The presence of extraganglionic fluorescent neurons in the myenteric plexus of the guinea pig small intestine

Fluorescent histochemical observations of the small intestine of the guinea pig demonstrated that single fluorescent cell bodies, separate from the ganglia, were present in the myenteric plexus. These cell bodies gave rise to single processes which entered the ganglia or the interganglionic strands. They were of a very small size, and the intensity of their fluorescence increased by pretreatment with L-DOPA and nialamide. Interruption of extrinsic nerve pathways to the small intestine caused a disappearance of the meshwork of fluorescent fibers in the myenteric plexus; but in some areas a fluorescent fiber which supplied its terminal to the ganglion was seen to remain. A photograph taken from the denervated myenteric plexus revealed that a long process arising from an extraganglionic cell entered the ganglion and ramified into terminal branches.     

The neural regulation of the mammalian esophageal motility and its implication for esophageal diseases

In contrast to the tunica muscularis of the stomach, small intestine and large intestine, the external muscle layer of the mammalian esophagus contains not only smooth muscle but also striated muscle fibers. Although the swallowing pattern generator initiates the peristaltic movement via vagal preganglionic neurons that project to the myenteric ganglia in the smooth muscle esophagus, the progressing front of contraction is organized by a local reflex circuit composed by intrinsic neurons similarly to other gastrointestinal tracts. On the other hand, the peristalsis of the striated muscle esophagus is both initiated and organized by the swallowing pattern generator via vagal motor neurons that directly innervate the muscle fibers. The presence of a distinct ganglionated myenteric plexus in the striated muscle portion of the esophagus had been enigmatic and neglected in terms of peristaltic control for a long time. Recently, the regulatory roles of intrinsic neurons in the esophageal striated muscle have been clarified. It was reported that esophageal striated muscle receives dual innervation from both vagal motor fibers originating in the brainstem and varicose intrinsic nerve fibers originating in the myenteric plexus, which is called ‘enteric co-innervation’ of esophageal motor endplates. Moreover, a putative local neural reflex pathway that can control the motility of the striated muscle was identified in the rodent esophagus. This reflex circuit consists of primary afferent neurons and myenteric neurons, which can modulate the release of neurotransmitters from vagal motor neurons in the striated muscle esophagus. The pathogenesis of some esophageal disorders such as achalasia and gastroesophageal reflux disease might be involved in dysfunction of the neural networks including alterations of the myenteric neurons. These evidences indicate the physiological and pathological significance of intrinsic nervous system in the regulation of the esophageal motility. In addition, it is assumed that the components of intrinsic neurons might be therapeutic targets for several esophageal diseases.     

Target specific organization and neuron types of the dog pelvic ganglia: a retrograde-tracing and immunohistochemical study

The major pelvic ganglion in both the rat and guinea pig has been extensively studied because of its anatomical simplicity. To clarify the target specific neural pathway in the diffusely distributed pelvic ganglia of larger animals, the pelvic plexus of the female dog was investigated by retrograde tracing and immunohistochemistry. The whole mount staining of the pelvic plexus with acetylcholinesterase histochemistry revealed 70-100 ganglia of varying sizes. Neurons retrogradely labeled from the rectum were mainly found in ganglia located in the dorso-caudal part of the plexus. The majority of these were non-catecholaminergic, immunoreactive for either calbindin (Calb) or neuropeptide Y (NPY), and characteristically associated with baskets of enkephalin (ENK)-immunoreactive varicose fibers. Neurons projecting to the utero-vaginal walls were distributed in ganglia located in the ventro-caudal part of the plexus. These mainly consisted of two major neuron groups: catecholaminergic Calb-immunoreactive neurons, and non-catecholaminergic neurons containing nitric oxide synthase (NOS) and/or vasoactive intestinal peptide (VIP), which were preferentially associated with a network of ENK-immunoreactive varicose fibers. Neurons retrogradely labeled from the urinary bladder mainly occurred in ganglia located around the junction between the ureter and the bladder. These consisted of catecholaminergic Calb neurons and noncatecholaminergic neurons containing Calb or NOS. Only a few ENK-immunoreactive fibers were found within the clusters of catecholaminergic neurons. These results indicate that organ specific neurons are located in separate ganglia and have both a distinctive composition of neuron types as well as different innervation by preganglionic fibers.     

Light and electron microscopic studies of pituitary adenylate cyclase-activating peptide (PACAP) – immunoreactive neurons in the enteric nervous system of rat small and large intestine

 Pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive (IR) neurons in the myenteric and submucosal plexus of the rat small and large intestine were examined by immunostaining with purified polyclonal antiserum against PACAP (1–15), using both light and electron microscopy. Many PACAP-IR neuronal cell bodies and fibers were found in the myenteric and submucosal plexus. Many of the PACAP-IR fibers originated from the cell bodies of the myenteric and submucosal ganglia. The ganglia were also innervated by PACAP-IR fibers. PACAP-IR fibers penetrated both the circular and longitudinal muscle layers, confirming the previous observations indicating that PACAP neurons act as motor neurons. Ultrastructural study demonstrated that PACAP-IR nerve terminals formed synaptic contacts with PACAP-IR nerve cell bodies or dendritic processes. This observation suggests that PACAP-IR neurons innervate other PACAP-IR neurons, and that PACAP neurons work as interneurons in the enteric nervous system. PACAP-IR nerve cells received not only PACAP-positive nerve terminal input also PACAP-negative nerve terminal input. It also suggests that PACAP neurons are regulated not only by PACAP-IR enteric neurons, but also by neurons originating elsewhere. Our observations support the view that PACAP-IR neurons are involved in the control of gut motility. Accepted: 20 April 1998     

Neurons with 5-hydroxytryptamine-like immunoreactivity in the enteric nervous system: Their projections in the guinea-pig small intestine

Changes in the distribution of 5-hydroxytryptamine-like immunoreactivity have been examined in enteric neurons at various times after microsurgical lesions of the enteric plexuses. In the myenteric plexus, varicose immunoreactive nerve fibres disappeared or were reduced in number in ganglia anal to an interruption of the myenteric plexus. Up to about 2 mm on the anal side, all varicose immunoreactive fibres disappeared from the ganglia. At about 14–16 mm below an interruption, there were about 50% of the normal number of fibres in the myenteric ganglia and at about 24 mm the innervation was normal. In the submucosa, fibres immunoreactive for 5-hydroxytryptamine were absent from an area on the anal side following interruption of the myenteric plexus. From consideration of the pattern of disappearance, it is deduced that some myenteric nerve cell bodies send immunoreactive axons in an anal direction to supply submucous ganglia. The axons run for about 8 mm in the myenteric plexus, enter the submucosa and then run for a further 4 mm approximately.Thus, varicose fibres immunoreactive for 5-hydroxytryptamine, which occur around the enteric ganglion cells of both plexuses arise from nerve cell bodies in the myenteric ganglia that send their axons in an anal direction.     

Effects of age on sympathetic innervation of the myenteric plexus and gastrointestinal smooth muscle of Fischer 344 rats

Loss of myenteric neurons with age is well documented, however little is known about age-related changes of the sympathetic innervation of the myenteric plexus and gastrointestinal smooth muscle. The goal of the present study, therefore, was to evaluate the influence of age on the sympathetic innervation of the myenteric plexus throughout the gastrointestinal tract. Ad libitum fed virgin male Fischer 344 rats at 3, 15–16, 24, and 27–28 months of age were sampled. Whole mounts of the stomach, small intestine, and large intestine were processed with an antibody to tyrosine hydroxylase (TH). Additionally, some specimens labeled for TH were stained for NADPH-diaphorase to selectively label the nitrergic subpopulation of neurons in the myenteric plexus. Age-related changes in the TH-positive axons occurred as early as 15–16 months and became more pronounced by 27–28 months. Changes included markedly swollen axons and terminals and a decrease in the intensity of TH staining in some of the surviving processes. Similarly, swollen NADPH-diaphorase-positive axons were found in the myenteric ganglia and secondary plexus between ganglia in the whole mounts of rats 15–28 months of age, but swollen nitrergic axons and dystrophic TH-positive axons were never present in the same ganglion or connective. Therefore, in the aged rat, deterioration of the sympathetic innervation of the myenteric plexus could be one possible mechanism for the age-related decline in gastrointestinal motor function evidenced in the elderly.     

Distribution and projections of nerves with enkephalin-like immunoreactivity in the guinea-pig small intestine

Whole mounts of guinea-pig small intestine were used to examine the distribution of neurons with enkephalin-like immunoreactivity and the effects of microsurgical lesions on these neurons. The enkephalin neurons are intrinsic to the intestine. Cell bodies are found in the myenteric ganglia; processes are in the myenteric plexus, circular muscle (including deep muscular plexus) and submucosa, but not in the mucosa. The cell bodies have one prominent process and several short processes, the latter occasionally are seen to give rise in turn to fine, faint processes. The prominent processes provide fibres to the circular muscle and deep muscular plexus beneath and just anal (up to about 2 mm) to the cell bodies. Fibres in the submucous ganglia come from the overlying myenteric plexus. Orally-directed processes (possibly dendrites) of myenteric cell bodies provide the varicose fibres in the myenteric ganglia. These processes are 3.5-4 mm long. The enkephalin neurons represent a population of enteric neurons, with a distinct distribution and projections, which does not correspond to any of the other populations of enteric neurons that have been studied.     

Projections of substance P-containing neurons within the guinea-pig small intestine

The origins of substance P immunoreactive axons in the small intestine of the guinea-pig were investigated with an immunohistochemical technique in whole mount preparations. Nerve pathways were interrupted either in vitro or in vivo to detect the accumulation of substance P proximal to the lesion and the disappearance of immunoreactive fibres resulting from the degeneration of the severed axons. Various operations, namely, extrinsic denervation, interruption of the myenteric plexus (myotomy) or removal of the myenteric plexus with the longitudinal muscle (myectomy), were performed prior to examination of substance P-containing neurons.There are several projections of substance P-containing neurons which supply the intestine. Extrinsic neurons are the sources of two projections, one to submucosal blood vessels and one to the submucous ganglia. Intrinsic neurons located in the submucous ganglia supply the villi. Five projections arise from the myenteric plexus, a very short projection ending either within the same row of ganglia or within the adjacent rows of ganglia on both sides, a longer projection within the myenteric plexus, a very short projection to the circular muscle, a projection to the submucous ganglia where the axons surround most of submucous nerve cell bodies, and a projection to the villi.It is likely that the highly organised patterns of innervation by different substance P-containing neurons have specific roles in the intestine. Some of these neurons may act as sensory neurons, others as interneurons, and yet others as motor neurons in nerve pathways within the enteric nervous system.     

Neurons with 5-hydroxytryptamine-like immunoreactivity in the enteric nervous system: their visualization and reactions to drug treatment

Immunoreactive nerve cell bodies and fibres in the intestine have been examined using three antibody preparations raised against 5-hydroxytryptamine. Cross reactivity studies indicate that the substance localized was an hydroxylated indoleamine. In the guinea-pig small intestine, nerve cell bodies were located in the myenteric plexus and varicose fibres were found in the ganglia of the myenteric and submucous plexus. The nerve cell bodies had prominent short, broad processes and a single long process. Similar nerve cells and fibres were found in the guinea-pig stomach and large intestine and areas of intestine that were examined in mice, rabbits and rats. Properties of the neurons were examined in the small intestine of the guinea-pig. The immunoreactive material was depleted by treatment with reserpine, but not by guanethidine or 6-hydroxydopamine in dose sufficient to deplete noradrenaline stores in axons in the intestine. No depletion of 5-hydroxytryptamine by the neurotoxin 5, 7-dihydroxytryptamine was observed. After depletion by reserpine, immunoreactivity of the neurons could be restored by application in vitro of 5-hydroxytryptamine, 5,7-dihydroxytryptamine or 5-hydroxytryptophan. The restoration by 5-hydroxytryptophan was prevented by the inhibitor of L-aminoacid decarboxylase, benserazide. After reserpine treatment, immunoreactivity was not restored by tryptophan. Uptake of 5, 7-dihydroxytryptamine into the nerves was antagonized by fluoxetine. The distribution of neurons with 5-hydroxytryptamine-like immunoreactivity was compared with the distribution of enteric amine-handling neurons that take up and decarboxylate L-dopa. This comparison indicated that there are two classes of aromatic amine neuron in the guinea-pig small intestine, the enteric 5-HT neurons and enteric, non-5-HT, amine handling neurons.     

Neuron density and distribution of NADPH-diaphorase positive neurons in the human stomach

Neuron density and distribution of the NADPH-diaphorase positive neurons were studied in the fundus, corpus and antrum of adult human stomach using cresyl violet staining and NADPH-diaphorase histochemistry. The submucous plexus contained significantly less neurons than the myenteric plexus. Submucous NADPH-d positive neurons were mostly located in ganglia close to the circular muscle layer. Myenteric NADPH-d positive neurons represented 50–60% of the neurons in all the three regions; their density, however, was significantly lower in the fundus. NADPH-d positive fibers formed a rich plexus in the innermost portion of the circular muscle layer of the corpus.     

Origins of synaptic inputs to calretinin immunoreactive neurons in the guinea-pig small intestine

Summary Calretinin immunoreactivity is almost completely confined to two classes of neuron in the myenteric plexus of the guinea-pig small intestine, longitudinal muscle motor neurons and ascending interneurons. Nerve cell bodies of the two classes can be readily identified by their sizes and positions in ganglia. The motor neurons, which are small Dogiel type I neurons, are about 20% and the interneurons, which are medium-sized Dogiel type I neurons, are about 5% of myenteric neurons. In the present work, we have also discovered a minor population (0.1%) of small filamentous neurons. In unoperated regions of intestine, at the light microscopic level, numerous calretinin immunoreactive nerve fibres were found in the tertiary plexus that innervates the longitudinal muscle and a medium density of varicose fibres formed pericellular endings in the myenteric ganglia. After double myotomy operations, in areas of plexus 0.5 to 1.5 mm wide which were isolated from ascending and descending inputs, calretinin-immunoreactive fibres of the tertiary plexus were unchanged, but the periceliular endings in the ganglia disappeared. Both the ascending interneurons and the longitudinal muscle motor neurons received ultrastructurally identified synapses and close axonal contacts that were calretinin-immunoreactive. These were counted in semi-serial sections from normal intestine and from regions between myotomy operations. In unoperated intestine, the proportions of calretinin-immunoreactive synapses on small, calretinin-immunoreactive, Dogiel type I nerve cells and small filamentous nerve cells were 30% and 0.1% respectively and on medium-sized Dogiel type I cells the proportion was 28%. Electron microscopy revealed an almost complete loss of immunoreactive inputs to the small Dogiel type I cells between double myotomies, but the number of unreactive inputs was the same as in normal intestine. This work demonstrates that the ascending calretinin-immunoreactive interneurons connect with one another to form ascending chains in the myenteric plexus and that they also provide about 1/3 of the inputs received by calretinin-immunoreactive longitudinal muscle motor neurons. Many of the remaining inputs to these motor neurons are local; we have deduced that these are mainly from primary sensory neurons.     

The origins,pathways and terminations of neurons with VIP-like immunoreactivity in the guinea-pig small intestine

We have analyzed changes in the distributions of terminals with vasoactive intestinal polypeptide (VIP)-like immunoreactivity, and accumulations in severed processes, that occur after lesions of intrinsic and extrinsic nerve pathways of the guinea-pig small intestine. The observations indicate that enteric vasoactive intestinal polypeptide immunoreactive neurons have the following projections. Nerve cell bodies in the myenteric plexus provide varicose processes to the underlying circular muscle; the majority of these pathways, if they extend at all in the anal or oral directions, do so for distances of less than 1 mm. Nerve cell bodies of the myenteric plexus also project anally to provide terminals to other myenteric ganglia. The lengths of the majority of these projections are between 2 and 10 mm, with an average length of about 6 mm. Processes of myenteric neurons also run anally in the myenteric plexus and then penetrate the circular muscle to provide varicose processes in the submucous ganglia at distances of up to 15 mm, the average length being 9–12 mm. In addition, there is an intestinofugal projection of myenteric neurons whose processes end around nerve cell bodies of the coeliac ganglia. A similar projection from the colon supplies the inferior mesenteric ganglia. The nerve cell bodies in submucous ganglia give rise to a subepithelial network of fibres in the mucosa and also supply terminals to submucous arterioles.It is concluded that vasoactive intestinal polypeptide is contained in neurons of a number of intrinsic nerve pathways, influencing motility, blood flow and mucosal transport. The myenteric neurons that project to prevertebral sympathetic ganglia may be involved in intestino-intestinal reflexes.     

豚鼠胃肠壁内神经丛的磷酸醋、醋类和神经递质代谢有关的酶组织化学观察

本文报道用光镜半定量和显微光度计定量分析研究了豚鼠胃肠壁内神经丛神经元的几种酶的组织化学反应。结果表明,神经元的碱性磷酸酶(AlP)、酸性磷酸酶(AcP)、5′-核苷酸酶(5′-Nase)、硫胺素焦磷酸酶(TPPase)、非特异性酯酶(NsE)和胆碱乙酰转移酶(ChAT)反应强弱明显不等。消化道不同节段或不同部位神经元的单胺氧化酶(MAO)、氨基肽酸(AP)和乙酰胆碱酯酶(AChE)反应虽有差别,但却显阳性反应,同一神经节内各神经元的反应比较近似。胃肠各段壁内神经丛中50～66%神经元呈ChAT强阳性反应,这些细胞可能为胆碱能神经元。整个消化道粘膜下丛与肠肌丛神经元相比,除NsE外,另几种酶均有高度显著差异。粘膜下丛神经元AcP和AP反应较强,肠肌丛神经元AlP、5′-Nase、TPPase、MAO、ChAT和AChE反应较强,胃壁内神经丛不如肠道的发达。尤其是胃粘膜下丛只见少数单个散在的神经元,它们的各种酶组织化学反应均较弱。各段肠中,以十二指肠和近端结肠壁内神经丛神经元的各种酶组织化学反应较强。上述结果表明,消化道不同部位以及同一部位不同类型的神经元在代谢和功能上有明显的差别。     

Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine.

The distribution of nitric oxide synthase (NOS) immunoreactivity was investigated in the guinea-pig small intestine. There were many immunoreactive nerve cell bodies in the myenteric plexus but very few in submucous ganglia. NOS immunoreactivity was not found in non-neuronal cells except for rare mucosal endocrine cells. Abundant immunoreactive nerve fibres in both myenteric and submucous ganglia, and in the circular muscle, arose from myenteric nerve cells whose axons projected anally along the intestine. NOS immunoreactivity coexisted with VIP-immunoreactivity, but not with substance P immunoreactivity. We conclude that nitric oxide synthase is located in a sub-population of enteric neurons, amongst which are inhibitory motor neurons that supply the circular muscle layer.     

The neurochemistry and innervation patterns of extrinsic sensory and sympathetic nerves in the myenteric plexus of the C57Bl6 mouse jejunum

In vitro anterograde tracing of axons in mesenteric nerve trunks using biotinamide in combination with immunohistochemical labelling was used to characterize the extrinsic nerve projections in the myenteric plexus of the mouse jejunum. Anterogradely-labelled spinal sensory fibres innervating the enteric nervous system were identified by their immunoreactivity for calcitonin gene-related peptide (CGRP), while sympathetic noradrenergic fibres were detected with tyrosine hydroxylase (TH), using confocal microscopy. The presence of these markers has been previously described in the spinal sensory and sympathetic fibres. Labelled extrinsic nerve fibres in the myenteric plexus were identified apposing enteric neurons that were immunoreactive for either calretinin (CalR), calbindin (CalB) or nitric oxide synthase (NOS). Of the total anterogradely labelled axons in the myenteric plexus, 20% were CGRP-immunoreactive. Labelled CGRP-immunoreactive varicosities were closely apposed to CalR-immunoreactive myenteric cells, many of which were Dogiel type I (40%; interneurons) or type II (20%; intrinsic sensory) neurons. Labelled CGRP-immunoreactive varicosities were also observed in close appositions to CalB-immunoreactive myenteric cell bodies, of which a small subset had type II morphology (18%; intrinsic sensory neurons). A further 43% of all biotinamide-filled fibres were immunoreactive for TH and these fibres were apposed to CalR-immunoreactive cell bodies (small-sized; excitatory motor neurons) and NOS-immunoreactive cell bodies (either type I or small neurons; inhibitory motor neurons and interneurons) in the myenteric plexus. The results provide a neurochemical and neuroanatomical basis for connections between dorsal root afferent neurons and myenteric neurons and suggest an anatomical substrate for the well-known modulation of enteric circuits from sympathetic nerves. No anterogradely-labelled fibres were stained for NOS-immunoreactivity, despite more than 60% of dorsal root ganglion (DRG) neurons retrogradely labelled from the jejunum showing NOS-immunoreactivity. This was due to a substantial, time-dependent, and apparently selective, loss of NOS from extrinsic axons under in vitro conditions. Lastly, a small population of non-immunoreactive biotinamide-filled fibres (<1%) gave rise to dense terminal structures around individual myenteric cell bodies lacking CalR, CalB or NOS. These specialized endings may represent vagal fibres or a subset of spinal sensory neurons that do not contain CGRP.     

γ-Aminobutyrate as an autonomic neurotransmitter: Release and uptake of [H]γ-Aminobutyrate in guinea pig large intestine and cultured enteric neurons using physiological methods and electron microscopic autoradiography

To provide further evidence that some enteric neurons use γ-aminobutyrate (GABA) as a neurotransmitter, we have demonstrated a depolarization-induced release of [³H]GABA from isolated myenteric ganglia in culture, and from segments of large intestine containing the myenteric plexus. In addition, light and electron microscopic autoradiography has been employed to visualize the putative GABAergic neurons and their projections, both in cultured ganglia and in sections from the gut wall.

Explant cultures of the guinea-pig myenteric plexus, containing only neurons and glia intrinsic to the gut, were incubated with 0.14μ M [³H]GABA, washed and then repeatedly depolarized by 62 mM K⁺. The depolarizations always evoked release of [³H]GABA. The evoked release was reversibly blocked by 5mM Co²⁺, suggesting a dependence on Ca²⁺ influx, a characteristic of neuronal transmitter release. Strips of the guinea-pig taenia coli, containing the myenteric plexus, were incubated with 0.14 or 0.7μM [³H]GABA, washed and subjected to electrical field stimulation. This caused release of [³H]GABA, which could be evoked successively on repeated stimulation. The release was of neuronal origin and Ca²⁺ dependent, since it was abolished by 3μ M tetrodotoxin and reversibly blocked by 10 mM Co²⁺. By combined electrophoresis and chromatography the released tritium was identified as being attached to GABA.

Autoradiography, following incubation with low concentrations of [³H]GABA, was used to identify specifically putative GABAergic neurons. Light microscopic autoradiography of cultured ganglia, and electron microscopic autoradiography of sections from the taenia coli including the myenteric plexus, were in good agreement, showing a selective and heavy labelling over a sub-population of neurons, and light labelling over glial cells. The majority of neurons and the non-neural cells were unlabelled. The electron microscopic autoradiographs also showed heavy labelling over some, but not all, axons in the fine axon bundles that innervate the longitudinal muscle of the taenia.

These results strongly support our previous suggestion that a population of myenteric neurons are GABAergic, although more work is needed before this is fully established. Our data also suggest that some of the putative GABAergic neurons project out to the gut musculature, where their axons, together with axons from other neurons, run in bundles to form the intramuscular nerves. We therefore propose that one of the roles of these neurons in the gut is the presynaptic modulation of transmitter released from neighbouring axons in these nerves.     

Excitatory synaptic potentials due to activation of neurons with short projections in the myenteric plexus

Intracellular microelectrodes have been used to examine the effects, on excitatory inputs to myenteric nerve cells, of lesions of intrinsic pathways in the myenteric plexus of the guinea-pig small intestine. The lesions consisted of circumferential cuts (myotomies) which severed the external musculature to the depth of the submucosa and thus interrupted pathways in the myenteric plexus. Sufficient time was allowed between creating the lesions and recording from the neurons for the endings of severed neurites to degenerate and this was confirmed histochemically by examining the distribution of varicose fibres with 5-hydroxytryptamine immunoreactivity in myenteric ganglia from which recordings were made. Two types of excitatory input, eliciting fast and slow excitatory post-synaptic potentials, respectively, were demonstrable in response to focal stimulation of nerves in the ganglia from which recordings were made. There were no differences in the proportions of neurons in which fast or slow excitatory synaptic potentials were evoked in unoperated preparations (controls), in islands 1.5-4 mm wide between myotomies, or within 1 mm on the oral or anal sides of myotomies. Possible differences in the amplitudes, durations at half amplitude, and threshold numbers of stimuli for initiation of slow excitatory synaptic potentials were analyzed. The only significant differences were found when data from control and oral areas were pooled and compared with combined data from island and anal areas (this assessed differences that could arise from severing nerve fibres running from oral to anal).(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical localization of galanin receptors (GAL-R1, GAL-R2, and GAL-R3) on myenteric neurons from the sheep and dog stomach

Galanin exerts its biological activities (inhibitory or excitatory) via three different G protein-coupled receptors. In the present study, double immunocytochemical labeling was used to localize GAL-R1, GAL-R2 and GAL-R3 on PGP 9.5-positive myenteric neurons from the dog and sheep stomach/forestomachs. In both species, the occurrence of galanin in neurons and nerve fibers of gastric ganglia was also studied. Myenteric ganglia of the dog stomach were supplied with numerous, mainly varicose, galanin-immunoreactive (IR) nerve terminals whereas the frequency of galanin-positive nerve fibers in myenteric ganglia of the ovine stomach and forestomachs was moderate. The number of PGP 9.5-IR/galanin-IR myenteric neurons was significantly lower in the dog stomach (12.3+/-1.3%) as compared to the sheep rumen (20.1+/-0.7%), omasum (19.5+/-2.9%), abomasum (23.8+/-1.2%) but not reticulum (8.1+/-0.8%). In the canine stomach the frequencies of GAL-R1, GAL-R2 and GAL-R3 expressing myenteric neurons were statistically equivalent (4.4+/-0.9%, 3.5+/-0.7% and 3.1+/-0.5%, respectively). Immunoreactivity to GAL-R1 was absent in myenteric ganglia from the ovine rumen, reticulum as well as omasum. GAL-R1 was localized on 0.5+/-0.3% of myenteric perikarya from the abomasum. GAL-R2 bearing myenteric neurons were localized in the ovine rumen (0.6+/-0.3%), reticulum (0.5+/-0.3%), omasum (1.0+/-0.2%) and abomasum (1.1+/-0.3%). The percentages of PGP 9.5-IR/GAL-R3-IR neurons were 0.8+/-0.2% in the rumen, 0.6+/-0.3% in the reticulum, 0.7+/-0.2% in the omasum and 0.9+/-0.3% in the abomasum. In all compartments of the sheep stomach, the proportions of GAL-R1, GAL-R2 and GAL-R3 expressing neurons were significantly lower when compared to analogous neuronal subpopulations present in the dog. It is suggested that, although endogenous galanin may potentially inhibit or stimulate the activity of sparse gastric enteric neurons, its general role in indirect mediation of gastric motility and/or secretion seems to be of minor importance.     

猪食管神经支配的神经化学研究——一氧化氮类及肽类成分在平滑肌中的分布(英文)

采用免疫荧光组织化学技术及迷走神经切断术,探讨猪食管一氧化氮类及肽类神经支配的神经化学特性。在光学显微镜下可观察到肌间神经丛及粘膜下神经丛中有部分神经元呈nNOS、VIP、GAL、NPY、PACAP、L-ENK、SP、5-HT及CB免疫阳性,但未见CGRP及SOM阳性神经元。nNOS及CB免疫阳性产物主要分布于不同的神经元胞体内。将PGP9.5作为神经元胞体的标记物,并采用免疫荧光免疫组织化学双重染色方法,分别观察了PGP9.5与nNOS、VIP、SP的双标情况。结果如下:(1)nNOS免疫阳性神经元约占PGP9.5标记神经元总数的63%,而VIP免疫阳性神经元约占36%,SP免疫阳性神经元约占28%;(2)神经节内神经元的平均数量呈现吻尾方向的递增趋势,且食管腹段神经丛内神经节数量明显高于食管其他部位;(3)食管肌层内VIP/GAL/NPY免疫阳性纤维分布最广,其中部分阳性纤维同时呈nNOS或PACAP免疫阳性;SP和/或L-ENK免疫阳性纤维在粘膜肌层的分布明显多于平滑肌层。CGRP阳性纤维非常少见,这一点不同于对其他动物的观察结果;(4)经一侧迷走神经切断后,肌间神经丛内PACAP及5-HT免疫阳性纤维明显减少,提示这些纤维可能来源于迷走神经;而平滑肌中VIP/GAL/NPY和/或nNOS免疫阳性纤维数量未发现明显变化,可能为内源性来源。