The Enteric Nervous System

Home , Gastric acid, Myenteric plexus, Submucosal plexus

John Barton Furness PhD, FAA Department of Anatomy and Cell Biology, University of Melbourne, Victoria, Australia

John Barton Furness PhD, FAA Department of Anatomy and Cell Biology, University of Melbourne, Victoria, Australia © 2006 John B. Furness Blackwell Publishing, Inc., 350 Main Street, Malden, Massachusetts 02148-5020, USA Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK Blackwell Publishing Asia Pty Ltd, 550 Swanston Street, Carlton, Victoria 3053, Australia The right of the Author to be identiﬁ ed as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. First published 2006 Library of Congress Cataloging-in-Publication Data

Furness, John Barton. The enteric nervous system / John B. Furness. p. ; cm. Includes bibliographical references and index. ISBN-13: 978-1-4051-3376-0 ISBN-10: 1-4051-3376-7 1. Gastrointestinal system--Diseases. I. Title. [DNLM: 1. Enteric Nervous System--physiology. 2. Neurons --physiology. WL 600 F988e 2006] RC817.F87 2006 616.3--dc22 2005024527

ISBN-13: 978-1-4051-3376-0 ISBN-10: 1-4051-3376-7 A catalogue record for this title is available from the British Library Set in 10/13½ Sabon by Sparks, Oxford – www.sparks.co.uk Printed and bound by Narayana Press, Odder, Denmark Commissioning Editor: Alison Brown Editorial Assistant: Saskia Van der Linden Development Editor: Rob Blundell Production Controller: Kate Charman For further information on Blackwell Publishing, visit our website: http://www.blackwellpublishing.com The publisher’s policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp processed using acid-free and elementary chlorine-free practices. Furthermore, the publisher ensures that the text paper and cover board used have met acceptable environmental accreditation standards. Contents

Preface, ix

Abbreviations, xi

1: Structure of the enteric nervous system, 1

The enteric plexuses, 3 Interconnections between the plexuses, 14 Extent of the ganglionated plexuses, 15 Intramural extensions of extrinsic nerves, 17 Electron microscope studies, 17 Enteric glia, 20 The structural similarities and functional differences between regions may have an evolutionary basis, 21 Development of the enteric nervous system, 23 Maturation of enteric neurons and development of function, 26 Changes in enteric neurons with aging, 27 Summary and conclusions, 28

2: Constituent neurons of the enteric nervous system, 29

Shapes of enteric neurons, 31 Cell physiological classiﬁ cations of enteric neurons, 43 Functionally deﬁ ned enteric neurons, 53 Neurons in human intestine with equivalence to those investigated in laboratory animals, 76 Summary and conclusions, 78

v vi CONTENTS

3: Reﬂ ex circuitry of the enteric nervous system, 80

Evolution of ideas about enteric circuitry, 80 Motility controlling circuits of the small and large intestine, 81 Intrinsic secretomotor and vasomotor circuits, 88 Assemblies of neurons, 93 Circuits in the esophagus and stomach, 96 Co-ordination of motility, secretomotor, and vasomotor reﬂ exes, 98 Circuits connecting the intestine, biliary system, and pancreas, 98 Sympathetic innervation of the gastrointestinal tract, 99 Summary and conclusions, 101

4: Pharmacology of transmission and sites of drug action in the enteric nervous system, 103

Chemical coding and multiple transmitters, 103 Transmitters of motor neurons that innervate the smooth muscle of the gut, 104 Transmitters at neuro-neuronal synapses, 111 Sites within the reﬂ ex circuitry where speciﬁ c pharmacologies of transmission can be deduced to occur, 120 Transmission from entero-endocrine cells to IPANs, 126 Roles of interstitial cells of Cajal in neuromuscular transmission, 127 Transmitters of secretomotor and vasodilator neurons, 128 Synapses in secretomotor and vasodilator pathways, 130 Transmitters of motor neurons innervating gastrin cells, 130 Summary and conclusions, 130

5: Neural control of motility, 132

Rhythmic activity of gastrointestinal muscle, 132 Structure and properties of interstitial cells of Cajal, 134 Relationship between slow wave activity and neural control, 138 Gastric motility, 140 Patterns of small intestine motility and their intrinsic neural control, 147 Motility of the colon, 157 Neural control of the esophagus, 159 Gall bladder motility, 160 Sphincters, 161 Muscle of the mucosa, 165 CONTENTS vii

Mechanism of sympathetic inhibition of motility in non-sphincter regions, 166 Sympathetic innervation of the sphincters, 169 Physiological effects of noradrenergic neurons on motility in undisturbed animals, 170 Reﬂ ex activities of sympathetic neurons that affect motility, 171 Summary and conclusions, 178

6: Enteric neurons and the physiological control of ﬂ uid secretion and vasodilation, 180

Water and electrolyte secretion in the small and large intestines, 180 Reﬂ ex control of water and electrolyte secretion, 182 Secretion of gastric acid, 189 Pepsinogen secretion, 194 Gastric secretion of bicarbonate, 195 Secretion into the gall bladder, 195 Pancreatic exocrine secretion, 196 Summary and conclusions, 198

7: Disorders of motility and secretion and therapeutic targets in the enteric nervous system, 200

Therapeutic endpoints for motility disorders, 201 Therapies for secretory diarrheas, 205 Enteric neuropathies involving neuronal loss or phenotypic changes, 206 Mitochondriopathies with intestinal manifestations, 207 Irritable bowel syndrome and plasticity of enteric neurons, 208 Summary and conclusions, 210

Epilogue: the future of enteric neurobiology, 211 References, 214 Index, 267

Preface

The enteric nervous system is of special interest because it is the only sub- stantial grouping of neurons outside the central nervous system that form circuits capable of autonomous refl ex activity. In humans it contains around 500 million neurons that fall into about 20 functional classes. Because of its size, complexity, and certain structural similarities, it has been likened to a second brain. Although the enteric nervous system was discovered almost 150 years ago, and several remarkably insightful hypotheses about its functions were made in the 19th century, a long period ensued in which progress was mea- gre in comparison to the effort made, because methods available were not adequate to determine the intrinsic circuitry of the enteric nervous system and the properties of its constituent neurons. In the last 20–30 years, new techniques, and excellent application of such techniques, have provided a wealth of information on the structural complexity, neuron types, and connectivity of the enteric nervous system and on the transmitters and cell physiology of enteric neurons. Beginning at an earlier time, and proceeding in parallel, have been investigations of the patterns of movement and secretory functions of the digestive tract, and their control. This book aims to integrate the detailed cellular knowledge of the enteric nervous system with the more macroscopic information that is provided by physiological studies of organs, especially in the living animal or human. In doing so, I have tried to deal with the emergence of knowledge in historical perspective, where possible by drawing on early information to acknowledge the contributions made by pioneers of enteric neurobiology, and in places to reproduce original illustrations from early publications. I hope that the reader will enjoy this approach. I have also created many new illustrations, especially of the organization of enteric nerve circuits, which I hope will provide an understanding of the enteric nervous system that the written word cannot easily convey. The fi rst four chapters lay the groundwork, by dealing with the structure of the enteric nervous system, the defi ning cell physiological, morphological,

ix x PREFACE and neurochemical properties that allow its neurons to be functionally clas- sifi ed, the enteric neurotransmitters and the intrinsic nerve circuits within the alimentary tract. This is followed by two chapters on gastrointestinal physiology, fi rst on the contractile activity of the muscular walls of the digestive tract and the second on secretory function. In these two chapters I try to develop an understanding of the roles of enteric neurons and how they perform these roles. I have also sought to relate control through enteric circuits to control exerted by the vagus and the sympathetic innervation of the digestive organs, and to a lesser extent through the pelvic nerves. The involvement of altered structure and function of the enteric nervous system in some disease states is well recognized. Nevertheless, how to use the new-found knowledge of the enteric nervous system to understand the rela- tions between changes in the neurons and clinical manifestations of disease is a challenge. Moreover, how the neurons might be manipulated by therapeutic compounds to ameliorate disorders of the digestive system is elusive, in many cases. The problems of understanding and treating digestive diseases that involve the enteric nervous system, or functions controlled by the enteric nervous system, are touched on throughout the book, and are specifi cally discussed in Chapter 7. In writing this book I have relied on the assistance and advice of many colleagues who have generously read and commented on parts of book, in some cases through several drafts. My special thanks go to Dr Paul Andrews, Dr Joel Bornstein, Dr Axel Brehmer, who also helped me with the interpreta- tion of some of the older literature published in German, Dr Nadine Clerc, Dr Helen Cox, Dr Roberto de Giorgio, Dr Giorgio Gabella, Dr Peter Holzer, Dr Terumasa Komuro, Dr Alan Lomax, Dr Kulmira Nurgali, Dr Michael Sche- mann, Dr Keith Sharkey, Dr Henrik Sjövall, Dr Werner Stach, who provided previously unpublished micrographs, Dr Jean-Pierre Timmermans, Dr Mar- cello Tonini and Dr Heather Young. For assistance in the preparation of the illustrations I am very grateful to Melanie Clarke, Anderson Hind, and Trung Nguyen, and for editorial help and assistance with the references, to Emma James. I would also like to thank the many colleagues who gave permission for illustrations to be included in the book. I hope that this book succeeds in linking the extensive knowledge of the structure and cell physiology of the enteric nervous system to an understanding of digestive physiology, and that in so doing it helps provide a rational basis for therapeutic intervention, and even reasons why some interventions may fail. I enjoyed writing the book, although at times it was a hard task. I hope that in reading the book you encounter only the enjoyment. John B Furness Melbourne, May 2005 Abbreviations

AC, adenylyl cyclase ACh, acetylcholine AChE, acetylcholine esterase ADP, after-depolarizing potential AH, designation of neurons having slow after-hyperpolarizing potentials AHP, after-hyperpolarizing potential AMP, adenosine monophosphate AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AP, action potential ATP, adenosine triphosphate BK, large-conductance potassium channel BMP, bone morphogenic protein BN, bombesin (the mammalian form also referred to as GRP, below) cAMP, cyclic adenosine monophosphate CCK, cholecystokinin CFTR, cystic ﬁ brosis transmembrane conductance regulator CGRP, calcitonin gene-related peptide ChAT, choline acetyltransferase CM, circular muscle CNS, central nervous system DAG, diacyl glycerol DMP, deep muscular plexus DMPP, dimethyl phenyl piperazinium DYN, dynorphin ECL, enterochromafﬁ n-like (cell) EJP, excitatory junction potential ENK, enkephalin EPSP, excitatory post-synaptic potential GABA, γ-aminobutyric acid GAL, galanin gCav, voltage-sensitive calcium conductance gK Ca2+-dependent K+ conductance Ca, + gNav, voltage-dependent Na conductance GRP, gastrin-releasing peptide (also known as mammalian bombesin)

xi xii ABBREVIATIONS

Gs, stimulating G-protein 5-HT, 5-hydroxytryptamine (serotonin) HCN, hyperpolarization activated non-speciﬁ c cation conductance HVA, high-voltage activated calcium current I AHP , AHP current IBS, irritable bowel syndrome I Cav, voltage-sensitive calcium current ICC, interstitial cell(s) of Cajal Ih, hyperpolarization-activated cation current IK, intermediate-conductance potassium channel I KAT P , ATP-dependent potassium current IPAN, intrinsic primary afferent neuron IPSP, inhibitory post-synaptic potential LM, longitudinal muscle MAP2, microtubule associated protein 2 MELAS, multisystem mitochondriopathy MMC, migrating myoelectric complex MNGIE, mitochondrial neurogastrointestinal encephalomyopathy MP, membrane potential Muc, mucosa L-NAME, L-nitro-arginine methyl ester nAChRs, nicotinic acetylcholine receptors NANC, non-adrenergic, non-cholinergic NFP, neuroﬁ lament protein Nic, nicotinic NK, neurokinin NO, nitric oxide NOS, nitric oxide synthase NPY, neuropeptide tyrosine, usually known as neuropeptide Y

P2X, purine receptor 2X

P2Y, purine receptor 2Y PACAP, pituitary adenylyl cyclase activating peptide PCR, polymerase chain reaction PDBu, phorbol dibutyrate PHI, peptide histidine isoleucine PHM, peptide histidine methionine PKA, protein kinase A PKC, protein kinase C PLC, phospholipase C PPADS, pyridoxal-phosphate-6-azophenyl-2΄,4΄-disulfonic acid PVG, prevertebral ganglion PYY, peptide tyrosine tyrosine Rin, input resistance RT, room temperature SAC, stretch activated channel SGLT1, Na+/glucose co-transporter 1 SK, small-conductance potassium channel ABBREVIATIONS xiii

SM, submucosa SOM, somatostatin SSPE, sustained slow post-synaptic potential STC, slow-transit constipation TEA, tetraethylammonium TK, tachykinin TRH, thyrotropin-releasing hormone TTX, tetrodotoxin I TTX-R NaV, TTX-resistant sodium current VIP, vasoactive intestinal peptide VPAC, vasoactive intestinal peptide; pituitary adenylyl cyclase activating peptide

1: Structure of the enteric nervous system

A vast amount of neural tissue, which constitutes the enteric nervous system, is embedded in the wall of the gastrointestinal tract. Within the enteric nervous system, nerve cells and supporting (glial) cells are grouped in small clusters, the enteric ganglia, which are interconnected by nerve fi ber bundles (Fig. 1.1). The individual ganglia are small, but are so numerous that the system as a whole contains millions of nerve cells. The processes of these nerve cells connect with other neurons and innervate the muscle, secretory epithelium, and blood vessels of the digestive tract, biliary system, and pancreas. Processes of nerve cells from outside the digestive tract also connect with enteric neurons, and intermingle with processes of enteric neurons. A remarkable aspect of the enteric nervous system is that its refl ex circuits are capable of directing the functions of the digestive system without relying on commands from the brain or spinal cord. This independence is modulated by the rich interchange of signals between the enteric and central nervous systems. The fi rst clear descriptions of ganglionated plexuses within the wall of the digestive tract were those of Meissner (1857), Billroth (1858), and Auerbach (1862a,b, 1864). Remak (1840, 1852) had earlier noted the presence of micro- scopic ganglia in the walls of the pharynx and stomach, but his descriptions do not suggest that he recognized a ganglionated plexus. Following their discovery, the enteric ganglia and plexuses attracted considerable attention and numerous descriptions of their organization were published, including those of Henle (1871), Drasch (1881), Dogiel (1895b, 1899), Cajal (1911), Kuntz (1913, 1922), Hill (1927), Schabadasch (1930a,b), Stöhr (1930), and Irwin (1931). These studies, and the contemporary literature they cite, provide detailed information on the sizes, arrangements and interconnections of the ganglia. The descriptions that Meissner, Billroth, and Auerbach provided of the general organization of the ganglionated plexuses, based on quite primitive techniques to reveal the nerve tissue, were not superseded by work in the subsequent 100 years and the descriptions of the arrangements of the enteric plexuses that

1 2 CHAPTER 1

are set out in the following pages were essentially established by the time of the reviews of Schabadasch (1930a,b) and Stöhr (1930). An English translation of Auerbach’s 1864 description has been published (Furness & Costa 1987). The enteric nervous system of the tubular digestive tract (the esophagus, stomach, and intestines) is formed of a number of interconnected networks, or plexuses, of neurons, their axons, and enteric glial cells (Fig. 1.1). In the

Fig. 1.1 The enteric plexuses as they are seen (A) in wholemounts and (B) in transverse section. The drawings depict the small intestine. There are two ganglionated plexuses, the myenteric and the submucosal plexuses, in addition to nerve ﬁ bers that innervate the muscle layers, the mucosa and intramural arterioles. Nerve ﬁ bers enter the intestine with mesenteric blood vessels in paravascular nerves (B). Adapted from Furness and Costa (1980).

1405133767_4_001.indd 2 31/08/2005 12:15:12 STRUCTURE OF THE ENTERIC NERVOUS SYSTEM 3 small intestine and colon, most nerve cells are found in two sets of ganglia, the ganglia of the myenteric (Auerbach’s) plexus and of the submucosal plexus (often referred to as Meissner’s plexus, but see below). The axons of these nerve cells innervate other ganglia and the tissues of the digestive organs, such as the muscle layers and the mucosa.

The enteric plexuses

Myenteric plexus

The myenteric plexus is a network of nerve strands and small ganglia that lie between the outer longitudinal and inner circular muscle layers of the external muscle coat of the intestine (Fig. 1.2). The network is continuous around the circumference and along the gastrointestinal tract (Fig. 1.3). The myenteric ganglia vary in size, shape, and orientation between animal species and from

Fig. 1.2 Drawing of a wholemount of the myenteric plexus of the human small intestine, prepared by Auerbach and published in Henle’s Text- book of Histology in 1871. Myenteric ganglia, internodal strands, and small nerve trunks of the secondary component of the myenteric plexus (arrows) can be seen. The secondary nerve strands supply ﬁ bers to the circular muscle and deeper layers. Calibration: 1 mm. 4 CHAPTER 1

Fig. 1.3 Schabadash’s (1930a) depiction of the myenteric plexus of the pyloric canal in the cat. The large dark areas are the ganglia, which are con- nected by nerve ﬁ ber bundles of various calibers. The continuity of the plexus around and along the gut wall can be clearly seen. Calibration: 2 mm.

one part of the intestine to another (Fig. 1.4), but the shape of the meshwork is usually characteristic and readily identifi ed in any major region from a particular species (Irwin 1931, Gabella 1981a). Although the pattern is easily recognized, considerable variation in the size of ganglia is encountered. In the ileum of the guinea-pig, ganglia range in size from 5 to over 200 nerve cell bodies. Single nerve cell bodies are occasionally encountered outside the main meshwork of the plexus, usually adjacent to a nerve strand. The ganglia are sometimes referred to as the nodes of the plexus because they lie at the junc- tions of nerve strands, which in turn are called internodal or interganglionic strands, or sometimes interganglionic connectives. The ganglia are deform- able and are distorted by the movements of the muscle. Thus measurement of such features as their shape and spatial density must take into account the state of contraction of the gut wall (Gabella & Trigg 1984). Three components of the myenteric plexus are described (Fig. 1.5): a primary plexus, a secondary plexus, and a tertiary plexus (Auerbach 1864, Schabadasch 1930a,b, Stöhr 1930, Li 1940, 1952). Together, the ganglia and internodal strands make up the primary meshwork of the myenteric plexus. Many of the nerve fi bers in an internodal strand do not enter the ganglion with which a strand connects, but pass over the ganglion, usually between the ganglion and the longitudinal muscle, and continue in another internodal strand. Finer nerve fi ber bundles, constituting the secondary component of the plexus, branch from the primary internodal strands or arise from ganglia, but do not usually link adjacent ganglia. The secondary strands run parallel to the circular muscle bundles and often cross internodal strands. They run on the inner aspect of the primary plexus and ganglia, between the primary plexus and the circular muscle (Schabadasch 1930b, Stöhr 1952). Auerbach (1864) traced nerve processes from the secondary strands to the circular muscle, a connection that has been confi rmed (Wilson et al. 1987). The secondary STRUCTURE OF THE ENTERIC NERVOUS SYSTEM 5

Fig. 1.4 Drawings of the myenteric plexus in different regions of the gastrointestinal tract of the guinea-pig: A, esophagus; B, pylorus; C, duodenum; D, ileum; E, colon; F, rectum. Note that patterns of the ganglia differ between regions. They also differ between species. All at the same magniﬁ cation, except B. The calibration lines are 1 mm apart. Reproduced from Irwin (1931). 6 CHAPTER 1

1 2 1

3 2

Fig. 1.5 The three components of the myenteric plexus found in small animals are shown in a drawing of a wholemount from the guinea-pig small intestine. Common to all species is the primary component of the plexus (1), consisting of the ganglia and internodal strands (interganglionic connectives), and the secondary component (2), consisting of nerve strands lying parallel to the circular muscle (across the page). The tertiary plexus (3) is found only where the longitudinal muscle is thin; in such regions, few nerve fi bers are found within the longitudinal layer. Neuron cell bodies are depicted as white ovals in the ganglia. Redrawn from Furness and Costa (1987). Calibration: 100 μm. strands can be seen in Auerbach’s drawing (Fig. 1.2). The tertiary meshwork (tertiary plexus) is made up of fi ne nerve bundles that meander in the spaces between the meshwork formed by the primary plexus (Richardson 1958, Llewellyn Smith et al. 1993, Furness et al. 2000) (Fig. 1.5). Nerve bundles of the tertiary plexus can be traced from primary internodal strands, ganglia and secondary strands. The defi nition of the tertiary plexus given here accords with that of Stöhr (1930), which is different from that given by Schabadasch and Li. The defi nitions of the latter authors combine the secondary and tertiary plexuses under the name secondary plexus and they call the tertiary plexus those fi ne fi bers that run parallel to and extend into the circular muscle and which join the deep muscular plexus. I refer to these fi bers as the circular muscle plexus, or simply as the circular muscle innervation.

Submucosal plexus

A submucosal ganglionated plexus is found in the small and large intestine (Figs 1.6, 1.7), and was ﬁ rst described in the mid-19th century by Meissner (1857) and Billroth (1858). Although scattered ganglia are found in the submucosal layer in the esophagus and stomach, these do not form a ganglionated plexus comparable to that of the intestines. In general, the interconnecting strands of the submucosal plexus are ﬁ ner and the ganglia are smaller than those of the myenteric plexus (Henle 1871, STRUCTURE OF THE ENTERIC NERVOUS SYSTEM 7

Fig. 1.6 Distribution of enteric ganglia in the tubular digestive tract. The gastrointestinal tract is represented schematically in longitudinal section to reveal the myenteric ganglia, which form a continuous plexus from the upper esophagus to the internal anal sphincter, and the submucosal plexus, which is prominent in the small and large intestines. Isolated ganglia occur in the gastric and esophageal submucosa and in the mucosa throughout the digestive tract. From Furness et al. (1991). 8 CHAPTER 1

Fig. 1.7 Drawing of the submucosal plexus of the small intestine of a 6-day-old child, published by Billroth (1858). It accurately depicts the ganglia, with nerve cells drawn as small circles, and the connecting strands. Note that Billroth depicts ganglia and nerve strands at two levels. Calibration (approx): 250 μm.

Goniaew 1875, Timmermans et al. 2001). The plexus is continuous around the circumference and along the length of the small and large intestines. The arrangements of ganglia in the submucosal plexus, and the functional types of neurons in these ganglia, differ between species (Scheuermann et al. 1987b,c, Hoyle & Burnstock 1989, Timmermans et al. 1990). In large animals, good examples being the pig and human, submucosal ganglia form distinct, but interconnected, plexuses that lie at different levels, as ﬁ rst clearly described by Schabadasch (1930b). Two or sometimes three layers of ganglia have been distinguished (Schabadasch 1930b, Gunn 1968, Hoyle & Burn- stock 1989, Timmermans et al. 2001). Ganglia at different depths contain different populations of neurons, these variations being apparent in the shapes and chemical natures of the constituent nerve cells. The inner ganglionated plexus (closer to the gut lumen) has been likened to the plexus described by Meissner (1857) and the outer has been identiﬁ ed with that described by Henle (1871) and Schabadasch (1930b). Because it is not completely clear who should be credited with the discovery of individual components of the submucosal plexus, it seems sensible to refer to the most obvious groupings as the inner and outer submucosal plexuses (Timmermans et al. 2001), the inner being that closer to the intestinal lumen, and the outer that closest to the circular muscle layer. Among the neurons of the outer plexus are some that supply innervation to the circular and even to the longitudinal muscle (Sanders & Smith 1986, Furness et al. 1990a, Timmermans et al. 1994, 1997, STRUCTURE OF THE ENTERIC NERVOUS SYSTEM 9

Porter et al. 1999). The outer submucosal plexus also supplies innervation to the mucosa. The inner submucosal plexus has few neurons that supply the muscle, but many that innervate the mucosa (Porter et al. 1999, Timmermans et al. 2001). In small mammals, typiﬁ ed by the guinea-pig, there is generally a single layer of submucosal ganglia, and these ganglia contain secretomotor neurons, but not motor neurons that supply the external muscle; in fact, in the guinea-pig there are four main types of neurons in the submucosal ganglia of the small intestine (Furness et al. 1984, 2003a). The ganglia of the submucosal plexus in small mammals most closely resemble those of the inner submucosal plexus of larger species.

Paucity of ganglia in the submucosa of the esophagus and stomach

Extensive submucosal ganglionated plexuses, such as those found in the small and large intestines, do not occur in the esophagus (Harting 1934, Schoﬁ eld 1960, Rash & Thomas 1962, Christensen & Rick 1985a, Izumi et al. 2002). Small groups of nerve cell bodies are occasionally found adjacent to submucosal mucus-secreting glands that are scattered along the esophagus, although some investigators have reported that there are no nerve cell bodies at all in the submucosa of the esophagus (Christensen & Rick 1985a). Submucosal ganglia are absent or extremely rare in the stomach of small animals (guinea-pig and rat) and are sparse, but clearly present, in larger mammals, such as dog, human, and cat (Schabadasch 1930a, Kyösola et al. 1975, Stach et al. 1975, Radke et al. 1978, Christensen & Rick 1985a, Fur- ness et al. 1991, Schemann et al. 2001, Colpaert et al. 2002) and denervation and tracing experiments show that the intrinsic innervation of the gastric mucosa is derived almost entirely from the myenteric ganglia (Furness et al. 1991, Pfannkuche et al. 1998). Submucosal nerve cells that do occur in the stomach are more common in the antrum. Some of the myenteric ganglia extend into the clefts (septa) between the large blocks of circular muscle in the stomach and can be mistaken for submucosal ganglia.

Ganglia in the mucosa

Small groups of nerve cell bodies occur in the lamina propria of the mucosa in the small and large intestine, and, rarely, in the stomach (Drasch 1881, Vau 1932, Stöhr 1934, Ohkubo 1936, Isisawa 1939, 1949, Lassmann 1975, Newson et al. 1979, Fang et al. 1993, Balemba et al. 1998). Stöhr (1934) has suggested that these are displaced (ectopic) submucosal ganglia. These nerve cells are almost always close to the muscularis mucosae, that is, they are close to the inner submucosal plexus. 10 CHAPTER 1

Subserosal plexus

This is a plexus of ﬁ ne nerve bundles that is found in the connective tissue layer at the surfaces of digestive organs, for example between the serosal lining of the peritoneal cavity and the external muscle of the intestine (Schaba- dasch 1930b). These nerve bundles connect extrinsic nerves and nerves of the deeper layers of the gut wall, as was recognized and described by Auerbach (1864). Small ganglia sometimes occur in the subserosal plexus, particularly in the esophagus and stomach and near the mesenteric attachment of the intestine and on the surface of the rectum. Some subserosal ganglia lie within or adjacent to the branches of the vagus nerves as they enter the walls of the stomach and esophagus.

Longitudinal muscle innervation and the tertiary plexus

The longitudinal muscle is innervated by a longitudinal muscle plexus, which consists of fi ne bundles of nerve fi bers that run parallel to and within the muscle, or by the tertiary component of the myenteric plexus, which consists of axons in bundles that lie against the inner surface of the muscle (Richard- son 1958). How the muscle is innervated seems to be simply determined by its thickness. In large animals, and in small animals where this muscle layer is thickened, for example in the teniae which occur in the large intestines of some species, a longitudinal muscle plexus is observed. Where the muscle layer is less than about 10 muscle cells thick, it is innervated exclusively by fi ne nerve bundles of the tertiary component of the myenteric plexus. These bundles are frequently found in small grooves at the inner surface of the muscle (Llewellyn Smith et al. 1993). The tertiary plexus is described in more detail above (see Fig. 1.5).

Circular muscle innervation

Fine nerve bundles that run parallel to the length of the muscle cells are found throughout the thickness of the circular muscle (Fig. 1.8). These bundles connect with the primary and secondary components of the myenteric plexus and with the deep muscular plexus in the small intestine. The nerve ﬁ ber bundles of the circular muscle plexus form a continuous meshwork both around the circumference of the intestine and, through oblique interconnecting nerve strands, along its length. In small mammals, most of the axons within the circular muscle plexus derive from motor neurons with cell bodies in the myenteric ganglia, but there are some ﬁ bers that come from nerve cells in the outer ganglia of the submucosal plexus. Fibers that originate from submucosal ganglia are more numerous in larger species (see above and Chapter 2). STRUCTURE OF THE ENTERIC NERVOUS SYSTEM 11

Fig. 1.8 Nerve ﬁ bers in the circular muscle. This micrograph is of a wholemount preparation of the circular muscle of the guinea-pig small intestine, stained with the Champy-Mail- let zinc iodide and osmium technique. The level of focus corresponds to the deep muscular plexus. Major nerve ﬁ ber bundles run approximately parallel to the long axes of the muscle cells and there are many connections between these bundles. Calibration: 50 μm.

Deep muscular plexus and submuscular plexus

An aggregation of nerve ﬁ ber bundles is found near the inner part of the circular muscle layer of the small intestine (Li 1937, 1940, Taxi 1965, Gabella 1972b, 1974) (Fig. 1.9) and also of the large intestine (Stach 1972, Faussone- Pellegrini & Cortesini 1984, Faussone-Pellegrini 1985, Christensen & Rick 1987b). These concentrations of innervation were described by Cajal (1895,

Fig. 1.9 Diagram to illustrate the nerve supply to the mucosa of the small intestine, as seen in histological section. The nerve ﬁ bers are in small bundles that form a continuous network in the connective tissue of the mucosa, the lamina propria (lp). The mucosal nerve network can be divided into interconnecting subglandular, periglandular, and villous components. Muscularis mucosae, mm; gland, gl. 12 CHAPTER 1

1911) who provided two descriptive names, plexus musculaire profond (deep muscular plexus) and plexus sous-musculeux (submuscular plexus) (Cajal 1911). A concentration of ﬁ bers near the inner surface of the circular muscle coat is not observed in the canine stomach (Furness et al. 1990a), although there is some degree of concentration of innervation in the human stomach (Faussone-Pellegrini et al. 1989). It is of interest that Cajal provided two names, because there is a subtle difference that these two names accommodate. In the small intestine, the plexus separates a thin layer of muscle cells from the bulk of the circular muscle, and here it has been generally referred to as the deep muscular plexus (Li 1937, Gabella 1974). In some regions, good examples being the dog and pig colon, there is no layer of muscle internal to the plexus, which lies at the ex- treme inner surface of the circular muscle, adjacent to the submucosa (Stach 1972, Christensen & Rick 1987b, Furness et al. 1990a). In this position, it can be called the submuscular plexus. The nerve bundles of the deep muscular and submuscular plexuses form continuous meshworks around the circumference and along the intestine. Their predominant orientation is parallel to the direction of the circular muscle, with frequent oblique connections between adjacent bundles (Fig. 1.8). The reason why part of the innervation of the circular muscle is concen- trated close to its inner surface is not known, but the axons have the same spectrum of neurochemical types as axons in the rest of the circular muscle. It may simply be that the circular muscle is innervated asymmetrically, just as the innervation of arteries is asymmetric (in arteries, the axons are primarily at the outer border of the muscle coat) and the innervation of the longitudinal muscle through the tertiary plexus is asymmetric. Interstitial cells of Cajal (ICC) lie in close proximity to nerve ﬁ bers of the deep muscular plexus and they have a critical role as intermediates in transmission between the axons of motor neurons and the smooth muscle cells (Chapters 4, 5).

Innervation of the muscularis mucosae

The layers of smooth muscle at the surface of the mucosa, adjacent to the submucosa, are known as the muscularis mucosae. In general, this consists of inner bundles of circularly disposed smooth muscle cells and outer longitu- dinally oriented smooth muscle, but in some places it is thin and has smooth muscle bundles at various orientations. In the esophagus, the muscle bundles are arranged primarily in a longitudinal direction. Fine nerve fi ber bundles that run parallel to the long axes of the muscle cells make up the innervation of the muscularis mucosae. In the small intestine, bundles of smooth muscle STRUCTURE OF THE ENTERIC NERVOUS SYSTEM 13 that are considered part of the muscularis mucosae make fi nger-like intru- sions into the cores of the villi and in the stomach similar slivers of muscle are found between gastric glands. These intra-mucosal muscle bundles are also accompanied by nerve fi bers.

Mucosal innervation

The structure of the mucosa varies more from one part of the gastrointestinal tract to another than do the structures of other layers. In the small intestine, it consists of the muscularis mucosae, the connective tissue (lamina propria), into which simple tubular glands protrude (the intestinal crypts, or glands of Lieberkuhn), and the villi. The lining of the glands and the surface of the villi are a single layer columnar epithelium. A dense network of fi ne interconnecting nerve bundles that is found throughout the connective tissue (lamina propria) of the mucosa (Fig. 1.9) makes up the mucosal innervation and was described in the 19th century (Billroth 1858, Drasch 1881, Müller 1892, Berkley & Baltimore 1893, Cajal 1895, 1911). The mucosal innervation in the small intestine can be divided into different components: a subglandular plexus, a periglandular plexus, a villous subepithelial plexus, and a plexus of the villous core. There is some specifi city in the nerve fi bers that contrib- ute to the different components. For example, in guinea-pig small intestine, calretinin immunoreactive secretomotor neurons selectively supply the subglandular and periglandular components (Brookes et al. 1991a, Clerc et al. 1998b). In the stomach and colon, nerve fi bers are found throughout the depth of the mucosa, and there is also a dense mucosal innervation in the gall bladder. A sparser plexus of nerve fi bers occurs adjacent to the mucosal epithelium in the esophagus, which is a protective stratifi ed epithelium that is devoid of secretory elements. The nerve fi bers that innervate the mucosa lie in the connective tissue of the lamina propria, they do not penetrate the epithelium, which is a single layer in the stomach, small intestine, and colon. Some nerve fi bers, which are believed to be sensory, penetrate the inner layers of the stratifi ed epithelium that lines the esophagus (Rodrigo et al. 1975, Clerc & Condamin 1987). Nerve fi bers in the mucosa inevitably come close to entero-endocrine cells of the gastric and intestinal epithelium. It is diffi cult to defi ne any special relationship with the entero-endocrine cells, even using electron microscopy. Nevertheless, there are functional interactions between nerve fi bers of the mucosal plexus and the endocrine cells (Chapter 2). Nerve fi bers in the mucosa also come close to cells of the immune system, e.g. lymphocytes, and to the lymph nodules (Peyer’s patches) that occur in the mucosa (Chapter 2).