Distribution of enteric nerve cell bodies and axons showing immunoreactivity for vasoactive intestinal polypeptide in the guinea-pig intestine

Immunoreactivity for vasoactive intestinal polypeptide has been localized in neurons in the guinea-pig ileum, colon and stomach. In the ileum, 2.5% of the nerve cell bodies of the myenteric plexus and 45% of those of the submucous plexus showed vasoactive intestinal polypeptide-like immunoreactivity. Varicose axons containing vasoactive intestinal polypeptide ramified amongst the nerve cell bodies of both plexuses and in some cases formed rings of varicosities around non-reactive nerve cells. Axons were traced from the myenteric plexus to the circular muscle and deep muscular plexus. There were numerous positive axons running in fine strands within the circular muscle, parallel to the muscle bundles. Axons containing vasoactive intestinal polypeptide were associated with mucosal blood vessels, but few supplied the vascular network of the submucosa; some immunoreactive axons also contributed to the periglandular plexus of the mucosa. There were no changes in the distribution of axons in the ileum after extrinsic denervation.The results are discussed in relation to the possible functional roles of neurons that contain vasoactive intestinal polypeptide in the intestine: the distribution of such nerve cells in the myenteric plexus and of axons in the circular muscle and sphincters is consistent with this polypeptide being a transmitter of enteric inhibitory neurons; it is also possible that vasoactive intestinal polypeptide is the enteric vasodilator transmitter.     

Projections of intestinal neurons showing immunoreactivity for vasoactive intestinal polypeptide are consistent with these neurons being the enteric inhibitory neurons.

Experiments were performed to determine if the distribution of vasoactive intestinal peptide(VIP)-like immunoreactivity in nerve cell bodies and axons of the myenteric plexus and circular muscle of the small intestine is consistent with VIP being the transmitter of enteric inhibitory neurons. Immunoreactivity for VIP was found in nerve cell bodies of the myenteric plexus and in axons within the myenteric plexus and circular muscle. When the axons in the myenteric plexus were interrupted, there was accumulation of material showing reactivity for VIP on the oral side, indicating that the neurons project in an anal direction. The VIP-like immunoreactivity in axons which supply the circular muscle disappeared after a myectomy in which the overlying myenteric plexus was removed, but remained intact when extrinsic nerves were served. The projections of VIP neurons from the myenteric plexus to the circular muscle correspond to the expected projections of enteric inhibitory neurons determined by functional studies.     

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.     

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.     

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.     

Mapping 5-HT inputs to enteric neurons of the guinea-pig small intestine

5-HT released by gastrointestinal mucosa and enteric interneurons has powerful effects on gut behavior. However, the targets of 5-HT-containing neurons within enteric circuits are not well characterized. We used antisera against 5-HT and selected markers of known enteric neuron types to investigate the connections made by 5-HT-containing neurons in the guinea-pig jejunum. Confocal microscopy was used to quantify the number of 5-HT-immunoreactive varicosities apposed to immunohistochemically identified cell bodies. Large numbers of varicosities were identified apposing cholinergic secretomotor neurons, immunoreactive for neuropeptide Y, in both myenteric and submucous plexuses. Subgroups of neurons identified by calretinin (ascending interneurons) and nitric oxide synthase (descending interneurons and inhibitory motor neurons) immunoreactivity were also apposed by many varicosities. Longitudinal muscle motor neurons (calretinin immunoreactive) and AH/Dogiel type II (sensory) neurons (calbindin immunoreactive) were apposed by small numbers of varicosities. Combined retrograde tracing and immunohistochemistry were used to identify excitatory circular muscle motor neurons; these were encircled by 5-HT-immunoreactive varicosities, but the appositions could not be quantified. We suggest that 5-HT-containing interneurons are involved in secretomotor pathways and pathways to subgroups of other interneurons, but not longitudinal muscle motor neurons. There also appear to be connections between 5-HT-containing interneurons and excitatory circular muscle motor neurons. Physiological evidence demonstrates a functional connection between 5-HT-containing interneurons and AH/Dogiel type II neurons, but few 5-HT-immunoreactive varicosities were observed apposing calbindin-immunoreactive cell bodies. Taken together these results suggest that neural 5-HT may have significant roles in excitatory pathways regulating both motility and secretion.     

An ultrastructural analysis of the developing enteric nervous system of the guinea-pig small intestine

Summary The developing enteric nervous system of the guinea-pig has been analysed ultrastructurally. In addition, electron microscope autoradiography, following incubation with tritiated 5-hydroxytryptamine ([³H]5-HT) or tritiated norepinephrine ([³H]NE) was used to locate the developing axons of enteric serotoninergic and adrenergic neurons respectively. Observations have been correlated with previous studies of the development of the various types of enteric neuron and the onset of intestinal neuromuscular function. Prior to 25 days of gestation no neurons can be recognized morphologically. Neurons first appear at 25 days' gestation, together with a primitive neuropil in neural islands within the outer gut mesenchyme. Ganglion cell precursors are primitive at first and resemble the cells in the surrounding mesenchyme. Growth cones are abundant but there are no terminal varicosities or synapses. The circular muscle also begins to form at this time. At 32 days' gestation the longitudinal layer of smooth muscle can be discerned and, within the myenteric plexus, terminal axonal varicosities appear containing small (about 50 nm in diameter) electron-lucent synaptic vesicles. The submucosal plexus appears to be derived from neurons and neurites that reach it from the earlier-developing myenteric plexus. The submucosal plexus can be recognized at 38 days of gestation but is not well developed until day 42. Synapses on ganglion cell somata first appear in the myenteric plexus on gestational day 38 and are numerous on day 42 when the first axo-dendritic synapses can be seen. Between days 42 and 48 the developing neural tissue and growing smooth muscle cells interdigitate but after day 48, the plexus becomes ensheathed by supporting cells and connective tissue and this interdigitation is lost. Prior to day 48 most varicosities contain small electron-lucent synaptic vesicles; however, after this time a variety of terminals appears. Between days 48 and 53 of gestation evidence of degenerating neuronal processes is common, indicating that cell death may occur. Electron microscopic autoradiography with [³H]5-HT reveals labelling of axons in the neuropil of the myenteric plexus at day 32 of gestation. Some primitive appearing cell bodies, however, are also labelled and these cells seem to be entering the myenteric plexus from the surrounding mesenchyme. After 42 days of gestation [³H]5-HT labels only axons of both nerve plexuses. Often, labelled terminals are apposed to ganglion cells or dendrites. In contrast, significant labelling by [³H]NE is not found until gestational day 48. Axons are labelled by [³H]NE and these tend to be located at the interface between the myenteric plexus and the surrounding connective tissue.     

Immunocytochemical analysis of potential neurotransmitters present in the myenteric plexus and muscular layers of the corpus of the guinea pig stomach

Recent electrophysiological studies of neurons of the myenteric plexus of the corpus of the guinea pig stomach have revealed that slow synaptic events are extremely rare. In contrast, they are commonly encountered in similar investigations of myenteric ganglia of the guinea pig small intestine. The current immunocytochemical analysis of the myenteric plexus and innervation of the muscularis externa of the corpus of the guinea pig stomach was undertaken in order to determine whether putative neurotransmitters capable of mediating slow synaptic events are present in gastric ganglia. A major difference between the small intestine and the stomach was found in the innervation of the musculature. Whereas the longitudinal muscle layer of the small intestine contains very few nerve fibers and is innervated mainly at its interface with the myenteric plexus, the longitudinal muscle of the corpus of the stomach contained as many varicose substance P (SP)-, vasocative intestinal polypeptide (VIP)-, and neuropeptide Y (NPY)-immunoreactive axons as the circular muscle layer. These putative neurotransmitters were also present in the ganglia of the myenteric plexus, where varicose SP-, VIP-, and NPY-immunoreactive fibers encircled nonimmunoreactive neurons. Varicose 5-hydroxytryptamine (5-HT)-immunoreactive terminal axons were essentially limited to the myenteric plexus and were found both in ganglia and in interganglionic connectives, where they were particularly numerous; 5-HT-immunoreactive neurons appeared to be more abundant in the stomach than in the small intestine. Tyrosine hydroxylase (TH)- and calcitonin-gene-related-peptide (CGRP)-immunoreactive axons were also more common in the myenteric plexus than in the musculature, but of these, only the TH-immunoreactive neurites tended, like those of the other putative transmitters, to encircle neurons in myenteric ganglia. Evidence was obtained that, as in the small intestine, at least some of the SP-, VIP-, NPY-, and 5-HT-immunoreactive fibers in the stomach are derived from intrinsic gastric myenteric neurons. In contrast, unlike the small intestine, gastric myenteric ganglia appeared to lack intrinsic CGRP-immunoreactive neurons; therefore, the CGRP-immunoreactive gastric axons are probably of extrinsic origin.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Ultrastructural identification of noradrenergic axons and their distribution within the enteric plexuses of the guinea-pig small intestine

Summary Noradrenergic axons in the enteric plexuses of the guinea-pig ileum have been identified at the ultrastructural level using three techniques: the chromaffin reaction, localization of dopamine--hydroxylase (DBH) with horseradish peroxidase-conjugated antibody, andin vivo andin vitro loading with 5-hydroxydopamine (5-OHDA).In the myenteric (Auerbach's) plexus from normal ileum all of these methods produced electron-dense deposits in a distinctive population of axonal varicosities that contained many flattened vesicles (usually more than 30% of the total number of vesicles), as well as oval or irregularly shaped vesicles. When noradrenergic axons to the small intestine had degenerated after surgical denervation, no profiles containing vesicles with electron-dense deposits were observed with the chromaffin reaction, DBH localization or loading with 5-OHDA. Pretreatment with 6-hydroxydopamine (6-OHDA) substantially reduced the number of noradrenergic axons identified by these three techniques. Axons with many flattened vesicles of similar dimensions but without dense cores were found in myenteric plexus from conventionally fixed intestine. These axons had the same distribution within the ganglia as cytochemically labelled noradrenergic terminals and disappeared after extrinsic denervation.In the normal submucous (Meissner's) plexus, both 5-OHDA loading and the chromaffin reaction produced electron-dense granules in small and large vesicles in some axon terminals. In ganglia labelled by these techniques, reactive terminals contained many small round vesicles and few flattened and large round vesicles as did a population of nonreactive terminals. In axon terminals of submucous plexus labelled with anti-DBH, flattened vesicles were found to be more numerous than with the other treatments. As in the myenteric plexus, all reactive axons disappeared from the submucous plexus after extrinsic denervation. In conventionally processed submucous ganglia, noradrenergic axon profiles could not be distinguished from some non-noradrenergic profiles on the basis of types and proportions of vesicles.In the myenteric plexus noradrenergic axon terminals were seen most often near the edges of ganglia. Noradrenergic varicosities also occurred near nerve cell bodies but were rarely found in internodal strands. In the submucous plexus noradrenergic terminals appeared to be randomly distributed throughout submucous ganglia. No axosomatic synapses formed by noradrenergic axons were found in either plexus, but synapses on nerve processes were occasionally encountered in submucous ganglia.     

The central projections of visceral primary afferent neurons of the inferior mesenteric plexus and hypogastric nerve and the location of the related sensory and preganglionic sympathetic cell bodies in the rat

Summary The central projections of visceral primary afferents of the inferior mesenteric plexus and hypogastric nerve of the rat were investigated using the transganglionic transport of horseradish peroxidase (HRP). In addition, the location of the corresponding spinal ganglion cells as well as the preganglionic sympathetic neurons is demonstrated.Labelled afferent axons were found in dorsal roots, dorsal root entry zone (preferentially in its lateral part), in all parts of the tract of Lissauer, and in the dorsolateral funiculus. Preterminal axons and/or terminals were distributed mainly to laminae I, IIa and the nucleus of the dorsolateral funiculus. Fewer afferents reached laminae IIb, III–V and X. Afferent projections are densest at L1 and 2 and the caudal T13, but extend up to T10 rostrally, and at least down to L4 caudally. A few visceral afferents ascend to the nucleus gracilis.The great majority of sensory and preganglionic sympathetic cell bodies is located at levels L1 and 2 bilaterally. A few cells are found in decreasing numbers rostrally up to T11.Preganglionic sympathetic neurons (PSN) are located in nucleus intermediolateralis (IML), n. intercalatus (IC) and n. commissuralis dorsalis (DCN). Axons of DCN and IC neurons run laterally, joining those of IML neurons on their way to the ventral roots. Dendrites of IML neurons ramify in all directions but preferentially to the dorsal horn and dorsolateral funiculus. Dendrites of IC and DCN neurons are distributed mainly mediolaterally, the latter also ventrally around the canalis centralis.     

The fine structure of the submucous plexus of the guinea-pig ileum. I. The ganglia, neurons, Schwann cells and neuropil

Summary A fine structural study was made of the ganglia, neurons, Schwann cells and neuropil of the submucous plexus of the guinea-pig ileum. The arrangement of the plexus as seen by light microscopy is briefly described. Submucous ganglia are small, containing an average of eight neurons per ganglion (compared with 43 in myenteric ganglia) and are connected with each other by fine nerve strands.The cell bodies of neurons and Schwann cells and a neuropil consisting of neuronal and Schwann cell processes form the ganglia. No other cell types or blood vessels are found within the ganglia. Ganglia are surrounded by a continuous basal lamina but lack a well-defined connective tissue investment. The glial investment of neurons is incomplete: many neurons lie directly beneath the basal lamina with no intervening Schwann cell processes, and the plasma membranes of adjacent neurons are often directly apposed over large areas. Other areas of apposition occur between the cell bodies and processes of neurons and Schwann cells. Desmosome-like membrane specializations may be seen between neurons and other neurons or Schwann cells. Submucous neurons could not be categorized according to size, shape, organelle content or types of processes. Processes emerging from nerve-cell bodies were placed into four broad categories on the basis of shape and microtubule content.Many bundles of closely apposed small nerve profiles lacking intervening Schwann processes are found in the neuropil in addition to a large number of vesiculated varicosities, some of which are directly apposed to the plasma membranes of nerve-cell bodies. A small proportion of vesiculated profiles form synapses with nerve cell bodies, their processes and profiles in the neuropil. From their structure, submucous neurons appear to form a more homogeneous population than myenteric neurons. Because of their incomplete investment they are more likely to be freely exposed to substances diffusing in the extraganglionic tissue than are neurons of sympathetic ganglia.     

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.     

Nitric oxide synthase immunoactivity and NADPH diaphorase enzyme activity in neurons of the gastrointestinal tract of the toad, Bufo marinus.

The presence of nitric oxide synthase (NOS) was demonstrated immunohistochemically, and NADPH diaphorase was demonstrated by enzyme histochemistry in neurons throughout the gastrointestinal tract of the anuran amphibian, Bufo marinus. Successive staining showed that NOS immunoreactivity and NADPH diaphorase activity occurred in precisely the same subgroup of enteric neurons. Subsequent detailed studies of the distribution of these neurons were made using NADPH diaphorase histochemistry. Numerous reactive nerve cell bodies and fibres were found in the myenteric plexus from the esophagus to the cloaca. A dense innervation of the longitudinal and circular muscle layers occurred throughout the gastrointestinal tract. The lamina muscularis mucosae was only prominent in the stomach, where it was sparsely innervated. Reactive nerve cell bodies were common in the submucosa of the large intestine, less common in the small intestine and extremely rare in the stomach and esophagus. Reactive fibres contributed to subepithelial plexuses in the esophagus, colon, rectum and cloaca. It is concluded that NOS/NADPH diaphorase is conserved amongst vertebrate classes and that NO is a likely neurotransmitter in the toad gastrointestinal tract.     

Distribution of enteric neurons showing immunoreactivity for substance P in the guinea-pig ileum

Substance P-like immunoreactivity has been localized in whole mount preparations of the isolated layers of the guinea-pig ileum. Axons containing substance P formed dense networks around the nerve cells and ran in the primary, secondary and tertiary nerve bundles of the myenteric plexus. 3.6% of the nerve cell bodies of the myenteric plexus and 11.3% of the cell bodies in the submucous plexus showed immunoreactivity for substance P. Axons ran in fine nerve bundles parallel to the longitudinal muscle, between this muscle and the myenteric plexus. Axons containing substance P also ran in small nerve trunks parallel to the circular muscle throughout its thickness and in the deep muscular plexus at the base of this muscle coat. In the submucosa, these axons ramified amongst ganglion cells of the plexus and ran in the internodal strands. In addition they formed a perivascular network around submucous arteries and contributed to the paravascular nerves following these arteries. Axons containing substance P formed a delicate plexus in the mucosa. After extrinsic denervation the nerves containing substance P that were associated with submucous arteries, and some in the submucous plexus, disappeared. The nerves in the other areas were not detectably different from normal.Comparison with the distribution of somatostatin, enkephalin and vasoactive intestinal polypeptide indicated the neurons containing substance P constitute a separate population within the enteric nervous system.     

Calcitonin gene-related peptide (CGRP)-immunoreactive neurons in the human cervico-thoracic paravertebral ganglia

The occurrence of CGRP-immunoreactive neurons in human paravertebral sympathetic ganglia was investigated and CGRP-immunoreactive perikarya correlated with the distribution of structures which were immunoreactive to other peptides and tyrosine hydroxylase, the key enzyme of catecholamine-synthesis. CGRP-immunoreactive neurons were present in all investigated ganglia (superior cervical ganglion, stellate ganglion, thoracic ganglia IV and VII). Most of the CGRP-immunolabelled cell bodies contained also vasoactive intestinal polypeptide-as well as somatostatin-immunoreactivity. Approximately 40 per cent of the CGRP-immunoreactive neurons were weakly tyrosine hydroxylase-immunolabelled. CGRP-immunoreactive cell bodies appear to be neither identical with the large population of neuropeptide Y-labelled perikarya nor with the large group of cell bodies which were surrounded by leuenkephalin-immunoreactive nerve fibres. Colocalization of CGRP- with vasoactive intestinal polypeptide- and somatostatin-immunoreactivity in postganglionic sympathetic neurons substantiates the suggestion of sympathetic origin of respective peptidergic nerve fibres in sweat glands.     

Ultrastructure and synaptic relationships of calbindin-reactive, Dogiel type II neurons, in myenteric ganglia of guinea-pig small intestine

Summary Immunoreactivity for calbindin D 28K was localized ultrastructurally in nerve cell bodies and nerve fibres in myenteric ganglia of the guinea-pig small intestine. Reactive cell bodies had a characteristic ultrastructure: the cytoplasm contained many elongate, electron-dense mitochondria, numerous secondary lysosomes that were peripherally located, peripheral stacks of rough endoplasmic reticulum and dispersed Golgi apparatus. The cells were generally larger than other myenteric neurons and had mainly smooth outlines. The cytoplasmic features of these neurons were shared by a small group of immunonegative cells, but the majority of negative cells had clearly different ultrastructural appearances. Of 310 cells from 16 ganglia that were systematically examined, 38% were immunoreactive for calbindin, 10% were unreactive but similar in ultrastructure to the calbindin-reactive neurons and 51% were unreactive and dissimilar in the appearance of their cytoplasmic organelles. Immunoreactive varicosities with synaptic specializations were found on most unreactive neurons, but were markedly less frequent on the calbindin-immunoreactive cell bodies. Non-reactive presynaptic fibres were also more common on non-reactive neurons than on the calbindin-positive cell bodies. Numerous reactive varicosities, some showing synaptic specializations, were found adjacent to other fibres in the neuropil. Light microscopic studies show calbindin immunoreactive neurons to have Dogiel type-II morphology. Thus the present work links distinguishing ultrastructural features to a specific nerve cell type recognized by light microscopy in the enteric ganglia for the first time.     

An immunohistochemical study of the distribution of enteric GABA-containing neurons in the rat and guinea-pig intestine

gamma-Aminobutyric acid (GABA) antiserum was applied to sections of rat and guinea-pig intestine which were subsequently processed to reveal any immunoreactivity using either fluorescence or peroxidase techniques. Immunopositive fibres were demonstrated in stomach, duodenum, ileum and colon of rat and guinea-pig intestine. Myenteric ganglia and nerve bundles in the circular muscle contained immunopositive nerve fibres, while the longitudinal muscle, submucosa and mucosa were only rarely innervated. In favourable sections, immunopositive fibres could be seen running from the myenteric plexus into the circular muscle, thus suggesting that the GABA-immunopositive nerves in the circular muscle originate from neurons in the myenteric plexus. In both rat and guinea-pig, immunoreactive nerve cell bodies were most numerous in the myenteric plexus of the colon. In the rat, immunopositive fibres in the circular muscle were most abundant in the ileum, whereas in the guinea-pig it was the colon circular muscle that was most richly innervated. The results demonstrate that neurons which show GABA immunoreactivity are present along the length of the gastrointestinal tract. Their distribution in both myenteric ganglia and circular muscle is heterogeneous both within and between the two species studied. It is probable that this heterogeneity reflects the diversity and specificity of function of this class of enteric neurons.     

Serotonin in the rabbit ileum: localization,uptake, and effect on motility

Repeated experiments to localise serotonin in the myenteric plexus of rabbit ileum failed. After preincubation in serotonin (10(-5) M), an extensive varicose fibre system was detected by immunocytochemical methods. Stained fibres left the myenteric plexus and ran to the muscle layers. Labelled cell bodies could not be found, even after pretreatment with colchicine or pargyline. Application of reserpine (10(-5) M) and fluoxetine (10(-5) M) prevented serotonin uptake. Antisera against tryptophan hydroxylase revealed a rich fibre system, including those processes that entered the tertiary plexus. These fibres were able to accumulate serotonin, but again the cell bodies could not be detected. Serotonin caused concentration-dependent contraction in the longitudinal muscle layer of the rabbit ileum. Pretreatment with tetrodotoxin strongly reduced the effect of serotonin. Preapplication of atropine caused a slight decrease of response evoked by serotonin. Combined administration of tetrodotoxin and atropine significantly reduced the responses to serotonin, but did not abolish them. At the same time, agonists of 5-HT(2) and 5-HT(4) receptors caused concentration-dependent contractions. Our studies show that: 1). Without pretreatment, serotonin cannot be detected in the myenteric plexus of rabbit ileum. 2). An extensive uptake system works in this plexus. If released from myenteric nerve fibres, serotonin may evoke contractions in indirect and direct ways. 3). There may be an extrinsic serotoninergic innervation from the mesenteric ganglia. 4). Serotonin exerts its effect through 5-HT(2) and 5-HT(4) receptors on smooth muscle cells and nerve elements.