Survival and neurotransmitter plasticity in cultured rat colonic myenteric neurons

The enteric nervous system is of great importance for maintenance and proper function of the gastrointestinal tract. The aim of this study was to quantify myenteric neuronal subpopulations expressing calcitonin gene-related peptide (CGRP), galanin, neuropeptide Y (NPY), somatostatin, vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in rat colon in vivo and after culturing. Further we investigated if culturing in the presence of CGRP, galanin, VIP, S-nitroso-N-acetyl-d,l-penicillamine (SNAP, a NO donor) or N-nitro-l-arginine methyl ester (l-NAME, a NOS inhibitor) affect neuronal survival.

After 4 days of culturing the proportions of neurons expressing CGRP, NPY, somatostatin or VIP increased as compared to in vivo, while the proportions of neurons expressing galanin or NOS did not change. Neuronal survival was unaffected after culturing in media enriched with CGRP, galanin, VIP, SNAP or l-NAME. Neither did addition of CGRP, galanin nor VIP to the cultures affect the relative numbers of neurons expressing CGRP, galanin or VIP respectively. Addition of SNAP or l-NAME did not change the percentage of neurons expressing NOS.

In conclusion, cultured rat colonic myenteric neurons increase their expression of CGRP, NPY, somatostatin and VIP, suggesting that these neuropeptides are of importance for neuronal survival.     

Differential effects of VIP and PACAP on survival of cultured adult rat myenteric neurons

Our knowledge of neuroprotective factors important for the adult enteric nervous system is poor. Changes in expression of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) in enteric neurons in response to neuronal injury or colchicine treatment, as well as in intestinal adaptation, have been described. Cultured myenteric neurons increase their expression of VIP; furthermore, culturing myenteric neurons in the presence of VIP enhances neuronal survival. The aims of this study were to evaluate possible changes in PACAP expression in dissociated and cultured myenteric neurons from adult rat small intestine, and to determine the ability of PACAP-38 and PACAP-27 to promote survival of cultured myenteric neurons, as compared with that of VIP. A marked decrease in the number of surviving neurons was noted during culturing. No difference in neuronal survival was found after culturing in the presence of PACAP-38 or PACAP-27, whereas VIP significantly increased neuronal survival. In contrast to the marked increase noted in the number of VIP-expressing neurons, culturing caused no change in the number of PACAP-expressing myenteric neurons. We were thus able to demonstrate that VIP, but not PACAP, promoted survival of myenteric neurons in culture. This suggests the presence of a VIP-specific receptor mediating neuroprotection in adult myenteric neurons.     

Extrinsic control of the release of galanin and VIP from intrinsic nerves of isolated, perfused, porcine ileum.

By immunohistochemistry galanin-like immunoreactivity and vasoactive intestinal polypeptide (VIP)-like immunoreactivity were found in nerve cell bodies mostly in the submucous plexus and in nerve fibres in the mucosa, submucosa and muscularis including the myenteric plexus of the porcine ileum and were found to co-exist in most of these structures. Using isolated, perfused porcine ileum we studied the release of galanin and VIP in response to electrical stimulation of the mixed periarterial nerves or to intraarterial infusions of different neuroactive agents. Nerve stimulation (4-10 Hz) inhibited the basal release of galanin and VIP from the ileum (to 69 +/- 6 and 62 +/- 6% of basal release). After infusion of the alpha-adrenergic blocker, phentolamine, (10(-6) M) electrical stimulation increased the release of both galanin and VIP (to 140 +/- 12 and 133 +/- 13% of basal output). This increase was abolished by atropine (10(-6) M) and by hexamethonium (3.10(-5) M). Infusion of norepinephrine (10(-6) M) inhibited, whereas acetylcholine (10(-6) M) stimulated the release of both peptides. The effect of the latter was abolished by atropine. The inhibitory effect of nerve stimulation was not influenced by atropine. Our results suggest that the galanin- and VIP-producing intrinsic neurons receive inhibitory signals by noradrenergic nerve fibers and stimulatory signals mediated by cholinergic nerves, possibly via a cholinergic interneuron.     

Vasoactive intestinal peptide rescues cultured rat myenteric neurons from lipopolysaccharide induced cell death

The role of the enteric nervous system in intestinal inflammation is not fully understood and the plethora of cellular activities concurrently ongoing in vivo renders intelligible studies difficult. In order to explore possible effects of bacterial lipopolysaccharide (LPS) on enteric neurons we utilised cultured myenteric neurons from rat small intestine. Exposure to LPS caused markedly reduced neuronal survival and increased neuronal expression of vasoactive intestinal peptide (VIP), while the expression of Toll-like receptor 4 (TLR4) was unchanged. TLR4 was expressed in approximately 35% of all myenteric neurons irrespective of if they were cultured in the presence or absence of LPS. In neurons cultured in medium, without LPS, 50% of all TLR4-immunoreactive neurons contained also VIP. Addition of LPS to the neuronal cultures markedly increased the proportion of TLR4-immunoreactive neurons also expressing VIP, while the proportion of TLR4 neurons devoid of VIP decreased. Simultaneous addition of LPS and VIP to the neuronal cultures resulted in a neuronal survival comparable to controls. CONCLUSIONS: LPS recognition by myenteric neurons is mediated via TLR4 and causes neuronal cell death. Presence of VIP rescues the neurons from LPS-induced neurodegeneration.     

Corticotropin releasing factor—Distribution in rat intestine and role in neuroprotection

Aims of the present study were to describe the distribution of corticotropin releasing factor (CRF) immunoreactivity in rat small and large intestines, to quantify the percentage of CRF-immunoreactive (CRF-IR) enteric neurons, to reveal possible CRF immunoreactivity in cultured myenteric neurons from rat ileum and to examine if additions of CRF, urocortin 1 (Ucn1), CRF antagonist or vasoactive intestinal peptide (VIP) affect neuronal survival in vitro. Co-localization of CRF- and VIP-immunoreactivity was examined, as well as a possible interplay between CRF and VIP in neuroprotection. Further we wanted to elucidate if mast cells affect neuronal survival via CRF signaling.Networks of CRF-containing nerve cell bodies and fibers were detected in rat intestine. CRF-IR neurons contained to a high degree also VIP. A low number of cultured myenteric neurons was CRF-IR. CRF, Ucn1 or CRF-antagonist did not promote neuronal survival of cultured myenteric neurons, while VIP significantly enhanced neuronal survival. Simultaneous presence of CRF attenuated the VIP mediated increase in neuronal survival. Co-culturing neurons and mast cells resulted in a marked reduction in neuronal survival, not executed via CRF signaling pathways. Conclusion: CRF is present in enteric neurons and counteracts the neuroprotective effect of VIP in vitro.     

Characterization of content and chromatographic forms of neuropeptides in purified nerve varicosities prepared from guinea pig myenteric plexus

Partially purified nerve varicosities (PV) prepared from guinea pig ileal myenteric plexus were found to contain, by radioimmunoassay, gastrin-releasing polypeptide (GRP), substance P (SP), galanin, Leu-enkephalin (LE), Met-enkephalin (ME), and vasoactive intestinal polypeptide (VIP). SP was present in the highest concentration followed by, in descending order, ME, LE, VIP, GRP and galanin. On reverse-phase HPLC, SP-, LE- and ME-like immunoreactivity in the PV preparation eluted at retention times similar to their synthetic analogues, galanin-like immunoreactivity eluted at a retention time different from that of synthetic porcine galanin and VIP-like immunoreactivity eluted at the retention time of synthetic guinea pig VIP. GRP-like immunoreactivity, on reverse-phase HPLC, eluted at retention times close to that of synthetic porcine GRP-(1-27) and its major oxidized form. Evidence was obtained for the presence of an alpha-neurokinin-like immunoreactive entity and an unidentified SP-like immunoreactive entity in guinea pig myenteric plexus.     

Lipopolysaccharide-Induced Loss of Cultured Rat Myenteric Neurons - Role of AMP-Activated Protein Kinase

Objective

Intestinal barrier function is vital for homeostasis. Conditions where the mucosal barrier is compromised lead to increased plasma content of lipopolysaccharide (LPS). LPS acts on Toll-like receptor 4 (TLR4) and initiates cellular inflammatory responses. TLR4 receptors have been identified on enteric neurons and LPS exposure causes neuronal loss, counteracted by vasoactive intestinal peptide (VIP), by unknown mechanisms. In addition AMP activated protein kinase (AMPK) stimulation causes loss of enteric neurons. This study investigated a possible role of AMPK activation in LPS-induced neuronal loss.

Design

Primary cultures of myenteric neurons isolated from rat small intestine were used. Cultures were treated with LPS (0.2–20 µg/mL) with and without TAK1-inhibitor (5Z)-7-Oxozeaenol (10⁻⁶ M) or AMPK inhibitor compound C (10⁻⁵ M). AMPK-induced neuronal loss was verified treating cultures with three different AMPK activators, AICAR (10⁻⁴−3×10⁻³ M), metformin (0.2–20 µg/mL) and A-769662 (10⁻⁵−3×10⁻⁴ M) with or without the presence of compound C (10⁻⁵ M). Upstream activation of AMPK-induced neuronal loss was tested by treating cultures with AICAR (10⁻³ M) in the presence of TAK1 inhibitor (5Z)-7-Oxozeaenol (10⁻⁶ M). Neuronal survival and relative numbers of neurons immunoreactive (IR) for VIP were evaluated using immunocytochemistry.

Results

LPS caused a concentration dependent loss of neurons. All AMPK activators induced loss of myenteric neurons in a concentration dependent manner. LPS-, AICAR- and metformin-,but not A-769662-, induced neuronal losses were inhibited by presence of compound C. LPS, AICAR or metformin exposure increased the relative number of VIP-IR neurons; co-treatment with (5Z)-7-Oxozeaenol or compound C reversed the relative increase in VIP-IR neurons induced by LPS. (5Z)-7-Oxozeaenol, compound C or A-769662 did not per se change neuronal survival or relative numbers of VIP-IR neurons.

Conclusion

AMPK activation mimics LPS-induced loss of cultured myenteric neurons and LPS-induced neuronal loss is counteracted by TAK1 and AMPK inhibition. This suggests enteric neuroimmune interactions involving AMPK regulation.     

Inhibitory effects of galanin on evoked [Ca2+]i responses in cultured myenteric neurons

Galanin modulates gastrointestinal motility by inhibiting the release of ACh from enteric neurons. It is, however, not known whether galanin also inhibits neuronal cholinergic transmission postsynaptically and whether galanin also reduces the action of other excitatory neurotransmitters. The aim of the present study was thus to investigate the effect of galanin on the evoked intracellular Ca(2+) concentration ([Ca(2+)](i)) responses in myenteric neurons. Cultured myenteric neurons from small intestine of adult guinea pigs were loaded with the Ca(2+) indicator fluo-3 AM, and the [Ca(2+)](i) responses following the application of different stimuli were quantified by confocal microscopy and expressed as a percentage of the response to high-K(+) solution (75 mM). Trains of electrical pulses (2 s, 10 Hz) were applied to stimulate the neuronal fibers before and after a 30-s superfusion with galanin (10(-6) M). Substance P (SP), 5-HT, 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP), and carbachol were used as direct postsynaptic stimuli (10(-5) M, 30 s) and were applied alone or after galanin perfusion. Galanin significantly reduced the responses induced by electrical fiber stimulation (43 +/- 2 to 35 +/- 3%, P = 0.01), SP (15.4 +/- 1 to 8.0 +/- 0.3%, P < 0.01), and 5-HT (26 +/- 2 to 21.4 +/- 1.5%, P < 0.05). On the contrary, galanin did not affect the responses induced by local application of DMPP and carbachol. We conclude that in cultured myenteric neurons, galanin inhibits the excitatory responses induced by electrical stimulation, SP, and 5-HT. Finally, the inhibitory effect of galanin on electrical stimulation, but not on DMPP- and carbachol-induced responses, suggests that, at least for the cholinergic component, galanin acts at the presynaptic level.     

SNV, a lipophilic superactive VIP analog, acts through cGMP to promote neuronal survival.

The current study explored whether the neuroprotective effects of vasoactive intestinal peptide (VIP) and its analog Stearyl-Nle17-VIP (SNV) were mediated through cGMP. SNV, was previously found to be 100-fold more potent than VIP in providing neuroprotection. Neuronal survival was assessed in rat cerebral cortical cultures. A cGMP antagonist (RP-8-pCPT-cGMPS, 10(-12)-10(-9) M) reduced the number of surviving neurons (40-60%), this decline was spared in the presence of SNV (10(-13)M). A cGMP agonist (Sp-8-pCPT-cGMPS, 10(-14)-10(-8)M) and SNV (10(-16)-10(-8)M) both provided significant neuroprotection against 10(-12) M of the cGMP antagonist. Immunoassays indicated that SNV induced increases in cGMP (two-threefold) in these cultures, whereas VIP was 1000-fold less potent. These results implicate cGMP as a second messenger for VIP/SNV-mediated effects on neuronal survival.     

Morphology of VIP/nNOS-immunoreactive myenteric neurons in the human gut

In this study, we characterized human myenteric neurons co-immunoreactive for neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP) by their morphology and their proportion as related to the putative entire myenteric neuronal population. Nine wholemounts (small and large intestinal samples) from nine patients were triple-stained for VIP, neurofilaments (NF) and nNOS. Most neurons immunoreactive for all three markers displayed radially emanating, partly branching dendrites with spiny endings. These neurons were called spiny neurons. The spiny character of their dendrites was more pronounced in the small intestinal specimens and differed markedly from enkephalinergic stubby neurons described earlier. Exclusively in the duodenum, some neurons displayed prominent main dendrites with spiny side branches. Of the axons which could be followed from the ganglion of origin within primary strands of the myenteric plexus beyond the next ganglion (70 out of 140 traced neurons), 94.3% run anally and 5.7% orally. Very few neurons reactive for both VIP and nNOS could not be morphologically classified due to weak or absent NF-immunoreactivity. Another six wholemounts were triple-stained for VIP, nNOS and Hu proteins (HU). The proportion of VIP/nNOS-coreactive neurons in relation to the number of HU-reactive neurons was between 5.8 and 11.5% in the small and between 10.6 and 17.5% in the large intestinal specimens. We conclude that human myenteric spiny neurons co-immunoreactive for VIP and nNOS represent either inhibitory motor or descending interneurons.     

Chemical sympathectomy and postganglionic nerve transection produce similar increases in galanin and VIP mRNA but differ in their effects on peptide content

Large changes in neuronal gene expression occur in adult peripheral neurons after axonal transection. In the rat superior cervical ganglion, for example, neurons that do not normally express vasoactive intestinal peptide (VIP) or galanin do so after postganglionic nerve transection. These effects of axotomy could result from a number of aspects of the surgical procedure. To test the idea that the important variable might be the disconnection of axotomized neuronal cell bodies from their target tissues, we examined the effects of producing such a disconnection by means of the compound 6-hydroxydopamine (6-OHDA), a neurotoxin that causes degeneration of sympathetic varicosities and avoids many of the complications of surgery. Two days after 6-OHDA treatment, VIP and galanin immunoreactivities had increased two- and 40-fold, respectively. Nevertheless, these increases were substantially smaller than the 30- and 300-fold changes seen after surgical axotomy. When expression of VIP and galanin was examined at the mRNA level, however, comparable increases were found after either procedure. The results indicate that chemical destruction of sympathetic varicosities produces an equivalent signal for increasing VIP and galanin mRNA as does axonal transection. The differences in the neuropeptide levels achieved suggests that peptide expression after nerve transection is regulated both at the mRNA and protein levels. © 1996 John Wiley & Sons, Inc.     

Pharmacological evidence for both neuronal and smooth muscular PAC1 receptors and a VIP-specific receptor in rat colon

The receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) were characterised in vitro on rat colon longitudinal smooth muscle with adherent myenteric ganglia. VIP, PACAP-38 and PACAP-27 all caused concentration-dependent relaxations. PACAP-27 and PACAP-38 were equipotent, while VIP was less potent. Tetrodotoxin (10(-6) M), L-NAME (10(-4) M), 7-NINA (10(-5) M) and ODQ (3 x 10(-6) M) reduced the amplitude of the relaxatory responses to PACAP-38 but did not affect relaxations induced by VIP or PACAP-27. Apamin (10(-6) M) almost totally abolished the PACAP-27- and PACAP-38-induced relaxations, while VIP-induced relaxations were only slightly reduced. Tetraethylammonium (TEA) reduced VIP- but not PACAP-27-induced relaxations, while charybdotoxin was ineffective. Cross-desensitisation between PACAP-27, PACAP-38 and VIP could be revealed to some extent. In conclusion: VIP, PACAP-27 and PACAP-38 are effective relaxants in rat colon longitudinal muscle. The receptors involved are classified as: (1) a neuronal PAC1 receptor localised on NO-synthesising neurones, the preferred ligand being PACAP-38. Activation of this receptor leads to an increased NO production. (2) A smooth muscle PAC1 receptor, the preferred ligand being PACAP-27. However, also PACAP-38 and, to a less extent, VIP activate this receptor. The relaxatory responses elicited by both PACAP-27 and PACAP-38 are abolished by apamin and thus mediated through small conductance Ca2+-activated K+ channels. (3) A VIP-specific receptor localised on smooth muscle cells. The mechanisms whereby this receptor elicits a relaxatory response involve, at least to some extent, TEA-sensitive K+ channels.     

Localization and inhibitory actions of galanin at the feline lower esophageal sphincter

Intrinsic reflexes of the lower esophageal sphincter (LES) are mediated by specific arrangements of excitatory and inhibitory nerves. We have previously described an excitatory reflex at the feline LES mediated by a bombesin-like peptide (BN) which causes release of substance P (SP) to directly contract the LES. Galanin is a neurotransmitter in the enteric nervous system which colocalizes in neurons containing vasoactive intestinal peptide (VIP). The aims of this study were to determine: (1) the distribution of galanin at the feline LES; (2) the effect of galanin on basal LES tone; (3) the effect of galanin on agonist-induced LES contractions by BN, SP and bethanechol; and (4) the effect of galanin on LES relaxation induced by esophageal distension and exogenous VIP. Galanin-like immunoreactivity (galanin-LI) was localized in neurons that were widely distributed throughout the LES and adjacent organs. Galanin-LI was most abundant in the circular muscle, muscularis mucosa and myenteric plexus of the LES. In anesthetized cats, intra-arterial galanin had no effect on basal LES pressure in a dose range of 10⁻¹¹ to 10⁻⁶ g/kg. Galanin (5 10⁻⁷ g/kg) reduced the LES contractile response to SP by 65 ± 8% (P = 0.0001). This galanin-mediated inhibition of SP was not blocked by tetrodotoxin. Galanin similarly decreased the LES contractile response to BN (63 ± 7%, P = 0.005) and bethanechol (55 ± 17%, P = 0.012). Galanin had no effect on the LES relaxation induced by esophageal distension or exogenous VIP. We conclude: (1) galanin-LI is present in neurons at the feline LES; (2) galanin has no effect on basal sphincter tone, but inhibits contractions of the LES by both direct and indirect agonists; and (3) galanin does not effect the LES relaxation induced by esophageal distension or VIP.     

Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse

Immunohistochemical techniques were used to examine the presence and co-localisation of a range of putative neurotransmitters and other neuronal markers in the myenteric plexus of the small and large intestine of the mouse. Distinct sub-populations of myenteric neurons were identified, based on the combinations of substances they contained and the distribution of their fibres. In the small intestine, there were two major classes of circular muscle motor neurons; one class was characterised by the presence of nitric oxide synthase, vasoactive intestinal peptide plus neuropeptide Y (NOS/VIP/NPY), and the second class contained calretinin plus substance P (CalR/SP). There were seven classes of neurons that innervated myenteric ganglia; these contained NOS, VIP, NOS/VIP, NPY, CalR/calbindin (CalB), SP or 5-HT. In the large intestine, there were five major classes of motor neurons that contained NOS, NOS/VIP, GABA, SP, or CalR/SP, and seven major classes of neurons that innervated myenteric ganglia and contained NOS, VIP, CalR/CalB, CalR, SP, GABA or 5-HT. Although some aspects of the patterns of co-localisation are similar to those in other species, this study re-inforces recent analyses that indicate significant species differences in neurochemical patterns in the enteric neurons of different species. Received: 28 August 1995 / Accepted: 30 November 1995     

PACAP 1-38 as neurotransmitter in the porcine antrum

The concentration of PACAP 1-38 in porcine antrum amounted to 15.4+/-7.9 and 20.3+/-8 pmol/g tissue in the mucosal and muscular layers. PACAP immunoreactive (IR) fibres innervated the muscular (co-localised with VIP) and submucosal/mucosal layers (some co-storing VIP and CGRP) including myenteric and submucosal plexus and blood vessels. Only myenteric nerve cell bodies contained PACAP-IR (co-storing VIP). In isolated perfused antrum, vagus nerve stimulation (8 Hz) and capsaicin (10(-5) M) increased PACAP 1-38 release. PACAP 1-38 (10(-9) M) increased substance P (SP), gastrin releasing peptide (GRP) and VIP release. PACAP 1-38 (10(-8) M) inhibited gastrin secretion and stimulated somatostatin secretion and motility dose-dependently. PACAP-induced motility was strongly inhibited by the antagonist PACAP 6-38 but also by atropine and substance P-antagonists (CP99994/SR48968) but PACAP 6-38 had no effect on vagus-induced secretion or motility. Conclusion: PACAP 1-38 may be involved in antral motility and secretion by interacting with cholinergic, SP-ergic, GRP-ergic and/or VIP-ergic neurones, and may also be involved in afferent reflex pathways.     

Galanin-immunoreactive neurons in the guinea-pig small intestine: their projections and relationships to other enteric neurons

Summary Galanin immunoreactivity was observed in nerve cell bodies and nerve fibres, but not in enteroendocrine cells, in the small intestine of the guinea-pig. Nerve terminals were found in the myenteric plexus, in the circular muscle, in submucous ganglia, around submucous arterioles, and in the mucosa. Lesion studies showed that all terminals were intrinsic to the intestine; those in myenteric ganglia arose from cell bodies in more orally placed ganglia. Myenteric nerve cells were also the source of terminals in the circular muscle. Galanin (GAL) was located in a population of submucous nerve cell bodies that also showed immunoreactivity for vasoactive intestinal peptide (VIP) and in a separate population that was immunoreactive for neuropeptide Y (NPY). Processes of the GAL/VIP neurons supplied submucous arterioles and the mucosal epithelium. Processes of GAL/NPY neurons ran to the mucosa. It is concluded that galanin immunoreactivity occurs in several functionally distinct classes of enteric neurons, amongst which are neurons controlling (i) motility, (ii) intestinal blood flow, and (iii) mucosal water and electrolyte transport.     

Chemical coding of neurons expressing δ- and κ-opioid receptor and type I vanilloid receptor immunoreactivities in the porcine ileum

Opioid drugs have profound antidiarrheal and constipating actions in the intestinal tract and are effective in mitigating abdominal pain. Mediators of intestinal inflammation and allergy produce increased mucosal secretion, altered bowel motility and pain due to their ability to evoke enteric secretomotor reflexes through primary afferent neurons. In this study, the distribution of delta- and kappa-opioid receptor (DOR and KOR, respectively) immunoreactivities in chemically identified neurons of the porcine ileum was compared with that of the capsaicin-sensitive type 1 vanilloid receptor (VR1). DOR and VR1 immunoreactivities were observed to be highly localized in choline acetyltransferase (ChAT)- and calcitonin gene-related peptide (CGRP)-positive neurons and nerve fibers of the submucosal and myenteric plexuses and both receptors exhibited frequent colocalization. In the inner submucosal plexus, they also were colocalized in substance P (SP)-positive neurons. Neurons in the outer submucosal plexus expressed DOR immunoreactivity alone or in combination with VR1. KOR-immunoreactive neurons were found only in the myenteric plexus; these cells coexpressed immunoreactivity to ChAT, CGRP, vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS). In addition, some KOR-positive neurons coexpressed immunoreactivities to DOR and VR1. Based on their neurochemical coding, opioid and vanilloid receptor-immunoreactive neurons in the submucosal and myenteric plexuses may include primary afferents and constitute novel therapeutic targets for the palliation of painful intestinal inflammatory, hypersensitivity and dysmotility states.     

Projections of neurochemically specified neurons in the porcine colon

The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5-HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics.     

What neurons hide behind calretinin immunoreactivity in the human gut?

Calretinin (CALR) is often used as an immunohistochemical marker for the histopathological diagnosis of human intestinal neuropathies. However, little is known about its distribution pattern with respect to specific human enteric neuron types. Prior studies revealed CALR in both myenteric and submucosal neurons, most of which colabel with choline acetyl transferase (ChAT). Here, we specified the chemical code of CALR-positive neurons in small and large intestinal wholemounts in a series of 28 patients. Besides other markers, we evaluated the labeling pattern of CALR in combination with vasoactive intestinal peptide (VIP). In colonic submucosa, CALR and VIP were almost completely colocalized in about three-quarters of all submucosal neurons. In the small intestinal submucosa, both the colocalization rate of CALR and VIP as well as the proportion of these neurons were lower (about one-third). In the myenteric plexus of both small intestine and colon, CALR amounted to 11 and 10 %, respectively, whereas VIP to 5 and 4 % of the whole neuron population, respectively. Colocalization of both markers was found in only 2 and 3 % of myenteric neurons, respectively. In section specimens, nerve fibers coreactive for CALR and VIP were found in the mucosa but not in the muscle coat. Summarizing the present and earlier results, CALR was found in at least one submucosal and two myenteric neuron populations. Submucosal CALR+/VIP+/ChAT± neurons innervate mucosal structures. Furthermore, CALR immunoreactivity in the myenteric plexus was observed in morphological type II (supposed primary afferent) and spiny type I (supposed inter- or motor-) neurons.