1989). However, there were a number of locations where inhibitory neurotransmission to vascular or non-vascular smooth muscle did not appear to be mediated by noradrenaline, acetylcholine, ATP or any known peptide. Furchgott and Zawadzki (1980) reported that the relaxant action of acetylcholine on in vitro preparations of rabbit aorta was mediated by a substance released from endothelial cells, which they termed EDRF (endothelium-derived relaxing factor). In 1987, using chemical assay and bioassay, it was shown by two different groups that EDRF is nitric oxide (NO) (Ignarro et al., 1987; Palmer et al., 1987). The similarities between EDRF and the unidentified inhibitory neurotransmitter in the rat anococcygeus muscle and the bovine retractor penis muscle was pointed out by Gillespie (1987) and Furchgott (1988). Using drugs that selectively block nitric oxide synthase (NOS), the enzyme responsible for the synthesis of NO, it was shown by a number of groups that NO is a major neurotransmitter mediating inhibitory transmission in the anococcygeus muscle of the rat (Gillespie et al., 1989; Li and Rand, 1989; Ramagopal and Leighton, 1989) and in the bovine retractor penis muscle (Liu et al., 1991). In fact, these experiments performed on peripheral neurones innervating the anococcygeus muscle were the first demonstration that NO acts as a neurotransmitter, in addition to its role as an endothelium-derived relaxing factor. Shortly after it was shown that NO acts as a neurotransmitter in the anococcygeus muscle, Bredt and Snyder (1990) demonstrated, using an antibody raised to NOS, that many neurones in the central and peripheral nervous systems are capable of producing NO.

Handbook of Chemical Neuroanatomv, Vol. 17." Functional Neuroanatomv of the Nitric Oxide System H.W.M. Steinbusch, J. De Vente and S.R. Vincent. editors (~) 2000 Elsevier Science B.V. All rights reserved.

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1.2. GENERAL PROPERTIES OF NO-MEDIATED NEUROTRANSMISSION The general properties of NO as a neurotransmitter, that have been reviewed in detail elsewhere (Bredt and Snyder, 1992, 1994; Rand, 1992; Sanders and Ward, 1992; Snyder, 1992; Stark and Szurszewski, 1992; Rand and Li, 1995), are summarised below. NO (and L-citrulline) are synthesised from L-arginine by NOS. There are three main isoforms of NOS neuronal, endothelial and macrophage NOS - - which are encoded by different genes and have different molecular weights. Both endothelial and neuronal NOS are dependent on Ca 2+, calmodulin and NADPH. Neuronal NOS is a 160 kDa cytoplasmic protein that is found as a homodimer (see Marietta, 1993). There is no evidence (or expectation) that stores of pre-formed NO exist in neurones, and it appears that NO is formed, by NOS, on demand and immediately released. The synthesis of NO can be inhibited by a number of arginine analogues, which are taken up by cells and competitively inhibit the synthesis of NO (see Moncada et al., 1991); these NOS inhibitors have been critical for determining whether NO plays a physiological role in different tissues. Since the formation of NO is Ca2+-dependent, it is likely that the increase in intracellular free Ca 2+ that occurs following the arrival of an action potential in a nerve terminal results in the activation of NOS and the consequent release of NO. Although other possible molecular targets are known, the major receptor for NO is soluble guanylyl cyclase, which unlike receptors for other neurotransmitters, is present within the cytoplasm, not at the cell membrane of the effector cell. Activation of soluble guanylyl cyclase results in an increase in the production of cGME which in turn activates cGMP-dependent kinases; increases in cGMP might also have other effects such as direct actions on ion channels. Cells containing NOS can be localised using immunohistochemistry, or using a histochemical reaction called the NADPH diaphorase reaction (Hope et al., 1991; Dawson et al., 1991; see Vincent, Chapter II of this volume). In the peripheral nervous system, there have been relatively few studies that have examined the equivalence of NOS immunoreactivity and NADPH diaphorase staining. However, those studies that have been performed have all shown that NOS immunoreactivity and NADPH diaphorase staining show identical locations in peripheral neurones. Consequently, throughout this review we have often not specified whether the presence of NOS was demonstrated immunohistochemically or histochemically. The only ambiguity that can arise is in the staining of nerve cell bodies. All cells contain NADPH-dependent enzymes, other than NOS, and thus if the NADPH diaphorase reaction is left to run for a long time, all cells will show some staining. It is therefore possible that there could be some false-positive reports of NOS in nerve cell bodies, based only on the presence of NADPH diaphorase staining. 1.3. SCOPE OF THIS REVIEW This review is not an exhaustive survey of every report of NOS in different regions of the peripheral autonomic nervous system of different mammalian species. We have concentrated on locations where anatomical studies showing the presence of NOS neurones have been backed up by physiological and pharmacological studies that have examined the function of neurally released NO. Moreover, the presence and role of NO neurones innervating the pelvic organs is only covered briefly (in Section 3.1.1 (c)), but is dealt with in detail in Chapter VIII of this volume.

216

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2. NO IN A U T O N O M I C G A N G L I A 2.1. NITRIC OXIDE AND SYMPATHETIC PRE- AND P O S T G A N G L I O N I C NEURONES

2.1.1. Presence of NOS in sympathetic pre- and postganglionic neurones The cell bodies of sympathetic preganglionic neurones are found in the spinal cord, usually between the first thoracic segment (T1) and, depending on the species, the upper to mid-lumbar segments. The axons of preganglionic neurones project in the ventral roots to innervate the cell bodies of postganglionic neurones in the paravertebral and prevertebral ganglia, and chromaffin cells in the adrenal medulla. Most sympathetic preganglionic neurones are located in nuclei on the lateral margins of the intermediate grey matter of the spinal cord, called the intermediolateral nuclei (Petras and Cummings, 1972). Synaptic transmission from sympathetic preganglionic neurones to postganglionic neurones is mediated primarily by acetylcholine, and although sympathetic preganglionic neurones contain many other putative neurotransmitters (Morris and Gibbins, 1992), non-cholinergic components of transmission are not easily demonstrated. NOS is present in many sympathetic preganglionic neurones (Fig. 1). Using NADPH diaphorase staining, NOS was first identified in rat sympathetic preganglionic neurones in 1992 (Anderson, 1992; Blottner and Baumgarten, 1992; Dun et al., 1992; Valtschanoff et al., 1992). An earlier study by Thomas and Pearse (1961) had reported intense NADPH diaphorase activity in the intermediolateral column of the spinal cord, but since the enzyme underlying the reaction was unidentified at the time, the significance of the staining was not recognised. Later, NADPH diaphorase-stained axons were found in the ventral roots of rats, but the axons were not definitely identified as arising from preganglionic neurones (Aimi et al., 1991). Since 1992, many studies have confirmed the presence of NOS in sympathetic preganglionic neurones of a wide range of species using immunohistochemistry and/or NADPH diaphorase staining (see Table 1), and the two techniques appear to show the same population of fibres and cell bodies (Anderson et al., 1993; Furness and Anderson, 1994). NOS is present in many, but not all, sympathetic preganglionic neurones in the species that have been examined in detail. Most is known about the distribution of NOS in rat sympathetic preganglionic neurones. In the upper thoracic segments in the rat, NOS is present in around

TABLE 1. Species in which NOS-containing syml~athetic preganglionic neurones have been demonstrated Species

References

Rat

Anderson (1992); Blottner and Baumgarten (1992): Dun et al. (1992. 1993a,b). Valtschanoff et al. (1992); Morris et al. (1993): Ceccatelli et al. (1994): Saito et al. (1994); Domoto et al. (1995); Okamura et al. (1995): Ando et al. (1996): Soinila et al. (1996); Chiba and Tanaka (1998); Tang et al. (1998) Brtining (1992); Dun et al. (1993b) Anderson et al. (1994); Furness et al. (1994) Hakim et al. (1995) Dun et al. (1993b) Hisa et al. (1995); Vizzard et al. (1997) Xu et al. (1996) Marley et al. (1995) Dun et al. (1993b) Smithson and Benarroch (1996)

Mouse Guinea pig Rabbit Cat Dog Sheep Cow Monkey Human

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Fig. 1. (A) Horizontal (longitudinal) section through the T8 thoracic segment of a rat stained for the NADPH diaphorase reaction. A compact mass of NADPH diaphorase-stained sympathetic preganglionic neurones is present in the intermediolateral nucleus (IML) on the margin between the grey matter (G) and white matter (W). The neurones extend dendrites along the IML as well as across the grey matter towards the intermediate grey above the central canal. The positively stained neurones in the midline are not sympathetic preganglionic neurones. Scale bar -- 100 Ixm. (B) Sympathetic preganglionic neurones in the intermediolateral nucleus of the TI2 spinal cord segment. The reaction product is homogeneous, not granular and is found throughout the neurone, including into the finest dendrites, but is not present within the nucleus. Scale bar = 25 l.tm. (C) Fluorescence micrograph of rat sympathetic preganglionic neurones in the intermediolateral nucleus tlML)of the TI3 spinal cord segment stained with an antiserum to NOS. The appearance is identical to that seen with the NADPH diaphorase reaction. W = white matter; G = grey matter. Scale bar = 100 It m.

85% o f all p r e g a n g l i o n i c n e u r o n e s . In m i d d l e to low thoracic s e g m e n t s , N O S is f o u n d in 6 5 - 7 5 % o f all p r e g a n g l i o n i c n e u r o n e s , and in a slightly h i g h e r p r o p o r t i o n ( 7 0 - 8 5 % ) in low thoracic and u p p e r l u m b a r levels ( A n d e r s o n ,

1992; G r k o v i c and A n d e r s o n ,

1997). T h e r e

are d i f f e r e n c e s in the p r o p o r t i o n s o f p r e g a n g l i o n i c n e u r o n e s c o n t a i n i n g N O S m e d i o l a t e r a l l y across the i n t e r m e d i o l a t e r a l c o l u m n . N O S - c o n t a i n i n g n e u r o n e s are m o r e c o m m o n laterally in the i n t e r m e d i o l a t e r a l c o l u m n than medially, in both the rat ( A n d e r s o n ,

1992) and the

g u i n e a pig ( F u r n e s s and A n d e r s o n , 1994). S y m p a t h e t i c p r e g a n g l i o n i c n e u r o n e s in the spinal 218

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autonomic nuclei innervate postganglionic neurones in sympathetic ganglia throughout the body. Successively more caudal sympathetic ganglia receive preganglionic inputs from successively more caudal spinal segments (see Strack et al., 1988). Experiments in which retrograde tracers were injected into different ganglia have revealed that there are small differences in the proportions of all preganglionic neurones that contain NOS that project to different paravertebral sympathetic ganglia; these differences largely parallel the differences in the occurrence of NOS-containing preganglionic neurones at different levels of the spinal cord (the proportion of total preganglionic neurones that project to the superior cervical, stellate and L5 lumbar sympathetic chain ganglia that contain NOS are 84%, 77% and 89%, respectively; all figures calculated from data in Grkovic and Anderson, 1996). In contrast, different prevertebral ganglia of the rat differ markedly in the proportion of their preganglionic neurones that contain NOS (superior mesenteric ganglion 43%, inferior mesenteric ganglion 85%; Grkovic and Anderson, 1996). There are similar differences between prevertebral ganglia in guinea pigs, with a larger percentage of preganglionic neurones projecting to the inferior mesenteric ganglion containing NOS (69%, Anderson et al., 1994) than to the coeliac ganglion (42%, Furness and Anderson, 1994). Sympathetic postganglionic neurones innervate a range of target tissues. Most of them use noradrenaline, ATP and a variety of peptides, particularly neuropeptide Y, as neurotransmitters. The overwhelming majority of studies have found very few NOS-containing neurones in the sympathetic paravertebral ganglia of the mouse and rat (Fig. 2A; Grozdanovic et al., 1992; Anderson et al., 1993; Dun et al., 1993a; Santer and Symons, 1993; Ceccatelli et al., 1994; Klimaschewski et al., 1994; Vanhatalo and Soinila, 1994: Morales et al., 1995; Handa et al., 1996; Lumme et al., 1996; Soinila et al., 1996), and they are only slightly more common in the guinea pig (Kummer et al., 1992; Fischer et al., 1993, 1996a). However, NOS neurones are common in sympathetic paravertebral ganglia of larger species including the cat (Fig. 2C; Anderson et al., 1995), dog (Hisa et al., 1995, 1997), pig (Modin et al., 1994), humans (Klimaschewski et al., 1996) and cattle (Sheng et al., 1993; Majewski et al., 1995). Little is known about the targets of the sympathetic NOS neurones in these larger animals except in the cat, where their targets include hindlimb vasculature, and sweat glands and blood vessels in paw pads (Fig. 2D; Anderson et al., 1995). In prevertebral ganglia, NOS-containing neurones are also present in low numbers in the coeliaco-mesenteric complex and in the inferior mesenteric ganglion of rats (Santer and Symons, 1993), guinea pigs (Furness and Anderson, 1994) and pigs (Kaleczyc et al., 1994). Sympathetic postganglionic neurones that contain NOS are all reported to lack noradrenergic markers (e.g. Kummer, 1992; Kummer et al., 1992; Modin et al., 1994; Majewski et al., 1995), with the exception of the bovine superior cervical ganglion, where a proportion of the NOS-containing neurones also contain dopamine [3-hydroxylase (Sheng et al., 1993).

2.1.2. Functionally identified subclasses of sympathetic preganglionic NOS neurones The targets of sympathetic preganglionic neurones include postganglionic neurones of different function and the chromaffin cells of the adrenal medulla. The presence of NOS in preganglionic neurones is likely to correlate with functionally distinct pathways. For instance, when a retrograde tracer is injected into the adrenal medulla of the rat, all labelled preganglionic neurones are NOS-immunoreactive (Grkovic and Anderson, 1997), even though at middle to low thoracic levels of the spinal cord, only around 70% of the preganglionic neutones contain NOS (see above). Determining the presence or absence of NOS in preganglionic neurones projecting to functionally identified subpopulations of postganglionic neurones relies 219

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Fig. 2. (A) NOS-immunoreactive nerve terminals outline the nerve cell bodies (asterisks) of postganglionic neurones in the rat superior cervical ganglion. The NOS nerve terminals arise from sympathetic preganglionic neurones. Note that there is no sign of immunoreactivity in the postganglionic cell bodies. Scale bar = 50 lxm. (B) Horizontal section through the sacral parasympathetic nucleus (IML) in the S1 spinal cord segment of a rat, stained for the NADPH diaphorase reaction. Stained parasympathetic preganglionic neurones are present oriented transversely across the lateral portion of the grey matter (G), close to the white matter (W). Scale bar -- 100 Ixm. (C) Section through the stellate ganglion of the cat stained for the NADPH diaphorase reaction. NADPH diaphorase-stained postganglionic neurones are present and they represent sympathetic cholinergic neurones that project to either blood vessels or to sweat glands (see Anderson et al.. 1995). Scale bar = 50 ~m. (D) Micrograph of a blood vessel from the paw of a cat, stained for the NADPH diaphorase reaction. The stained nerve terminals are likely to be collaterals from sympathetic cholinergic neurones that also innervate sweat glands in the paw pad. Scale bar = 50 lxm.

on the identification o f s u b p o p u l a t i o n s of p o s t g a n g l i o n i c n e u r o n e s b a s e d on the p r e s e n c e o f u n i q u e c o m b i n a t i o n s o f i m m u n o h i s t o c h e m i c a l l y d e t e c t a b l e s u b s t a n c e s a n d / o r the injection o f r e t r o g r a d e t r a c e r into p a r t i c u l a r p e r i p h e r a l targets. T h e f u n c t i o n a l l y identified p o s t g a n g l i o n i c n e u r o n e s are then e x a m i n e d to d e t e r m i n e if they are s u r r o u n d e d by N O S - i m m u n o r e a c t i v e n e r v e t e r m i n a l s . In the rat, N O S is p r e s e n t in p r e g a n g l i o n i c n e u r o n e s i n n e r v a t i n g p o s t g a n glionic n e u r o n e s p r o j e c t i n g to s w e a t glands, s u b m a n d i b u l a r salivary gland, heart, skeletal m u s c l e , b l o o d v e s s e l s and b r o w n fat ( G r k o v i c and A n d e r s o n , 1995, 1997; H a n d a et al., 1996; 220

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Anderson, 1998; Chiba and Tanaka, 1998). NOS is absent from the preganglionic neurones innervating postganglionic neurones projecting to the iris (CRA, unpubl.). Other functional groups of preganglionic neurones almost certainly contain NOS.

2.1.3. Presence of NOS in parasympathetic pre- and postganglionic neurones NOS is also present in many parasympathetic preganglionic (Fig. 2B) and postganglionic (Figs. 3B,C, 5A and 7A) neurones. Parasympathetic preganglionic neurones are located in the brain and lumbosacral spinal cord. Cranial parasympathetic preganglionic neurones are found in distinct nuclei throughout the brain stem and their axons run in the cranial nerves to innervate postganglionic neurones in the ciliary, sphenopalatine, submandibular and otic ganglia, as well as many neurones within organs in the head and within the thoracic and abdominal cavities. Lumbosacral parasympathetic preganglionic neurones are present in the sacral intermediolateral nuclei, two parallel longitudinal nuclei in the lateral aspects of the intermediate grey matter analogous to the intermediolateral nuclei of the thoracic spinal cord (Petras and Cummings, 1972), and they project via ventral roots to innervate postganglionic neurones in ganglia associated with the pelvic viscera. Transmission from all parasympathetic preganglionic neurones to postganglionic neurones is cholinergic, and NO is not a primary neurotransmitter. The presence of NOS in pelvic parasympathetic postganglionic neurones is discussed in Chapter VIII of this volume. Cranial preganglionic NOS neurones. The presence of NOS-containing nerve terminals in the major parasympathetic ganglia has been reasonably well studied, but the presence of NOS terminals in ganglia within structures in the head, thorax and abdomen has not been systematically examined. If NOS expression is confined to specific pathways, as it is in the sympathetic nervous system, then NOS-containing preganglionic nerve terminals may not necessarily be present in all parasympathetic ganglia. As most parasympathetic ganglia contain nerve cell bodies and dendrites strongly immunoreactive for NOS or reactive for NADPH diaphorase (see Section 3.1.1 (a,c), Sections 5.2 and 6.1), NOS-containing preganglionic inputs to these cells may be obscured. Notwithstanding, in the rat and mouse, the terminals of parasympathetic preganglionic neurones projecting to cranial ganglia rarely seem to contain NOS. Postganglionic neurones in the sphenopalatine ganglion of the rat are not innervated by preganglionic neurones that contain NOS (Ceccatelli et al., 1994). Similarly, the nerve cell bodies in the submandibular ganglia of mice and rats are not innervated by NOS-containing preganglionic neurones (Grozdanovic et al., 1992). In contrast, following injection of a retrograde tracer into the rabbit sphenopalatine ganglion, 75% of the labelled preganglionic neurones in the brain stem are immunoreactive for NOS (Zhu et al., 1997). A similar study in which retrograde tracer was injected into the submandibular ganglion of the rabbit reported that 100% of the retrogradely labelled preganglionic neurones as NOS-immunoreactive (Zhu et al., 1996). A subpopulation of axons in the vagus nerve contain NOS; some of these are the peripheral extensions of vagal sensory neurones (Fischer et al., 1993, 1996a), and some are efferent fibres that originate in the dorsal motor nucleus and project to the gastrointestinal tract and the heart. Efferent vagal NOS fibres are also very likely to project to other organs, but this is yet to be demonstrated. Following intraperitoneal injections of fluorogold, which will label all preganglionic neurones (Anderson and Edwards, 1994), only about 10% of the labelled nerve cell bodies in the dorsal motor nucleus of the vagus of the rat are stained for NADPH diaphorase (Krowicki et al., 1997). In the guinea pig, at least some of the vagal preganglionic neurones containing NOS innervate intrinsic cardiac ganglia, because there are 221

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guinea-pig, pial Vessel

Fig. 3. (A) Whole-mount preparation of a pial vessel from a guinea pig stained for the NADPH diaphorase reaction. Stained nerve terminals are abundant in the perivascular plexus of this vessel. The background staining is due to the presence NOS in endothelial cells of this vessel. Scale bar = 25 [tm. (B) Section through the sphenopalatine (pterygopalatine) ganglion of a rat after processing for neuronal NOS histochemistry. There are many NOS-immunoreactive nerve cell bodies in the ganglion and some sparse NOS-containing nerve fibres. Scale bar = 50 gm. (C, D) Paired micrographs of a section through the sphenopalatine ganglion of a rat that had been processed for both NOS (C) and choline acetyltransferase (D, CHAT) immunohistochemistry. Most of the NOS-immunoreactive nerve cell bodies (arrows indicate two examples) are also ChAT-positive. There are a small number of cells that are ChAT-immunoreactive (asterisk). but not NOS-positive (the location of the cell in C appears as a black hole). Note the high density of ChAT-positive nerve terminals, which presumably arise from parasympathetic preganglionic neurones, surrounding the nerve cell bodies. Scale bar = 25 Itm.

NOS nerve fibres in the heart that degenerate following vagotomies (Tanaka and Chiba, 1998; see Section 3.2). In the mouse, some of the vagal NOS-containing nerve fibres originating in the dorsal motor nucleus project to the myenteric plexus of the oesophagus, but none of the neurones in the nucleus ambiguus, which innervate the striated muscle of the oesophagus, contain NOS (Sang and Young, 1998b; Sang et al., 1999). However, the NOS neurones comprise only about one third of the neurones projecting from the dorsal motor nucleus to the mouse oesophagus (Sang and Young, 1998b). 222

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Sacral preganglionic NOS neurones. NOS-containing neurones in the sacral spinal cord have been identified as preganglionic neurones, based on their location in the intermediolateral nucleus or by the use of retrograde tracer, in the mouse (Brtining, 1992; Briining and Mayer, 1996), rat (Anderson, 1992; Valtschanoff et al., 1992; Vizzard et al., 1993; Saito et al., 1994; Burnett et al., 1995), cat (Dun et al., 1993b) and dog (Vizzard et al., 1997). The sacral parasympathetic preganglionic outflow innervates postganglionic neurones that control a range of pelvic organs. There is little systematic knowledge of the functional pelvic pathways that may include NOS-containing parasympathetic preganglionic neurones. Virus-tracing experiments have shown that at least some preganglionic neurones projecting to the parasympathetic ganglia of the bladder in the rat contain NOS (Papka et al., 1995). Parasympathetic postganglionic NOS neurones. Many cranial and sacral parasympathetic postganglionic neurones contain NOS. The presence of NOS in cranial postganglionic neurones is discussed in Section 3.1.1 (a) and Section 6.1, and the presence of NOS in pelvic ganglia is discussed briefly in Section 3.1.1 (c) and in Chapter VIII of this volume.

2.2. NITRIC OXIDE AND GANGLIONIC TRANSMISSION Given the widespread occurrence of NOS in sympathetic preganglionic neurones, it is reasonable to expect that NO might influence transmission from the preganglionic terminals to postganglionic nerve cells. The primary transmitter at these synapses is acetylcholine which acts through nicotinic receptors to generate fast excitatory postsynaptic potentials (EPSPs). In the isolated rat superior cervical ganglion, NO donors increase the size of postganglionic compound action potentials when the preganglionic nerve supplying the ganglion is electrically stimulated (Briggs, 1992). The effect is likely to be mediated by increases in cGMP, as application of 8-bromo cGMP, the membrane permeant analogue of cGMP, has identical effects to nerve stimulation (Briggs, 1992). Ganglionic cGMP levels are elevated following either electrical stimulation of the preganglionic nerves or exposure of the ganglion to high potassium concentrations (Quenzer et al., 1980; Volle et al., 1981, 1982; Ando et al., 1983; Volle and Quenzer, 1983; De Vente et al., 1987a,b; Sheng et al., 1992) and this effect can be blocked by NOS inhibitors (Sheng et al., 1992), or by prior preganglionic denervation (Volle et al., 1982). Other studies, using patch-clamping techniques, have demonstrated that NO donors enhanced Ca 2+ currents in postganglionic neurones in the rat superior cervical ganglion (Chen and Schofield, 1993, 1995). The effect is again mediated by cGMP, as it is mimicked by direct application of cGMP analogues and inhibited by methylene blue. Thus, NO is concluded to augment cholinergic neurotransmission in the superior cervical ganglion. In contrast to paravertebral ganglia, the effect of NO release in prevertebral ganglia is to diminish the effectiveness of transmission. In the mouse superior mesenteric ganglion, NO donors directly hyperpolarised the membrane of two thirds of the neurones impaled (Mazet et al., 1996). In this study, stimulation of the colonic nerves in the presence of a NOS inhibitor led to an increase in the size of the slow EPSP, suggesting that under normal circumstances, NO released by nerve stimulation inhibited the slow EPSP. The fact that this effect was present when the colonic nerves were stimulated suggests that the origin of the NOS nerve fibres involved was in the gut (intestinofugal neurones, see Section 4.8), or perhaps collaterals of dorsal root ganglion neurones (Domoto et al., 1995), rather than from preganglionic neurones. In the rabbit coeliac ganglion, prolonged stimulation of the splanchnic nerves led to a decreased probability of a presynaptic impulse firing an action potential in the coeliac ganglion neurones (Quinson et al., 1998). The progressive decrease in the probability of firing an action potential was due to the release of NO, as it was prevented by NOS inhibitors 223

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and NOS scavengers. The NO was presumably acting via increases in cGMP levels to diminish nicotinic transmission, as the probability of firing an action potential was increased by inhibitors of cGMP production and decreased by inhibitors of phosphodiesterases.

3. ROLE OF NO IN THE NEURAL CONTROL OF THE VASCULATURE AND THE HEART

3.1. ROLE OF NEURALLY DERIVED NO IN THE CONTROL OF THE VASCULATURE NO is a potent vasodilator. NOS is present in vascular endothelial cells, and NO is produced by these cells under basal conditions and in response to a variety of stimuli, including acetylcholine released from autonomic nerve terminals (Furchgott, 1996). NO derived from endothelial cells is thus an essential component of many of the signalling pathways mediating relaxation of blood vessels. However, some blood vessels are also innervated by nerve fibres containing NOS, and neurally released NO can also mediate vasodilation. It can be difficult to determine the relative physiological importance of the two sources of NO, and prior to the recent development of drugs that selectively block endothelial NOS or neuronal NOS, the only way that the contribution of neurally derived NO could be distinguished from that of endothelial-derived NO was by pharmacological examination of the relaxations with and without the endothelium. In this section, we will only consider the role of neurally derived NO in the regulation of vascular tone. Although neural control of blood flow through most blood vessels is mediated primarily by sympathetic vasoconstrictor neurones, blood vessels in some tissues can be innervated by three types of neurones: sympathetic vasoconstrictor neurones, parasympathetic, enteric or sympathetic vasodilator neurones, and the peripheral processes of some dorsal root ganglion neurones which mediate vasodilation (see Morris et al., 1995). All vasoconstrictor neurones are thought to contain noradrenaline, and to use various combinations of noradrenaline, ATP and neuropeptide Y as neurotransmitters. Since NO is a potent vasodilator, not surprisingly, NOS does not appear to be present in any vasoconstrictor neurones (Kummer et al., 1992; Hohler et al., 1995). 3.1.1. Autonomic vasodilator neurones

Populations of non-noradrenergic vasodilator neurones are present in cranial parasympathetic ganglia, some sympathetic ganglia, pelvic ganglia and enteric ganglia. (a) Vasodilator neurones in cranial parasympathetic ganglia. Vasodilator neurones play a prominent role in the regulation of blood flow through many blood vessels in the head (see Lee et al., 1975; Faraci and Brian, 1994; Toda and Okamura, 1996). Thus, in addition to sympathetic nerve fibres, the arteries supplying the brain are innervated by parasympathetic vasodilator nerve fibres arising from cranial parasympathetic ganglia, primarily the sphenopalatine (pterygopalatine) ganglion (Chorobski and Penfield, 1932; Hara and Weir, 1986; Goadsby, 1990). In addition, blood vessels supplying other structures in the head such as the salivary glands, the tongue, and the nasal mucosa are innervated by parasympathetic neurones that are located either in intrinsic ganglia or in major cranial parasympathetic ganglia. Recent studies indicate that NOS is present in the terminals of vasodilator neurones in the perivascular plexuses of arteries supplying the brain and other structures in the head, including the eye, salivary glands, tongue and nasal mucosa (Fig. 3A; 224


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Bredt et al., 1990; Bredt and Snyder, 1992; Ceccatelli et al., 1992; Grozdanovic et al., 1992; Iadecola et al., 1993; Suzuki et al., 1993; Yamamoto et al., 1993; Minami et al., 1994; Saito and Goto, 1994; Yoshida et al., 1994; Lohinai et al., 1995; Toda et al., 1995: Goadsby et al., 1996; Toda and Okamura, 1996; Young et al., 1997; Zhu et al., 1997). The role of NO in axons innervating blood vessels supplying the salivary glands and nasal mucosa is discussed in Sections 6.1 and 6.3. Using NOS immunohistochemistry and/or NADPH diaphorase histochemistry, NOS-containing nerve cell bodies have been found to comprise up to 90% of neurones in cranial parasympathetic ganglia, including the sphenopalatine and ciliary ganglia, and intrinsic ganglia within the salivary glands and the tongue of a variety of species (Fig. 3B; Grozdanovic et al., 1992; Yamamoto et al., 1993; Yoshida et al., 1993; Ceccatelli et al., 1994; Minami et al., 1994; Sun et al., 1994; Lee et al., 1995; Goadsby et al., 1996; Hu et al., 1996; Hanazawa et al., 1997; Jeon et al., 1997; Zhu et al.. 1997; Lacroix et al., 1998). Studies in rats and dogs that have examined the origin of the NOS-containing nerve fibres innervating cerebral arteries have shown that many of them arise from the sphenopalatine ganglion. In the rat, sectioning of fibres leaving both sphenopalatine ganglia (Nozaki et al., 1993) or unilateral removal of a sphenopalatine ganglion (Minami et al., 1994) result in a marked decrease in the density of NOS-immunoreactive nerve fibres associated with vessels forming the rostral circle of Willis. Moreover, most of the labelled neurones in the sphenopalatine ganglion labelled by application of retrograde tracer to the middle cerebral artery are NADPH diaphorase-positive (Minami et al., 1994). In the dog, damaging the sphenopalatine ganglion, or sectioning of the nerve fibres leaving it, results in a dramatic reduction or total loss of NOS-immunoreactive nerve fibres in the ipsilateral middle and posterior cerebral arteries, demonstrating that the sphenopalatine ganglion is the major source of NOS-containing nerve fibres innervating these vessels (Nozaki et al., 1993; Yoshida et al., 1993). Cholinergic nerve terminals, revealed by choline acetyltransferase (CHAT) immunohistochemistry, form a perivascular plexus around the cerebral and basilar arteries of a range of animals (Saito et al., 1985). These cholinergic nerve terminals also contain VIP (Hara et al., 1985). Many of the NOS-containing neurones innervating cerebral blood vessels also contain VIP (Ceccatelli et al., 1992; Minami et al., 1994; Goadsby et al., 1996), and thus the cerebral vasodilator neurones are cholinergic/NOS/VIP neurones (Fig. 3C,D). Although acetylcholine appears to be the main mediator of the nerve-mediated vasodilation of cerebral vessels (Forbes and Cobb, 1938), there is also an endothelium-independent dilatory response to nerve stimulation that is not mediated by acetylcholine (Lee, 1980; Lee et al., 1984). There is convincing pharmacological evidence from organ-bath experiments and from whole-animal studies that NO is an important mediator of vasodilation in cerebral arteries (for reviews see: Iadecola, 1993; Faraci and Brian, 1994), but most studies have not distinguished between endothelial-derived and neurally derived NO. However, experiments on cerebral arteries from which the endothelium has been removed show that neurally released NO contributes to the vasodilator responses. For example, segments of pig basilar artery (Lee and Sarwinski, 1991), monkey cerebral and temporal arteries (Toda and Okamura, 1990; Yoshida et al., 1994), guinea-pig middle cerebral artery (Saito and Goto, 1994) and dog cerebral artery (Toda and Okamura, 1990, 1991) from which the endothelium has been removed respond to transmural electrical stimulation with relaxations that are abolished by tetrodotoxin, and abolished or dramatically reduced by NOS inhibitors. Thus, although parasympathetic vasodilator nerves innervating the cerebral vessels contain at least three potential neurotransmitters, NO appears to the be dominant neurotransmitter that is responsible for nerve-mediated endothelium-independent vasodilation, and acetylcholine is the predominant neurotransmitter responsible for the nerve-mediated endothelium-dependent vasodilation. 225

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(b) Vasodilator neurones in sympathetic ganglia. In many species, a small population of sympathetic vasodilator neurones innervate the arteries supplying skeletal muscle, and they contain acetylcholine-related markers and a variety of peptides (Lindh et al., 1989; Gibbins, 1992; Morris et al., 1998). The presence of NOS and other peptides in sympathetic vasodilator neurones varies between species. In the guinea pig, sympathetic vasodilator neurones contain VIP, but not NOS (Morris et al., 1998), and thus any involvement of NO in vasodilation must be exclusively that derived from the endothelium. Similarly, in the pig, there are no NOS-immunoreactive nerve fibres associated with blood vessels supplying skeletal muscle (Modin et al., 1994). In the rat the situation is unclear. Following exposure of the rat to air-jet stress (a noxious stimulus), there is a marked vasodilation of the hindquarter vasculature, which is thought to be part of a 'defence reaction' (Folkow, 1982). Davisson et al. (1994, 1997) have reported that neurally released NO is responsible for the air-jet stress-induced vasodilation in the rat. However, a previous report (Anderson et al., 1993) had shown that there are no NADPH diaphorase-positive or NOS-immunoreactive nerve cell bodies in lumbar sympathetic ganglia of the rat, and so the source of the NOS nerve fibres that were concluded to innervate the rat hindlimb vasculature is unclear. In the cat, vasodilator neurones innervating the hindlimb vasculature contain both NOS and VIP, but not CGRP (Anderson et al., 1995). Arterioles in the cat paw pad are also innervated by NOS nerve fibres (Fig. 2D), but the contribution of neurally released NO to vasodilatory responses in the hindlimb and paw pad arteries of the cat has yet to be examined. (c) Vasodilator neurones in peh,ic ganglia. Like cerebral arteries, arteries supplying erectile tissue and the uterus are densely innervated by vasodilator neurones. Vasodilation of pelvic arteries is important during the later stages of pregnancy and for sexual function (Bell, 1972). Within the penis of many mammalian species, the penile arteries, veins and cavernous tissue are surrounded by a plexus of nerve fibres showing NOS immunoreactivity and NADPH diaphorase staining (Burnett et al., 1992, 1993; Keast, 1992; Alto et al., 1993; Ding et al., 1993; Schirar et al., 1994; Dail et al., 1995: Hedlund et al., 1995; Tamura et al., 1995). Retrograde tracing studies have shown that the vast majority of these NOS-containing nerve fibres originate from pelvic ganglia (Keast, 1992; Ding et al., 1993, 1995; Domoto and Tsumori, 1994; Schirar et al., 1994, 1997; Vizzard et al., 1994; Vanhatalo and Soinila, 1995). A retrograde tracing study in which tracer was injected into the penile shaft revealed some labelled, NOS-immunoreactive neurones in the intermediolateral column of the upper lumbar levels of the spinal cord, suggesting that some NOS preganglionic neurones may project directly to the penis (Vanhatalo and Soinila, 1995). However, it is also possible that the tracer was taken up by the cell bodies of neurones along the pelvic nerves, which are part of the pelvic plexus. Lumbar level dorsal root ganglia may also be sources of some of the NOS-containing nerve terminals within the penis (McNeill et al., 1992). A large proportion of the NOS neurones in the pelvic plexus that innervate the penile vasculature also contain acetylcholine-related molecules and VIE but none contain tyrosine hydroxylase (Ding et al., 1995; Hedlund et al., 1995; Ehmke et al., 1995; Tamura et al., 1995; Dail, 1996; Vanhatalo et al., 1996). There is substantial evidence showing that both endothelial-derived and neurally derived NO contribute to relaxation of vascular smooth muscle necessary for penile erection (see Burnett, 1995, 1997). Electrical field stimulation induces relaxations of the smooth muscle of the corpus cavernosum that are abolished by tetrodotoxin and markedly reduced by NOS inhibitors (Ignarro et al., 1990; Kim et al., 1991; Knispel et al., 1992a,b; Rajfer et al., 1992; Wang et al., 1994; Hedlund et al., 1995; Hayashida et al., 1996). Relaxations can be elicited from both endothelium-denuded and endothelium-intact preparations, and in both types of preparations, the relaxations induced by low-frequency stimulation are abolished by NOS 226

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inhibitors, whereas higher-frequency stimulation induces relaxations that are partly resistant to NOS inhibition; thus neurally derived NO plus another unidentified molecule appear to contribute to penile vasodilation (Kim et al., 1991" Simonsen et al., 1995, 1997). Although VIP is co-localised with NOS in many of the axons innervating penile blood vessels, no role for VIP in the regulation of muscle tone has been detected (Hayashida et al., 1996). Mice with a targetted deletion of the neuronal NOS gene (Huang et al., 1993" Burnett et al., 1996) or the endothelial NOS gene (Huang et al., 1995) breed normally, suggesting that neither endothelial NOS nor neuronal NOS are, by themselves, essential for penile function. However, in the neuronal NOS knockout mice, penile erections can be blocked by non-isoform-specific NOS inhibitors such as L-NAME, and since the levels of endothelial NOS present within the endothelium of these mice are significantly higher than in wild-type mice, it is believed that NO released from the endothelium mediates vasodilation during erections in mice lacking neuronal NOS (Burnett et al., 1996). Hence, compensatory mechanisms may prevent the normal physiological role of each isoform of NOS from being revealed in the knockout mice. Little is known about the role in NO in erectile tissue (clitoris and bulbs) of females. Neuronal NOS immunoreactivity is present in nerve fibres, and endothelial NOS immunoreactivity is present in vascular and sinusoidal endothelial cells of the human clitoris (Burnett et al., 1997b). However, to our knowledge there have been no functional studies examining the role of NO in this tissue. The uterine artery is innervated by vasodilator neurones, and a significant component of the vasodilator response in this vessel is non-cholinergic (Bell, 1969) and endothelium-independent (Morris, 1993" Nelson et al., 1995). NOS-immunoreactive nerve fibres form a plexus around the uterine arteries of humans and guinea pigs (Kummer et al., 1992; Toda et al., 1994; Anderson et al., 1997). In the guinea pig, the vasodilator neurones innervating the uterine artery arise from pelvic (paracervical) ganglia and they contain five potential inhibitory neurotransmitters or their synthetic enzymes: acetylcholine, VIP, CGRP, neuropeptide Y and NOS (Morris et al., 1985" Morris and Gibbins, 1987" Anderson et al., 1997). Pharmacological studies have revealed endothelial-independent vasodilations of the uterine artery in guinea pigs and humans that are blocked by tetrodotoxin and inhibited, but not abolished, by NOS inhibitors (Morris, 1993" Toda et al., 1994; Nelson et al., 1995). Thus both neurally released NO plus another neurotransmitter mediate endothelium-independent vasodilation in the uterine artery. In the guinea pig, the other neurotransmitter is likely to be a peptide such as VIP or CGRP, because the non-NO component of the response is trypsin-sensitive (Morris, 1993). Both the endothelium-dependent and endothelium-independent vasodilatory responses observed in uterine arteries become more pronounced during pregnancy (Nelson et al., 1995, 1998; Jovanovic et al., 1997). (d) Vasodilator neurones in the enteric nervous system. Submucosal arterioles are densely innervated by axons of extrinsic vasoconstrictor neurones that arise exclusively from sympathetic ganglia, axons arising from extrinsic sensory (dorsal root ganglia) neurones that cause vasodilation, and intrinsic vasodilator neurones with cell bodies in enteric ganglia (see Vanner and Surprenant, 1996). The vasodilatory responses that occur following the ingestion of food appear to be mediated largely by intrinsic neurones, whereas extrinsic sensory neurones mediate vasodilation principally during inflammatory states. Although endothelial NOS immunoreactivity and NADPH diaphorase activity are present in the endothelial cells of submucosal arterioles (Nichols et al., 1992, 1993" O'Brien et al., 1995), there have been no reports of NOS-containing nerve fibres associated with these vessels, and neurogenic vasodilation in submucosal arterioles appears to be mediated by acetylcholine, VIP and possibly other peptides (Vanner and Surprenant, 1996). 227

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(e) Blood vessels innervated by NOS nerve fibres of unknown origin and role. In the human, guinea pig and ferret, the blood vessels supplying the airways are surrounded by a plexus of NOS-immunoreactive nerve fibres (Dey et al., 1993" Fischer et al., 1993; Fischer and Hoffmann, 1996). However, the source and the role of these perivascular NOS fibres in the airways have yet to be examined. Since there are no intrinsic NOS-immunoreactive nerve cell bodies in the guinea-pig trachea, the perivascular NOS fibres in the guinea-pig airways are likely to arise from nerve cell bodies in the oesophagus, non-noradrenergic sympathetic neurones in the stellate ganglion (Section 5) or extrinsic sensory neurones (Fischer et al., 1993, 1996a). In humans and ferrets, NOS-immunoreactive nerve cell bodies are present in intrinsic ganglia of the trachea, and hence at least some of the perivascular NOS fibres could arise from this source (Section 5). There do not appear to have been any pharmacological studies which have examined the role of NO in the neural control of the airway vasculature. 3.1.2. Sensory vasodilation

Perivascular sensory nerve fibres (arising from dorsal root ganglia and vagal sensory ganglia) are associated with many blood vessels (Furness et al., 1982; Gibbins et al., 1985, 1987), and release a variety of neurotransmitters that can regulate vascular function (see Holzer and Maggi, 1998). The actions of sensory neurones can be distinguished from autonomic vasodilator neurones by their sensitivity to capsaicin, which acutely causes excitation, and in the long-term, degeneration of their nerve endings (Holzer, 1991). When stimulated, perivascular sensory fibres cause a vasodilation that is mediated by a number of different neurotransmitters, primarily CGRP, although ATP and tachykinins can also be involved (for reviews see: Holzer et al., 1995a; Maggi, 1995). NOS is present in subpopulations of sensory neurones (Fig. 4), and the role of sensory neurone-derived NO in vasodilation varies between vascular beds and species (Holzer et al., 1995b). For example, in mesenteric arterioles of the rabbit, neurally released ATP and CGRP fully account for the capsaicin-sensitive relaxation, although endothelial-derived NO is involved in the response (Kakuyama et al., 1998). In the cerebral cortex of the cat, the increase in blood flow induced by stimulation of the nasociliary nerve (which stimulates the fibres of trigeminal neurones) is unaffected by NOS inhibition, and the cerebral vasodilatation appears to be mediated largely by CGRP (Edvinsson et al., 1998). However, NO derived from primary sensory neurones does appear to mediate vasodilation in the mesenteric artery of the guinea pig. In segments of mesenteric artery of the guinea pig from which the endothelium has been removed and which have been pre-treated with guanethidine (to block neurotransmitter release from sympathetic nerve terminals), vasodilatory responses are reduced by over 50% by the NOS inhibitor, L-NNA, and nerve terminals containing both NOS and substance P are found in the perivascular plexus of this artery (Zheng et al., 1997). In addition to acting directly on the vascular smooth muscle, there is also evidence in some vascular beds that NO can facilitate the release of CGRP from afferent nerve fibres (Holzer et al., 1995b). 3.1.3. Role of neurally released NO in regulation of blood vessels --- summary

(i) NO released from cranial parasympathetic neurones innervating cerebral blood vessels, and other blood vessels supplying structures in the head, is an important mediator of nerve-stimulated, endothelial-independent vasodilation. (ii) NO released from the terminals of pelvic parasympathetic neurones innervating the uterine artery and blood vessels supplying the penis is an important mediator of vasodilation. 228


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Fig. 4. (A) Frozen section through the nodose ganglion of a rat, showing scattered, NOS-immunoreactive neurones in the ganglion. Scale bar -- 100 ~tm. (B) Scattered NOS-immunoreactive neurones are also present in the dorsal root ganglia (DRG) of the rat. The ganglion illustrated is from T12 (thoracic level). Scale bar = 25 ~tm. (C, D) Paired, high-power micrograph showing co-localisation between NOS (C) and calcitonin gene-related peptide (D, CGRP) in a T12 dorsal root ganglion of the rat. Some CGRP-positive cells (asterisks) do not show NOS immunoreactivity. Scale bar -- 25 ~tm. (iii) In some species only, sympathetic vasodilator neurones innervating skeletal muscle m a y use N O as a neurotransmitter. (iv) Within the gastrointestinal tract, neurally derived NO plays no role in the vasodilation of submucosal arterioles mediated by enteric vasodilator neurones. (v) Neurally mediated vasodilation in some vascular beds is mediated by NO released from subpopulations of perivascular sensory nerve fibres arising from dorsal root ganglia. 3.2. R O L E OF N E U R A L L Y D E R I V E D NO IN T H E C O N T R O L OF T H E H E A R T The heart is innervated by sympathetic (excitatory) neurones, vagal parasympathetic (inhibitory) pathways and extrinsic sensory neurones. The sympathetic nerve fibres arise from extrinsic neurones located in the cervical and upper thoracic sympathetic ganglia, and the sen229

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sory innervation arises from dorsal root ganglia and vagal sensory ganglia. An early study by Schmiedeberg and Koppe (1869) showed that the effects of muscarine and vagal stimulation on the heart are similar, and both are antagonised by atropine. Loewi (1921) demonstrated that the slowing of the heart in response to vagal activation is due to the release of a substance (which he called 'Vagusstoff'), whose action was blocked by atropine. Although the vagal parasympathetic preganglionic neurones that innervate the heart are cholinergic, the acetylcholine that inhibits the cardiac muscle is actually released from intrinsic neurones that are located in small ganglia within the heart and its surrounding tissues (King and Coakley, 1958), and which receive synaptic input from vagal nerve terminals. Consistent with the pharmacological data, all of the intrinsic cardiac neurones in the guinea-pig heart are ChAT-immunoreactive, and thus cholinergic (Mawe et al., 1996). However, the cardiac neurones show considerable electrophysiological, morphological and neurochemical diversity (Baluk and Gabella, 1989; Hassall et al., 1992; Steele et al., 1994; Edwards et al., 1995). Subpopulations of the cardiac neurones contain a range of neuropeptides including somatostatin, tachykinins, neuropeptide Y and pituitary adenylate cyclase-activating polypeptide (PACAP) (Baluk and Gabella, 1989; Steele et al., 1994; Braas et al., 1998). In addition, a subpopulation of the neurones in the heart of a range of mammalian species, including the rat, guinea pig, human, monkey and dog, contains NOS, as demonstrated by immunohistochemistry or NADPH diaphorase staining (Fig. 5A; Hassall et al., 1992; Klimaschewski et al., 1992; Tanaka et al., 1993; Steele et al., 1994; Armour et al., 1995; Mawe et al., 1996; Sosunov et al., 1996; Yoshida and Toda, 1996). In the guinea pig and rat, less than 10% of the cardiac neurones contain NOS (Hassall et al., 1992; Klimaschewski et al., 1992; Steele et al., 1994; Mawe et al., 1996), but in the dog, 30-40% of the neurones are NADPH diaphorase-positive (Armour et al., 1995). In close apposition with nerve cell bodies within the cardiac ganglia are nerve terminals that are immunoreactive for ChAT (Mawe et al., 1996), NOS (Fig. 5B; Sosunov et al., 1996), tyrosine hydroxylase or substance P plus CGRP (Steele et al., 1994). Most of the cholinergic nerve terminals probably arise from vagal preganglionic neurones but some are also likely to arise from intrinsic cholinergic neurones, the tyrosine hydroxylase-

Fig. 5. (A, B) Whole-mount preparation from the guinea-pig atrium showing two cardiac ganglia which are adjacent to nerve bundles. The ganglion shown in A has many NOS-immunoreactive nerve cell bodies, whereas the ganglion shown in B has no NOS-positive nerve cell bodies, but varicose, NOS-containing nerve terminals are present in the ganglion. Scale bar = 50 ~m. Preparation kindly dissected by David Hirst.

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immunoreactive nerve terminals arise from sympathetic neurones and the substance P/CGRP nerve terminals arise from extrinsic sensory neurones (Steele et al., 1994; Mawe et al., 1996). The source of NOS-immunoreactive nerve terminals within the heart has only recently been examined. Tanaka and Chiba (1998) examined the origin of NOS-immunoreactive nerve terminals in different parts of the guinea-pig heart by performing immunohistochemistry following unilateral cervical vagotomies. They found that the baskets of NOS nerve terminals that are present around intracardiac nerve cell bodies in ganglia associated with the left atrium and the interatrial septum arise from neurones that project to the heart via the left vagus nerve, since they completely disappear following a left-side vagotomy. The authors concluded that these vagal NOS fibres must be parasympathetic preganglionic nerve fibres, although it is also possible that some were extrinsic sensory fibres, since there are many NOS neurones in the nodose ganglion. The NOS-immunoreactive nerve terminals that form baskets around ganglion cells in the atrioventricular nodal region, or that form a sparse plexus covering the fight atrium, are unaffected by vagotomies of the right or left side, and thus must arise from intrinsic, NOS-containing neurones. The physiological role of NO in the neural control of heart rate has been examined in a number of studies (Han et al., 1994; Conlon et al., 1996, 1997; Elvan et al., 1997; Sears et al., 1998a,b). All of the studies have reported some effect of NOS inhibitors and NO donors on the sympatho-vagal control of heart rate. However, most of the studies did not use isoform-specific NOS inhibitors, and since non-isoform-specific NOS inhibitors, such as L-NA, affect baseline heart rate and mean arterial pressure, the results can be difficult to interpret. However, a study was recently published which used the specific neuronal NOS inhibitors, 1-(2-trifluoromethylphenyl) imidazole (TRIM) and 7-nitroindazole (7-NiNa), to examine the effect of neuronal NOS inhibition on sympathetic and vagal control of heart rate (Sears et al., 1998b). Unlike non-isoform-specific NOS inhibitors, TRIM was found to have no significant effect on mean arterial blood pressure or baseline heart rate in cardiac sympathectomised and vagotomized anaesthetised rabbits. Following unilateral sympathetic stellate ganglion stimulation, TRIM caused a significant increase in the cardioacceleration, which was reversed by L-arginine, indicating that neurally released NO inhibits the heart rate response to sympathetic nerve stimulation. Since TRIM had no effect on the change of heart rate observed following intravenous infusion of isoprenaline, the neurally released NO appears to inhibit the heart rate response to sympathetic nerve stimulation by acting presynaptically on sympathetic nerve terminals, rather than directly on the cardiac muscle (Sears et al., 1998b). In contrast, TRIM, 7-NiNa or even the non-isoform-specific inhibitor L-NA, had no effect on the size of the heart rate response to vagal nerve stimulation in either the anaesthetised rabbit or in the isolated guinea-pig atria (Sears et al., 1998b). However, in the ferret, both TRIM (Conlon et al., 1997) and the non-isoform-specific NOS inhibitors, L-NAME and L-NOARG (Conlon et al., 1996, 1998), reduce the heart rate response to vagal nerve stimulation, demonstrating that in some species, neurally released NO can facilitate the vagal transmission to the heart. To summarise, in the rat and rabbit, neurally released NO acts presynaptically on sympathetic nerve terminals to inhibit the heart rate response to sympathetic nerve stimulation (Sears et al., 1998b). In the ferret (Conlon et al., 1996, 1997, 1998), but not in the rat and rabbit (Sears et al., 1998b), neurally released NO also facilitates the vagal effects on the heart. Thus, overall, neurally released NO has cardiodepressor actions by decreasing sympathetic transmission and, in some species, enhancing vagal transmission. It remains unclear whether the neuronal NO that modulates the sympathetic and vagal control of heart rate is released predominantly from the terminals of vagal preganglionic neurones or from intrinsic cardiac neurones. 231

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4. ROLE OF NO IN THE NEURAL CONTROL OF THE GASTROINTESTINAL TRACT 4.1. INTRODUCTION The gastrointestinal tract is innervated by intrinsic (enteric) neurones, and extrinsic neurones with cell bodies in sympathetic, parasympathetic and sensory ganglia. Within the enteric nervous system are different functional types of neurones, including sensory neurones, interneurones, secretomotor neurones, vasodilator neurones (see Section 3.1.1 (d)) and excitatory and inhibitory muscle motor neurones (Fumess et al., 1999). The evidence that NO is synthesised by subpopulations of enteric neurones, and has roles in enteric transmission, is supported by abundant data (Rand, 1992; Sanders and Ward, 1992; Stark and Szurszewski, 1992; Brookes, 1993). These data indicate that NOS is contained in inhibitory enteric muscle motor neurones of all mammals that have been investigated, and that transmission from enteric inhibitory neurones to gastrointestinal muscle is antagonised when the production or action of NO is compromised. Interneurones that form synapses in the ganglia of the gastrointestinal tract also contain NOS, but NO seems not to have a substantial role in enteric neuro-neuronal transmission. NOS is also in intrinsic neurones that supply one component of the innervation of motor endplates in the oesophagus. In addition, NOS is in neurones supplying the gastric mucosa, in enteric neurones that project from the gut to sympathetic ganglia (intestinofugal neurones), and in neurones of the gallbladder and pancreas. Each of these is discussed below, but greatest emphasis is given to the role of NO in neuromuscular transmission. Although a role of NO in neuromuscular transmission is not in dispute, there are several theories to suggest how this transmission is mediated. 4.2. NO IS A NEUROTRANSMITTER OF ENTERIC INHIBITORY MOTOR NEURONES The enteric inhibitory neurones relax gut muscle. They innervate both the longitudinal and circular layers of the external musculature, and the muscularis mucosae. In functional terms, it is the innervation of the circular muscle that seems the most important because mixing and propulsion of the gut contents depends on contractile activity in this muscle coat. The neurones relax the circular muscle in order to aid the passage of food along the gut and through its sphincters. Although enteric inhibitory neurones were known to physiologists from the beginning of the century, identification of their transmitter(s) has proved difficult, even controversial. In the early 1970s it was proposed that ATP is the transmitter, but soon after evidence for VIP was advanced, then came the evidence that NO participates, followed by evidence for PACAP and carbon monoxide (Burnstock, 1972; Fahrenkrug, 1979; Rand, 1992; Furness et al., 1995). The proposition that NO is a transmitter of enteric inhibitory neurones arose almost simultaneously from six laboratories, all of whom took advantage of the advent of NOS inhibitors and the simplicity of isolated gut muscles for studies of the pharmacology of transmission from these neurones (Gillespie et al., 1989; Li and Rand, 1989; Ramagopal and Leighton, 1989; Bult et al., 1990; Gibson et al., 1990; Hata et al., 1990). In each case, it was found that inhibition of NOS substantially reduced the amplitude of the relaxation of the muscle caused by stimulation of the inhibitory neurones. In the next two to three years, more than 20 papers were published, each showing, in circular and longitudinal muscle, including sphincter muscle, from several mammalian species, that inhibition of NOS or inactivation of NO by oxyhaemoglobin attenuated transmission from the enteric inhibitory neurones 232


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(Sanders and Ward, 1992; Stark and Szurszewski, 1992). The hypothesis that NO is an enteric neurotransmitter was consolidated by the histological demonstration of NOS in enteric neurones, either by NOS immunohistochemistry or by the NADPH diaphorase reaction, which label identical neurones in the gut (Fig. 6A,B; Branchek and Gershon, 1989; Bredt et al., 1990; Belai et al., 1992; Costa et al., 1992; Furness et al., 1992; Grozdanovic et al., 1992; Nichols et al., 1992; Ward et al., 1992; Young et al., 1992). Many more papers describing the distribution of enteric nerve cells and nerve fibres containing NOS were published in subsequent years. Consistently in all species, NOS nerve cells are in the myenteric plexus of the small and large intestines, and nerve fibres are found in the fibre bundles innervating the circular and longitudinal muscle (Fig. 6C). The projections and chemistries of the NOS neurones that innervate the muscle indicate them to be enteric inhibitory motor neurones (Costa et al., 1992; Furness et al., 1992; McConalogue and Furness, 1993; Ekblad et al., 1994a,b; Timmermans et al., 1994; Costa et al., 1996; Porter et al., 1997; Sang et al., 1997; Pfannkuche et al., 1998). The NOS nerve fibres in the circular muscle arise from myenteric nerve cells that send their axons to the underlying and more anally located muscle. VIP immunoreactivity was previously identified as a marker of enteric inhibitory motor neurones (see Furness et al., 1995) and is co-localised with NOS in the neurones that innervate the muscle (Costa et al., 1992, 1996; Furness et al., 1992; Ekblad et al., 1994a,b; Keef et al., 1994; Ward et al., 1994b; Sang and Young, 1996). NO is released from enteric neurones when they are stimulated (Boeckxstaens et al., 1991; Wiklund et al., 1993a; Shuttleworth et al., 1995; Chakder and Rattan, 1996), although this release cannot be attributed specifically to the inhibitory motor neurones; it could be from NOS interneurones. In summary, there is little reason to doubt that NO is a transmitter of the enteric inhibitory motor neurones. However, it is not the only transmitter of these neurones, and the mechanism by which information is transmitted from the neurones to the muscle has been the subject of several theories. 4.3. ROLE OF NO IN CO-TRANSMISSION FROM ENTERIC INHIBITORY MOTOR NEURONES It became apparent in the 1980s that there are separable components of transmission from enteric inhibitory neurones. Niel et al. (1983) reported that the inhibitory junction potential (IJP), elicited by stimulation of these neurones, consisted of two phases of hyperpolarisation in the muscle of the guinea-pig intestine, a fast component blocked by apamin (a blocker of small-conductance potassium channels), and a slow component resistant to this drug. Crist et al. (1992) showed that the fast component of the IJP was blocked by ~,13-methylene ATE thereby consolidating the earlier proposal that ATP is a transmitter. A survey of inhibitory transmission throughout the guinea-pig intestine, using mechanical rather than electrical recording, indicated that the relative contribution of an apamin-sensitive component varied considerably between gut regions (Costa et al., 1986). For example, apamin almost completely abolished inhibitory transmission in the longitudinal muscle of the ileum, but was without effect in the circular muscle of the fundus. Other comparisons of transmission that confirmed the involvement of several transmitters have been reviewed (Furness et al., 1995). In addition to NO and ATP, substances that possibly contribute to muscle relaxation include VIP (Fahrenkrug, 1979; Grider et al., 1985a,b), PACAP (Schworer et al., 1992; Gilder et al., 1994; Jin et al., 1994; McConalogue and Furness, 1993; Katsoulis et al., 1996) and carbon monoxide (Rattan and Chakder, 1993; Ny et al., 1995; Zakhary et al., 1997). 233

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Following the initial identification of NO as an inhibitory transmitter, studies were undertaken to determine which component(s) NO contributed to the transmission process, and how the actions of NO were related to those of the other transmitters. It was found

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that NOS inhibition blocked the slow component of transmission in the guinea-pig ileum (Lyster et al., 1992; He and Goyal, 1993). This seemed to lead to a simple interpretation, the existence of a fast apamin-sensitive component of transmission mediated by ATP, and a slow component mediated by NO. However, it was also reported that the slow component was blocked by the VIP antagonist, VIP 10-28 (Crist et al., 1992). This led to a cascade hypothesis of transmission, in which inhibitory neurones were proposed to release VIP and PACAP (which act on the common VPAC receptor; Harmar et al., 1998), and these transmitters in turn release NO from muscle, which acts back on the muscle, causing relaxation (Grider et al., 1992; Murthy and Makhlouf, 1994). This hypothesis itself is confounded to some extent by the observation that NO-mediated inhibitory transmission, at least in some regions, is mediated via the interstitial cells of Cajal (ICC). The ICC are spindle-shaped cells that are at the surfaces of, and within, the muscle, and are closely apposed by the axons of excitatory and inhibitory motor neurones (Sanders, 1996). Stimulation of inhibitory motor neurones elicited IJPs of reduced amplitude when the ICC were dissected away (Huizinga et al., 1990). A similar result was obtained by comparison of inhibitory transmission in the lower oesophageal sphincter, stomach and pyloric sphincter of normal mice and mutants ( W / W ~') lacking ICC in the circular muscle of these regions (Burns et al., 1996: Ward et al., 1998). The component of transmission that was mediated via NO was absent in the mutants. In a study in which the ICC were disrupted by treating neonatal mice with antibodies to Kit (this specifically damages ICC), excitatory and inhibitory transmission to the circular muscle of the ileum were impaired, and neural transmission to colonic circular muscle was abolished (Torihashi et al., 1995). Data published by Publicover et al. (1993) indicate that the ICC can produce NO. This could possibly amplify transmission to the muscle. Thus, NO might be involved in transmission at three levels: (1) NO can be produced by inhibitory motor neurones and act both directly on muscle and on ICC. (2) The ICC can in turn inhibit the muscle through electrical connections, and perhaps through the facilitated release of NO. (3) NO derived from muscle may amplify inhibitory transmission. The hypothesis that NO is produced by ICC is supported by the immunohistochemical localisation of one of the non-neuronal isoforms of NOS, endothelial NOS (eNOS) in ICC (Xue et al., 1994). Similarly, RT-PCR and Northern analysis indicates that intestinal muscle cells express eNOS (Teng et al., 1998). There is something unusual about the eNOS in intestinal ICC and muscle, in that they are not revealed by N A D P H diaphorase staining (which should reveal all isoforms of NOS), and not by all antibodies against eNOS (Young et al., 1997). There are few experiments which bear on the question of whether NO acts in a primarily serial fashion, via ICC, or mainly in a parallel fashion, both via ICC and directly on the

Fig. 10. (A) Cross-section of the ventral horn of the spinal cord from a normal rat. (B, C) Cross-sections of the ventral horn of the spinal cord from experimental rat (B) or guinea pig (C) 2 weeks following root avulsion. Sections were stained with NADPH-d histochemistry and counterstained with neutral red. Normal spinal motoneurons do not express NOS and are stained by neutral red into red in color (A). By 2 weeks alter the injury, the perikarya of NOS-positive motoneurons is shrunken and the normal multipolar cell body transforms and becomes round as compared to the normal motoneurons (arrows in B). Compared with the normal motoneurons in A, the cell body of these NOS-positive motoneurons is smaller (B). These changes more likely resemble apoptosis. In contrast, injured motoneurons in the guinea pig show the different morphological changes. Many of these NOS-positive motoneurons show characteristics of necrosis which include the swelling of the cell body (open arrows in C), breakdown of the cell membranes (many cells in C without a clear nuclear membrane, asterisk in C indicates a motoneuron with disrupted membrane). Still, some motoneurons in guinea pig is shrunken and the cell body of these cells becomes round and smaller compared with the normal motoneurons (arrows in C). Bar = 20 gm.

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to any particular subcellular organelle. The formazan, or NOS immunoreactivity, is scattered throughout the whole cytoplasm. Although there is more labeling associated with smooth and rough endoplasmic reticulum and near the Golgi apparatus, it is absent from the mitochondria and cell nuclei (Mizukawa et al.. 1988; Llewellyn-Smith et al., 1992). However, other investigators have reported that NOS reactivity is found in the nuclear envelope, inner and outer mitochondria membranes, Golgi apparatus, and endoplasmic reticulum (Calka et al., 1994). Different distribution of NOS in the subcellular organelles reported in various studies may be due to different staining methods used. With either NADPH-d or NOS immunocytochemistry, we have found that NOS reactivity is not specifically associated with any subcellular organelle or plasma membrane if Triton X-100 is added to the reactive solution. Without Triton X-100 in the reactive solution, NOS reactivity can be observed in some membrane structures, such as endoplasmic reticulum (Wu, unpubl, data). The subcellular distribution of injury-induced NOS in injured neurons is similar to that of normal NOS-containing neurons. Injury-induced NOS is found throughout the cytoplasm but not the nucleus, and is not specifically associated with any subcellular organelles (Fig. 11A,B). Morphologically, these injury-induced NOS-positive neurons seem normal in some areas of the nervous system (Fig. 11A), but abnormal in others (Fig. 11B). Neuronal death following injury is often assumed under two major forms, i.e. necrosis or apoptosis (Ellis et al., 1991; Oppenheim, 1991 ; Cohen and Duke, 1992). Whether expression of NOS plays any role in either necrosis or apoptosis is not clear and may vary in different neuronal populations, developmental ages, and animal species. The expression of NOS in the rat spinal motoneurons may be more likely associated with necrosis, especially during the early stage of the injury when NOS concentration is in a higher level. This is based on the following observations. (1) Injury of the peripheral nerve in the developing animals causes apoptosis in spinal motoneurons without NOS expression (Li et al., 1998). (2) Expression of NOS in adult spinal motoneurons following root avulsion is associated with a necrotic type of cell death as characterized by the disintegration of membrane (Fig. l lC) and the activation of macrophages in the ventral horn of the spinal cord (Fig. 12). However, injury-induced NOS-positive motoneurons have mixed morphology, showing characteristics of both apoptosis and necrosis. As shown in the earlier section of this review, different animal species may show different characteristics of degeneration, either apoptosis or necrosis, following a same injury. Even in the same animal species, morphological characteristics of NOS-positive neurons could be different at different post-injury stages. For instant, injured rat motoneurons show necrotic damage during the early stage of root avulsion lesion (as shown earlier in this section). However, at the late stage after injury, these motoneurons exhibit features of apoptosis, including increased electron density in both the cytoplasm and nucleus with intact cell and nuclear membranes, the shrunken nucleus, and the reduced soma size of the cell (Fig. 13A). Occasionally, apoptotic bodies are found in these cells (Fig. 13B). In the cerebellar cortex, the size of injury-induced NOS-positive Purkinje cells appears to decrease gradually after lesion. The average diameter of reactive Purkinje cells 6 weeks post injury is about 38% smaller than that observed 3 days post injury (Chen and Aston-Jones, 1994). These changes seem to resemble apoptosis. In contrast, subcellular structures of injury-induced NOS-positive neurons in the hypothalamus following hypophysectomy seem normal, morphologically and functionally (Wu and Scott, 1993; Scott et al., 1995). No evidence of necrosis or apoptosis is observed in those hypothalamic neurons (Scott et al., 1995). These data indicate that the role of NOS in injured neurons is likely complex and expression of injury-induced NOS in different populations of neurons may play different roles in neuronal injury. 332

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De Vente J, Steinbusch HWM (1997): cGMP-immunocytochemistry. In: Rayne RJ (Ed), Neurotransmitter Methods. Methods in Molecular Biolog), Vol 72. Totowa, NJ: Humana Press, pp 125-143. De Vente J, Garssen J, Tilders FJH, Steinbusch HWM, Schipper J (1987a): Single cell quantitative immunocytochemistry of cyclic GMP in the superior cervical ganglion of the rat. Brain Res 411:120-128. De Vente J, Steinbusch HWM, Schipper J (1987b): A new approach to immunocytochemistry of 3',5'-cyclic guanosine monophosphate: preparation, specificity, and initial application of a new antiserum against formaldehyde-fixed 3',5'-cyclic guanosine monophosphate. Neuroscience 22:361-373. De Vente J, Bol JGJM, Steinbusch HWM (1989a): Cyclic GMP-producing atrial natriuretic factor-responding cells in the rat brain: an immunocytochemical study. EurJ Neurosci 1:436-460. De Vente J, Bol JGJM, Steinbusch HWM (1989b): Localization of cGMP in the cerebellum of the adult rat: an immunohistochemical study. Brain Res 504:332-337. De Vente J, Schipper J, Steinbusch HWM (1989c): Formaldehyde fixation of cGMP in distinct cellular pools and their recognition by different cGMP-antisera. An immunocytochemical study into the problem of serum specificity. HistochemistiT 91:401-412. De Vente J, Bol JGJM, Berkelmans HS, Schipper J, Steinbusch HWM (1990): Immunocytochemistry of cGMP in the cerebellum of the immature, adult, and aged rat: the involvement of nitric oxide. A micropharmacological study. Eur J Neurosci 2:845-862. De Vente J, Hopkins DA, Markerink-Van Ittersum M, Steinbusch HWM (1996): Effects of the 3',5'-phosphodiesterase inhibitors isobutylmethylxanthine and zaprinast on NO-mediated cGMP accumulation in the hippocampus slice preparation: an immunocytochemical study. J Chem Neutvanat 10:241-248. De Vente J, Hopkins DA, Markerink-Van Ittersum M, Emson PC, Schmidt HHHW, Steinbusch HWM (1998a): Distribution of nitric oxide synthase and nitric oxide-receptive, cyclic GMP-producing structures in the rat brain. Neuroscience 87:207-241. De Vente J, Hopkins DA, Markerink-Van Ittersum M, Steinbusch HWM (1998b): Nitric oxide-mediated cGMP production in the islands of Calleja in the rat. Brain Res 789:175-178. De Vente J, Markerink-Van Ittersum M, Van Staveren W, Steinbusch HWM (1999): YC-1 potentiates nitric oxide activation of soluble guanylyl cyclase in rat brain slices: characterization of the cGMP response in cholinergic, GABAergic and glutamatergic neurons. Soc Neu~vsci Abstr 481:12. De Vente J, Markerink-Van Ittersum M, Van Abeelen J, Emson PC, Axer H, Steinbusch HWM (2000): NO-mediated cGMP synthesis in cholinergic neurons in the rat forebrain: effects of lesioning dopaminergic or serotonergic pathways on nNOS and cGMP synthesis. EurJ Neulvsci 12:507-519. Denninger JW, Marietta MA (1999): Guanylate cyclase and the NO/cGMP signaling pathway. Biochim Biophys Acta 1411:334-350. Derer P, Derer M (1993): Ontogenesis of NADPH-diaphorase neurons in the mouse forebrain. Neurosci Lett 152:21-24. Detre JA, Nairn AC, Aswad DW, Greengard P (1984): Localization in mammalian brain of G-substrate, a specific substrate for guanosine 3',5'-cyclic monophosphate-dependent protein kinase. J Neurosci 4:2843-2849. Dierks EA, Hu S, Vogel KM, Yu AE, Spiro TG, Burstyn JN (1997): Demonstration of the role of scission of the proximal histidine-iron bond in the activation of soluble guanylyl cyclase through metalloporphyrin substitution studies. J Am Chem Soc 1•9:7316-7323. Diket AL, Pierce MR, Munshi UK, Voelker CA, Eloby-Childress S, Greenberg SS, Zhang XJ, Clark DA, Miller MJS (1994): Nitric oxide inhibition causes intrauterine growth retardation and hind-limb disruptions in rats. Am J Obstet Gvnecol 171:1243-1250. Dinerman JL, Dawson TM, Schell MJ, Snowman A, Snyder SH (1994): Endothelial nitric oxide synthase localized to hippocampal pyramidal cells: implications for synaptic plasticity. Proc Natl Acad Sci USA 91:4214-4218. Egberongbe YI, Gentleman SM, Falkai R Bogerts B, Polak JM, Roberts GW (1994): The distribution of nitric oxide synthase immunoreactivity in the human brain. Neurosciem'e 59:561-578. EI-Husseini AED, Bladen C, Vincent SR (1995a): Molecular characterization of a type II cyclic GMP-dependent protein kinase expressed in the rat brain. J Neurochem 64:2814-2817. E1-Husseini AED, Bladen C, Vincent SR (1995b): Expression of the olfactory cyclic nucleotide gated channel (CNG1) in the rat brain. Neuroreport 6:1459-1463. Eliasson MJL, Blackshaw S, Schell MJ, Snyder SH (1997): Neuronal nitric oxide synthase alternatively spliced forms: prominent functional localizations in the brain. P~w" Natl Acad Sci USA 94:3396-3401. Endoh M, Maiese K, Wagner JA (1994): Expression of the neural form of nitric oxide synthase by CA1 hippocampal neurons and other central nervous system neurons. Neuroscience 63:679-689. Fedele E, Raiteri M (1999): In vivo studies of the cerebral glutamate receptor NO/cGMP pathway. Prog Neurobiol 58:89-120. 408

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Wang H, Morris JF (1996): Presence of neuronal nitric oxide synthase in the suprachiasmatic nuclei of mouse and rat. Neuroscience 74:1059-1068. Wang X, Robinson PJ (1997)" Cyclic GMP-dependent protein kinase and cellular signaling in the nervous system. J Neurochem 68:443-456. Wedel BJ, Garbers DL (1997): New insights on the functions of the guanylyl cyclase receptors. FEBS Lett 410:29-33. Wedner HJ, Hoffer BJ, Battenberg E, Steiner AL. Parker CW, Bloom FE (1972)" A method Ior detecting intracellular cyclic adenosine monophosphate by immunofluorescence. J Histochem Cvtochem 20:293-299. Wei JW, Chang CM, Chang LW (1993): Differential responses in cyclic GMP Ik)rmation induced by excitatory amino acids (EAA) and sodium nitroprusside (SNP) in various regions of the rat brain and of rats of varied age. hit J Biochem 25:1579-1585. Weinberg RJ, Valtschanoff JG, Zabel U. Schmidt HHHW (1998): Immunocytochemical localization of soluble guanylyl cyclase in the rat brain. Soc Neurosci Abstr 24:242.4. Williams CV, Nordquist D, McLoon SC (1994): Correlation of nitric oxide synthase expression with changing patterns of axonal projections in the developing visual system. J Neurosci 14:1746-1755. Wood J, Garthwaite J (1994): Models of the diffusional spread of nitric oxide: implications for neural nitric oxide signalling and its pharmacological properties. Neuropharmacology 33:1235-1244. Wood PL, Emmett MR, Wood JA (1994): Involvement of granule, basket and stellate neurons but not Purkinje or Golgi cells in cerebellar cGMP increases in vivo. L(fe Sci 54:613-620. W6rl J, Wiesand M, Mayer B, Gresk6tter KR. Neuhuber WL (1994)" Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation. Histochemistrv 102:353-364. W6rtwein G, Gustafson B, Hansen KL, Mogensen J (19971: Behavioral symptoms in adult rats after postnatal L-nitro-arginine. Int J Dev Neurosci 15:147-154. Wu HH, Williams CV, McLoon SC (1994)- Involvement of nitric oxide in the elimination of a transient retinotectal projection in development. Science 265:1593-1596. Xu ZQD, De Vente J, Steinbusch HWM, Grillner S. H6kfelt T (1998): The NO-cGMP pathway in the rat locus coeruleus" electrophysiological, immunohistochemical and in situ hybridization studies. E u r J Neurosci 10:3508-3516. Yah C, Bentley JK, Sonnenburg WK, Beavo JA (1994): Differential expression of the 61 kDa and 63 kDa calmodulin-dependent phosphodiesterases in the mouse brain. J Neurosci •4:973-984. Yah XX, Garey LJ, Jen LS (19941): Development of NADPH-diaphorase activity in the rat neocortex. Dev Brain Res 79:29-38. Yang Q, Paskind M, Bolger G, Thompson WJ, Repaske DR, Cutler LS, Epstein PM (1994): A novel cyclic GMP stimulated phosphodiesterase from rat brain. Biochem Biophys Res Commtm 205:1850-1858. Zabel U, Weeger M, La M, Schmidt HHHW (1998)" Human soluble guanylate cyclase: functional expression and revised isoenzyme family. Biochem J 335:51-57. Zabel U, H~iusler C, Weeger M, Schmidt HHHW (1999) Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione S-transferase affinity tag. J Biol Chem 2 74 18149-18152. Zhuo M, Laitinen JT, Li XC, Hawkins RD (1998): On the respective roles of nitric oxide and carbon monoxide in long-term potentiation in the hippocampus. Learn Mem 5:467-480. Zufall E Shepherd GM, Barnstable CJ (1997): Cyclic nucleotide gated channels as regulators of CNS development and plasticity. Curr Opin Nelovsci 7:404-412. Zwiller J, Ghandour MS, Revel MD, Basset P (1981): Immunohistochemical localization of guanylate cyclase in rat cerebellum. Neurosci Lett 23:31-36.

415


CHAPTER XI

Invertebrate models for studying NO-mediated signaling N.L. SCHOLZ AND J.W. TRUMAN

1. INTRODUCTION For more than half a century, research on invertebrate nervous systems has made important contributions to our general understanding of how individual neurons and neural networks give rise to animal behavior. The large number of invertebrate phyla represent a rich diversity of body forms and behavior. Correspondingly, invertebrate nervous systems range from the simple nerve nets of cnidarian jellyfish to the elaborate brains of cephalopod molluscs (squid and octopus), which rival their fish competitors in terms of total numbers of neurons and complex network organization. The nervous systems of invertebrates offer the twin advantages of numerical simplicity and the presence of comparatively large and identifiable nerve cells. These 'identified neurons' can be reliably located in different animals, which makes it possible to analyze the specific function of a given neuron in differing developmental and physiological contexts. This approach has been a valuable tool in the study of the cellular aspects of nervous system function and development, and it will be a common theme in the discussion of NO-mediated signaling in invertebrates that follows. NO is an ancient signaling molecule, presumably evolving before the radiation of the metazoans (Feelish and Martin, 1995). There is remarkable conservation of function for NO-mediated signaling, even among organisms that belong to different kingdoms. For example, NO plays a key role in disease resistance in both plants (Delledonne et al.. 1998) and animals (Nathan, 1995). Comparative studies have found NO-producing neurons in animals from all the major phyla, suggesting that cellular signaling using NO is an ancestral character of both vertebrate and invertebrate nervous systems. For example. the NO/cGMP pathway has been identified in the coelenterate HTdru i~ilgaris,the most primitive organism known to possess a nervous system (Colasanti et al., 1995). In mammals, the NO signaling pathway was first identified in the search for the agent that caused the relaxation of vascular smooth muscle (Palmer et al., 1987). It was subsequently discovered that NO is also an important anterograde as well as retrograde intercellular signaling molecule in the mammalian brain (Bredt and Snyder, 1992; Garthwaite and Boulton, 1995). Comparative studies soon found nitric oxide synthase (NOS) enzymatic activity in molluscs (Elofsson et al., 1993; Gelperin, 1994) and arthropods (Radomski et al., 1991: Elphick et al., 1993; Ribeiro et al., 1993; Johansson and Carlberg. 1994). In parallel with mammalian studies, initial research in invertebrates focused on roles for NO in olfaction (Gelperin, 1994; Muller and Bicker, 1994) and learning and memory (Robertson et a].. 1994).

417

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N.L. Scholz and J.W. Truman

Research has expanded in recent years and widespread roles for NO in cell-cell signaling are now established for most of the major phyla. Moreover, specific roles lbr NO have been extended into other aspects of nervous system function and into development. Most of the work continues to focus on molluscs and arthropods, and comprehensive reviews by Jacklet (1997), MOiler (1997), and Bicker (1998) have recently surveyed the function of NO in these animals. We will not try to repeat these reviews here. Rather, we will consider emerging research that is providing mechanistic insights into the roles of NO in the physiology and development of the nervous system.

2. COMPONENTS OF THE NO SIGNALING PATHWAY IN INVERTEBRATES

Nitric oxide is produced when NOS converts arginine into citrulline and NO. A widely used tool in assessing the presence of NOS in invertebrates has been the distribution of fixation-insensitive, NADPH-diaphorase staining. This histochemical procedure identified NOS activity in vertebrate systems (Bredt et al.. 1991" Hope et al., 1991). In invertebrate systems diaphorase activity was subsequently shown to copurify with NOS in both bees and Drosophila (Mtiller, 1994). However, considering the great diversity in the invertebrates, patterns of diaphorase staining should be taken as an indication, not a proof, of the presence of NOS. There may be forms of NOS that do not show up by diaphorase staining, and likewise, some diaphorase staining may not be due to NOS. Despite these cautions, however, diaphorase staining has been a valuable tool in establishing the range of NOS distributions in animals. The biochemical studies on the brains of locusts (Elphick et al., 1993) and of the marine molluscs Pleurobranchia and Aplysia (Moroz et al., 1996) demonstrate NOS enzymatic activity that shows the requirements for 6-NADPH and calmodulin that characterize some mammalian NOSs. The large size of invertebrate neurons, especially those from molluscs, coupled with capillary electrophoresis, has subsequently allowed the detailed examination of NO metabolism from single, identified neurons (Moroz et al., 1999). A gene for NOS was isolated from Drosophila (Regulski and Tully, 1995). It has 43% sequence identity with rat neuronal NOS and contains the putative FMN, FAD, NADPH, heine and calmodulin binding sites that are general features of constitutive NOS isoforms. NOS has since been cloned from other insects (the moth Mand, ca sexta, Nighorn et al., 1998; the grasshopper Locusta, Ogunshola et al., 1995) and from the snail Lym,aea stagnalis (Korneev et al., 1998). In vertebrates, NO acts on target cells via multiple pathways. The best characterized pathway is through activation of and the production of cGMP (Schuman and Madison, 1994; Garthwaite and Boulton, 1995). More recently, production of poly-ADP-ribose has been shown to mediate some NO actions (Zhang et al., 1994) as have redox pathways involving nitrosylation of cellular proteins (Stamler et al., 1997). Actions through the latter two pathways are still largely unexplored in invertebrates, and the only target of NO signaling that has been well characterized are the soluble guanylate cyclases (sGCs). Both Drosophila and the moth Mand, ca have homologs of sGCal and sGC[31. These function as an obligate heterodimer that is stimulated by NO (Shah and Hyde, 1995; Nighorn et al., 1998). In addition to the NO stimulatable sGCs, both the lobster (Prabhakar et al., 1997) and Mand,ca (Nighorn et al., 1999) have an additional cytoplasmic cyclase that is not NO-sensitive. In Mand, ca this is a novel 6 subunit, MsGCI33, that has little or no NO sensitivity and which needs not form a heterodimer for activity. The MsGC[33 lacks some of the residues thought to be needed for heme binding and it is insensitive to the sGC inhibitor 418

Invertebrate models for studying NO-mediated signaling

Ch.X/

1H-[1,2,4]oxadiazolo[4,3-0~]quinoxalin-l-one (ODQ; Garthwaite et al., 1995). The function of this novel cyclase has not been established but it may be involved in a peptide signaling pathway that involves the peptide eclosion hormone (Morton, 1997).

3. DEVELOPMENTAL ROLES FOR NO/cGMP SIGNALING IN INVERTEBRATES The developmental role of the NO/cGMP signaling pathway has been studied primarily in molluscs and arthropods. Developmental studies in molluscs are in their early phases and represented by reports of changes in NADPH-diaphorase histochemistry during embryonic and metamorphic development of the gastropods llvanassa obsoleta (Lin and Leise, 1996) and Lvmnaea stagnalis (Serfozo et al., 1998). More extensive data are available for the arthropods, and these animals are the main focus of this section. 3.1. NO SIGNALING AND PROLIFERATION CONTROL The role of NO in the control of proliferation is an emerging area of developmental research in invertebrates. The first evidence that NO regulates cell division came from the study of the growth of imaginal discs in Dpvsophila (Kuzin et al., 1996). These structures grow in the larva to give rise to adult structures such as the legs, wings, genitalia, etc. NADPH-diaphorase staining indicated that NOS activity appears in imaginal discs early in the last larval stage, as these structures are entering their rapid phase of growth. Pharmacological inhibition of NOS in larvae resulted in excessive growth of the discs and hypertrophied structures in the adult. By contrast, the ectopic expression of NOS resulted in undergrown structures. These findings suggest that NO acts as an antiproliferative agent during Drosophila development. A role for NO in proliferation control is also evident during neurogenesis in the brain outer proliferation zone (OPZ) in the tobacco hornworm moth Manduca sexta. This zone is a band of proliferating stem cells that produce the interneurons of the outer two layers of the optic lobe (the lamina and medulla). Early in metamorphosis, proliferation in the OPZ requires the presence of the steroid hormone, 20 hydroxyecdysone (20E) (Champlin and Truman, 1998). Importantly, cells in the zone respond to steroid in concert, such that all of the cells are either cycling or quiescent depending on steroid concentration. This coordinated proliferation suggests that there must be a non-cell-autonomous aspect to the ecdysteroid response that allows the system to respond as a unit. This coordination appears to be established via NO signaling (D. Champlin and J. Truman, unpubl, data). The OPZ of Manduca shows intense NADPH-diaphorase staining at the beginning of metamorphosis. In addition, cells in the proliferation zone stain with an antibody directed against a conserved region of NOS. The possibility that NO coordinates mitotic activity within the proliferation zone was tested in vitro using brains cultured in subthreshold levels of 20E (20 ng/ml), which arrested their proliferation. Brains w~'re then shifted to threshold levels of 20E (60 ng 20E/ml) with or without the NOS inhibitor, L-NAME. In the absence of L-NAME, the proliferation zone showed the expected all-or-none response to the steroid. In the presence of L-NAME, by contrast, the proliferation zone responded asynchronously, with a scattering of neuroblasts cycling independently within each optic lobe (D. Champlin and J. Truman, unpubl, data). Importantly, proliferation was still steroid-dependent in the presence of L-NAME, but L-NAME reduced the threshold concentration at which the steroid could initiate scattered proliferation. These results argue 419

Ch. XI


that the NO diffusion serves to coordinate cell division within the proliferation zone and that it does so through a suppression of steroid-induced proliferation. Interestingly, in both Drosophila and Manduca NO may not act via the production of cGMP to regulate cell proliferation. For the imaginal discs of Drosophila, the cells do not produce detectable cGMP in response to NO donors as measured using cGMP immunocytochemistry (Wildemann and Bicker, 1999a). The same is true for the neuroblasts in the OPZ in Manduca (D. Champlin and J. Truman, unpubl, data). Moreover, in the latter, the inhibition of sGC activity using 1H-[1,2,4]oxadiazolo[4,3-~]quinoxalin-l-one (ODQ; Garthwaite et al., 1995) had no effect on coordinating proliferation within the zone. Therefore, NO may be acting through another pathway, such as protein nitrosylation (Stamler et al., 1997). 3.2. NO SENSITIVITY DURING NEURONAL DEVELOPMENT After their birth, neurons progress through a number of discrete developmental phases, starting with axonal outgrowth and growth cone navigation, progressing through target recognition and branching over its target, and ending with synaptogenesis. Studies on a number of developing arthropods, including embryonic grasshoppers (Truman et al., 1996; Ball and Truman, 1998), Drosophila (Wildemann and Bicker, 1999a), and Manduca (Grueber and Truman, 1999) as well as metamorphic lobsters (Scholz et al., 1998), moths (Schachtner et al., 1998), and flies (Gibbs et al., 2000) have shown that many neurons exhibit a transient phase of NO sensitivity during their development. This sensitivity is defined by the ability of the neuron to produce levels of cGMP, that can be detected by immunocytochemistry, in response to a challenge with an NO donor in the presence of IBMX. The relationship between the appearance of NO sensitivity and the progression of neuronal development has been precisely established for two systems: developing motor neurons in the embryonic nervous system of grasshoppers (Truman et al., 1996; Ball and Truman, 1998) and the developing photoreceptors in the visual system of Drosophila (Gibbs and Truman, 1998). Grasshopper embryos have been a model system for studying neurodevelopment for almost 25 years, and the early development of many identified neurons is known in great detail (e.g., Goodman et al., 1984). As seen in Fig. 1, filleted embryos exposed to an NO donor and IBMX show a dramatic production of cGMP throughout their peripheral and central nervous systems (Truman et al., 1996; Ball and Truman, 1998). These embryos are especially convenient for studying early aspects of neuronal development because abdominal segments are produced sequentially, with about a 6-h delay between successive segments. Consequently, in a single embryo one can examine the properties of a neuron at progressively earlier times in development by examining cells in more posterior segments. As seen in Fig. 2, for particular identified motor neurons, such as RP2 and aCC, the onset of NO responsiveness is relatively abrupt, appearing over the space of a few hours. At the time the cell first becomes

Fig. 1. Photomicrograph of a filleted grasshopper embryo at 57c~ of embryonic development, showing cGMP immunoreactivity that was induced by incubation with a NO donor and an inhibitor of phosphodiesterase (IBMX). The embryo was cut down the dorsal midline, gutted, and the body wall pinned out to provide maximal access to the nervous system by bathing chemicals, cGMP production was confined to neurons in the central and peripheral nervous systems. Lines and letters down the right of the figure indicate the boundaries between the subesophageal (S), thoracic (T), and abdominal (A) regions of the embryo. Inset shows a higher magnification view of the cell bodies and processes of neurons that are responding in the fourth abdominal ganglion. Scale bar = 300 mm for main figure. Modified from Ball and Truman. 1998. 420


Ch. XI

NO-sensitive, it has the form of a 'balloon on a string', having a cell body and long axon but with little or no central or peripheral branches. Dendritic branches begin to be elaborated soon after the cell becomes NO-sensitive (Fig. 2).

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N.L. Scholz, and J.W. Truman

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Fig. 2. Drawings showing the changes in the central arbor of grasshopper motor neurons RP2 and aCC after they become NO-responsive. The abdominal segments (A I-A6) form sequentially with an approximate 6 h delay between segments. Therefore, the most posterior neurons are developmentally the youngest. When the cells first respond to NO, they already have an axon that extends to their target muscle but only a very weak cGMP response is observed in the cell body. Cells that are 6 h older in development show complete staining of their axon and initial process but these are relatively unadorned. Branches and filopodia begin to extend soon thereafter. The drawing of the axon is truncated in all cases. From Ball and Truman (1998).

The relationship between pre- and post-synaptic cells during the time of NO sensitivity can be best appreciated in the case of motor neurons at the developing neuromuscular junction. This is illustrated for neuron VML1 and its target, muscle 187 (Fig. 3). Detectable NO sensitivity first appears when the motor neuron's peripheral axonal growth cone is on its target muscle but still has a compact shape and lacks branches (Fig. 3A). Shortly thereafter, the growth cone begins to branch and spreads over the developing muscle target (Fig. 3 B - E ) . The photoreceptors in the developing eye of Drosophila also show an abrupt onset of NO sensitivity. The birth of photoreceptor neurons starts midway through the last larval stage as a morphogenetic furrow moves across the developing eye imaginal disc (Wolff and Ready, 1993). The photoreceptor axons grow into the optic lobe towards targets in the forming lamina or medulla. When they reach these targets, they then arrest their development until the onset of metamorphosis. Soon after the start of metamorphosis the photoreceptors terminate their arrest and their growth cones begin to move laterally within their neuropilar layer to make contact with the interneurons that will make up the lamina 422


Ch. XI

Fig. 3. Photomicrographs of the right ventral body wall in segment A4 from progressively older grasshopper embryos showing the spread of the axonal arbor of neuron VML1 over its target muscle (number 187). The arbor was revealed by its cGMP production after the filleted embryo was exposed to a NO donor and IBMX. The period spans the interval from the beginning of the peripheral cGMP response in segment A4 at about 50c~ of embryonic development (% E) (A) to the fadeout of cGMP expression at approximately 90c~ E (I). A-E span from 50 to 60% of embryonic development, whereas F-I span from about 65c~ to 90c~ E. Two prominent branch points are indicated in successive frames by the asterisk and the arrow. The body wall and muscle grow through this period so the branch point indicated by the asterisk is out of the image alter part (E). Scale bar = 25 mm in A, 50 mm in 1. From Ball and Truman (1998).

c a r t r i d g e s a n d the m e d u l l a c o l u m n s ( M e i n e r t z h a g e n a n d H a n s o n ,

1993). It is at this p o i n t

that the a x o n s o f the p h o t o r e c e p t o r s b e c o m e r e s p o n s i v e to N O (Fig. 4; G i b b s a n d T r u m a n , 1998). 423

Ch. XI


Fig. 4. Photomicrographs of whole mounts of Drosophila brains that have been immunostained for cyclic GMP after treatment with an NO donor in the presence of IBMX. The axons of the developing photoreceptors are not yet responsive to NO donors at A 10 h after puparium formation (APF) but are so by B 16 h APF (arrows). Many neurons in the medial region of the brain are already NO-responsive by 10 h APE E -- photoreceptors clusters in the developing compound eye; OL = optic lobes. Modified from Gibbs and Truman, 1998.

A number of points can be made by a comparison of the grasshopper and fly examples. NO sensitivity is not evident in the neurons as they are undergoing axonal extension and navigation. It appears abruptly, though, when they begin to interact with their synaptic targets, a situation that might require an active dialog between pre- and post-synaptic cells. Although the data are incomplete, a similar relationship appears to hold for a variety of neuronal types (e.g., sensory neurons and interneurons) in a number of organisms (Truman et al., 1996; Schachtner et al., 1998; Scholz et al., 1998). Interestingly, for interneurons that do not have an obvious axon, the cell becomes NO-sensitive after it produces a 'basic skeleton' of its arbor but before it makes extensive branches and elaborations (Ball and Truman, 1998). In parallel with the situation found in motor neurons and sensory neurons, this initial 'skeleton' may be a product of pathfinding mechanisms while the higher-order branching would then be established through an interactive dialog with synaptic partners. The latter would involve NO signaling. The timing of appearance of NO sensitivity in a circuit may provide an anatomical marker for the onset of the interactions between per- and postsynaptic targets. For example, in the developing visual system of Drosophila, the first cells to become NO-responsive are the photoreceptors and the interneurons in the last synaptic neuropil, the lobula. Interneurons in the intermediate visual layers, the lamina and medulla, showed NO sensitivity about 20 to 30 h later. This pattern suggests that wiring first starts from the input and output portions of this brain region and the intermediate area is filled in later (Gibbs and Truman, 1998). A similar relationship is evident in the developing antennal system in the moth, Manduca sexta. A subset of antennal lobe interneurons become NO-responsive at the time of major synapse formation within the antennal lobe (Schachtner et al., 1998, 1999). In the larval lobster, one finds that NOS is upregulated in the accessory lobes of the brain just before the animal undergoes metamorphosis (Fig. 5; Scholz et al., 1998). NOS staining is most intense in the developing cortex and glomeruli, which presumably reflects a role for NO in the progressive wiring of synaptic contacts between deuterocerebral interneurons and projection neurons. The signals that cause a developing neuron to become NO-responsive are poorly understood. Target contact may be necessary for sensitivity to occur but this alone appears not to be sufficient (Ball and Truman, 1998: Gibbs and Truman, 1998). For example, in the fly visual system the first-born photoreceptors grow into the lamina and medulla regions but then wait for about 24 h before the appropriate hormonal signals, provided by 20E, cause them 424

Ch. XI


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Fig. 5. Developmental upregulation of NOS in the brain of a lobster just before metamorphosis. Panels Al-A3 represent single confocal sections, dorsal to ventral, through the deuterocerebrum. Panels Bl and B2 are highermagnification images corresponding to A2 and A~, respectively. Anti-NOS immunoreactivity becomes intense in the developing glomeruli of the accessory lobes arrowhead in A l and Bi at this stage, a time at which synaptic connections are presumably forming between midbrain interneurons and projection neurons. Note that NOS is expressed in both the cortex (arrowheads in Bi) and the glomeruli (B2). NOS label is also present in the deuterocerebral commissure neuropil (arrows in Al). Scale bar = 100 mm in A. 25 mm in B. From Schoiz et al., 1998.

to become NO-sensitive (Gibbs and Truman, 1998). For the developing antennal system in

Manduca, the steroid hormone 20E is also important for shifting developing neurons into an NO-sensitive state. The premature exposure of neurons to high levels of 20E results in the early appearance of NO sensitivity (Schachtner et al., 1999). For both the eye and antennal systems, though, it is not known if steroid exposure alone is sufficient or if the cells need both steroid and contact with potential targets. Although NO sensitivity is a dominant theme for developing neurons in the insect CNS, it is not a feature of every neuron. In the developing antennal lobe of Manduca, for example, only a subset of the antennal interneurons show NO sensitivity during metamorphosis (Schachtner et al., 1999). Also, in grasshopper embryos, many of the body wall muscles receive multiple innervation, but only one or two of the neurons projecting to a given muscle become NO-sensitive (Ball and Truman, 1998). The order by which axons arrive at their targets does not appear to determine NO sensitivity. It may relate instead to the types of synapses that are to be made. For example, among the motor neurons that innervate the extensor tibiae muscle in grasshoppers, the slow extensor tibiae motoneuron (SETi) becomes NO-responsive during its development whereas the fast extensor tibiae motoneuron (FETi) does not (Ball and Truman, 1998). Likewise, in Drosophila very few of the motor neurons are NO-responsive during embryonic development but they become so later when they innervate muscles in the adult (Wildemann and Bicker, 1999a; Gibbs et al., 2000). 425

Ch. XI

N.L. Schol:~ and J.W. Truman

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Fig. 6. Nuclear localization of anti-cGMP immunoreactivity in two different neurons in the ventral nerve cord of a lobster at metamorphosis. Scale bar = 25 mm in B, 10 mm in A. From Scholz et al., 1998.

For many neurons, NO sensitivity is a transient feature of their development. It occurs while synaptic sites are being selected but then wanes as synapses mature (Fig. 3; Truman et al., 1996; Ball and Truman, 1998: Gibbs and Truman, 1998). For many of these developing neurons, their cGMP distribution after a challenge with NO is unusual in that it includes a prominent accumulation of cGMP within the nucleus (e.g., Fig. 6) (Truman et al., 1996; Scholz et al., 1998). This nuclear targeting of cGMP may be a feature of a developmental, rather than a physiological function of NO/cGMP signaling. This conclusion comes from the examination in grasshopper embryos of neurons, like the 'H' cell, that are NO-sensitive during their early development but then continues to be NO-responsive as a mature neuron. Treatment of the immature cell results in accumulation of cGMP in the nucleus whereas in the mature cell cGMP is excluded from the nucleus and is associated with cytoplasmic or cell membrane sites (Truman et al., 1996). In invertebrates, it is not known which potential cGMP target is responsible for the nuclear localization of cGMP. One of the mammalian cGMP-dependent protein kinases translocates to the nucleus after binding cGMP (Gudi et al., 1997). A similar kinase may play a role in developing invertebrate neurons and be responsible for the nuclear cGMP that is observed. This possibility is exciting because it provides a direct intercellular signaling pathway between a neuron and the nucleus of its target cell. As described below, some neurons may continue to be NO-sensitive during post-embryonic life because NO signaling constitutes part of their mature physiology. In other cases, though, it may reflect a continuing developmental plasticity. The latter is thought to be the case for bristle sensory neurons in the grasshopper. During embryogenesis, these sensory neurons show a window of NO sensitivity after their axons enter the CNS. This sensitivity transiently reoccurs, though, as the insect subsequently undergoes each of its nymphal molts (Truman et al., 1996). A possible reason for this reoccurrence comes from studies on the related cricket, in which bristle afferents are known to shift their post-synaptic targets as the animal grows and this changing of targets involves retrograde signaling (Murphey and Davis, 1994). Similar shifts probably also occur in growing grasshoppers and these periods of NO sensitivity may show when the retrograde signaling is taking place to allow these changes. In grasshoppers, NO sensitivity of motor neurons is essentially lost at hatching. However, in larval stages of both Manduca (Truman, unpubl, data) and Drosophila (Wildemann and 426


Ch.X~

Bicker, 1999a) motor neurons continue to show persistent NO sensitivity as the larva grows. The larvae of both species grow rapidly, a process accompanied by the rapid enlargement of body wall muscles. Post-embryonic NO sensitivity in these animals may be involved with a need to adjust synaptic strengths as the target muscle grows. The proposal of NO signaling at the larval neuromuscular junction is supported by the experiments in Drosophila, which show that both NO and membrane-permeant cGMP can induce vesicle release at the neuromuscular junction (Wildemann and Bicker, 1999b). This section has focussed primarily on neurons that can show cGMP production in response to NO donors. In many of the systems, though, the nature of the normal signaling molecule is not known. It is likely NO in the case of the Drosophila visual system, since the optic neuropils exhibit both NADPH-diaphorase staining and NOS-like immunoreactivity (Gibbs and Truman, 1998). The diaphorase activity in the optic lobes appears to be dependent on the presence of the retinal axons (Atkinson and Panni, 1999). Also, as described below, the inhibition of NO synthesis, or the presence of NO scavengers, disrupts neuronal projections (Gibbs and Truman, 1998). For the developing neuromuscular system, though, the nature of the signaling molecule is unclear. There is no diaphorase activity associated with developing or growing muscle in either grasshopper or Dtvsophila (Truman et al., 1996; Wildemann and Bicker, 1999a). Possibly, this tissue may contain a form of NOS that lacks fixation-insensitive diaphorase activity. Alternatively, another signaling molecule, such as CO, may be involved. 3.3. FUNCTIONAL SIGNIFICANCE OF NO/cGMP SIGNALING The correlation of NO sensitivity with a particular phase of neuronal development suggests that NO/cGMP signaling is required for some aspect of synapse formation. The first experimental test of such a function in invertebrates was for the development of the visual system in Drosophila. As described above, the photoreceptor axons become NO-responsive at about 10 h after puparium formation (APF), when they begin to seek out contact with the visual interneurons in the lamina and medulla (Meinertzhagen and Hanson, 1993). Also, NOS immunoreactivity is highest in the developing optic ganglia from this time through to the midpoint of metamorphosis (Gibbs and Truman, 1998). The brain/eye imaginal disc complexes from Dtvsophila larvae can be cultured and respond to ecdysteroids by the production of an appropriately layered optic lobe. Pharmacological manipulations of the NO/cGMP pathway including inhibition of NOS activity (L-NAME treatment), the scavenging of NO (using PTIO), and the inhibition of sGC (with ODQ) all caused a disorganization of the retinal projections into the medulla (Gibbs and Truman, 1998). Photoreceptor axons often moved out of their normal synaptic layers and extended into deeper layers of the optic lobe or even into the central brain (Fig. 7). The disruptive effects of L-NAME were not observed when we used the inactive form, D-NAME. Also, the disorganizing effects of k-NAME were rescued by adding a 10-fold excess of arginine. Most interestingly, though, the abnormal development provoked by 1 mM L-NAME was prevented by the addition of 8-bromo-cGMP to the cultures. Thus, the developmental effects of blocking NO production can be rescued by a proposed, down-stream effector. This strongly supports the interaction of NO and cGMP in visual system development. The effects of the NOS inhibitors provide some insight into a role for NO/cGMP signaling in neuronal development. NO sensitivity appears at the time when the growth cone is switching its behavior from responding to cues important for pathfinding to interactive associations with potential synaptic partners. When NO signaling is blocked, the growth cone 427

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Fig. 7. Photomicrographs of the left optic lobe from brain-eye complexes that were cultured in various concentrations of the NOS inhibitor, L-NAME, and then immunostained with an antibody against chaoptin, which is an antigen expressed on photoreceptor axons. The staining shows the projection of photoreceptor axons into the lamina (1) and the medulla (m). Numbers refer to control complexes (0). and complexes that show progressively greater disruption of the projection pattern due to the presence of the NOS inhibitor (1 to 4). From Gibbs and Truman, 1998.

resumes extension into deeper brain layers rather than branching in its appropriate synaptic layer. Hence, the local action of NO at this time may serve to maintain the growth cone in the appropriate neighborhood as it selects synaptic partners. After these contacts are made and synaptogenesis has begun, such an arresting agent would no longer be necessary. This relationship is especially interesting considering recent work with cultured neurons showing that cyclic nucleotides change the manner by which growth cones respond to signaling molecules (Song et al., 1998).

4. THE ROLE OF NO IN FEEDING BEHAVIOR

Recent mechanistic studies have found that NO has certain conserved functions, even among animals that are only distantly related. A prominent example is the selective role of NO in feeding. This topic has been reviewed by Jacklet (1997), and we will only briefly consider recent studies linking NO to feeding-related behaviors in animals from different phyla. Nitric oxide mediates the chemosensory-induced activation of feeding behavior in 14ydra (Colasanti et al., 1997), the most primitive animal known to possess a nervous system. H~'dra express a calcium-dependent but calmodulin-independent NOS. When a feeding tentacle comes in contact with a prey item, transmitter-evoked calcium influx is thought to trigger local NO synthesis (Colasanti et al., 1997). NO is apparently involved in triggering the discharge of some of the nematocytes (Salleo et al., 1996). It also rapidly diffuses to neighboring tentacles thereby recruiting these appendages into the feeding response. An NO-sensitive guanylate cyclase is also expressed in this primitive organism (Colasanti et al., 1995) and NO-activated cGMP resets the system by inhibiting the feeding response on longer timescales. Thus, the selective association between NO/cGMP signaling and chemosensory function (e.g., Breer and Shepherd, 1993) appears to have been conserved throughout nervous system evolution. NO has also been shown to trigger feeding behaviors in molluscs and crustaceans. In the gastropod mollusc Lymnaea, NO is an anterograde transmitter between chemoreceptor neurons in the lip and the feeding motor network in the central nervous system (Elphick et al., 1995). NO release within the nervous system triggers feeding movements of the buccal mass (Moroz et al., 1993; Elphick et al., 1995). Similarly, in the crab Cancer productus the release of NO into the stomatogastric nervous system underlies the dynamic assembly of a feeding 428

Invertebrate models for studring NO-mediated signaling

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motor network (Scholz et al., 1997). As in Hydra, NO acts at least in part via the stimulation of a sGC in the feeding networks of molluscs and crustaceans alike. The role of NO in the feeding behaviors of other invertebrates is less well known. Among echinoderms, NOS-like immunoreactivity has been detected in the neurons that innervate the cardiac stomach of the starfish Marthasterias glacialis (Martinez et al., 1994). Starfish feed by everting their stomach, thereby encapsulating and digesting the soft tissues of their prey. Recently, Elphick and Melarange (1998) found that NO relaxes the smooth muscle of the starfish cardiac stomach, indicating that NO may play a role in feeding in these animals as well. Interestingly, the NO-induced relaxation of muscle tone in starfish is similar to NO-mediated non-adrenergic, non-cholinergic (NANC) neurotransmission in the mammalian gut (reviewed by Boeckxstaens and Pelckmans, 1997). Anatomical studies of the feeding systems of other invertebrates have generally revealed an enrichment of NO-synthesizing neurons in the networks associated with food intake or the gut. For example, in the planarian Dugesia tigrina NOS is almost exclusively expressed in the pharynx (Eriksson, 1996). A similar profile was observed for the nematode Ascaris suum (Bascal et al., 1995). Curiously, the leech Hirudo medicinalis seems to be an exception to this rule. Leake and Moroz (1996) found little or no NOS expression in the leech gut, although it was found in other annelids. The authors speculate that this may be an adaptation to episodic feeding on hemoglobin-rich blood. Since hemoglobin is an NO scavenger, the perfusion of the gut with blood would be expected to limit or entirely block NO-mediated neurotransmission.

5. THE ROLE OF NO IN REGULATING RHYTHMIC MOTOR NETWORKS In molluscs and crustaceans, the neurons which drive rhythmic feeding behaviors (e.g., rasping, chewing, and swallowing) are often assembled into central pattern-generating (CPG) networks. The NO/cGMP pathway has been identified in a few of the more extensively studied model systems, and electrophysiological studies have provided considerable insights into the role of NO in the organization of motor behaviors. This section will compare NO-mediated signaling in the feeding circuit in gastropod molluscs to its function in the stomatogastric ganglion of crustaceans. Subsequently, these networks will be contrasted to a non-feeding CPG, the cardiac ganglion which drives the neurogenic heart of crabs. 5.1. THE GASTROPOD FEEDING CIRCUIT NO activates a feeding motor program in the gastropod mollusc Lymnaea stagnalis (Moroz et al., 1993; Elphick et al., 1995), a freshwater pond snail that feeds via the scraping movements of a toothed radula. The behavioral elements involve a simple three-phase activity cycle that includes an extension of the radula, followed by a rasp, and then swallowing (Rose and Benjamin, 1979). These movements are driven by a CPG network, the components of which have been well-studied (reviewed by Benjamin and Elliott, 1989). The CPG consists of a network of interneurons that coordinate the activity of feeding motor neurons in the buccal ganglia. The CPG, in turn, is modulated by central interneurons in the buccal and cerebral ganglia. The feeding motor program can be directly activated by chemosensory inputs from the lips or by stimulating the modulatory interneurons. Many neurons in the buccal ganglion contain NOS, as shown in studies using NADPHdiaphorase histochemistry (Elofsson et al., 1993; Moroz et al., 1993) and anti-NOS immunocytochemistry (Moroz et al., 1994). Moreover, exogenous applications of the NO donor 429

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N.L. Schol:~ and J.W. Truman

S-nitrosocysteine or L-arginine elicit movements of the buccal mass, which led to the early conclusion that the NO-producing neurons in the buccal ganglion activate the CPG (Moroz et al., 1993). It was subsequently shown that lip chemosensory afferents are the primary source of NO in the feeding motor system (Elphick et al., 1995). Bipolar neurons in the lip stain intensely with NADPH-diaphorase histochemistry. These cells project to the cerebral ganglia and arborize in close proximity to modulatory interneurons in the feeding network. Chemosensory stimulation of the lips evokes a fictive feeding motor pattern in in vitro preparations of the Lymnaea nervous system. NO donors and a cGMP analogue mimic this effect when applied directly to the isolated central nervous system (Elphick et al., 1995). In addition, chemosensory-induced activation of the CPG is blocked when extracellular NO is scavenged with hemoglobin. NO thus appears to act as an anterograde neurotransmitter between sensory neurons in the lip and the CPG. The central release of NO presumably activates a guanylate cyclase in an as yet unidentified group of target neurons within the CNS. While central NO diffusion is required to initiate motor output from the CPG, it does not modulate synaptic interactions within the motor network itself (Elphick et al., 1995). 5.2. THE CRUSTACEAN STOMATOGASTRIC GANGLION The crustacean stomatogastric ganglion (STG) is a model system for studying how neuromodulatory transmitters act on a common pool of neurons to specify different functional networks. There are only ---30 neurons in the STG, and the physiological and synaptic properties of these cells have been extensively studied for more than 30 years (Harris-Warrick et al., 1992). STG neurons are arrayed into two functionally distinct motor networks which direct the movements of feeding structures in the foregut. These two networks, the gastric mill and pyioric, drive the grinding teeth of the gastric mill and the contractions of the pylorus, respectively. Rhythmic motor output from the STG is not fixed. Instead, the functional configuration of each circuit depends upon neuromodulatory and hormonal inputs to the system. At present, more than 20 different biogenic amines and neuropeptides are thought to be released into the STG neuropil (Harris-Warrick et al., 1992). Each transmitter evokes complex changes in the cellular and synaptic properties of individual neurons within the ganglion. These changes range from subtle modifications of an individual cell's activity pattern to the complete reorganization of an entire circuit. It has only recently become possible to study the distribution of NOS in the crustacean nervous system. The localization of crustacean NOS proteins has lagged behind parallel studies in other invertebrates, in part because conventional NADPH-d histochemistry does not label the majority of NOS-containing cells in the crustacean nervous system (Scholz et al., 1998). This may reflect an unusual sensitivity of the crustacean enzyme to paraformaldehyde fixation, as has been shown for the endothelial form of vertebrate NOS (Dinerman et al., 1994). However, the introduction of specific antibodies to universal NOS (uNOS) and citrulline (a product of NO biosynthesis) has now made it possible to detect NO-producing neurons in crustaceans. The anatomical distribution of key enzymes in the NO/cGMP signaling pathway suggests that NO acts as an anterograde transmitter in the STG. NOS-like proteins have been identified in the STG of the crab Cancer productus using an antibody to uNOS (Scholz et al., 1997). Citrulline accumulates in the terminals of neurons that project to the STG (Scholz et al., 1997), suggesting that modulatory inputs release NO directly into the synaptic neuropil of the ganglion. NO donors activate cGMP synthesis (Scholz et al., 1996) and cGMP immunocytochemistry reveals a distinct subset of sGC-expressing cells in the ganglion 430


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l Fig. 8. NO-induced sGC activation in the crab stomatogastric ganglion. The surrounding glial sheath was removed and the ganglion was treated with an NO donor (SNP) in the presence of IBMX. Following NO stimulation, the ganglion was fixed, processed for anti-cGMP immunocytochemistry, and imaged on a confocal microscope. As previously reported (Scholz et al., 1996), NO activates cGMP synthesis in --~13 of the 30 neurons in the ganglion. Note that cGMP staining intensity varies among individual neurons. Within a neuron, staining is diffusely distributed in somata, the central neuropil (the principal site of synaptic contacts in the ganglion), and in the axons that project out to the muscles that line the foregut. Scale bar = 50 ram. (N.L. Scholz, unpublished results.)

(Fig. 8). These NO-sensitive neurons (~ 13) innervate both gastric and pyloric muscles (Scholz et al., 1997), providing little clue as to how NO might modulate STG networks. Importantly, NO/cGMP signaling specifies the temporary assembly of a complete functional network within the adult STG. Electrophysiological studies from in vitro preparations of the STG have shown that a tonic release of NO within the ganglion is required for the production of gastric mill behavior (Scholz et al., 1997). Blocking anterograde NO diffusion 431

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5s Fig. 9. Inhibition of an NO-sensitive sGC results in the functional reorganization of the gastric mill motor circuit in the crab stomatogastric ganglion. Under basal conditions, the DG motor neuron (as recorded extracellularly from the dorsal gastric nerve, or dgn) and the GM motor neurons (as recorded from the anterior ventricular nerve, or avn) exhibit rhythmic bursts of coordinated activity. The motor pattern rpidly breaks down when the ganglion is bathed in ODQ, a specific inhibitor of NO-sensitive sGC. The DG neuron falls silent, and the GM cells switch pattern and begin firing in time with a separate network (the pyloric circuit). The effect is reversible, and the gastric motor pattern reforms when the ganglion is returned to normal saline. (N.L. Scholz, unpublished results.)

within the synaptic neuropil results in the rapid and selective disassembly of the gastric mill CPG. Some gastric mill neurons fall silent while others switch pattern and begin firing in phase with the pyloric CPG. The STG is similarly reconfigured when the NO-sensitive sGC is blocked by the inhibitor ODQ (Fig. 9). When PTIO or ODQ treatments are alternated with normal saline, the gastric mill network is selectively dismantled and reassembled over the span of a few seconds. This can be repeated multiple times during the course of a single experiment. Therefore, NO-releasing inputs can functionally rewire an active neural circuit by selectively modulating neurons that share a common diffusional space. NO-mediating signaling in the crab STG differs in some respects from the situation in the gastropod feeding network. In both systems NO originates from external inputs and serves to activate a feeding motor rhythm. However, in molluscs NO simply initiates the feeding motor program and is not involved in the central organization of the behavior (Elphick et al., 1995). By contrast, NO triggers an extensive reorganization of central circuitry in the STG, as manifest by pattern-switching in the gastric mill neurons (Scholz et al., 1997). Although specific mechanisms are unknown, network assembly is likely to involve complex changes in the cellular and synaptic properties of each of the 13 NO-sensitive neurons in the ganglion. In both molluscs and crustaceans, NO subserves specific functions that can be readily traced to the behavior of the intact animal. In crustaceans, NO-induced network partitioning in vitro closely resembles dynamic changes in motor output that have been recorded from freely behaving animals. Chronic recordings from intact lobsters have shown that while the pyloric rhythm is produced continuously, the gastric mill rhythm rapidly switches on and off (Clemens et al., 1998). Similarly, neurons that switch pattern in response to NO/cGMP neuromodulation are the same ones that switch between networks in vivo (Heinzel et al., 1993). We propose that the central release of NO triggers the dynamic assembly of the gastric mill motor network, which in turn drives the teeth of the gastric mill during bouts of feeding. This arrangement is likely to remain in place only as long as there is food in the gut. Upon the cessation of feeding, NO/cGMP signaling would be expected to terminate, returning the STG to a unitary network configuration. There is an intriguing relationship between NO-mediated signaling and the development of STG networks. In the lobster (genus Homarus) STG, the temporal appearance of the NO-sensitive sGC coincides with the first production of gastric mill motor behavior (Scholz et al., 1998). Both appear relatively late during post-embryonic development. At earlier 432


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stages (e.g., in newly hatched larvae) the foregut lacks a gastric mill. This condition persists throughout the larval stages of development, a period when the animal feeds on zooplankton in the water column. During this time the planktonic larva shreds prey tissues using its mandibles (Factor, 1981). The teeth of the gastric mill first appear at metamorphosis when the animal begins its transition down to the seafloor. However, the complete formation and calcification of the teeth is a gradual process that spans several developmental stages. While STG neurons are born and innervate their respective muscles in the embryo (Casasnovas and Meyrand, 1995), motor output from the ganglion is limited to a simple, unique rhythm throughout embryonic and larval stages of development. Adult STG networks begin to elaborate from this larval network at metamorphosis, in parallel with the gradual formation of the gastric mill. Interestingly, NO-responsiveness in the STG first appears in a single neuron at this time (Scholz et al., 1998). Additional neurons become NO-responsive as the teeth form and the gastric motor pattern takes shape (Fig. 10). The close developmental association between NO/cGMP signaling, the formation of the gastric mill, and the elaboration of adult STG networks further supports a key role for NO in the specification of gastric mill motor behavior. 5.3. THE CRUSTACEAN CARDIAC NETWORK The crustacean heart contains a very high level of NOS enzymatic activity relative to other tissues, including both skeletal muscles and the nervous system (Labenia et al., 1998). This raises the possibility that NO regulates cardiac rhythmicity in certain invertebrates, perhaps by mechanisms that are similar to those identified in vertebrates. NO plays an extensive and complex role in the regulation of vertebrate cardiac function, where it acts as a second messenger in the cholinergic control of pacemaking activity in mammalian sinoatrial myocytes (Han et al., 1994, 1995). Unlike mammals, the crustacean heart is neurogenic. The underlying cardiac nervous system consists of only nine neurons: five large motor neurons and four small pacemaking cells contained within the cardiac ganglion (Alexandrowicz, 1932). Together these cells generate stable bursts of activity which drive the rhythmic contractions of the cardiac muscles. Basic mechanisms of pattern generation have been studied in the crustacean cardiac ganglion for more than 50 years (reviewed by Cooke, 1988). Inhibitory and excitatory synaptic inputs originate from a pair of nerves which represent the only connection between the ganglion and the rest of the nervous system. The cardiac network is arrayed along the dorsal lumen of the heart and therefore also receives extensive modulatory input from blood-borne neurohormones. Although the oscillatory properties of crustacean cardiac and stomatogastric neurons are similar (Benson and Cooke, 1984), NO appears to play a very different role in the two nervous systems. Whereas NO acts as an anterograde transmitter in the STG, it appears to serve as an intracellular messenger within the cardiac ganglion. All nine cardiac cells label with an antibody to uNOS (Labenia et al., 1998). Citrulline accumulates in the somata and axons alike (Fig. 11), indicating a distributed release of NO throughout the entire "cardiac network. Moreover, all neurons show detectable cGMP accumulation under basal conditions, and the total cGMP content of the ganglion can be elevated by treatment with NO donors. Surprisingly, basal cGMP levels are --~10 times higher in the cardiac ganglion than in the STG. While overall cyclase activity is due in part to the NO-sensitive sGC, there is also a significant contribution from an NO-insensitive enzyme, the one that has been previously identified in the crustacean nervous system (Prabhakar et al., 1997). Taken together, the anatomical and biochemical data suggest that cardiac neurons both synthesize and respond to NO. 433

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Preliminary electrophysiological studies support a role for NO as an intracellular cardioinhibitor (Labenia et al., 1998). PTIO, which scavenges extracellular NO and was very effective on the crab STG, has no effect on the cardiac motor rhythm. However, blocking sGC activation with O D Q accelerates the firing frequency of the large motor neurons. Intrinsic NO production appears to be sufficient to saturate the physiological system since NO donors and c G M P analogs have no effect on firing activity. NO presumably modulates the oscillatory properties of individual cardiac neurons or synaptic transmission within the ganglion, and may be intermediate to excitatory or inhibitory synaptic inputs from the central nervous system. However, specific mechanisms have yet to be determined. Based on anatomical evidence, NO rarely acts as an intracellular messenger in the crustacean nervous system (Scholz et al., 1998). In this respect cardiac neurons are more similar to mammalian sinoatrial myocytes than to stomatogastric neurons. This raises the interesting possibility that NO may play an evolutionarily conserved functional role in cardiac pacemaking. In both mammals (Han et al., 1994) and crabs, NO acts as an inhibitory messenger. In mammalian primary pacemaker cells, acetylcholine activates N O / c G M P signaling, which in turn inhibits an I_-type calcium current via crosstalk with the c A M P pathway (Han et al., 1995). While a similar mechanism may be in place in the cardiac ganglion, the upstream activator is probably not acetylcholine since this transmitter increases rhythmic burst frequency in the network (Sullivan and Miller, 1990). In summary, N O / c G M P signaling serves different functions in the three model systems surveyed here. The specific role for NO ranges from an anterograde network activator (Lymnaea feeding circuit) to a network reorganizer (crab STG) to an intracellular messenger (carb cardiac ganglion). In all three cases, NO is likely to be a cotransmitter or a parallel modulator in a simple circuit that receives multiple chemical inputs. Therefore, the variation may arise both from subtle differences in circuitry and the presence of other transmitters. Together these models provide an opportunity to compare different mechanisms of N O / c G M P neuromodulation in networks that give rise to discrete behaviors.

6. NO AND NOCICEPTION N O / c G M P signaling is involved in the nociceptive pathway in the rat somatosensory system (e.g., Salter et al., 1996). This pathway may also be involved in nociception in invertebrates. In the moth Manduca sexta, early in metamorphosis the transection of peripheral nerves results in a dramatic production of c G M P in a small set of central neurons, many of which are serotonergic (Schachtner et al., 1998). This c G M P increase in response to injury is suppressed by the presence of L-NAME (J. Schachtner and J. Truman, unpubl, data), indicating the involvement of NO in mediating this response.

Functional Neuroanatomy of the Nitric Oxide System (Handbook of Chemical Neuroanatomy)

Functional Neuroanatomy of the Nitric Oxide System

Glutamate (Handbook of Chemical Neuroanatomy)

Atlas of Functional Neuroanatomy

Functional Neuroanatomy of Pain

Functional Neuroanatomy of Pain

Atlas of Functional Neuroanatomy