Slow synaptic excitation (slow EPSP) in enteric neurones is recorded as a slowly activating depolarization of the membrane potential in specific populations of enteric neurones when neurotransmitters are released experimentally by focal electrical stimulation of presynaptic axons in the myenteric and submucosal plexuses (reviewed by Surprenant, 1989;Wood, 1994;Galligan, 1998;Gershon, 1998). Mediators released to the enteric nervous system in paracrine fashion from non-neuronal cell types (e.g. histamine and cytokines from enteric mast cells) can evoke responses that mimic slow synaptic excitation (Wood, 1992;Liu et al. 2003). Two kinds of slow EPSPs are recorded in enteric neurones. An increase in input resistance is associated with the depolarization and augmented excitability for one kind of slow EPSP. The input resistance decreases or remains unchanged during the depolarization and augmented excitability of the second kind. Slow EPSPs with increased input resistance are found generally in AH-type neurones with multipolar Dogiel Type II morphology. Most evidence suggests that the principal ionic mechanism for this type of slow EPSP is suppression of resting Ca 2+ -dependent K + conductance that accounts for the membrane depolarization, increased input resistance, and suppression of the Ca 2+ component of the rising phase of the action potential (e.g. Grafe et al. 1980). Signal transduction for the slow EPSP with increased input resistance involves coupling of metabotropic receptors through heterotrimeric G proteins to adenylate cyclase, and elevation of intraneuronal cyclic adenosine monophosphate (Palmer et al. 1986(Palmer et al. , 1987.Whereas slow EPSPs characterized by increased input resistance during the depolarizing response predominate in AH-type neurones in the myenteric plexus, slow EPSPs characterized by decreased input resistance are routinely found in S-type uniaxonal neurones in the small and large intestinal submucosal plexus. Likewise, application of putative neurotransmitters and paracrine mediators (e.g. serotonin, ATP and substance P) evoke slowly activating depolarizing responses associated with decreased input resistance in S-type neurones in the submucosal plexus.This report presents evidence that synaptically released ATP acts at P2Y 1 purinergic receptors to evoke slow EPSPs that are characterized by decreased input resistance in the submucosal plexus. The evidence suggests that the signal transduction cascade for the submucosal P2Y 1 receptor includes activation of phospholipase C, release of inositol 1,4,5-trisphosphate and elevation of cytosolic free Ca

Hu proteins, together with neurone-specific enolase (NSE), protein gene product 9.5 (PGP-9.5), microtubule-associated protein-2 (MAP-2) and tubulin beta III isoform, were evaluated immunohistochemically as neuronal markers in whole-mount preparations and cultures obtained from the myenteric plexus of guinea-pig small intestine. Anti-Hu immunostaining marked the ganglion cell somas and nuclei without staining of the neuronal processes in the whole-mounts and cultures. The ganglion cell bodies were not obscured by staining of multiple neuronal fibres and this facilitated accurate counting of the neurones. MAP2 immunostaining also provided clear images of individual neurones in both whole mounts and cultures. Immunoreactivity for NSE, PGP-9.5 and tubulin beta III isoform provided sharp images of the ganglion cells in culture, but not in whole-mount preparations. Strong staining of the neuronal processes in the whole-mount preparations obscured the profiles of the ganglion cell bodies to such an extent that accurate counting of the total neuronal population was compromised. Anti-Hu immunostaining was judged to be an acceptable method for obtaining reliable estimates of total numbers of myenteric neurones in relation to other specific histochemical properties such as histamine binding.

Reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiological recording, and intraneuronal injection of the neuronal tracer biocytin were integrated in a study of the functional expression of corticotropin-releasing factor (CRF) receptors in the guinea pig enteric nervous system. RT-PCR revealed expression of CRF1 receptor mRNA, but not CRF2, in both myenteric and submucosal plexuses. Immunoreactivity for the CRF1 receptor was distributed widely in the myenteric plexus of the stomach and small and large intestine and in the submucosal plexus of the small and large intestine. CRF1 receptor immunoreactivity was coexpressed with calbindin, choline acetyltransferase, and substance P in the myenteric plexus. In the submucosal plexus, CRF1 receptor immunoreactivity was found in neurons that expressed calbindin, substance P, choline acetyltransferase, or neuropeptide Y. Application of CRF evoked slowly activating depolarizing responses associated with elevated excitability in both myenteric and submucosal neurons. Histological analysis of biocytin-filled neurons revealed that both uniaxonal neurons with S-type electrophysiological behavior and neurons with AH-type electrophysiological behavior and Dogiel II morphology responded to CRF. The CRF-evoked depolarizing responses were suppressed by the CRF1/CRF2 receptor antagonist astressin and the selective CRF1 receptor antagonist NBI27914 and were unaffected by the selective CRF2 receptor antagonist antisauvagine-30. The findings support the hypothesis that the CRF1 receptor mediates the excitatory actions of CRF on neurons in the enteric nervous system. Actions on enteric neurons might underlie the neural mechanisms by which stress-related release of CRF in the periphery alters intestinal propulsive motor function, mucosal secretion, and barrier functions.

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Melanin-concentrating hormone (MCH) is expressed primarily in the hypothalamus and has a positive impact on feeding behavior and energy balance. Although MCH is expressed in the gastrointestinal tract, its role in this system remains elusive. We demonstrate that, compared to wild type, mice genetically deficient in MCH had substantially reduced local inflammatory responses in a mouse model of experimental colitis induced by intracolonic administration of 2,4,6 trinitrobenzene sulfonic acid (TNBS). Likewise, mice receiving treatments with an anti-MCH antibody, either prophylactically or after the establishment of colitis, developed attenuated TNBS-associated colonic inflammation and survived longer. Consistent with a potential role of MCH in intestinal pathology, we detected increased colonic expression of MCH and its receptor in patients with inflammatory bowel disease. Moreover, we found that human colonic epithelial cells express functional MCH receptors, the activation of which induces IL-8 expression. Taken together, these results clearly implicate MCH in inflammatory processes in the intestine and perhaps elsewhere.experimental colitis ͉ IL-8 ͉ inflammatory bowel disease ͉ neuropeptides ͉ MCH deficient mice M elanin-concentrating hormone (MCH) is a 17-to 19-aa cyclic neuropeptide conserved from fish to human (1) and predominantly localized in the brain (2). Several pharmacological and genetic studies revealed a role for this peptide in the regulation of feeding behavior and energy expenditure toward a positive energy balance (3-5). More recent studies extended the physiological functions of MCH as a broad regulator of cognitive and autonomic aspects related to rewarding behaviors (6, 7). Outside the brain, MCH is localized in the pancreas (8), skin (9), and gastrointestinal tract (10). It has been also found in tissular and circulating immune cells (11)(12)(13)(14). However, the physiological role of MCH in these peripheral tissues has yet to be established.In humans, two G-protein-coupled receptors for MCH have been identified, MCHR1 (also known as SLC1 or GPR24) (15-18) and MCHR2 (19-21), whereas rodents express only MCHR1. In the rodent brain, MCHR1 is expressed in areas important for feeding, learning and motivated behavior, integration of sensory and gustatory inputs, autonomic control, and arousal (22, 23). MCHR1 mRNA is also expressed in the thyroid, kidney, adipose tissue, lung, testes, and tongue (23), whereas functional MCHR1 is also present in lymphocytes (12,14), insulin-producing cell lines (24), and mouse and human pancreatic islets (8).Several neuropeptides that are part of the neuroendocrine system exhibit important immunomodulatory effects and mediate inflammation in various organs, including the intestine (25, 26). There is little evidence to indicate expression of MCHR of either type in the intestine of animals or humans, and the role of MCH in inflammatory responses in the gut or elsewhere has not been evaluated. Based on these considerations and because MCH is also expressed in immune c...