Progress in Neurobiology 72 (2004) 143–164

Intrinsic primary afferent neurons and nerve circuits within the intestine John B. Furness a,∗ , Clare Jones b , Kulmira Nurgali a , Nadine Clerc b a

Department of Anatomy & Cell Biology and Centre for Neuroscience, University of Melbourne, Parkville, Vic. 3010, Australia b Lab ITIS, UMR CNRS-Univ Méditerranée, Marseille, France Received 30 August 2003; accepted 3 December 2003

Abstract Intrinsic primary afferent neurons (IPANs) of the enteric nervous system are quite different from all other peripheral neurons. The IPANs are transducers of physiological stimuli, including movement of the villi or distortion of the mucosa, contraction of intestinal muscle and changes in the chemistry of the contents of the gut lumen. They are the first neurons in intrinsic reflexes that influence the patterns of motility, secretion of fluid across the mucosal epithelium and local blood flow in the small and large intestines. In the guinea pig small intestine, where they have been characterized in detail, IPANs have Dogiel type II morphology, that is they are large round or oval neurons with multiple processes, some of which end close to the luminal surface of the intestine, and some of which form synapses with enteric interneurons, motor neurons and with other IPANs. The IPANs have well-defined ionic currents through which their excitability, and their functions in enteric nerve circuits, is determined. These include voltage-gated Na+ and Ca2+ currents, a long lasting calcium-activated K+ current, and a hyperpolarization-activated cationic current. The IPANs exhibit long-term changes in their states of excitation that can be induced by extended periods of low frequency activity in synaptic inputs and by inflammatory mediators, either applied directly or released during an inflammatory challenge. The IPANs may be involved in pathological changes in enteric function following inflammation. © 2003 Elsevier Ltd. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Neural control of intestinal function: types of neurons that form enteric nerve circuits . . Intrinsic primary afferent neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Why use this terminology? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. What properties are expected of primary afferent neurons? . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Primary afferent neurons: intrinsic and extrinsic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Characteristics of intrinsic primary afferent neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Chemosensitive IPANs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. IPANs sensitive to stretch or distortion at the level of the myenteric plexus . . . 2.4.3. Mucosal mechanoreceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Polymodal nature of IPANs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: A1 , adenosine 1 receptor; AC, adenylyl cyclase; ACh, acetylcholine; AHP, afterhyperpolarizing potential; BK, large-conductance potassium channel; BB, bombesin; CCK, cholecystokinin; CGRP, calcitonin gene related peptide; ChAT, choline acetyltransferase; CNS, central nervous system; COX-2, cyclooxygenase-2; DAG, diacyl glycerol; ENK, enkephalin; EPSP, excitatory post-synaptic potential; GABA, ␥-aminobutyric acid; GAL, galanin; CaV , voltage-dependent Ca2+ conductance; gKCa , Ca2+ -dependent K+ conductance; gNaV , voltage-dependent Na+ conductance; GRP, gastrin releasing peptide (the mammalian form of bombesin); H2 , histamine 2 receptor; HVA, high voltage-activated Ca2+ channel; 5-HT, 5-hydroxytryptamine; IA , A-type K+ current; IAHP , AHP current; IBS, irritable bowel syndrome; ICaV , voltage-dependent Ca2+ current; Ih, hyperpolarization-activated cation current; IK, intermediate-conductance potassium channel; IL, interleukin; IP3, inositol triphosphate; IPAN, intrinsic primary afferent neuron; IR, immunoreactivity; M2 , muscarinic 2 receptor; NFP, neurofilament protein; NK, neurokinin; NOS, nitric oxide synthase; NPY, neuropeptide Y; PACAP, pituitary adenylyl cyclase-activating peptide; PDBu, phorbol dibutyrate; PG, prostaglandin; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; Rin, input resistance; SK, small-conductance potassium channel; SOM, somatostatin; SSPE, sustained slow post-synaptic potential; TEA, tetraethylammonium; TK, tachykinin; TNBS, trinitrobenzene sulfonic acid; TNF, tumor necrosis factor; TTX, tetrodotoxin; TTX-R INaV , TTX-resistant sodium current; VIP, vasoactive intestinal peptide ∗ Corresponding author. Tel.: +61-3-83448859; fax: +61-3-93475219. E-mail address: [email protected] (J.B. Furness). 0301-0082/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.pneurobio.2003.12.004

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Synaptically-mediated changes in IPAN excitability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1. Slow excitatory post-synaptic potentials (slow EPSPs) . . . . . . . . . . . . . . . . . . . . . . . 2.6.2. Sustained slow post-synaptic excitation (SSPE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Synaptic interactions between IPANs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. IPANS are activated in groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9. Changes in IPAN excitability caused by inflammatory mediators . . . . . . . . . . . . . . . . . . . . . 2.10. Are IPANs involved in neuropathologies? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Enteric nerve circuits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Involvement of IPANs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Inter-species differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Circuits for motility control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Different patterns of motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Secretomotor and vasomotor reflexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.

1. Introduction The enteric nervous system is involved in many physiological and pathophysiological processes in the gastrointestinal tract. It is essential to normal life, as indicated by the high morbidity or mortality associated with congenital or acquired abnormalities of the enteric nervous system (De Giorgio et al., 2000; Sharkey and Lomax, 2001). Pathological conditions involving enteric neuronal abnormalities include achalasia and chronic intestinal pseudo-obstruction, that cause dysmotilities, and conditions such as infantile hypertrophic pyloric stenosis and Hirschsprung’s disease that are potentially fatal. By contrast, animals and humans have low morbidity after comprehensive interference with the sympathetic or parasympathetic divisions of the autonomic nervous system, such as removal of the sympathetic chains (Cannon et al., 1929), or total infracardiac vagotomy (Kuntz, 1945). For a long time after their discovery in the mid-19th century, the internal structures of the intrinsic ganglia of the gastrointestinal tract and the circuits that they form remained a mystery. However, with the advent of new technologies in the last 20 years, the organization of enteric circuits has been substantially unraveled, and in very recent years many important advances in understanding the roles of individual neurons in the circuits have been made. Each of the major neuron types, intrinsic primary afferent neurons, interneurons and motor neurons, has been characterized. Of these, the identification of the intrinsic primary afferent neurons has been the most recent, and a significant portion of this review relates to these neurons and their properties. 1.1. Neural control of intestinal function: types of neurons that form enteric nerve circuits Substantial agreement exists between laboratories concerning the classification of neurons in the small and large intestines of the guinea pig (Costa et al., 1996; Furness et al.,

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2000; Brookes, 2001). There are 14 types of neuron in the small intestine (Fig. 1, Table 1), although some of these types can be subdivided. For example, the inhibitory neurons to the circular muscle can be divided into subgroups with short and with long axons, which have slightly different chemical coding.

2. Intrinsic primary afferent neurons 2.1. Why use this terminology? Cervero (1994) has provided a valuable summary of the conceptual differences between the afferent innervation of tissues and organs and their sensory innervation. As he points

Fig. 1. The types of neurons in the small intestine of the guinea pig, all of which have been defined by their functions, cell body morphologies, chemistries, key transmitters and projections to targets. The numbers adjacent to the neurons correspond to the numbers in Table 1, which lists each of the neuron types by their functions and provides data on the their chemistries and the percentages of their cell bodies in the myenteric or submucosal ganglia. LM: longitudinal muscle; MP: myenteric plexus; CM: circular muscle; SM: submucosal plexus; Muc: mucosa.

Table 1 Types of neurons in the enteric nervous system Chemical coding

Function/comments

Myenteric neurons Excitatory circular muscle motor neurons (6)

12%

Short: ChAT/TK/ENK/GABA; long: ChAT/TK/ENK/NFP

Inhibitory circular muscle motor neurons (7)

16%

Excitatory longitudinal muscle motor neurons (4) Inhibitory longitudinal muscle motor neurons (5)

25% About 2%

Short: NOS/VIP/PACAP/ENK/NPY/GABA; long: NOS/VIP/PACAP/dynorphin/BB/NFP ChAT/calretinin/TK NOS/VIP/GABA

Ascending interneurons (1) Descending interneurons local reflex (8)

5% 5%

ChAT/calretinin/TK ChAT/NOS/VIP ± BB ± NPY

Descending interneurons (secretomotor reflex) (9)

2%

ChAT/5-HT

Descending interneurons (migrating myoelectric complex) (10) Myenteric intrinsic primary afferent neurons (2) Intestinofugal neurons (3) Excitatory motor neurons to the muscularis mucosae* Inhibitory motor neurons to the muscularis mucosae*

4% 26% <1% N/A N/A

ChAT/SOM ChAT/calbindin/TK/NK3 receptor/P2X2 receptor ChAT/BB/VIP/CCK/ENK N/A N/A

Motor neurons to gut endocrine cells*

N/A

N/A

To all regions, primary transmitter ACh, cotransmitter TK Several cotransmitters with varying prominence: NO, ATP, VIP, PACAP Primary transmitter ACh, cotransmitter TK Several cotransmitters with varying prominence: NO, ATP, VIP, PACAP (local reflex) Primary transmitter ACh, ATP may be a cotransmitter Primary transmitters ACh, 5-HT (at 5-HT3 receptors) Primary transmitter ACh Primary transmitters TK and ACh Primary transmitter ACh The primary transmitter is ACh Pharmacology of transmission appears to be similar to other enteric muscle motor neurons For example, neurons innervating gastrin cells

45%

VIP/PACAP/GAL

Cholinergic secretomotor/vasodilator neuron (13) Cholinergic secretomotor (non-vasodilator) neurons (14)

15% 29%

ChAT/calretinin/dynorphin ChAT/NPY/CCK/SOM/CGRP/dynorphin

Submucosal intrinsic primary afferent neurons (11)

11%

ChAT/TK/calbindin

Submucosal neurons Non-cholinergic secretomotor/vasodilator neurons (12)

Primary transmitter VIP/PACAP. A small proportion of these have cell bodies in myenteric ganglia Primary transmitter ACh Primary transmitter ACh. A small proportion of these have cell bodies in myenteric ganglia Calbindin-IR, seen with some antisera only. Primary transmitter ACh, may be TK contribution

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Proportion

This Table lists the neuron types that are found in the guinea pig small intestine, some of their defining characteristics, and percentages of occurrence in each of the ganglionated plexuses. We have also listed three types of motor neuron that are found in other parts of the tubular digestive tract, marked by asterisks. The numbers in brackets are the identifying numbers for the neurons in Figs. 1 and 11.

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out, the activation of visceral receptors (such as arterial chemoreceptors, stretch receptors in the lung and osmoreceptors in the liver) evokes no sensation, and therefore the term afferent is most appropriate to designate the neurons that carry this information. On the other hand, some neurons innervating the viscera do give rise to sensation, such as pain and warmth, and these are properly called sensory neurons. Sherrington (1900) and Langley (1903) advanced similar arguments. Sherrington used the distinction when referring to the production of pain by activation of fibres that normally do not cause sensation. He wrote: “The impulses from visceral fibres on the central nervous system appear hardly at all to elicit conscious sensations. When abnormally they do so, it is as though particular afferent nerves, which usually are not in the strict meaning of the term sensory nerves at all, can on occasion become sensory”. The important difference between visceral afferent neurons in general, and the subset of visceral afferents that mediate conscious sensation from the viscera, extends to the afferent neurons that reside within the intestine, and when the first clues about the identities of these intrinsic afferent neurons were being gathered, Kirchgessner and Gershon (1988) were careful to call them intrinsic primary afferent neurons (IPANs), a nomenclature that was followed by other authors (Furness et al., 1998). Thus, when for convenience or economy of words, the IPANs are referred to as sensory, it should be remembered that they do not actually convey any sensation from the intestine. Afferent pathways, for example those that enter the central nervous system via the dorsal roots, involve neurons that connect in series and that have been referred to as primary, secondary and higher order afferent neurons. In accord with this nomenclature, the intrinsic neurons that detect the state of the intestine are referred to as primary afferent neurons because they are the first neurons of the reflex pathways in the intestine. Afferent neurons are neurons that carry information towards reflex centers or to integrating nerve circuits, whereas efferent neurons carry information away from reflex or integrating centers. Included amongst efferent neurons are motor neurons to muscles and glands. Matters are not quite as simple as this, because there are many examples of neurons that have more than one function. A good example is the motor (efferent) neuron to skeletal muscle. These neurons have collaterals that connect with neurons within the spinal cord (Renshaw, 1946; Eccles et al., 1954). It would obscure their main function if these were to be called interneurons instead of being called motor neurons. A further example is spinal primary afferent neurons, which, through transmitter release from their peripheral endings, cause vasodilatation and plasma extravasation (Lewis, 1927). Thus, these primary afferent neurons, including those that supply the gut, also have efferent effects (Holzer et al., 1991; Holzer and Maggi, 1998). IPANs probably also have efferent effects when they release transmitter onto the mucosal epithelium (see below: secretomotor and vasomotor reflexes). The

IPANs receive excitatory synapses and provide outputs to other neurons; in this context they can be considered to be interneurons. Another example of primary afferent neurons that receive synapses to their somas is trigeminal primary afferent neurons that have their cell bodies in the trigeminal mesencephalic nucleus (Honma et al., 2001). So, like the IPANs, these trigeminal primary afferent neurons could be considered to be interneurons. 2.2. What properties are expected of primary afferent neurons? Primary afferent neurons transduce and encode information about the chemical environment and physical state of the tissues that they innervate, and convey this information to integrative circuitry through which the functional states of organs can be modified (Martin, 1981). A fundamental property is that the primary afferent neurons respond to adequate (that is, physiologically appropriate) stimuli in a manner that codes the intensity of stimuli. Investigation of the IPANs is less complete than is investigation of other primary afferent neurons, such as those of the skin and joints. Nevertheless, it has been shown that the IPANs react to stimuli that are at the threshold for eliciting enteric reflexes, for example they respond to puffs of nitrogen gas applied to the mucosa (Gershon and Kirchgessner, 1991) and have a low level of activity even when no deliberate stimulus is applied (Kunze et al., 1997). The activity of IPANs has been detected by intracellular and patch electrodes and by activity-dependent dyes. With these methods, IPANs have been identified that are responsive to mechanical distortion of the mucosa, to distortion of their processes within the myenteric plexus and to various chemicals applied to the mucosa (Gershon and Kirchgessner, 1991; Kirchgessner et al., 1992; Bertrand et al., 1997, 1998; Kunze et al., 1998, 1999, 2000). As expected of primary afferent neurons, the responses of IPANs are graded with stimulus strength (Kunze et al., 1998). In addition to transducing adequate physiological stimuli, IPANs may sometimes function as nociceptors, since their activation by noxious stimuli triggers protective responses. In the small intestine and colon, protective responses are initiated by irritants that are included in enemas, by bacterial products that cause diarrhea, and by parasitic infestations that induce expulsion of the parasites (Collins, 1996; Lundgren, 2002). 2.3. Primary afferent neurons: intrinsic and extrinsic Detection of the state of the gastrointestinal tract involves three systems: primary afferent neurons, entero-endocrine cells and immune cells (Fig. 2). Each of these detecting systems is more extensive than those of non-digestive organs (Furness et al., 1999). About 20% of neurons in the enteric nervous system, which contains of the order of 108 neurons, are primary afferent neurons, and more than 50,000 nerve

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Fig. 2. Afferent signals that originate from the gastrointestinal tract. Endocrine messages are carried by hormones released from entero-endocrine cells in the mucosal epithelium. The hormones enter the circulation, and can act at remote sites. They also act locally, on nerve endings, on the epithelium and on cells of the immune system. Immune messages are conveyed by circulating lymphocytes, that are activated by antigens presented to them from the lumen. Immune cells and cells of tissue defense, such as mast cells and macrophages, also release substances that act locally, within the gut wall. Messages are conveyed by neurons whose receptive endings are in the lamina propria, beneath the mucosal epithelium, in the muscle and in enteric ganglia. Some afferent neurons have cell bodies in the gut wall (IPANs and intestinofugal neurons) and the cell bodies of others are in extrinsic ganglia (extrinsic primary afferent neurons, see Fig. 3).

processes reaching the gut through the vagus and splanchnic nerves are also afferents. Furthermore, the gastroenteropancreatic endocrine system contains thousands of enteroendocrine cells, many of which react to their local environment, and from which more than 20 identified hormones are released. Finally, the gut immune system detects immunogens and contains 70–80% of the body’s immune cells. There is extensive interaction between these three systems, and physiological responses commonly involve actions through neurons, endocrine cells and immune cells. Two broad classes of primary afferent neurons are associated with the gut: IPANs with cell bodies, processes and synaptic connections in the gut wall and extrinsic primary afferent neurons (Fig. 3). Extrinsic primary afferent neurons have cell bodies in nodose and jugular ganglia (vagal afferents) or in dorsal root ganglia (spinal afferents). In addition, signals are carried by intestinofugal neurons that have cell bodies in the gut but send processes to neurons outside the gut wall. Intestinofugal neurons are in afferent pathways, but it is possible that they are not the first neurons in reflex pathways that pass between gut regions (Sharkey et al., 1998; Szurszewski et al., 2002).

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Fig. 3. The afferent neurons of the digestive tract. Two classes of intrinsic primary afferent neuron (IPAN) have been identified: myenteric IPANs that respond to distortion of their processes in the external muscle layers and, via processes in the mucosa, to changes in luminal chemistry, and submucosal IPANs that detect mechanical distortion of the mucosa and luminal chemistry. Extrinsic primary afferent neurons have cell bodies in dorsal root ganglia (spinal primary afferent neurons) and vagal (nodose and jugular) ganglia. Spinal primary afferent neurons supply collateral branches in prevertebral (sympathetic) ganglia. Intestinofugal neurons are parts of the afferent limbs of entero-enteric reflex pathways. LM, longitudinal muscle; CM, circular muscle; MP, myenteric plexus; SM, submucosa; Muc, mucosa. Nerve endings in the mucosa can be activated by hormones released from entero-endocrine cells (arrows). Adapted from Furness et al., 1998.

Monitoring and control of the digestive system by neurons is hierarchical. The enteric nervous system is capable of generating appropriate reflex responses to the gut lumen contents. For example, local reflexes generate mixing movements of the muscle, local changes in blood flow, and secretion of water and electrolytes. The enteric nervous system also participates in reflexes between organs, for example, between the duodenum and stomach, in this case to regulate gastric emptying. Furthermore, signals are conveyed from the digestive organs via extrinsic primary afferents to the central nervous system, which can trigger reflexes that act back on the digestive system. Some afferent signals to the CNS mediate co-ordination with other body systems, and some relate to sensations including discomfort, nausea, pain and satiety. 2.4. Characteristics of intrinsic primary afferent neurons IPANs that have been identified to date are round or oval in profile and are multi-axonal or pseudounipolar, with one or more axons that lead to and ramify in the lamina

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Fig. 4. Diagram of a myenteric intrinsic primary afferent neuron. The intrinsic primary afferent neurons are multipolar. Action potentials initiated by physiologically appropriate stimuli can traverse the cell bodies (transcellular conduction) or can be conducted to output synapses via an axon reflex (axon reflex conduction). Conduction across the cell body can be modified by the synaptic inputs that it receives. The myenteric IPANs make synaptic connections with other neurons in the myenteric and submucosal ganglia. Adapted from Furness et al., 1998.

propria of the mucosa, just beneath the absorptive epithelium. This distinctive shape is known as Dogiel type II morphology (Fig. 4). Interestingly, when Dogiel (1899) originally described these neurons, he guessed that they would be primary afferent neurons. Myenteric IPANs have axons that supply terminals around several types of nerve cells in myenteric ganglia, including other IPANs, interneurons and motor neurons, and also project to submucosal ganglia. They receive synapses on their somas and on the initial parts of their axons (Pompolo and Furness, 1988). Submucosal Dogiel type II neurons connect with other neurons in the submucosal ganglia (Furness et al., 2003a) and with neurons in myenteric ganglia (Kirchgessner and Gershon, 1988). In addition to their unique shapes and projections, these neurons have distinct electrophysiological properties. They have broad action potentials that are carried by both sodium and calcium currents and are followed by early and late (slow) afterhyperpolarizing potentials (AHPs). The electrophysiological properties of IPANs in the guinea pig are influenced by the recording conditions. Under conditions in which background synaptic transmission is suppressed, they exhibit the late AHP that identifies them as AH neurons in the terminology of Hirst (Hirst et al., 1974), but when IPANs are acted upon by neurotransmitters or hormones, the late AHP can be suppressed or obliterated (Wood and Mayer, 1979a; Morita and North, 1985; Clerc et al., 1999). Homologous neurons in the pig (Cornelissen et al., 2000) and mouse (Nurgali et al., 2004) often do not exhibit an AHP. Whether this is because the AHP is suppressed or that it is intrinsically less prominent is unknown. When IPANs are in a state of low excitability they do not fire more than 1–2 action potentials to a 500 ms intracellular

depolarizing pulse, and they have a resting membrane potential of around −60 to −65 mV. The resting conductance is low, with input resistance of about 200 M measured by intracellular electrodes and about 500 M measured with whole cell patch recording. Background K+ and probably Cl− currents contribute to the resting conductance. Some contribution to the background conductance is made by the hyperpolarization-activated cation current, Ih. Whole cell patch recording estimates the contribution of Ih at about 20% of the cell conductance (Rugiero et al., 2002). There is also evidence for a contribution of a Ca2+ -dependent K+ conductance (gKCa). Charybdotoxin (20 nM), which blocks the gKCa of intermediate conductance K+ (IK) and large conductance K+ (BK) channels, depolarized AH neurons from −57 to −49 mV, and increased input resistance from 190 to 260 M, in experiments using intracellular recordings (Kunze et al., 1994). Consistent with this, AH neurons depolarize by 4–15 mV in Ca2+ -free solution, and their input resistance increases by approximately 15% (North and Tokimasa, 1987). IPANs also have an A-type K+ current (IA ) that may have a low level of activation at rest (Starodub and Wood, 2000b). Chloride currents occur in IPANs, but there is no evidence to suggest that they make a significant contribution to resting conductance (Bertrand and Galligan, 1994; Starodub and Wood, 2000a). The action potential in the IPAN soma has a large amplitude (about 80–90 mV, measured by intracellular electrodes) and a half width of about 2.5 ms at 33 ◦ C; at 37 ◦ C the half width is about 2.0 ms (Jones et al., 2003). Two inward currents underlie the action potential in the soma, tetrodotoxin (TTX)-sensitive Na+ current (INaV ) and a TTX-insensitive Ca2+ current (ICaV ) (North, 1973; Hirst et al., 1985a). A TTX-resistant Na+ current (TTX-R INaV ) also occurs in these neurons (Rugiero et al., 2003), but there is no evidence that this contributes in any significant way to the action potential in physiological conditions. The properties of TTX-R INaV are consistent with it being due to the expression of NaV 1.9; transcripts corresponding to this channel, but not to NaV 1.8, were obtained by single cell RT-PCR of AH neurons (Rugiero et al., 2003). In the presence of TTX, the Ca2+ current is still sufficient for action potential generation in the soma, but active conduction of action potentials in the processes of the IPANs is prevented by TTX. The ICaV is responsible for an inflection (hump) on the repolarizing phase of the action potential. The hump on the action potential persists in the presence of the L-type calcium channel blockers nicardipine (3 ␮M) (Kunze et al., 1994) or nifedipine (1 ␮M) (North and Tokimasa, 1987). The identity of the high voltage-activated (HVA) Ca2+ channels has been investigated in experiments in which the contribution of Ca2+ to the action potential was enhanced by adding tetraethylammonium (TEA). The Ca2+ plateau, which occurs in the presence of this K+ channel blocker and nicardipine, was almost blocked by ␻-conotoxin GVIA or ␻-conotoxin MVIIA, both blockers of N-type channels, but not by ␻-agatoxin GIVA, which is a blocker of P/Q-type channels (Rugiero et al.,

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2002). There may be a small contribution of R channels to the ICaV (Rugiero et al., 2002). Consistent with N-type channels being the predominant type of HVA channel of IPANs, immunoreactivity for the ␣1B (N-type channel) calcium channel subunit is localized to IPANs, whereas only weak ␣1A (P/Q-type channel) immunoreactivity was found (Kirchgessner and Liu, 1999). Properties of the TTX-sensitive Na+ current have been analyzed in cultured myenteric IPANs of adult guinea pigs (Zholos et al., 2002). This current was activated between −50 and −40 mV and peaked at −10 mV. Inactivation kinetics were fast and the steady state half-inactivation potential was −56 mV. The time constant decreased as the holding potential depolarized, with a maximum of 161 ms at −70 mV and a minimum of 2.3 ms at −30 mV. Recovery from inactivation was also rapid with time constants between 7 and 44 ms for holding potentials of −100 and −60 mV, respectively. The early AHP is continuous with the falling phase of the action potential and lasts about 50–100 ms. The currents of the early AHP have been investigated in some detail (Furness et al., 1998) and include a contribution from large conductance potassium channels (BK channels). This is a post-spike hyperpolarization similar to that observed in many types of neurons in the central nervous system and in autonomic ganglia. Following the early AHP, there is a depolarization that is followed by the slow AHP. This gives the misleading impression that the onset of the slow AHP current is delayed. However, when the underlying channel activity is recorded it is clear that the onset is rapid, within a few ms (Vogalis et al., 2002a). The transient depolarization that occurs between the early and late AHPs is due to a Ca2+ -activated depolarizing cation current (Vogalis et al., 2002b). The slow AHP can last from about 2 to about 30 s (Hirst et al., 1974, 1985b). Two currents are activated and act in opposition during the late AHP: the late AHP current (IAHP ), a KCa current present in all Dogiel type II neurons, generates a prolonged hyperpolarization, and Ih (present in about 80% of these neurons), which is increased by IAHP -induced hyperpolarization. Ih generates a sustained depolarization (Galligan et al., 1990; Rugiero et al., 2002) (Fig. 5). The Ca2+ -dependence of the K+ current underlying the late AHP was first established by ion substitution (Hirst and Spence, 1973; Hirst et al., 1974). While addition of TTX has no effect, the absence of external Ca2+ , or addition of calcium channel blockers such as Co2+ , Mn2+ or Mg2+ , suppress the depolarization-evoked gKCa (Morita et al., 1982; Hirst et al., 1985b; North and Tokimasa, 1987). As mentioned above, enhancing the Ca2+ contribution to the action potential by adding TEA allows gKCa to be amplified without altering its onset kinetics (Hirst et al., 1985a,b; North and Tokimasa, 1987). IAHP increases progressively when the cell is dialyzed with caffeine included in a patch pipette (Rugiero et al., 2002). It is also substantially reduced by ryanodine and after Ca2+ store

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Fig. 5. The action potential and afterpotentials that occur in the cell bodies of intrinsic primary afferent neurons. The potentials are shown in diagrammatic form, not to scale. Soma action potentials are caused by the opening of voltage-activated sodium conductances (gNaV ) and a calcium conductance (gCaV ), that is carried by N-type Ca2+ channels. The major component of action potential that is carried by Na+ is blocked by TTX. There is also a TTX-resistant sodium conductance, gNaV (non-TTX). The calcium current outlasts the sodium current and is responsible for the hump on the falling phase of the action potential. The action potential is terminated by the decline in the Na+ and Ca2+ conductances and activation of at least three K+ conductances that contribute to an early AHP. This is followed by a late AHP that is carried by a calcium-dependent potassium conductance (gKCa ), the ion channels for which are IK channels. In about 80% of intrinsic primary afferent neurons, the hyperpolarization of the late AHP triggers a depolarizing non-selective cation conductance, g(K, Na), that reduces the amplitude of the late AHP. An afterdepolarizing potential (ADP), due to Ca2+ activation of a mixed cation conductance (gCAN) occurs between the early and late AHPs. Adapted from Furness et al., 1998.

depletion by long-term application of caffeine (Hillsley et al., 2000; Vogalis et al., 2001). It is thus deduced that the activation of the channel depends on Ca2+ -induced Ca2+ release from intracellular stores via a ryanodine-sensitive receptor (Fig. 6). The repolarization of the AHP is dependent on removal of Ca2+ by mitochondria, because block of mitochondrial Ca2+ uptake or of mitochondrial respiratory enzymes greatly prolongs the AHP (Vanden Berghe et al., 2002). IAHP is Ca2+ -activated, very weakly voltage-sensitive and not affected by apamin. These properties indicate that the channels responsible are not conventional BK (highly voltage-sensitive) or SK (apamin-sensitive channels). Recent evidence suggests that they are IK channels (Vogalis et al., 2002a), which were not thought to be expressed in neurons (Sah and Faber, 2002). These channels have been localized to Dogiel type II neurons, and the channel protein has been detected in extracts of external muscle including myenteric ganglia (Furness et al., 2003b). IK channels have consensus sites for protein kinase A (PKA) and protein kinase C (PKC) binding, and phosphorylation by either of these kinases causes a reduction in their opening probability (Wulf and Schwab, 2002; Del Carlo et al., 2003; Vogalis et al., 2003). Consistent with this, the PKC stimulant, phorbol dibutyrate (PDBu; 1 nM–1 ␮M), caused excitability increases, membrane depolarization and

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Fig. 6. Control pathways for IK channels of intrinsic primary afferent neurons. The opening probabilities of channels are increased by Ca2+ and reduced by phosphorylation, through the actions of both PKA and PKC. PKC is linked to the so far unidentified transmitter of sustained slow excitation (the SSPE) and probably to the transmitter of slow EPSPs. The action potential admits Ca2+ through the soma membrane. This Ca2+ then triggers Ca2+ -induced Ca2+ release from intracellular stores, which opens IK channels (probably by causing their dephosphorylation). The IK current generates the late afterhyperpolarizing potential (AHP). Removal of Ca2+ by mitochondria is important for terminating the AHP.

increased input resistance in a concentration-dependent manner (Pan et al., 1997; Kawai et al., 2003). A catalytic subunit of PKA, added to inside-out patches from IPANs, reduced the opening probability of the IK channels, and in whole cell recording, its application quickly suppressed the IAHP (Vogalis et al., 2003). The IK channels are probably dynamically controlled by partial phosphorylation of the channel population, as addition of alkaline phosphatase increases channel activity (Vogalis et al., 2003). IPANs of the guinea pig small intestine contain PKA and several isoforms of PKC, including the PDBu-activatable, Ca2+ -sensitive, PKC␥, which is only in this class of enteric neuron (Poole et al., 2003). 2.4.1. Chemosensitive IPANs Intracellular records from nerve cell bodies in the guinea pig small intestine show that IPANs respond to chemicals, such as inorganic acid and short chain fatty acids at neutral pH, applied to the luminal surface of the mucosa of the small intestine (Kunze et al., 1995; Bertrand et al., 1997). Activation of submucosal and myenteric neurons by glucose applied to the mucosa has been reported (Kirchgessner et al., 1996). Detection of changes in the chemical content of the gut lumen may be indirect, via the release of hormones from entero-endocrine cells. This is presumed because the mucosal epithelium separates the nerve endings from the luminal environment. There is evidence that 5-hydroxytryptamine (5-HT), which is a potent stimulant of the endings of IPANs, is an intermediate in enteric reflexes (Kirchgessner et al., 1992; Bertrand et al., 1997, 2000). 5-HT is released when the mucosa is mechanically stimulated to elicit motility reflexes and the reflex responses are antagonized by drugs that block

Fig. 7. Mechanism of activation of mechanosensitive IPANs by muscle contraction in the wall of the intestine. Stretch opens stretch-activated channels (SACs) in the muscle membrane, which results in muscle contraction. The contracting muscle distorts the IPAN processes. If the muscle SACs are blocked by gadolinium, or if contraction is prevented by isoprenaline or nicardipine, excitation of IPANs by stretch ceases. On the other hand, when the muscle is contracted by opening L-type Ca2+ channels with BK 8644, IPANs are activated. The muscle pulls on IPAN processes through connective tissue which can be weakened by dispase, preventing IPAN activation in response to stretch. The distortion of processes of IPANs opens Gd3+ -insensitive SACs, which cause depolarization and action potential initiation. IPANs can also be activated by direct distortion of their processes.

5-HT3 and/or 5-HT4 receptors (Neya et al., 1993; Foxx Orenstein et al., 1996; Grider et al., 1996). Furthermore, mechanical stimulation of the mucosa causes c-Fos induction in IPANs in submucosal ganglia, and this induction is blocked by N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, an antagonist of 5-HT1P receptors (Kirchgessner et al., 1992). Recent data indicate that ATP could be also involved in communicating excitation from entero-endocrine cells to the mucosal endings of IPANs (Bertrand and Bornstein, 2002). Other hormones that are contained in gut endocrine cells, such as cholecystokinin (CCK) and motilin, are released by nutrients and act on neurons, but have not been tested for their possible roles as intermediates in enteric reflexes. 2.4.2. IPANs sensitive to stretch or distortion at the level of the myenteric plexus IPANs possess mechanosensitive ion channels (Kunze et al., 2000) that allow them to transduce distortion of their processes to action potential firing (Wood, 1973; Kunze et al., 1998, 2000). In experimental conditions, distortion can be applied directly to the processes or can be caused by muscle movements, because the muscle is adherent to the processes via collagen fibers. From experiments performed on longitudinal muscle-myenteric plexus preparations, with some residual attached circular muscle, from the small intestine (Kunze et al., 1999), the mechanism through which these neurons are activated by muscle movement has been deduced (Fig. 7). Recordings have been taken from IPANs while pressure was applied to their processes within the myenteric plexus, close to the nerve cell body, with a fine probe. Generator potentials were recorded at distances of 0.1–0.5 mm from

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the cell body (Kunze et al., 2000). Generator potentials and action potentials initiated by pressing the myenteric processes of IPANs (Kunze et al., 2000), as well as action potentials generated by stretching the muscle (Kunze et al., 1999), were not affected by substantially lowering Ca2+ in the external solution. In addition, action potentials generated by contracting the longitudinal muscle (see below) were unaffected by gadolinium, which blocks mechanosensitive channels in intestinal muscle cells (Kunze et al., 1999). The neurons fire action potentials phasically, when their processes are directly deformed, or at the beginning of stretch applied to the longitudinal muscle (Kunze et al., 2000). During maintained stretch, the muscle contracts more-or-less rhythmically, and IPANs continue to discharge (Kunze et al., 1998). The rate of discharge is proportional to the degree of distension. However, the discharge of action potentials was abolished if the muscle contraction was prevented by muscle relaxants, either isoprenaline (an agonist of ␤-receptors for catecholamines) or nicardipine. This indicates that active tension in the muscle contributes to the excitation of the tension-sensitive IPANs (Fig. 7). The involvement of the muscle is interesting because it has long been known that intestinal muscle cells are directly sensitive to stretch and respond to it by contracting (Bülbring, 1955). This reaction of the smooth muscle may be integral to the response of IPANs during sustained stretch. When the tissue was exposed to a proteolytic enzyme, to weaken connective tissue links between the muscle and the IPANs, activation of IPANs by muscle contraction was prevented (Kunze et al., 1999). Interestingly, the IPAN cell bodies are hyperpolarized when an area of soma membrane is stretched (Kunze et al., 2000). This occurs through the opening of BK-type K+ channels on the cell soma, which appear to be directly distortion-sensitive. Myenteric nerve cell bodies are deformed by muscle movement (Gabella and Trigg, 1984), and it has been speculated that sufficient pressure in the wall of the intestine may reduce IPAN excitability, as part of a protective mechanism that limits the strength of reflex contraction of the intestine (Kunze et al., 2000). There is evidence that neurons other than Dogiel type II neurons are mechanosensitive. Kunze et al. (1999) recorded from one Dogiel type I neuron that responded directly to stretch. In flat sheet preparations of guinea pig colon in which the circular muscle was maintained attached to the myenteric plexus, stretching the circular muscle triggers reflexes via mechanosensory neurons that are activated even when the smooth muscle is paralyzed by nifedipine (Spencer et al., 2002). Excitatory junction potentials oral and inhibitory junction potentials anal to the stimulus were synchronized only in preparations longer than 7 mm, and they were not triggered in preparations 3 mm long. From these results, it was deduced that Dogiel type II neurons were not the mechanosensitive neurons through which the reflexes were initiated, because these would be expected to have outputs to motor neurons in short preparations, and because, by extrapolation from the small intestine, their activity was

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predicted to be blocked by nifedipine (Spencer et al., 2002). Spencer et al. (2002) suggested that the mechanosensory neurons were ascending interneurons. However, the projections of Dogiel type II neurons, their connections and their responsiveness to mechanical distortion in the colon have not been determined by direct recording. 2.4.3. Mucosal mechanoreceptors Functional evidence for IPANs with cell bodies in submucosal ganglia comes from experiments in which activity-dependent induction of c-Fos and activity-dependent uptake of dyes have been localized to enteric neurons. C-Fos immunoreactivity was detected in submucosal nerve cells after the mucosa had been distorted by puffs of nitrogen gas that were ejected from a pipette (Kirchgessner et al., 1992). The c-Fos expression was abolished by TTX, but not by the nicotinic receptor blocker, hexamethonium, suggesting that it was produced in the cell bodies of IPANs that had processes in the mucosa. Styryl dyes, which are taken up by the endings of active neurons and transported back to the cell bodies, have also been used to identify IPANs that are mucosal mechanoreceptors (Kirchgessner et al., 1996). Distortion of the villi by puffs of nitrogen gas caused styryl dye labelling in neurons of numerous submucosal ganglia and in a few myenteric ganglia in the presence of hexamethonium. Fibers in myenteric ganglia, presumed to be the axons of submucosal nerve cells, were labeled. These results suggest that cell bodies of mucosal mechanoreceptors are in submucosal ganglia and project to the myenteric plexus (Fig. 3). The activation of mucosal mechanoreceptors is mostly indirect, through the release of 5-HT from enterochromaffin cells in the mucosa (Pan and Gershon, 2000). Distension stimuli can activate both mucosal mechanoreceptors and distension-sensitive neurons (myenteric IPANs). This can explain why both myenteric and submucosal neurons were revealed by styryl dyes after distension (Kirchgessner et al., 1996). Whether cell bodies of stretch-sensitive neurons are present in the submucosal ganglia, as well as in myenteric ganglia, has not yet been determined. The axons of submucosal IPANs project more-or-less directly to the underlying mucosa and thus recording direct responses of submucosal IPANs to mechanical stimulation of the mucosa has not been reported. However, recordings have been made from second order submucosal neurons (Pan and Gershon, 2000). These respond to mechanical stimulation of the mucosa with fast and slow EPSPs. 2.5. Polymodal nature of IPANs IPANs are polymodal, although it is possible that subgroups of IPANs respond more strongly to one type of stimulus than to another. For example, IPANs in myenteric ganglia that respond to chemicals applied to the mucosa can also respond to mechanical stimulation of the mucosa (Bertrand et al., 1997, 1998), whereas, under some circumstances, mechanical stimulation of the mucosa excites

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only neurons with cell bodies in submucosal ganglia (Kirchgessner et al., 1996). In the guinea pig small intestine, all myenteric IPANs project to the mucosa (Song et al., 1991, 1994). In order to record from these neurons, it is necessary to remove part of the mucosa (Bertrand et al., 1997). About 50% of the neurons respond to electrical stimulation of the remaining, intact mucosa, confirming that their mucosally projecting axons survived the dissection. Of the neurons that had intact projections to the mucosa, 60% responded to chemicals (acid, base or fatty acid) applied to the mucosa (Bertrand et al., 1997). If it is assumed that the cells with intact mucosal projections following partial removal of the mucosa are representative of the whole population, then about 60% of myenteric IPANs are chemoreceptive. In tissue that was distended to excite stretch-activated IPANs with cell bodies in the myenteric plexus, about 80% of neurons responded when the intestine was stretched in the circumferential direction (Kunze et al., 1998). About 70% of the neurons that responded to stretch did so directly. Thus, the data indicate that some IPANs are activated by both mucosal chemical stimulation and stretch. 2.6. Synaptically-mediated changes in IPAN excitability The myenteric IPANs respond to brief stimulation of their synaptic inputs at 10–20 Hz with slow excitatory post-synaptic potentials (EPSPs) and to low frequency (1 Hz) stimulation applied for several minutes with sustained slow post-synaptic excitation (SSPE; Clerc et al., 1999). In contrast to some other regions and species, such as the pig (Cornelissen et al., 2001), fast excitatory synaptic inputs to these neurons are rarely seen in the guinea pig small and large intestine (Bornstein et al., 1994; Nurgali et al., 2003) or in the mouse colon (Furukawa et al., 1986; Nurgali et al., 2004), and when recorded, fast EPSPs are of low amplitude (Iyer et al., 1988; Bornstein et al., 1994; Tamura et al., 2001). There does however appear to be an increase in fast synaptic input to IPANs during inflammation of the intestine (see below). Morphological and pharmacological data support the presence of somatic nicotinic and purinergic P2X receptors that would be expected to mediate fast synaptic responses in IPANs. First, nicotinic acetylcholine receptors have been detected immunohistochemically on the surface membranes of myenteric IPANs (Dogiel type II morphology, calbindin positive) in the guinea pig small intestine (Kirchgessner and Liu, 1998), and, second, nicotine application leads to a fast depolarization of myenteric IPANs via a direct action (it occurs in the presence of TTX and in low Ca2+ , high Mg2+ solution) on post-synaptic nicotinic receptors (Schneider and Galligan, 2000). Myenteric IPANs are also immunoreactive for P2X2 receptors (Castelucci et al., 2002). Furthermore, synapses on the neurons are immunoreactive for the vesicular acetylcholine transporter, which is generally believed to be a reliable marker of cholinergic nerve endings (Li and Furness, 1998). It is

therefore not clear why fast EPSPs are not usually recorded from these neurons in response to synaptic stimulation. 2.6.1. Slow excitatory post-synaptic potentials (slow EPSPs) Slow EPSPs, elicited by stimulation of interganglionic connectives at 10–20 Hz for 1 s, are characterized by a depolarization of IPAN membrane potential, an increase in input resistance, blockade of the late AHP and an increase in somatic excitability, as exemplified by an increase in the number of spikes elicited by an intracellular 500 ms depolarizing pulse. The effects commonly peak within 10–15 s, last for 1–4 min (Morita and North, 1985) and are primarily due to a reduction in resting K+ conductance and gKCa (North and Tokimasa, 1983). This is reported to be combined in some cases with activation of a Cl− conductance (Bertrand and Galligan, 1994; Starodub and Wood, 2000a). The change in the late AHP appears not to be caused by reduced Ca2+ entry, as the principal effect of nerve stimulation is a reduction in late AHP duration but not amplitude (Morita and North, 1985). One possible intermediate in causing the change in the AHP is the activation of protein kinases and subsequent inactivation of the IK channels (see Fig. 6 and Vogalis et al., 2003). Slow EPSPs in IPANs are mimicked by the neurokinin-3 (NK3 ) receptor agonist, senktide (Bertrand and Galligan, 1995) and are partially blocked by the NK3 receptor antagonist, SR142801 (Alex et al., 2001). These slow synaptic inputs are proposed to come from neighboring IPANs that form a self-reinforcing network (see below) and contain tachykinins (Kunze et al., 1993; Brookes, 2001). Acetylcholine (ACh), acting via muscarinic receptors, also elicits slow depolarizing responses in myenteric IPANs, but as the majority of cells retain stimulation-evoked slow EPSPs in the presence of muscarinic antagonists, ACh is unlikely to be the principal slow neurotransmitter (Morita and North, 1985). Slow EPSPs are mimicked by activators of the adenylyl cyclase (AC)-PKA pathway, such as forskolin (Nemeth et al., 1986; Bertrand and Galligan, 1995), CCK (Palmer et al., 1987), histamine (Nemeth et al., 1984), gastrin releasing peptide (GRP or mammalian bombesin; Zafirov et al., 1985), pituitary adenylyl cyclase-activating peptide (PACAP; Christofi and Wood, 1993) and vasoactive intestinal peptide (VIP; Williams and North, 1979). One line of evidence that these act via stimulation of AC and subsequent increase in intracellular levels of cAMP is their inhibition by adenosine-induced stimulation of G␣I , a G-protein that is negatively coupled to AC (Palmer et al., 1987) (see Fig. 8). However, the slow EPSP is also mimicked by activators of the phospholipase C (PLC)-diacyl glycerol (DAG)- PKC pathway, such as calcitonin gene related peptide (CGRP) and tachykinins (including substance P, acting through NK3 receptors). Although 5-HT causes slow excitation principally through a PKC pathway, it also activates PKA to a small extent (Wood and Mayer, 1979b; Palmer et al., 1987; Pan et al., 1997).

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Fig. 9. Change in neuronal excitability during a sustained slow post-synaptic excitation (SSPE) recorded from an IPAN of the guinea pig distal colon. Stimulation of synaptic inputs to the neuron at 1 Hz for 4 min (bar) caused depolarization of the membrane potential (MP), increased input resistance (Rin) and increased excitability (measured as the numbers of action potentials elicited by 500 ms depolarizing pulses: AP/500 ms). Increased excitability lasted for more than 1 h. Fig. 8. Transmitters, hormones and inflammatory mediators are able to induce slow EPSP-like effects in myenteric IPANs, some via activation of adenylyl cyclase (AC), and others via phospholipase C (PLC). These pathways culminate in the activation of protein kinase A (PKA) and protein kinase C (PKC) which can then phosphorylate intracellular proteins, including IK channels. The closure of the IK channels causes depolarization and increased excitability. Slow EPSPs can be mimicked by forskolin, an AC activator. The principal neurotransmitter implicated in slow EPSPs in IPANs is a tachykinin acting via NK3 receptors, although acetylcholine (ACh) and CGRP also act at receptors coupled to PLC in IPANs. For other abbreviations, see list at the beginning of this article.

Two lines of evidence suggest that the PLC-DAG-PKC pathway, rather than the AC-PKA pathway, mediates the slow EPSP. First, the PLC blocker, D-609, inhibits the slow EPSP in IPANs (Bertrand and Galligan, 1995). Second, adenosine does not inhibit slow EPSPs, suggesting that the receptors primarily mediating slow EPSPs are not coupled to AC by Gs (Pan et al., 1997). Thus, receptors coupled to inositol phospholipid hydrolysis and the PKC pathway (Fig. 8), such as NK3 receptors (Guard et al., 1988), are probably the principal mediators of these slow excitatory responses in the guinea pig ileum (Alex et al., 2001). However, antagonists of receptors for CCK, which acts on IPANs through the AC-PKA pathway (Palmer et al., 1987), partly block slow EPSPs recorded from IPANs (Schutte et al., 1997). While a substantial amount is now known about the chemical coding of the various neuronal subtypes in the myenteric plexus, relatively little is known about the distribution of receptors expressed by individual neurons. It is therefore not always possible to determine whether the effects of applied agonists are direct on the IPANs themselves or whether the responses are the consequence of agonist-induced release of a neurotransmitter, such as substance P, which is known to activate post-synaptic NK3 receptors on IPANs. The increasing use of primary neuronal culture and the

availability of more selective pharmacological tools combined with antibodies against specific receptor subtypes should allow further elucidation of the mechanisms behind the slow excitatory responses of IPANs. 2.6.2. Sustained slow post-synaptic excitation (SSPE) A prolonged synaptic event, the SSPE, is observed in IPANs (Clerc et al., 1999; Alex et al., 2001, 2002; Nurgali et al., 2003). The SSPE outlasts nerve stimulation by many minutes (Fig. 9), and successive SSPEs show substantial facilitation. The increased excitation during a facilitated SSPE can be maintained after stimulation for as long as 4 h. The SSPE is induced by repeated, low frequency (0.5–2 Hz) trains of stimulation of synaptic inputs (Clerc et al., 1999; Alex et al., 2001). It involves increased somatic excitability that facilitates with successive stimuli. During the stimulation, the soma excitability of the IPANs slowly increases, they depolarize, and their input resistance increases markedly, often by over two-fold. This magnitude of increase, accompanied by depolarization, implies that K+ currents are inhibited. During the SSPE, late AHPs are reduced in amplitude and duration and can eventually be suppressed. In addition, anodal break action potentials occur more often after hyperpolarizing pulses (Clerc et al., 1999), which suggests that Ih could be increased during the SSPE. With sufficient depolarization of the neurons, invasion by antidromic action potentials is suppressed. Spontaneous action potentials were rare and the occurrence of fast EPSPs was not reported during the heightened excitability caused by the SSPE (Clerc et al., 1999). The suppression of the late AHP suggests that IK channels are targeted during the SSPE, possibly by phosphorylating enzymes (Fig. 6). Consistent with this, the PKC stimulant, PDBu (1 nM–1 ␮M), caused somatic excitability increases,

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membrane depolarization and increased input resistance in a concentration-dependent manner, mimicking the SSPE (Pan et al., 1997; Kawai et al., 2003). PDBu suppressed the late AHP, and the effects of PDBu (10 nM) on the late AHP were indistinguishable from those observed during the SSPE (Clerc et al., 1999; Kawai et al., 2003). It seems that PDBu mimics the sustained excitation caused by low frequency stimulation of synaptic inputs to IPANs by closing IK channels responsible for the AHP or restricting their opening by Ca2+ , and reducing the current carried by K+ channels that are active at rest. An obvious candidate neurotransmitter for the SSPE could be a tachykinin. However, our results (Alex et al., 2001, 2002) demonstrate that this is unlikely, because the SSPE persists after block of NK1 and NK3 tachykinin receptors on enteric neurons. 2.7. Synaptic interactions between IPANs Both physiological and ultrastructural studies indicate that IPANs synapse with other IPANs (Pompolo and Furness, 1988; Kunze et al., 1993). They also receive many other synaptic inputs whose origins have not been determined (Pompolo and Furness, 1988). The axons of IPANs give rise to very dense networks of varicose terminals, which appear to surround all nerve cell bodies in their own and adjacent ganglia (Furness et al., 1990b, 2003a; Bornstein et al., 1991). Electron microscope studies show that these terminals form synapses on nerve cell bodies, including the cell bodies of IPANs (Pompolo and Furness, 1988). Moreover, when the cell bodies of two IPANs were impaled with microelectrodes, and action potentials were evoked by stimulus pulses passed through one electrode, the other IPAN responded with slow EPSPs (Kunze et al., 1993). Pharmacological experiments support the conclusion that one origin of slow EPSPs in IPANs is synapses made on them by other IPANs. In the guinea pig small intestine, Dogiel type II neurons (IPANs) are immunoreactive for tachykinins, and antagonists of tachykinin receptors partially block the slow EPSPs in these cells (Alex et al., 2001). The conclusion that slow EPSPs in IPANs arise from other IPANs is consistent with experiments in which the axons of ascending and descending interneurons were severed (Bornstein et al., 1984). In areas between lesions, where the severed axons of interneurons had degenerated but endings of IPANs remained, slow EPSPs of usual amplitude were recorded in the cell bodies of IPANs. Thus, data from several experimental approaches all indicate that the IPANs form interconnected networks, and, because transmission at the connections between IPANs is excitatory, these networks would be self-reinforcing (Wood, 1994; Bertrand et al., 1997; Kunze and Furness, 1999). 2.8. IPANS are activated in groups In the guinea pig small intestine, all IPANs project to the mucosa (Song et al., 1991, 1994). A 1 mm length of guinea pig small intestine contains about 650 myenteric IPANs.

Because the stimuli giving rise to intestinal reflexes (luminal chemicals, distension, mucosal distortion) are not spatially confined to sub-millimeter distances, it can be deduced that reflexes are usually initiated by the activation of a population of several hundred primary afferent neurons. Moreover, many IPANs, whether they were directly activated or not, would be excited synaptically. IPANs have considerable overlap in their receptive fields; retrograde tracing indicates that each villus is supplied by the axons of about 65 IPANs with cell bodies in myenteric ganglia (Song et al., 1994). By mapping regions of mucosa from which IPANs can be electrically activated, it was concluded that each projects to a strip of mucosa, about 2 mm in oral to anal width and 7 mm in circumferential length (Bertrand et al., 1998), which contains about 80–120 villi. It is thus concluded that assemblies of several thousand IPANs respond together when changes in muscle tension and luminal chemistry occur. 2.9. Changes in IPAN excitability caused by inflammatory mediators IPANs are excited by the inflammatory mediators histamine, prostaglandins (PG), leukotrienes, interleukins, activation of proteinase-activated receptors and 5-HT (Nemeth et al., 1984; Dekkers et al., 1997; Palmer et al., 1998; Linden et al., 2001, 2002; Gao et al., 2002; Manning et al., 2002; Liu et al., 2003). Each of these inhibits the AHP current, IAHP , thus causing an increase of somatic excitability, and in most cases, they depolarize the membrane potential and increase Rin. The mechanisms leading to decrease of IAHP differ according to the mediator, and also from the actions of the SSPE in some respects. The inflammatory mediators may also act on other targets to affect excitability of IPANs. After infection with Trichinella spiralis, jejunal myenteric and submucosal IPANs exhibit an increase in somatic excitability, notably expressed by repetitive spike discharge during intrasomal injection of depolarizing current (Frieling et al., 1994; Palmer et al., 1998). This repetitive spike discharge was reversibly suppressed by H2 histamine receptor blockers in submucosal IPANs and might thus reflect an endogenous release of histamine in the T. spiralis-sensitized intestine (Frieling et al., 1994). Activation of H2 histamine receptors probably suppresses an IA -type current that is well known to modulate the firing frequency of neurons (see Starodub and Wood, 2000a). In IPANs, this current might also contribute to the resting potential (Starodub and Wood, 2000b). Thus, IA blockade in IPANs might result in increased action potential frequency and contribute to both depolarization and increase in Rin. An additional effect of histamine is the activation of a Cl− conductance via H2 receptors in about half of the myenteric IPANs (Starodub and Wood, 2000a), which would depolarize the neuron. In agreement with this putative role of IA in the T. spiralis infection model, the somas of the IPANs exhibit a significant depolarization and an increase of input resistance (Frieling

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et al., 1994; Palmer et al., 1998). In parallel, the AHP was suppressed in submucosal (Frieling et al., 1994) and myenteric (Palmer et al., 1998) IPANs. In myenteric IPANs, these effects are described as being accompanied by a small but significant decrease in the action potential duration (that indicates a decrease of Ca2+ entry) and by the occurrence of anodal break action potentials (Palmer et al., 1998). Decreased Ca2+ entry and increased Ih could both contribute to reducing the amplitude and the duration of the late AHP. In addition, spontaneous action potentials occurred in both submucosal and myenteric IPANs (Frieling et al., 1994; Palmer et al., 1998), and the neurons exhibited fast EPSPs that routinely reached suprathreshold depolarization and caused action potential discharge (Frieling et al., 1994; Palmer et al., 1998). When inflammation was induced by administration of trinitrobenzene sulfonic acid (TNBS) into the lumen of the distal colon (Linden et al., 2003), the somatic excitability of myenteric IPANs increased although no changes of membrane potential or membrane input resistance were noted. As in the T. spiralis infection model, the late AHP was decreased, cells were more likely to possess spontaneous activity and generate anodal break action potentials, and about 70% of the sampled neurons exhibited fast EPSPs. This increased neuronal excitability and reduction in the AHP were proposed to be principally due to an increased activation of Ih in the inflamed compared to control colon, rather than alterations in gKCa . Action potential shape, and therefore Ca2+ entry into the cell, appeared to be unaffected by TNBS-induced inflammation. 2.10. Are IPANs involved in neuropathologies? There is increasing evidence that IPANs are affected in inflammation of the small intestine and colon (see above) and that changes in IPAN properties might contribute to abnormalities of sensory signalling and enteric reflex control, including abnormalities that follow inflammation (Sharkey and Kroese, 2001; Sharkey and Mawe, 2002). A question that needs to be adequately answered is whether the actions of inflammatory mediators on IPANs are related to long-term sequelae of inflammation. One disorder that could involve IPANs is irritable bowel syndrome (IBS), and it has been suggested that they could be targets for therapeutic intervention in IBS (Mayer and Marvizon, 1999; Buéno et al., 2000; Clerc et al., 2002). The incidence of IBS increases following inflammation such as that induced by bacterial enteritis (Gwee et al., 1996; Neal et al., 1997), and symptoms can persist for over 6 years after an infective episode (Neal et al., 2002). In animals, the responses of the intestine itself to agonists that cause contraction and to nerve stimulation are altered (Barbara et al., 1997; Venkova et al., 1999), and the response of the animals to a painful distension of the gut are exaggerated (Al-Chaer et al., 2000) after a period of inflammation, implying that there is sensitization both of

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the gut itself and of the afferent signalling pathways from the gut. The hypothesis that IPANs are involved is given support by animal experiments that have shown changed enteric neuron function following intestinal inflammation (see above). IPANs exhibit somatic excitability increases that persist when the neurons are investigated in vitro following in vivo inflammation or exposure to inflammatory mediators, which is described above for T. spiralis infection and TNBS exposure. Prolonged exposure to PG also caused sustained increases in enteric neuron excitability (Manning et al., 2002). In mice, increased excitability, measured by recording from jejunal muscle strips in vitro, persisted for 4 weeks after inflammation was induced (Barbara et al., 1997), although direct recordings from enteric neurons after inflammation have not been made in this species. Enhanced, nerve-mediated, transmucosal fluid secretion also occurs following inflammation (Shea-Donohue et al., 2001). A common feature of intestinal inflammation is the upregulation of cyclooxygenase-2 (COX-2), leading to the increased synthesis of eicosanoids, including PGE2 . In human patients, COX-2 mRNA levels in the myenteric plexus are increased six to eight-fold when the gut is inflamed (Roberts et al., 2001), an increase that persists for several weeks after inflammation (Barbara et al., 2001). PGE2 excites myenteric neurons from guinea pig, an effect that is antagonized by the prostanoid receptor antagonist, AH-6809 (Dekkers et al., 1997; Manning et al., 2002). Enhancement of enteric reflexes that is caused by repeated distension of the rat colon is blocked by the COX inhibitor, indomethacin (Furness et al., 2002). COX inhibition also reduces the increased contractility of the external muscle that follows inflammation in mice (Barbara et al., 2001). Thus, one of the mechanisms for enhanced enteric neuron excitability following inflammation could be induction of COX-2 and production of greater than normal levels of PGE2 . However, other COX-2 products, and other inflammatory mediators, probably contribute. For example, in the rat gut, inflammation is associated with increased mRNA and protein for interleukin (IL)-1␣ and -1␤, IL-6 and tumor necrosis factor (TNF)-␣ in the longitudinal muscle-myenteric plexus preparation (Khan and Collins, 1994). In combination, IL-1␤ and IL-6 cause a two-fold increase in PGE2 levels in this preparation (Rühl et al., 1995), suggesting that they could indirectly excite myenteric neurons through stimulation of PGE2 production. Moreover, both IL-1␤ and IL-6 directly excite myenteric neurons (Xia et al., 1999; Kelles et al., 2000). Other arachidonic acid products, leukotrienes C4, D4 and E4, which can be expected to be increased during inflammation, also excite enteric neurons (Liu et al., 2003), as does the mast cell product, histamine (Nemeth et al., 1984). As summarized above, H2 receptor blockers suppress T. spiralis-induced increased spike discharge of submucosal IPANs (Frieling et al., 1994). Inflammatory mediators that excite or otherwise change enteric neuron properties can be products of activated

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macrophages that occur in myenteric ganglia after inflammation (Rühl et al., 1995; Vallance et al., 1999), of invading lymphocytes (Bradley et al., 1997; Vallance et al., 1999) or of mast cells, neurons, glia or muscle cells. The involvement of CD4-positive T lymphocytes is implied by the observation of Vallance et al. (1999), who found, in CD4 T cell deficient mice, a reduction in the enhanced muscle contraction and propulsive activity that is caused by T. spiralis infection. Greater reduction was found in athymic mice (Vallance et al., 1999). A recent human study (Törnblom et al., 2002) has provided powerful evidence that there may be an involvement of changes in enteric neurons in post-infection IBS. Full thickness intestinal biopsies were taken from 10 IBS patients. In each case, there was some degeneration of neurons in myenteric ganglia, and in 9 of 10 cases the ganglia were infiltrated with CD3-positive T lymphocytes. The authors conclude that inflammatory damage to enteric neurons leads to the dysmotility that is characteristic of IBS.

bodies in the submucosal ganglia (Cassuto et al., 1983; Diener and Rummel, 1990; Frieling et al., 1992; Cooke and Reddix, 1994). The secretomotor neurons stimulate the epithelial cells to pump chloride ions, which are accompanied by water, into the lumen. Local vasodilator reflexes in the small intestine are caused by mechanical or chemical irritation of the mucosa, and substantial evidence indicates that the vasodilator neurons are intrinsic to the intestine and transmission from them is predominantly non-cholinergic (Vanner et al., 1993; Vanner and Surprenant, 1996). It is presumed that the first neurons in these reflexes are the IPANs, but this has not been directly shown. In fact, of the reflexes in the intestine, the vasomotor reflexes are least studied. The same motor neurons have axons that branch to supply both the secretory epithelium and arterioles, thus some secretomotor and vasodilator reflexes may share the same final neurons. This makes physiological sense, as at least part of the secreted water and electrolyte comes indirectly from the vasculature (see below).

3. Enteric nerve circuits

3.2. Inter-species differences

3.1. Involvement of IPANs

Detailed studies have now been made of enteric neurons and nerve circuits in guinea pig, and a significant amount of information also exists for rat, mouse, human and porcine intestines. There are also data on other species. Thus, although the guinea pig has provided the model system, much of the information from that species appears to be applicable to others. The cell bodies of functionally analogous neurons may have different locations in different species, and analogous neurons may have different chemical coding. The possibility of differences in location is highlighted by comparison between the pig and guinea pig small intestines. In the pig, there are two prominent and distinguishable sets of ganglia in the submucosa, whereas there is one type in the guinea pig. In the pig and dog, some of the cell bodies of neurons that supply the circular muscle are in submucosal ganglia (Furness et al., 1990a; Timmermans et al., 1992), whereas in guinea pig they are all in the myenteric ganglia (Wilson et al., 1987).

Intrinsic reflexes that affect motility, water and electrolyte secretion and blood flow all occur in the intestine. Each of these is evoked by similar stimuli, although it is not known whether the same, different or overlapping populations of IPANs contribute to motility, secretomotor, and vasomotor reflexes. Muscle motor reflexes that have been studied as stereotyped responses of the circular muscle, i.e. excitation oral and relaxation anal, can be evoked by distension of the muscle (which was carried out without distorting the mucosa), by chemicals applied to the mucosal surface, and by mucosal distortion (Hukuhara, 1951; Furness and Costa, 1987; Yuan et al., 1991; Spencer et al., 2003). If the intestine is chronically denervated, so that endings of extrinsic neurons degenerate, and then removed from the animal, the stereotyped ascending excitatory and descending inhibitory reflexes, elicited by distension or mucosal distortion, are unaffected (Langley and Magnus, 1905; Crema et al., 1970; Furness et al., 1995). Thus, these reflexes occur after degeneration of the endings of extrinsic afferent neurons, and are the consequences of activation of IPANs (and down-stream neurons) by physiological stimuli. Secretomotor reflexes are initiated physiologically by chemical or mechanical interaction of luminal contents with the mucosa, or pathologically by toxins, such as cholera toxin or enterotoxins, in the lumen (Frieling et al., 1992; Cooke and Reddix, 1994). Enteric reflexes also cause bicarbonate secretion in response to duodenal acidification (Flemström, 1994). The enteric secretomotor circuits consist of IPANs with their endings in the mucosa and nerve circuits that pass through the myenteric and submucosal plexuses and impinge on secretomotor neurons with cell

3.3. Circuits for motility control The circuits for motility reflexes (Fig. 10) have been deduced from a combination of physiological, morphological and pharmacological studies (Furness et al., 2000). Many strategies have been used to reduce the complex movements of the intestine to underlying stereotyped responses to defined stimuli so that circuits can be deduced. The often unstated assumptions are that the complex patterns that are observed in vivo are the consequence of many superimposed responses to local variations in the volume and physicochemical properties of the contents of the intestine and that the local reflexes, by themselves, are relatively simple. Simplified approaches have permitted conclusions to be drawn about the natural stimuli that elicit reflexes, and the

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itself, and as digestive enzymes do their work. Moreover, the movements elicited by the reflexes have local effects on tension-sensitive IPANs. In the small intestine, the local reflexes spread and mix the contents, so it is not surprising that peristaltic movements progress for only short distances.

Fig. 10. The pathways for propulsive reflexes in the intestine. A short segment of intestine is represented, on which the first parts of descending inhibitory and ascending excitatory reflex pathways are depicted. The circuitry for the ascending and descending pathways have similar patterns. Intrinsic primary afferent neurons (IPANs) are circumferentially oriented and form self-reinforcing networks. They provide outputs to ascending and descending interneurons and monosynaptic connections to motor neurons. The interneurons form descending and ascending chains and also provide outputs to motor neurons. Ascending reflex pathways supply inputs to excitatory longitudinal muscle motor neurons and excitatory circular muscle motor neurons. A set of descending interneurons (bottom part of diagram), that is involved in conducting the migrating myoelectric complex along the small intestine, receives very little input from IPANs, but connects with motor neurons.

stereotyped responses of the musculature to stationary stimuli. In most of these studies, any propulsion of contents that involves luminal constriction has been referred to as peristalsis, or as a peristaltic reflex. Although peristalsis is thus rather loosely defined, it is a useful shorthand for propulsive activity. Bayliss and Starling (1899) defined peristalsis as consisting of a contraction of the circular muscle oral to a bolus in the lumen (later referred to as the ascending excitatory reflex) and relaxation on the anal side (the descending inhibitory reflex). Mall (1896) deduced that irritation of the mucosa by a bolus was the stimulus for peristalsis. This was demonstrated under more defined conditions by stroking the mucosa of the opened intestine, which elicited contraction and depolarization of the circular muscle oral to the stimulus plus relaxation and hyperpolarization on the anal side (Hukuhara et al., 1958; Smith and Furness, 1988; Neya et al., 1993). The same types of responses can be elicited by distorting the villi by gentle pressure (Yuan et al., 1991). When care is taken to avoid mechanical stimulation of the mucosa, polarized reflexes are also elicited by local application to the mucosa of hypertonic salt solutions, short chain fatty acids, bile salts or inorganic acid (Hukuhara et al., 1958; Baldwin and Thomas, 1975). Thus, three types of stimuli, distension, mechanical distortion of the mucosa and change in luminal chemistry can independently elicit polarized reflex responses in the intestine. In vivo, all of these stimuli are present at the same time, and will affect successive sites in the intestine to different degrees, and with varying intensities over time, as the intestine mixes and absorbs its contents, as more material arrives from the stomach, pancreas, gall bladder and the intestine

3.3.1. Different patterns of motility Several patterns of motility are observed in the intestine. These must rely on the same neurons whose roles have been determined in simplified experiments. It is logical that the same nerve circuits can be programmed to produce different outcomes, defined as migrating motor complexes, mixing movements and peristaltic movements. Migrating motor (myoelectric) complexes are observed in carnivores (including human) in the unfed state, and occur at all times in continuous eaters, which includes most or all herbivores. The migrating complexes involve regions of intestine in rhythmic contractile activity that moves slowly along the intestine. The complexes are dependent on activity in neural circuits and are blocked or substantially delayed in their times of occurrence by inhibiting synaptic transmission in the intestine, or by antagonizing excitatory transmission to the muscle (Borody et al., 1985; Galligan et al., 1986; De Vos, 1993). Progress of the migrating complexes is prevented by local infusion of TTX into an artery supplying a small region of the intestine (Sarna et al., 1981). Irregular contractile activity (mixing activity) occurs in the intervals between the migrating motor complexes and in the fed state in carnivores. The irregular contractile activity in the intestine is attenuated by pharmacological inhibition of excitatory transmission to enteric neurons or muscle (Quigley et al., 1934; Mellander et al., 1995; Hasler, 2003). The irregular activity that is observed in the fed state is probably the consequence of many reflex stimuli whose effects are superimposed on the rhythmic activity of the intestine that is generated by the muscle (Hasler, 2003). Although the reflexes are usually described in terms of ascending excitation of the circular muscle and descending inhibition, the types of responses that are observed are often more complex than this, and include relaxation at the site of distension (the accommodation reflex), contraction of the longitudinal muscle, local contractions of the circular muscle and contractions of the circular muscle that propagate anally (Cannon, 1911; Smith and Robertson, 1998; Spencer et al., 2002, 2003; Hasler, 2003). It had been observed by Bayliss and Starling (1899) that the longitudinal and circular muscles contract together. There had been some controversy about this observation (see Hasler, 2003), but it has been recently confirmed that during propulsive activity in the small intestine and colon the muscle layers do contract in synchrony (Smith and Robertson, 1998; Spencer et al., 2003). Shortening of the longitudinal muscle during circular muscle contraction prevents the circular muscle contraction being translated into a lengthening of the intestine, and directs the contractile force towards occlusion of the lumen.

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There have been extensive studies of the transmitter types in enteric neurons of motility controlling pathways, and of the receptors that are acted upon by the transmitters (Table 1). An analysis of the transmitter and receptor types at neuro-neuronal synapses in the enteric nervous system has been recently published (Galligan, 2002). 3.4. Secretomotor and vasomotor reflexes Circuits for secretomotor reflexes are illustrated in Fig. 11 (see also Furness et al., 2000). Distension, mechanical stimulation of the mucosa and chemicals applied to the mucosa, evoke both secretomotor and vasodilator reflexes (Diener and Rummel, 1990; Frieling et al., 1992; Vanner et al., 1993; Sidhu and Cooke, 1995). The secretomotor neurons stimulate the epithelial cells to pump Cl− into the lumen, which takes with it counter ions, mostly Na+ , and water. Histochemical studies in the guinea pig small intestine indicate that two of the final motor neurons for secretion are also motor neurons for vasodilatation (Fig. 11), that is, these secretomotor neurons may cause a physiologically appropriate vasodilation, concomitant with secretion, through collaterals to submucosal arterioles (Furness et al., 1987; Jiang et al., 1993; Evans et al., 1994; Li et al., 1998). Thus, there may not be separate reflexes for secretion and vasodilation,

Fig. 11. Schematic diagram of the neural circuits for secretomotor and vasodilator control in the guinea pig small intestine. Secretomotor responses are mediated both through antidromic action potential invasion of the mucosal endings of intrinsic primary afferent neurons, and through reflex activation of secretomotor and vasodilator neurons. Both locally absorbed fluid and fluid from the blood circulation, whose supply is modified by vasodilation, is utilized for secretion. ACh and VIP/PACAP are transmitters for secretion and vasodilation. Tachykinins (TK) released from mucosal terminals of IPANs also cause secretion. The numbers on the neurons refer to the descriptions in Table 1.

although a third type of secretomotor neuron does not project to the vasculature (Fig. 11). In the small intestine, an important physiological stimulus for secretion appears to be the presence or active uptake of nutrients. Nutrients such as glucose that are absorbed by a Na+ co-transporter draw in Na+ along with Cl− and water. At the same time, glucose or its uptake stimulates the enteric secretomotor reflex to return electrolyte to the lumen (Sjövall et al., 1984). Enteric reflexes also cause bicarbonate secretion in response to duodenal acidification, although other acid-sensitive mechanisms, including a neurally-independent stimulation of prostaglandin production, also release bicarbonate (Flemström, 1994). Secretomotor reflexes can also be initiated pathologically by toxins in the lumen, such as cholera toxin or enterotoxins. The IPANs can be considered to be acting as nociceptors when they are activated by toxins to cause secretion and diarrhea, whose physiological effect is to rid the intestine of the offending toxin. IPANs themselves may have secretomotor functions. IPANs are immunoreactive for tachykinins and their varicose processes are immunoreactive for the vesicular acetylcholine transporter (Li and Furness, 1998). Thus, their mucosal endings are likely to release acetylcholine and tachykinins, both of which cause secretion. It has been directly shown that action potentials in one process of an IPAN traverse the cell body to invade other processes (Hendriks et al., 1990) and the pattern of branching of the neurons indicates that action potentials could be conducted, as an axon reflex, between terminals that branch within the mucosa (Bertrand et al., 1998). Interestingly, secretory responses to distension and to mucosal stroking in the guinea pig colon are reduced by TTX and by atropine (which blocks the acetylcholine receptors on the epithelium), but not by an antagonist of cholinergic fast neuro-neuronal transmission, mecamylamine (Frieling et al., 1992; Sidhu and Cooke, 1995). The concentration of mecamylamine that was used was shown to block nicotinic receptors of submucosal neurons in the colon (Sidhu and Cooke, 1995). Moreover, the responses to stroking were not reduced by extrinsic denervation, indicating that they are dependent on activation of intrinsic neurons (Cooke et al., 1997). Thus, there is good evidence that acetylcholine released by axon reflex, or by mononeuronal reflexes crossing the soma, contributes to secretory responses (Fig. 11). Polyneuronal enteric secretomotor circuits consist of IPANs and an integrating circuitry in the myenteric and submucosal plexus that feeds back to secretomotor neurons with cell bodies in the submucosal ganglia (Fig. 11). In some cases, the reflex pathways involve the myenteric ganglia; this is so for cholera toxin-induced secretion (Jodal et al., 1993), whereas reflexes initiated by mechanical stimulation of the mucosa can be mediated entirely through the submucosal plexus (Frieling et al., 1992; Cooke and Reddix, 1994). Within the submucosal plexus, the only neurons that make synaptic connections with other submucosal neurons appear to be the IPANs (Lomax et al., 2001; Reed and

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Vanner, 2001; Furness et al., 2003a). Therefore, the submucosal reflexes are monosynaptic. Intrinsic connections within the submucosa give rise to fast cholinergic synaptic potentials (Bornstein et al., 1988; Reed and Vanner, 2001), which is consistent with the cholinergic phenotype of submucosal IPANs (Li and Furness, 1998). Elegant experiments in which recordings were taken from submucosal neurons, in preparations of guinea pig small intestine with no myenteric plexus present, showed that cholinergic fast EPSPs are recorded from second order neurons when mechanical stimuli are applied to the mucosa (Pan and Gershon, 2000). Slow EPSPs were also recorded, implying that there may be co-transmission from submucosal IPANs. Inputs to submucosal neurons from the myenteric plexus are from neurons that run anally in the myenteric plexus (Fig. 11). When these neurons are stimulated, cholinergic fast EPSPs are recorded in submucosal neurons (Moore and Vanner, 2000). There are two types of secretomotor neurons: cholinergic and non-cholinergic. The non-cholinergic neurons appear to mediate most of the local reflex response, and utilize VIP, or a related peptide (for example PACAP), as their primary transmitter (Jodal and Lundgren, 1989; Cooke and Reddix, 1994; Reddix et al., 1994). Two types of submucosal cholinergic neuron occur in the guinea pig small intestine, those that also contain NPY (neuropeptide Y) (and other peptides) and those that contain calretinin. The ACh/calretinin neurons preferentially innervate the glands at the base of the mucosa (Brookes et al., 1991; Clerc et al., 1998) and have collaterals to submucosal arterioles, whereas the ACh/NPY neurons do not appear to innervate the arterioles. The presence of three classes of secretomotor neurons, two of which also provide vasodilator collaterals, may provide a mechanism to balance secretion and vasodilatation in a way that is appropriate to the digestive state (Fig. 11). Secretory reflexes in the small intestine are involved with regulation of whole body water and electrolyte status, and with flushing material from the mucosal surface. In an equilibrium state, the amount of fluid lost via the kidneys, respiration and perspiration should be matched by absorption from the alimentary tract. If more fluid is absorbed with nutrients or across the gastric mucosa, some of that can be passed back under the control of secretomotor reflexes. Thus, the source of secreted fluid in the small intestine can be a mixture of serum electrolyte and electrolyte absorbed from the lumen. We postulate that local computation of the need for vasodilatation and local absorption to supply electrolyte for secretion determines the relative activation of vasodilator and non-vasodilator secretomotor neurons.

4. Conclusions IPANs have pivotal roles in the gastrointestinal tract as the first neurons of intrinsic reflex pathways controlling motility, fluid movement across the luminal epithelium and local blood flow in the small and large intestines. IPANs

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are responsive to physiologically appropriate mechanical stimuli and to the chemical constituents of the gut lumen. They form self-reinforcing networks and make synapses with interneurons and motor neurons. In the guinea pig small intestine, where they have been studied most intensely, IPANs have Dogiel type II morphology and can be identified electrophysiologically by the presence of a Ca2+ component to the action potential, a TTX-resistant Na+ current, a hyperpolarization-activated cation current and a prominent, post-action potential, late AHP. The late AHP and Ih currents can be modulated synaptically and by inflammatory mediators, via second messenger cascades, to produce long-term increases in neuronal excitability. There is increasing evidence from animal models, and from human studies, that enteric neuronal damage and phenotypic alterations that follow inflammation are involved in intestinal dysfunction observed in disorders such as post-infective IBS. IPANs are therefore of great interest for understanding digestive tract physiology and pathology and as possible therapeutic targets for treating functional bowel disorders.

Acknowledgements Current studies that are referred to in this work are supported by the National Health and Medical Research Council of Australia, the Centre National de la Recherche Scientifique (CNRS), GlaxoSmithKline (UK) and Pfizer Global Research and Development. We would like to thank Dr. Keith Sharkey for his helpful comments and Trung Van Nguyen for assistance with the figures. References Al-Chaer, E.D., Kawasaki, M., Pasricha, P.J., 2000. A new model of chronic visceral hypersensitivity in adult rats induced by colon irritation during postnatal development. Gastroenterology 119, 1276–1285. Alex, G., Kunze, W.A.A., Furness, J.B., Clerc, N., 2001. Comparison of the effects of neurokinin-3 receptor blockade on two forms of slow synaptic transmission in myenteric AH neurons. Neuroscience 104, 263–269. Alex, G., Clerc, N., Kunze, W.A.A., Furness, J.B., 2002. Responses of myenteric S neurons to low frequency stimulation of their synaptic inputs. Neuroscience 110, 361–373. Baldwin, M.V., Thomas, J.E., 1975. The intestinal intrinsic mucosal reflex: a possible mechanism of propulsive motility. In: Friedman, M.H.F. (Ed.), Functions of the stomach and intestine. University Park Press, Baltimore, pp. 75–91. Barbara, G., Vallance, B.A., Collins, S.M., 1997. Persistent intestinal neuromuscular dysfunction after acute nematode infection in mice. Gastroenterology 113, 1224–1232. Barbara, G., De Giorgio, R., Deng, Y., Vallance, B., Blennerhassett, P., Collins, S.M., 2001. Role of immunologic factors and cyclooxygenase 2 in persistent postinfective enteric muscle dysfunction in mice. Gastroenterology 120, 1729–1736. Bayliss, W.M., Starling, E.H., 1899. The movements and innervation of the small intestine. J. Physiol. (Lond.) 24, 99–143. Bertrand, P.P., Bornstein, J.C., 2002. ATP as a putative sensory mediator: activation of intrinsic sensory neurons of the myenteric plexus via P2X receptors. J. Neurosci. 22, 4767–4775.

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