Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.     

Immunohistochemical localisation of cholinergic markers in putative intrinsic primary afferent neurons of the guinea-pig small intestine

Antibodies against choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) were used to determine whether neurons that have previously been identified as intrinsic primary afferent neurons in the guinea-pig small intestine have a cholinergic phenotype. Cell bodies of primary afferent neurons in the myenteric plexus were identified by their calbindin immunoreactivity and those in the submucous plexus by immunoreactivity for substance P. High proportions of both were immunoreactive for ChAT, viz. 98% of myenteric calbindin neurons and 99% of submucosal substance P neurons. ChAT immunoreactivity also occurred in all nerve cell bodies immunoreactive for calretinin and substance P in the myenteric plexus, but in only 16% of nerve cells immunoreactive for nitric oxide synthase. VAChT immunoreactivity was in the majority of calbindin-immunoreactive varicosities in the myenteric ganglia, submucous ganglia and mucosa and also in the majority of the varicosities of neurons that were immunoreactive for calretinin and somatostatin and that had been previously established as being cholinergic. We conclude that the intrinsic primary afferent neurons are cholinergic and that they may release transmitter from their sensory endings in the mucosa.     

Choline acetyltransferase immunoreactivity of putative intrinsic primary afferent neurons in the rat ileum

The colocalisation of choline acetyltransferase (ChAT) with markers of putative intrinsic primary afferent neurons was determined in whole-mount preparations of the myenteric and submucosal plexuses of the rat ileum. In the myenteric plexus, prepared for the simultaneous localisation of ChAT and nitric oxide synthase (NOS), all nerve cells were immunoreactive (IR) for ChAT or NOS, but seldom for both; only 1.6 +/- 1.8% of ChAT-IR neurons displayed NOS-IR and, conversely, 2.8 +/- 3.3% of NOS-IR neurons were ChAT-IR. In preparations double labelled for NOS-IR and the general nerve cell marker, neuron-specific enolase, 24% of all nerve cells were immunoreactive for NOS, indicating that about 75% of all nerve cells have ChAT-IR. All putative intrinsic primary afferent neurons in the myenteric plexus, identified by immunoreactivity for the neurokinin 1 (NK1) receptor and the neurokinin 3 (NK3) receptor, were ChAT-IR. Conversely, of the ChAT-IR nerve cells, about 45% were putative intrinsic primary afferent neurons (this represents 34% of all nerve cells). The cell bodies of putative intrinsic primary afferent neurons had Dogiel type II morphology and were also immunoreactive for calbindin. All, or nearly all, nerve cells in the submucosal plexus were immunoreactive for ChAT. About 46% of all submucosal nerve cells were immunoreactive for both neuropeptide Y (NPY) and calbindin; 91.8 +/- 10.5% of NPY/calbindin cells were also ChAT-IR and 99.1 +/- 0.7% were NK3 receptor-IR. Of the nerve cells with immunoreactivity for ChAT, 44.3 +/- 3.8% were NPY-IR, indicating that about 55% of submucosal nerve cells had ChAT but not NPY-IR. Only small proportions of the ChAT-IR, non-NPY, nerve cells had NK3 receptor or calbindin-IR. It is concluded that about 45% of submucosal nerve cells are ChAT/calbindin/NPY/VIP/NK3 receptor-IR and are likely to be secretomotor neurons. Most of the remaining submucosal nerve cells are immunoreactive for ChAT, but their functions were not deduced. They may include the cell bodies of intrinsic primary afferent neurons.     

Enkephalin-immunoreactive subpopulations in the myenteric plexus of the guinea-pig fundus project primarily to the muscle and not to the mucosa

Enkephalin (ENK) immunoreactivity was localised in different neuronal subpopulations of the myenteric plexus in the guinea-pig gastric fundus using immunohistochemistry for neurone-specific enolase (NSE), ENK, choline acetyltransferase (ChAT), substance P (SP), neuropeptide Y (NPY), calretinin (CALRET), and somatostatin (SOM). NADPH-diaphorase staining was used to label nitric oxide synthase (NOS)-containing neurones. ENK was observed in 44% of the myenteric neurones. The major ENK-positive subpopulations were ChAT/ENK (35% of ENK-positive neurones), ChAT/SP/ENK (26%), NOS/NPY/ENK (22%) and ChAT/SP/ENK/CALRET (9%). The projection pathways of these ENK-positive subpopulations to the circular muscle and the mucosa were determined using retrograde labelling with DiI in organ culture followed by immunohistochemistry. Of myenteric neurones retrogradely labelled from the mucosa and the circular muscle, 13% and 48% exhibited ENK immunoreactivity, respectively. Three major ENK-positive subpopulations innervating the mucosa or circular muscle were identified: ascending ChAT/SP/ENK (7% of all mucosa neurones; 24% of all circular muscle neurones), ascending ChAT/ENK (4%; 15%) and descending NOS/NPY/ENK (1%; 8%) neurones. Only very few CALRET- or SOM-positive neurones projected to the mucosa or circular muscle. ChAT/SP/ENK and ChAT/ENK neurones might function as ascending excitatory muscle motor neurones, whereas NOS/NPY/ENK neurones are most likely descending inhibitory muscle motor neurones. The relatively few ENK-positive mucosa neurones do not favour a major involvement of ENK-positive myenteric neurones in the control of gastric mucosa activity.     

Calretinin-immunoreactive neurons and their projections in the guinea-pig colon

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.     

Calbindin immunoreactivity of enteric neurons in the guinea-pig ileum

Previous studies have identified Dogiel type II neurons with cell bodies in the myenteric plexus of guinea-pig ileum to be intrinsic primary afferent neurons. These neurons also have distinctive electrophysiological characteristics (they are AH neurons) and 82-84% are immunoreactive for calbindin. They are the only calbindin-immunoreactive neurons in the plexus. Neurons with analogous shape and electrophysiology are found in submucosal ganglia, but, with antibodies used in previous studies, they lack calbindin immunoreactivity. An antiserum that is more effective in revealing calbindin in the guinea-pig enteric nervous system has been reported recently. In the present work, we found that this antiserum reveals the same population that was previously identified in myenteric ganglia, and does not reveal any further population of myenteric nerve cells. In submucosal ganglia, 9-10% of nerve cells were calbindin immunoreactive with this antiserum. The submucosal neurons with calbindin immunoreactivity were also immunoreactive for choline acetyltransferase, but not for neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP). Small calbindin-immunoreactive neurons (average profile 130 microm2) were calretinin immunoreactive, whereas the large calbindin-immunoreactive neurons (average profile 330 microm2) had tachykinin (substance P) immunoreactivity. Calbindin immunoreactivity was seen in about 50% of the calretinin neurons and 40% of the tachykinin-immunoreactive submucosal neurons. It is concluded that, in the guinea-pig ileum, only one class of myenteric neuron, the AH/Dogiel type II neuron, is calbindin immunoreactive, but, in the submucosal ganglia, calbindin immunoreactivity occurs in cholinergic, calretinin-immunoreactive, secretomotor/vasodilator neurons and AH/Dogiel type II neurons.     

Calretinin immunoreactivity in cholinergic motor neurones,interneurones and vasomotor neurones in the guinea-pig small intestine

Summary Immunoreactivity for calretinin, a calcium-binding protein, was studied in neurones in the guinea-pig small intestine. 26±1% of myenteric neurones and 12±3% of submucous neurones were immunoreactive for calretinin. All calretinin-immunoreactive neurones were also immunoreactive for choline acetyltransferase and hence are likely to be cholinergic. In the myenteric plexus, two subtypes of Dogiel type-I calretinin-immunoreactive neurones could be distinguished from their projections and neurochemical coding. Some calretinin-immunoreactive myenteric neurones had short projections to the tertiary plexus, and hence are likely to be cholinergic motor neurones to the longitudinal muscle. Some of these cells were also immunoreactive for substance P. The remaining myenteric neurones, immunoreactive for calretinin, enkephalin, neurofilament protein triplet and substance P, are likely to be orad-projecting, cholinergic interneurones. Calretinin immunoreactivity was also found in cholinergic neurones in the submucosa, which project to the submucosal vasculature and mucosal glands, and which are likely to mediate vasodilation. Thus, calretinin immunoreactivity in the guinea-pig small intestine is confined to three functional classes of cholinergic neurones. It is possible, for the first time, to distinguish these classes of cells from other enteric neurones.     

Ruminal muscle of sheep is innervated by non-polarized pathways of cholinergic and nitrergic myenteric neurones

The motility patterns of the reticulorumen evoke mainly mixing of the ingesta. So far unknown, intrinsic neural circuits of the enteric nervous system are involved in the control of these motility patterns. The aim of the study was to characterize neurochemically sheep ruminal myenteric neurones, in particular the neural pathways innervating the ruminal muscle layers. Cell bodies within the myenteric plexus projecting to the longitudinal or circular muscle layer were retrogradely labelled by direct application of the fluorescent tracer 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) onto the circular or longitudinal muscle. The neurochemical code of myenteric neurones was identified by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). According to their neurochemical code, ruminal myenteric neurones were divided into three populations: ChAT/SP (68% of all myenteric neurones), NOS/VIP (26% of all myenteric neurones) and ChAT/- (5% of all myenteric neurones). Application of DiI onto the circular or longitudinal muscle revealed on average 64 or 44 labelled cell bodies in the myenteric plexus, respectively. DiI-labelled neurones expressed the code ChAT/SP or NOS/VIP. In the pathways to circular or longitudinal muscle, ChAT/SP-positive neurones outnumbered NOS/VIP-immunoreactive neurones by 5:1 and 2:1. Pathways to the circular or longitudinal muscle did not exhibit any pronounced polarized innervation patterns. This study demonstrated specific projections of myenteric neurones to the ruminal muscle. Neurones expressing the code ChAT/SP might function as excitatory muscle motor neurones, whereas NOS/VIP neurones are likely to act as inhibitory muscle motor neurones.     

Different subpopulations of cholinergic and nitrergic myenteric neurones project to mucosa and circular muscle of the guinea-pig gastric fundus

Since the stomach lacks a well-developed ganglionated submucous plexus, the somata of enteric neurones innervating the muscle or the mucosa have to be localised within the myenteric plexus. The aim of this study was to determine the projection pathways and the neurochemical coding of myenteric neurones innervating these different targets in the gastric fundus. Myenteric cell bodies projecting to the mucosa or the circular muscle were retrogradely labelled by mucosa or muscle application of the fluorescent tracer DiI and subsequently characterised by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and/or neuropeptide Y (NPY). On average 143±91 and 89±49 myenteric neurones were labelled from the mucosa and the circular muscle, respectively. DiI-labelled neurones were either ChAT- or NOS-positive. DiI-labelled ChAT-positive neurones were mainly ascending and outnumbered NOS-positive neurones, which were mainly descending (79.3±6.2% vs 20.7±6.2% for mucosa neurones; 69.3±11.1% vs 30.7±11.1% for muscle neurones). Three ChAT-positive subpopulations (ChAT/–, ChAT/SP, ChAT/NPY) and two NOS-positive subpopulations (NOS/–, NOS/NPY) were found. ChAT/SP neurones projected mainly to the circular muscle (36.1±11.9% of the cholinergic muscle neurones; mucosa projection: 8.0±2.1%), whereas ChAT/NPY neurones projected mainly to the mucosa (38.1±9.2% of the cholinergic mucosa neurones; muscle projection: 5.7±2.4%). NOS/– cells projected predominantly to the muscle. This study demonstrates polarised pathways in the myenteric plexus consisting of ascending ChAT and descending NOS cells that innervate the circular muscle and the mucosa of the gastric fundus. The ChAT/SP neurones might function as circular muscle motor neurones, whereas ChAT/NPY neurones might represent secretomotor neurones.     

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats

The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons; in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.     

Nitric oxide synthase immunoreactivity in the enteric nervous system of the developing human digestive tract

We have investigated indirectly the presence of nitric oxide in the enteric nervous system of the digestive tract of human fetuses and newborns by nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In the stomach, NOS immunoactivity was confined to the myenteric plexus and nerve fibres in the outer smooth musculature; few immunoreactive nerve cell bodies were found in ganglia of the outer submucous plexus. In the pyloric region, a few nitrergic perikarya were seen in the inner submucous plexus and some immunoreactive fibres were found in the muscularis mucosae. In the small intestine, nitrergic neurons clustered just underneath or above the topographical plane formed by the primary nerve strands of the myenteric plexus up to the 26th week of gestation, after which stage, they occurred throughout the ganglia. Many of their processes contributed to the dense fine-meshed tertiary nerve network of the myenteric plexus and the circular smooth muscle layer. NOS-immunoreactive fibres directed to the circular smooth muscle layer originated from a few NOS-containing perikarya located in the outer submucous plexus. In the colon, caecum and rectum, labelled nerve cells and fibres were numerous in the myenteric plexus; they were also found in the outer submucous plexus. The circular muscle layer had a much denser NOS-immunoreactive innervation than the longitudinally oriented taenia. The marked morphological differences observed between nitrergic neurons within the developing human gastrointestinal tract, together with the typical innervation pattern in the ganglionic and aganglionic nerve networks, support the existenc of distinct subpopulations of NOS-containing enterice neurons acting as interneurons or (inhibitory) motor neurons.     

Identification of neuron types in the submucosal ganglia of the mouse ileum

The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found. This work was supported by a grant from the National Health and Medical Research Council of Australia (grant no. 400020) and an Australian Research Council international linkage grant (no. LZ0882269) for collaboration between the Melbourne and Bologna laboratories.     

Neurochemical identification of enteric neurons expressing P2X(3) receptors in the guinea-pig ileum

It was hypothesised that P2X(3) receptors, predominantly labelling spinal and cranial sensory ganglionic neurons, are also expressed in intrinsic sensory enteric neurons, although direct evidence is lacking. The aim of this study was to localise P2X(3) receptors in the enteric nervous system of the guinea-pig ileum, and to neurochemically identify the P2X(3)-expressing neurons. In the submucous plexus, cholinergic neurons expressing calretinin (CRT), were immunostained for P2X(3). These neurons made up about 12% of the submucous neurons. In the myenteric plexus, approximately 36% of the neurons expressed P2X(3). Half of the latter neurons were immunoreactive for CRT, whereas about 20% were immunoreactive for nitric oxide synthase (NOS). Based on earlier neurochemical analysis of enteric neurons in the guinea-pig, the myenteric neurons exhibiting P2X(3)/CRT immunoreactivity were identified as longitudinal muscle motor neurons, and those expressing P2X(3)/NOS immunoreactivity as short inhibitory circular muscle motor neurons. In both plexuses, no colocalisation was observed between P2X(3) and calbindin, a marker for intrinsic sensory neurons. Multiple staining with antisera raised against somatostatin, neuropeptide Y, substance P or neurofilament protein did not reveal any costaining. It can be concluded that in the guinea-pig ileum, intrinsic sensory neurons do not express P2X(3) receptors. However, this does not negate the possibility that extrinsic sensory nerves expressing P2X(3) are involved in a purinergic mechanosensory transduction pathway as demonstrated in other organs.     

Immunohistochemical analysis of intracardiac ganglia of the rat heart

The neurochemistry of intracardiac neurons in whole-mount preparations of the intrinsic ganglia was investigated. This technique allowed the study of the morphology of the ganglionated nerve plexus found within the atria as well as of individual neurons. Intracardiac ganglia formed a ring-like plexus around the entry of the pulmonary veins and were interconnected by a series of fine nerve fibres. All intracardiac neurons contained immunoreactivity to PGP-9.5, choline acetyl transferase (ChAT) and neuropeptide Y (NPY). Two smaller subpopulations were immunoreactive to calbindin or nitric oxide synthase. Furthermore, a subpopulation (approximately 6%) of PGP-9.5/ChAT/NPY-immunoreactive cells lacking both calbindin and nitric oxide synthase (NOS) was surrounded by pericellular baskets immunoreactive to ChAT and calbindin. Vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activated peptide (PACAP), substance P and tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibres within the ganglion, but never in neuronal somata. Furthermore, immunoreactivity for NPY was not observed in pericellular baskets surrounding intracardiac neurons, despite being present in all intrinsic neuronal cell bodies. Taken together, the results of this study indicate a moderate level of chemical diversity within the intracardiac neurons of the rat. Such chemical diversity may reflect functional specialisation of neurons in the intracardiac ganglia.This work was supported by a grant-in-aid (G00M0670) from the National Heart Foundation of Australia     

Transient expression of neuronal nitric oxide synthase by neurons of the submucous plexus of the mouse small intestine

Although neurons containing neuronal nitric oxide synthase (NOS) are abundant in the myenteric plexus of the small intestine of all mammalian species examined to date, NOS-containing neurons are sparse in the submucous plexus, and there does not appear to be an innervation of the mucosa by nerve fibres containing NOS. In this study, we used immunohistochemical techniques to examine the presence of neuronal NOS in the mouse intestine during development. At embryonic day 18 and postnatal day 0 (P0), about 50% of the neurons in the submucous plexus of the small intestine showed strong immunoreactivity to NOS, and NOS-immunoreactive nerve fibres were present in the mucosa. By P7, there was a gradation in the intensity of NOS immunostaining exhibited by submucosal neurons, varying from intense to extremely weak. During subsequent development, the proportion of submucous neurons showing NOS immunoreactivity decreased, and immunoreactive nerve fibres were no longer observed in the mucosa. In adult mice, NOS neurons comprised only 3% of neurons in the submucous plexus, which is significantly less than at P0. In contrast to the submucous plexus, the percentage of neurons that showed NOS immunoreactivity in the myenteric plexus did not change significantly during development.     

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.     

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.     

Characterization of excitatory and inhibitory motor neurons to the human gastric clasp and sling fibers

The aim of this study was to determine the morphology and position of the excitatory and inhibitory motor neurons to the human gastric sling and clasp fibers. Motor neurons were identified by retrograde staining with 1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate (DiI), and choline acetyltransferase (ChAT) or nitric oxide synthase (NOS) immunoreactivity was then determined in these motor neurons. In the sling preparations, 46% of the DiI-stained cells were aboral motor neurons, 43% were local motor neurons, and only 10% were descending motor neurons. Overall, 58% were immunoreactive for ChAT, and 36% for NOS (P = 0.042). Sixty-two percent of local, and 66% of aboral DiI-stained motor neurons were immunoreactive for ChAT. In the clasp preparations, 52% of the DiI-stained cells were descending motor neurons, 45% were local motor neurons, and only 3% were aboral neurons. Overall, 31% were immunoreactive for ChAT and 65% for NOS (P = 0.039). Eighty-five percent of the DiI-stained descending motor neurons were immunoreactive for NOS. All of the cells that were labeled adequately had a single axon and a number of filamentous or flattened lobular dendrites, and fitted into the broad category of Dogiel type I neurons. In conclusion, the majority of the motor neurons to the sling fibers were ChAT-positive excitatory neurons from the myenteric plexus of the stomach and the local region, and to the clasp were predominantly NOS-positive inhibitory neurons from the esophagus.     

Adenylyl cyclase co-distribution with the CaBPs, calbindin-D28 and calretinin, varies with cell type: assessment with the fluorescent dye, BODIPY forskolin, in enteric ganglia

The aims of the present study were: (1) to evaluate BODIPY forskolin as a suitable fluorescent marker for membrane adenylyl cyclase (AC) in living enteric neurons of the guinea-pig ileum; (2) to test the hypothesis that AC is distributed in several subpopulations of enteric neurons; (3) to test the hypothesis that the distribution of AC in the myenteric plexus is not unique to AH/Type 2 neurons. BODIPY forskolin was used to assess the co-distribution of AC in ganglion cells expressing the specific calcium-binding proteins (CaBPs), calretinin, calbindin-D₂₈, and s-100. Cultured cells or tissues were incubated with 10?μM BODIPY forskolin for 30?min and fluorescent labeling was monitored by using laser scanning confocal microscopy. BODIPY forskolin stained the cell soma, neurites, and nerve varicosities of Dogiel Type I or II neurons. About 99% of myenteric and 27% of submucous ganglia contained labeled neurons. About 14% of myenteric and 3% of submucous glia with immunoreactivity for s-100 protein displayed BODIPY forskolin fluorescence. BODIPY forskolin differentially labeled myenteric neurons immunoreactive for calbindin-D₂₈ (80%) and calretinin (17%). The majority (63%) of BODIPY forskolin-labeled myenteric neurons displayed no immunoreactivity for either CaBP. In submucous ganglia, the dye labeled 44.6% of calretinin-immunoreactive neurons, representing 21% of all labeled neurons; it also labeled varicose nerve fibers running along blood vessels. AC thus exists in myenteric Dogiel type II/AH neurons, enteric cholinergic S/Type 1 neurons, and other unidentified non-cholinergic S/Type 1 neurons. Our data also support the hypothesis that AC is expressed in distinct functional subpopulations of AH and S neurons in enteric ganglia, and show that BODIPY forskolin is a suitable marker for AC in immunofluorescence co-distribution studies involving living cells or tissues.