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.     

Quantitative estimation of putative primary afferent neurons in the myenteric plexus of human small intestine

This study aimed at estimating the proportion of human myenteric Dogiel type II neurons, putative intrinsic primary afferent neurons (IPANs), in relation to the entire myenteric neuron population. Since, at present, there is no known single marker, which specifically labels these neurons, we tried to identify the most appropriate marker combination based on the results of an earlier study. For this purpose, 10 wholemounts derived from human small intestinal segments were immunohistochemically triple-stained for calretinin (CALR), somatostatin (SOM) and neurofilaments (NF) and 9 were stained for substance P (SP), SOM and NF. In each wholemount, 15 ganglia selected randomly were evaluated. On the basis of their NF-reactivity, neurons reactive for one or co-reative for both of the other two markers, respectively, were morphologically classified as type II or non-type II neurons. We found that the majorities of neurons co-reactive for CALR/SOM and SP/SOM, respectively, were type II neurons whereas this was not the case for neurons, which were reactive for only one of the two markers. One of the statistical parameters estimated was the positive predictive value, the probability that a neuron displaying CALR/SOM- or SP/SOM-co-reactivity, respectively, is a type II neuron. This value was 97% in case of CALR/SOM- and 95% in case of SP/SOM-co-staining. Although the difference of the statistical parameters between the two stainings was not significant, CALR and SOM were used to estimate indirectly the proportion of type II neurons, in wholemounts co-stained with the pan-neuronal marker neuronal protein HuC/HuD (HU). In these wholemounts, altogether 9.1% of neurons were coreactive for CALR/SOM. We suggest that the proportion of myenteric type II neurons in the human small intestine is related to the proportion of CALR/SOM-co-reactive neurons and may be near to one tenth of the total myenteric neuronal population.     

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.     

Substance P- and choline acetyltransferase immunoreactivities in somatostatin-containing, human submucosal neurons

The submucous layers of human small and large intestines contain at least two separate neuron populations. Besides morphological features, they differ in their immunoreactivities for calretinin (CALR) and somatostatin (SOM), respectively. In this study, submucosal wholemounts of 23 patients or body donors (including all segments of small intestine and colon) were immunohistochemically quadruple stained for CALR and SOM as well as for substance P (SP) and choline acetyltransferase (ChAT). We found that all SOM-positive neurons co-stained for ChAT and the majority for SP [between 50 % in the small intestinal external submucosal plexus (ESP) and 75 % in the colonic ESP]. In contrast, a majority of CALR-neurons contained ChAT (between 77 % in the small intestinal ESP and 92 % in the large intestinal ESP) whereas less than 4 % of CALR-neurons were co-immunoreactive for SP. Another set of wholemounts was co-stained for peripherin, a marker enabling morphological analysis. Where identifiable, both SOM alone- and SOM/SP-neurons displayed a uniaxonal (supposed pseudouniaxonal) morphology. We suggest that the chemical code of SOM-immunoreactive, human submucosal neurons may be “ChAT+/SOM+/SP±”. In additional sections double stained for SOM and SP, we regularly found double-labelled nerve fibres only in the mucosa. In contrast, around submucosal arteries mostly SOM alone- fibres were found and the muscularis propria contained numerous SP-alone fibres. We conclude that the main target of submucosal SOM(/SP)-neurons may be the mucosa. Due to their morpho-chemical similarity to human myenteric type II neurons, we further suggest that one function of human submucosal SOM-neurons may be a primary afferent one.     

Morphology of enkephalin-immunoreactive myenteric neurons in the human gut

The aim of this study was the morphological and further chemical characterisation of neurons immunoreactive for leu-enkephalin (leuENK). Ten wholemounts of small and large intestinal segments from nine patients were immunohistochemically triple-stained for leuENK/neurofilament 200 (NF)/substance P (SP). Based on their simultaneous NF-reactivity and 3D reconstruction of single NF-reactive cells, 97.5% of leuENK-positive neurons displayed the appearance of stubby neurons: small somata; short, stubby dendrites and one axon. Of these leuENK-reactive stubby neurons, 91.3% did not display co-reactivity for SP whereas 8.7% were SP-co-reactive. As to their axonal projection pattern, 50.4% of the recorded leuENK stubby neurons had axons running orally whereas in 29.4% they ran anally; the directions of the remaining 20.2% could not be determined. No axons were seen to enter into secondary strands of the myenteric plexus. Somal area measurements revealed clearly smaller somata of leuENK-reactive stubby neurons (between 259±47 m² and 487±113 m²) than those of putative sensory type II neurons (between 700±217 m² and 1,164±396 m²). The ratio dendritic field area per somal area of leuENK-reactive stubby neurons was between 2.0 and 2.8 reflecting their short dendrites. Additionally, we estimated the proportion of leuENK-positive neurons in comparison to the putative whole myenteric neuron population in four leuENK/anti-Hu doublestained wholemounts. This proportion ranged between 5.9% and 8.3%. We suggest leuENK-reactive stubby neurons to be muscle motor neurons and/or ascending interneurons. Furthermore, we explain why we do not use the term Dogiel type I neurons for this population.     

Pattern of lipofuscin pigmentation in nitrergic and non-nitrergic,neurofilament immunoreactive myenteric neuron types of human small intestine

Lipofuscin, an autofluorescent age pigment, occurs in enteric neurons. Due to its broad excitation and emission spectra, it overlaps with commonly used fluorophores in immunohistochemistry. We investigated the pattern of lipofuscin pigmentation in neurofilament (NF)-reactive nitrergic and non-nitrergic human myenteric neuron types. Subsequently, we tested two methods for reduction of lipofuscin-like autofluorescence. Myenteric plexus/longitudinal muscle wholemounts of small intestines of five patients undergoing surgery for carcinoma (aged between 18 and 69 years) were double stained for NF and neuronal nitric oxide synthase (nNOS). Lipofuscin pigmentation patterns were semiquantitatively evaluated by using confocal laser scanning microscopy with three different excitation wave lengths (one for undisturbed lipofuscin autofluorescence and two for specific labellings). Two pigmentation patterns could be detected in the five NF-reactive neuron types investigated. In nitrergic/spiny as well as in non-nitrergic/stubby neurons, coarse, intensely autofluorescent pigment granules were prominent. In non-nitrergic type II, III and V neurons, a fine granular, diffusely distributed and less intensely autofluorescent pigment was obvious. After incubation of wholemounts in either CuSO₄ or Sudan black B solutions, unspecific autofluorescence could be substantially reduced whereas specific NF and nNOS fluorescence remained largely unaffected. We conclude that NF immunohistochemistry is useful for morphological representation of subpopulations of human myenteric neurons. The lipofuscin pigmentation in human myenteric neurons reveals at least two different patterns which can be related to distinct neuron types. Incubations of multiply stained whole mounts in both CuSO₄ or Sudan black B are suitable methods for reducing autofluorescence thus facilitating discrimination between specific (immunohistochemical) and non-specific (lipofuscin) fluorescence.     

Effects of vasoactive intestinal peptide and galanin on survival of cultured porcine myenteric neurons

Enteric neuronal plasticity is probably fundamental in order to withstand injury or changes in intestinal activity. The role of the neuropeptides in neuroprotection is still enigmatic. The expression of galanin and vasoactive intestinal peptide (VIP) and the effects of the two peptides on survival of small intestinal porcine myenteric neurons cultured for 6 days were studied. Immunocytochemistry and cell counting were used to evaluate the numbers of surviving neurons and their expression of galanin and VIP. To reflect the in vivo situation, cryostat sections of porcine mid-jejunum were used. A concentration-dependent and marked increase in neuronal survival was noted when neurons were grown in the presence of VIP (10(-8)-10(-6) M), whereas addition of galanin (10(-8)-10(-6) M) slightly decreased neuronal survival. A dramatic increase in the proportions of myenteric neurons containing VIP or galanin immunoreactivity occurred during culturing. The presence of VIP further increased the number of galanin-expressing neurons. A majority of the galanin-immunoreactive neurons lacked VIP, while all VIP-immunoreactive neurons contained galanin. In conclusion, culturing porcine myenteric neurons in the presence of VIP increases, while the presence of galanin reduces, survival. Culturing significantly increased the proportion of neurons expressing VIP and/or galanin; the presence of VIP further increased the number of galanin-expressing neurons.     

Partial,selective survival of nitrergic neurons in chagasic megacolon

One frequent chronic syndrome of Chagas’ disease is megacolon, an irreversible dilation of a colonic segment. Extensive enteric neuron loss in the affected segment is regarded as key factor for deficient motility. Here, we assessed the quantitative balance between cholinergic and nitrergic neurons representing the main limbs of excitatory and inhibitory colonic motor innervation, respectively. From surgically removed megacolonic segments of four patients, each three myenteric wholemounts (from non-dilated oral, megacolonic and non-dilated anal parts) was immunohistochemically triple-stained for choline acetyltransferase, neuronal nitric oxide synthase (NOS) and the panneuronal human neuronal protein Hu C/D. Degenerative changes were most pronounced in the megacolonic and anal regions, e.g. bulked, honeycomb-like ganglia with few neurons which were partly enlarged or atrophic or vacuolated. Neuron counts from each 15 ganglia of 12 megacolonic wholemounts were compared with those of 12 age- and region-matched controls. Extensive neuron loss, mainly in megacolonic and anal wholemounts, was obvious. In all three regions derived from megacolonic samples, the proportion of NOS-positive neurons (control: 55%) was significantly increased: in non-dilated oral parts to 61% (p = 0.003), in megacolonic regions to 72% (p < 0.001) and in non-dilated anal regions to 78% (p < 0.001). We suggest the chronic dilation of megacolonic specimens to be due to the preponderance of the nitrergic, inhibitory input to the intestinal muscle. However, the observed neuronal imbalance was not restricted to the dilated regions: the non-dilated anal parts may be innervated by ascending, cholinergic axons emerging from less affected, more anally located regions.     

Neurotrophin-3 and neurotrophin receptor immunoreactivity in peptidergic enteric neurons

In the rat small intestine, neurotrophin-3 immunoreactivity was identified in ganglion cells and in processes mostly innervating the mucosa and occasionally the muscle layer and vasculature. The vast majority of neurotrophin-3 immunoreactive neurons contained vasoactive intestinal polypeptide (VIP), but not substance P or related tachykinin (SP/TK). Neurotrophin receptors visualized by pan-trk immunoreactivity were found in numerous ganglion cells of both plexuses and in nerve processes in the intestinal wall. Pan-trk submucosal neurons contained VIP (36%) or SP/TK-IR (47%). Pan-trk myenteric neurons contained VIP-IR (57%) or SP/TK (27%). Our data suggest that neurotrophin-3 and neurotrophin receptors may be involved in the maintenance of enteric neuronal circuits, transmission and phenotypic expression.     

ChAT and NOS in human myenteric neurons: co-existence and co-absence

Most myenteric neurons contain one of the two generating enzymes for major excitatory and inhibitory neurotransmitters: choline acetyltransferase (ChAT) or neuronal nitric oxide synthase (NOS). Two minor groups of myenteric neurons contain either both enzymes or neither. Our study had two aims: (1) to compare the proportions of neurons stained for ChAT and/or NOS in human small and large intestinal whole-mounts by co-staining with an antibody against the human neuronal protein Hu C/D (HU); (2) to characterize these neurons morphologically by co-staining with a neurofilament (NF) antibody. In small intestinal whole-mounts co-stained with HU, we counted more ChAT-positive (ChAT+) than NOS+ neurons (52% vs. 38%), whereas the large intestine exhibited fewer ChAT+ than NOS+ neurons (38% vs. 50%). Neurons co-reactive for both ChAT and NOS accounted for about 3% in both regions, whereas neurons negative for both enzymes accounted for 7% in the small intestine and 8% in the large intestine. Co-staining with NF revealed that, in both small and large intestine, ChAT+/NOS+ neurons were either spiny (type I) neurons or displayed smaller perikarya that were weakly or not NF-stained. Of all spiny neurons, almost one third was co-reactive for ChAT and NOS, whereas nearly two thirds were positive only for NOS. Neurons negative for both ChAT and NOS were heterogeneous in size and NF reactivity. Thus, neither the co-existence nor the co-absence of ChAT and NOS in human myenteric neurons is indicative for particular neuron types, with several qualitative and quantitative parameters showing a wide range of interindividual variability.     

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.     

Chagasic megacolon: enteric neurons and related structures

Megacolon, the irreversible dilation of a colonic segment, is a structural sign associated with various gastrointestinal disorders. In its hereditary, secondary form (e.g. in Hirschsprung’s disease), dilation occurs in an originally healthy colonic segment due to an anally located, aganglionic zone. In contrast, in chronic Chagas’ disease, the dilated segment itself displays pathohistological changes, and the earliest and most prominent being found was massive loss of myenteric neurons. This neuron loss was partial and selective, i.e. some neurons containing neuronal nitric oxide synthase and/or vasoactive intestinal peptide (VIP) were spared from neuron death. This disproportionate survival of inhibitory neurons, however, did not completely correlate with the calibre change along the surgically removed, megacolonic segments. A better correlation was observed as to potentially contractile muscle tissue elements and the interstitial cells of Cajal. Therefore, the decreased densities of α-smooth muscle actin- and c-kit-immunoreactive profiles were estimated along resected megacolonic segments. Their lowest values were observed in the megacolonic zones itself, whereas less pronounced decreases were found in the non-dilated, transitional zones (oral and anal to dilation). In contrast to the myenteric plexus, the submucosal plexus displayed only a moderate neuron loss. Neurons co-immunoreactive for VIP and calretinin survived disproportionately. As a consequence, these neurons may have contributed to maintain the epithelial barrier and allowed the chagasic patients to survive for decades, despite their severe disturbance of colonic motility. Due to its neuroprotective and neuroeffectory functions, VIP may play a key role in the development and duration of chagasic megacolon.     

Spiny versus stubby: 3D reconstruction of human myenteric (type I) neurons

We have compared the three-dimensional (3D) morphology of stubby and spiny neurons derived from the human small intestine. After immunohistochemical triple staining for leu-enkephalin (ENK), vasoactive intestinal peptide (VIP) and neurofilament (NF), neurons were selected and scanned based on their immunoreactivity, whether ENK (stubby) or VIP (spiny). For the 3D reconstruction, we focused on confocal data pre-processing with intensity drop correction, non-blind deconvolution, an additional compression procedure in z-direction, and optimizing segmentation reliability. 3D Slicer software enabled a semi-automated segmentation based on an objective threshold (interrater and intrarater reliability, both 0.99). We found that most dendrites of stubby neurons emerged only from the somal circumference, whereas in spiny neurons, they also emerged from the luminal somal surface. In most neurons, the nucleus was positioned abluminally in its soma. The volumes of spiny neurons were significantly larger than those of stubby neurons (total mean of stubbies 806 ± 128 μm³, of spinies 2,316 ± 545 μm³), and spiny neurons had more dendrites (26.3 vs. 11.3). The ratios of somal versus dendritic volumes were 1:1.2 in spiny and 1:0.3 in stubby neurons. In conclusion, 3D reconstruction revealed new differences between stubby and spiny neurons and allowed estimations of volumetric data of these neuron populations.     

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.     

Neuro-Anatomical Evidence Indicating Indirect Modulation of Macrophages by Vagal Efferents in the Intestine but Not in the Spleen

Background

Electrical stimulation of the vagus nerve suppresses intestinal inflammation and normalizes gut motility in a mouse model of postoperative ileus. The exact anatomical interaction between the vagus nerve and the intestinal immune system remains however a matter of debate. In the present study, we provide additional evidence on the direct and indirect vagal innervation of the spleen and analyzed the anatomical evidence for neuroimmune modulation of macrophages by vagal preganglionic and enteric postganglionic nerve fibers within the intestine.

Methods

Dextran conjugates were used to label vagal preganglionic (motor) fibers projecting to the small intestine and spleen. Moreover, identification of the neurochemical phenotype of the vagal efferent fibers and enteric neurons was performed by immunofluorescent labeling. F4/80 antibody was used to label resident macrophages.

Results

Our anterograde tracing experiments did not reveal dextran-labeled vagal fibers or terminals in the mesenteric ganglion or spleen. Vagal efferent fibers were confined within the myenteric plexus region of the small intestine and mainly endings around nNOS, VIP and ChAT positive enteric neurons. nNOS, VIP and ChAT positive fibers were found in close proximity of intestinal resident macrophages carrying α7 nicotinic receptors. Of note, VIP receptors were found on resident macrophages located in close proximity of VIP positive nerve fibers.

Conclusion

In the present study, we show that the vagus nerve does not directly interact with resident macrophages in the gut or spleen. Instead, the vagus nerve preferentially interacts with nNOS, VIP and ChAT enteric neurons located within the gut muscularis with nerve endings in close proximity of the resident macrophages.     

Distribution of nitric oxide synthase and vasoactive intestinal polypeptide immunoreactivity in the sphincter of Oddi and duodenum of the possum

The nitrergic innervation of the sphincter of Oddi (SO) and duodenum in the Australian brush-tailed possum and the possible association of this innervation with the neuropeptide vasoactive intestinal polypeptide (VIP) were investigated by using immunohistochemical localisation of nitric oxide synthase (NOS) and VIP, together with the general neuronal marker, protein gene product 9.5 (PGP9.5). Whole-mount preparations of the duodenum and attached SO without the mucosa, submucosa and circular muscle (n=12) were double- and triple-labelled. The density of myenteric nerve cell bodies of the SO in the more distal region (duodenal end) was significantly higher than that in the more proximal region. In the SO, approximately 50% of all cells were NOS-immunoreactive (IR), with 27% of the NOS-IR cells being VIP-IR. Within the duodenal myenteric plexus, NOS immunoreactivity was present in about 25% of all neurons, with 27% of these NOS-IR neurons also being VIP-IR, a similar proportion to that in the SO. Varicose nerve fibres with NOS and VIP immunoreactivity were present within the myenteric and submucous plexuses of the SO and duodenum, and in the circular and longitudinal muscle layers. The NOS-positive cells within both the SO and duodenum were unipolar, displaying a typical Dogiel type I morphology. The myenteric plexuses of the SO and duodenum were in direct continuity, with many interconnecting nerve trunks, some of which showed NOS and VIP immunoreactivity. Thus, the possum possesses an extensive NOS innervation of the SO and duodenum, with a significantly higher proportion of NOS-IR neurons within the SO, a subset of which contains VIP.     

Intrinsic choroidal neurons in the chicken eye: chemical coding and synaptic input

Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-β-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and α-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.     

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     

Investigation of general and cytoskeletal markers to estimate numbers and proportions of neurons in the human intestine

An important requirement in pathological diagnostics in the human enteric nervous system (ENS) is the estimation of the total numbers of neurons and of proportions of distinct subpopulations. In this study, we compared the suitability of two suggested panneuronal markers, cuprolinic blue (CB) and anti-Hu-protein (HU), for staining and counting human myenteric neurons in wholemounts, derived from small and large intestinal samples. Furthermore, the proportional expression of three cytoskeletal intermediate filaments, alpha-internexin (IN), neurofilament 200 (NF) and peripherin (PE), was correlated with both CB and HU. In 8 CB- and HU-stained wholemounts, 93.3% of all neurons were double labeled, 3.3% of neurons were stained only with CB whereas 3.3% were immuno-stained only for HU. Thus, both markers were comparably reliable in representing the putative total human myenteric neuron population in our material. The wholemounts double stained for IN/CB or IN/HU revealed between 56.2 and 71.5% of neurons to be IN-reactive. Between 42.8 and 50.9% of neurons were immunoreactive for NF whereas 53.9 to 62.4% of neurons were reactive for PE. Although our sample number was too small to allow final conclusions, we suggest that the variations in proportions of intermediate filament expression we observed may be due to individual circumstances rather than to correlation with age or region. The proportions of neurons positive for IN, NF or PE but unstained by CB histochemical or HU immunohistochemical techniques was between 0 and 2.2%. We conclude that both CB and HU techniques are suitable methods for representation of almost all myenteric neurons in the human gut and that the differential expression of the cytoskeletal proteins investigated has to be included in the classification of enteric neurons in pathological diagnostics of human gastrointestinal diseases.     

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.