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.     

A reciprocal adrenergic-cholinergic axoaxonic synapse in the mammalian gut.

Simultaneous stimulation of perivascular nerves inhibited the release of acetylcholine from stimulated cholinergic nerves of the rabbit jejunum. Adrenergic nerves were responsible for this inhibition because it did not occur in animals previously injected with 6-hydroxydopamine. Acetylcholine inhibited the release of transmitter from stimulated adrenergic axons; this effect was blocked by atropine. Since atropine enhanced the release of adrenergic transmitter when both adrenergic and cholinergic nerves were activated simultaneously (at 4.0 Hz), it seems likely that cholinergic nerves also inhibit release of norepinephrine (NE). Radioautographic examination of the myenteric plexus, incubated with tritiated NE, revealed a striking marginal distribution of adrenergic axons around the periphery of the myenteric plexus. Ultrastructural studies, with KMnO4 used to identify adrenergic terminal varicosities, confirmed this distribution and also revealed complexes formed between the terminal varicosities of adrenergic and probable cholinergic axons. The component varicosities forming these complexes contacted one another with no intervening Schwann cell elements. It is concluded that there is a reciprocal axoaxonic synapse between adrenergic and cholinergic neurons in the mammalian myenteric plexus.     

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.     

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.     

Autoradiographic study of the distribution of [3H]gamma-aminobutyrate-accumulating neural elements in guinea-pig intestine: evidence for a transmitter function of gamma-aminobutyrate

High affinity uptake, and the distribution of 3H-radiolabelled gamma-aminobutyrate (GABA), cis-3-aminocyclohexanecarboxylic acid, beta-alanine, proline, and leucine have been examined autoradiographically in laminar preparations of the myenteric plexus from the guinea-pig intestine. Following labelling with [3H]proline and [3H]leucine, which are incorporated into neurons, silver grains were concentrated over recognisable perikarya in the ganglia and meshworks of the plexus, whilst [3H]GABA labelled a smaller proportion of neurons and their processes. Specificity of labelling in the sites of [3H]GABA-uptake was established using combinations of labelled and unlabelled GABA, beta-alanine, and cis-3-aminocyclohexanecarboxylic acid, substrates for glial or neuronal high affinity GABA uptake systems. Only myenteric neurons and their processes were labelled significantly by [3H]GABA and its analogue cis-3-[3H]aminocyclohexanecarboxylic acid. Using autoradiographs of laminar preparations and paraffin sections, [3H]GABA labelling was found over nerve fibre bundles that could be traced from their ganglionic origins through the interconnecting meshworks of the myenteric plexus into the innervation of the deep muscular plexus of the circular muscle layer where GABA is evidently concerned with prejunctional modulation of transmitter release. The extensive but selective distribution of [3H]GABA high affinity uptake sites in neural elements of the guinea-pig myenteric plexus is consistent with GABA being an enteric neurotransmitter.     

Autoradiographic localization of [3H]gamma-aminobutyric acid in the myenteric plexus of the guinea-pig small intestine

Localization of [³H]GABA in the guinea-pig myenteric plexus has been studied using light microscopic autoradiography. In the presence of β-alanine, 10^?3 M, an inhibitor of glial cell high affinity GABA transport, [³H]GABA was transported by a high affinity uptake system into neuronal elements of the plexus. Scattered neurones accumulating [³H]GABA showed localization of silver grains over the soma and axonal processes. In addition a large population of uptake sites for [³H]GABA was found within the secondary and tertiary meshworks of the plexus so that dense accumulations of silver grains were observed localized over distinct ‘tracts’ within all three meshworks of the plexus. These results are considered to provide strong evidence for GABAergic neurones in the enteric nervous system.     

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.     

Reinnervation of dopamine neurons by regenerating serotonin axons in the rat medial zona incerta

Summary Cellular relationships between serotonin (5-HT) axons and tyrosine hydroxylase (TH)-containing neurons were examined by combined (³H)5-HT uptake radioautography and TH immunocytochemistry in the medial zona incerta (ZI) of adult rats, 7 and 50 days after an intracerebral injection of 5,7-Dihydroxytryptamine (5,7-DHT). Seven days post-lesion, only rare, scattered (³H)5-HT-labeled axon terminals were apparent in the zone of the medial ZI accessible to intraventricularly injected (³H)5-HT. In contrast, in sham-injected animals (³H)5-HT-labeled varicosities were numerous and often observed adjacent to TH-immunoreactive perikarya and dendrites. Fifty days post 5,7-DHT injection, the density of (³H)5-HT-labeled terminals approximated that seen in sham-treated animals. At the ultrastructural level, these regenerated 5-HT axons were similar in size, shape and content to those observed in sham-operated rats. Also, as in sham, some of the (³H)5-HT-labeled axons were directly apposed to TH-immunopositive labeled profiles. The latter included large dendritic shafts and dendritic spines, but only rare perikarya. In both sham- and 5,7-DHT-treated animals a few of the contacts between (³H)5-HT-labeled and TH-immunoreactive profiles exhibited an asymmetric synaptic differentiation. These results indicate that 5-HT fibers in the medial ZI, following regeneration, can reestablish normal relationships and even synapses with a given population of chemically identified cells.     

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.     

Ultrastructural relationships of serotonin and GABA terminals in the rat suprachiasmatic nucleus. Evidence for a close interconnection between the two afferent systems

Summary Serotonin (5-HT) and -aminobutyric acid (GABA) nerve endings were identified in the rat suprachiasmatic nucleus (SCN) by combined [³H]5-HT uptake radioautography and glutamate decarboxylase (GAD) immunocytochemistry at the electron microscope level. In areas of good overlap between radioautographic and immunocytochemical labellings, there were no axonal varicosities exhibiting both labellings, indicating that 5-HT and GABA are not co-localized in the SCN. The systematic survey in these areas of all profiles that had accumulated [³H]5-HT and of all GAD-immunoreactive varicosities allowed the analysis of 247 of the former and 896, i.e. an almost four-fold greater number, of the latter. This seems concordant with the view that GABA endings would be the most numerous of all classes of nerve terminals so far identified in the SCN. More than 22% of the [³H]5-HT labelled profiles showed the membrane specialization typically associated with synap'tic junctions. Thereby, it was possible to evaluate that about 45% of the 5-HT terminals actually form a synapse in the SCN. Some 37% of the GAD-positive varicose profiles which could be formally interpreted also showed well differentiated synaptic contacts, suggesting that the GABAergic innervation of the SCN could be entirely junctional. Whereas 5-HT terminals usually innervated only one dendritic or somatic element, a convergence of several GABAergic terminals onto the same postsynaptic target also receiving a 5-HT input was frequently observed. Of all the [³H]5-HT labelled varicose profiles scanned, as much as 41% were directly apposed to at least one GAD-immunoreactive profile, indicating that these 5-HT/GABA axonal interfaces could well represent privileged sites of interactions between the two transmitters. Taken together, these data could be of potential value in determining the neurochemical mechanisms subserving cellular integration of rhythmic signals in the SCN.     

Release of endogenous 5-hydroxytryptamine from resting and stimulated enteric neurons

Physiological and biochemical evidence has indicated that there may be serotoninergic neurons in the enteric nervous system. A critical step in the identification of a neurotransmitter is the demonstration of the release of the substance upon nerve stimulation. We now report the release of endogenous 5-hydroxytryptamine from enteric neurons. Segments of guinea-pig small intestine were everted and perfused in vitro through the newly created serosal lumen. Tests with [³H]5-hydroxytryptamine revealed the existence of a tissue barrier preventing diffusion of mucosal (enteroendocrine cell) 5-hydroxytryptamine into the perfusate; thus, all 5-hydroxytryptamine in the perfusate was of neural origin. The gut was stimulated electrically. 5-Hydroxytryptamine in the perfusate and in the myenteric plexus was assayed by a specific radioenzymatic method. 5-Hydroxytryptamine was present in the myenteric plexus; it was released into the perfusate spontaneously and the release was enhanced by electrical stimulation. The stimulated, but not the spontaneous, release of the amine was Ca²⁺-dependent. Comparison with the release of newly taken up [³H]5-hydroxytryptamine showed that the specific radioactivity of electrically released 5-hydroxytryptamine was higher than that of either the spontaneously released or tissue amine. Stimulation also increased the release of 5-hydroxytryptamine more than that of its metabolites in the perfusate.These results indicate that 5-hydroxytryptamine is an endogeneous constituent of the enteric nervous system, that it is released by electrical field stimulation of enteric nerves, and that newly taken up 5-hydroxytryptamine is released preferentially by these neurons.     

Vagal afferents innervating the gastrointestinal tract and CCKA-receptor immunoreactivity.

A large body of evidence derived from electrophysiological recording and pharmacological/behavioral experiments suggests the presence of CCKA-receptors on vagal primary afferent fibers innervating the gastrointestinal tract. With the availability of antibodies specific for the CCKA-receptor, we wanted to demonstrate its presence and distribution on identified vagal afferent fibers and different types of terminals in the mucosa, myenteric plexus, and external muscle layers of the stomach and duodenum. In the duodenal mucosa, neither a C-terminal (Ab-1) nor an N-terminal (Ab-2) specific antibody produced any specific staining; in the myenteric plexus, non-vagal enteric neurons and their processes, but not vagal intraganglionic laminar endings (IGLEs), exhibited CCKAR-immunoreactivity. Similarly, in the gastric myenteric plexus, a population of enteric neurons and their processes, but not identified vagal IGLEs, were labeled by both antibodies. In both external muscle layers of the stomach, CCKAR-immunoreactive axons were in close register with labeled vagal afferent intramuscular arrays, but the two labels were not contained in the same varicosities. Ab-1 immunoreactivity was found in the cell membrane of vagal afferent perikarya in the nodose ganglia and in pancreatic acinar cells. The failure to detect CCKAR-immunoreactivity in peripheral vagal afferent terminals cannot be due to methodological problems because it was present in enteric neurons in the same sections, and because it did not stain structures resembling IGLEs in material without the potentially masking vagal afferent label. We conclude that CCKA-receptors on vagal afferent terminals: 1) are below the immunohistochemical detection threshold, 2) exhibit a conformation or affinity state inaccessible to the two antibodies, or 3) are not transported to the peripheral terminals.     

Ultrastructural organization of regenerated serotonin axons in the dorsomedial hypothalamus of the adult rat

Summary The ultrastructural organization of regenerated serotonin (5-HT) axons was examined in the dorsomedial hypothalamus (DMH) of the adult rat using high-resolution radioautography after intraventricular infusion of [³H]5-HT. An analysis of the microenvironment of the [³H]5-HT-labelled terminals in the DMH was made 30 and 50 days after unilateral injection of 5,7-dihydroxytryptamine (5,7-DHT) or vehicle solution into the dorsolateral hypothalamus. In sham-treated animals [³H]5-HT-labelled axons were small, contained many small clear vesicles, one or more large granular vesicles, and showed only rare synaptic specializations. In 5,7-DHT-treated animals the internal organization of [³H]5-HT-labelled profiles resembled that of sham-treated animals. A tendency toward increased synaptic frequency was found for [³H]5-HT-labelled terminals in the 5,7-DHT-treated group 50 days post-lesion, and an increase in the number of [³H]5-HT-labelled terminals abutting unlabelled perikarya was found in both 30- and 50-day post-lesion groups as compared to sham-treated groups. No other differences in ultrastructural environment were found between sham- and 5,7-DHT-treated animals at either 30 or 50 days post-lesion. These results suggest that 5-HT fibres in the hypothalamus regenerate with a great deal of cellular specificity.     

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)     

Neural and non-neural activation of electrogenic secretion by 5-hydroxytryptamine in the rat ileum in vitro

5-hydroxytryptamine (5-HT) stimulates electrogenic Cl^?secretion in rat ileum stripped of its outer smooth musculature and myenteric plexus. The myenteric plexus, however, is a site of 5-HT synthesis in the gut, and the plexus mediates electrogenic ion secretion activated by luminal enterotoxin STa and taurocholate. Thus, we investigated the role of the myenteric plexus in 5-HT-induced electrogenic secretion in vitro by measuring short-circuit current (Isc, microamps) with voltage-clamp apparatus as an index of electrogenic Cl^?secretion in rat ileum which was either stripped of the myenteric plexus or was left intact. Serosally added 5-HT stimulated electrogenic Cl^?secretion in muscle-stripped and intact ileum in a concentration-dependent manner. Pre-treatment of stripped ileum with atropine (1 μm), hexamethonium (100 μm), tetrodotoxin (1.25 μm) and capsaicin (1 μm) for 15 min did not effect the maximum Isc induced by 5-HT which would implicate a direct action on the enterocyte. In intact ilea, however, tetrodotoxin (TTX) and capsaicin reduced significantly the maximum values of Isc stimulated by 5-HT, and the nitric oxide synthase inhibitor Nω-nitro- L -arginine methyl ester ( L -NAME) caused a significant decrease in the maximum response to 5-HT. These results suggest that electrogenic secretion induced by 5-HT in rat ileum in vitro occurs partly by activation of a non-neural pathway probably involving a direct interaction with the enterocyte, and partly via a nitrinergic-myenteric secretory reflex activated by sensory afferent fibres. These data highlight the danger of characterising intestinal secretory activity from in vitro experiments by using muscle-stripped tissue only.     

Infection with the cestode Hymenolepis diminuta induces changes in acetylcholine metabolism and muscarinic receptor mRNA expression in the rat jejunum

Total and neuron-specific uptake of [³H] choline into smooth muscle/myenteric plexus (SM/MP) preparations from the jejunum of rats infected with five Hymenolepis diminuta for 30 days compared to uninfected rats was significantly increased, as was choline acetyltransferase activity and acetylcholine biosynthesis. Although acetylcholinesterase and total cholinesterase activity levels in SM/MP preparations from infected rats were not significantly different from uninfected animals, pseudocholinesterase activity was significantly elevated in infected rats. Infection resulted in a significant elevation in the relative expression of muscarinic 2 (M2) receptor mRNA in jejunum compared to uninfected rats. Conversely, in rats infected with 50 worms for 30 days, the relative expression of muscarinic 1 (M1) receptor mRNA in the jejunum was significantly depressed, while the expression of M2 receptor mRNA was not significantly different from that in five worm infections. The relative expression of muscarinic 3 receptor mRNA was unaffected by infection. The present study shows that infection of rats with low numbers of an enteric cestode leads to a significant modulation of the cholinergic components of the myenteric plexus and M2 receptor mRNA, and that large number of worms result in suppression in the relative expression of M1 receptor mRNA.     

Vagal afferents innervating the gastrointestinal tract and CCKA‐receptor immunoreactivity

A large body of evidence derived from electrophysiological recording and pharmacological/behavioral experiments suggests the presence of CCKA‐receptors on vagal primary afferent fibers innervating the gastrointestinal tract. With the availability of antibodies specific for the CCKA‐receptor, we wanted to demonstrate its presence and distribution on identified vagal afferent fibers and different types of terminals in the mucosa, myenteric plexus, and external muscle layers of the stomach and duodenum. In the duodenal mucosa, neither a C‐terminal (Ab‐1) nor an N‐terminal (Ab‐2) specific antibody produced any specific staining; in the myenteric plexus, non‐vagal enteric neurons and their processes, but not vagal intraganglionic laminar endings (IGLEs), exhibited CCKAR‐immunoreactivity. Similarly, in the gastric myenteric plexus, a population of enteric neurons and their processes, but not identified vagal IGLEs, were labeled by both antibodies. In both external muscle layers of the stomach, CCKAR‐immunoreactive axons were in close register with labeled vagal afferent intramuscular arrays, but the two labels were not contained in the same varicosities. Ab‐1 immunoreactivity was found in the cell membrane of vagal afferent perikarya in the nodose ganglia and in pancreatic acinar cells. The failure to detect CCKAR‐immunoreactivity in peripheral vagal afferent terminals cannot be due to methodological problems because it was present in enteric neurons in the same sections, and because it did not stain structures resembling IGLEs in material without the potentially masking vagal afferent label. We conclude that CCKA‐receptors on vagal afferent terminals: 1) are below the immunohistochemical detection threshold, 2) exhibit a conformation or affinity state inaccessible to the two antibodies, or 3) are not transported to the peripheral terminals. Anat Rec 266:10–20, 2002. © 2002 Wiley‐Liss, Inc.     

An immunohistochemical study of the projections of somatostatin-containing neurons in the guinea-pig intestine

Somatostatin-like immunoreactivity was localized in nerves in whole mount preparations of the separated layers of the guinea-pig intestine. The directions in which the neurons project were determined by examining the accumulation of somatostatin-like immunoreactivity after axonal flow was interrupted. In some experiments this was done by crushing or cutting the nerves in isolated preparations which were then maintained in oxygenated Krebs solution for 3–5 h. In other experiments, the nerves were cut in vivo and the animals allowed to survive for 4–8 days before the intestine was examined.Somatostatin immunoreactive nerve cell bodies were found in both the myenteric plexus, where they represented 4.7% of the total population of neurons, and in the submucous plexus, where they formed 17.4% of the total population. The axons of the somatostatin-containing neurons in the submucosa are not polarized while those of the somatostatin-containing neurons in the myenteric plexus of the small intestine project in the anal direction for 8–12 mm to form pericellular baskets around other enteric neurons, some of which are reactive for somatostatin.It is postulated that somatostatin-containing neurons in the myenteric plexus are interneurons in a descending nerve pathway, possibly the one involved in the descending inhibitory reflex of peristalsis.     

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.     

Serotoninergic axon terminals in the rat dorsal accessory olive: Normal ultrastructure and light microscopic demonstration of regeneration after 5,6-dihydroxytryptamine lesioning

Summary The serotoninergic innervation of the lateral portion of the dorsal accessory nucleus of inferior olive (DAO) was studied using high resolution radioautography after intraventricular or intracisternal administration of tritiated serotonin ([³H]5-HT). In normal adult rats, the axonal varicosities labelled with [³H]5-HT were of round-elongate shape and averaged 0.6–1.0m in diameter. They contained microcanaliculi (15–25 nm in diameter), tubular-vesicular elements (25–40 nm) and large granular vesicules (80 nm), as well as mitochondria and smooth endoplasmic reticulum. Among 750 thin-sectional profiles of labelled varicosities, only 5% showed a differentiated area of membrane specialization which suggests that not more than one out of 7–8 varicosities was engaged in a synaptic junction. This serotoninergic innervation was therefore categorized as non-junctional. There were nevertheless certain structural features suggestive of polarity even in the non-synaptic serotoninergic varicosities of the lateral DAO: many labelled profiles exhibited aggregation of their microcanalicular and tubular-vesicular organelles against a part of their membrane apposed to a dendritic process, while the remainder of their periphery was usually surrounded by glia. These observations reinforced the supposition that non-junctional as well as junctional serotoninergic varicosities might release transmitter.Counts in light microscope radioautographs indicated that the lateral DAO of normal rat receives about 4.5 million serotoninergic varicosities per mm³. Denervation by intraventricular 5,6-dihydroxytryptamine (5,6-DHT) was severe, leaving only 0.45 million per mm³ after five days. It was followed by rapid regrowth, however, since an approximately normal number of serotoninergic varicosities was again measurable two months after 5,6-DHT. The growth process continued further, and six months after 5,6-DHT, the lateral DAO exhibited a hyperinnervation of 12.6 million varicosities per mm³. This re-establishment of a serotoninergic innervation was in line with our hypothesis concerning the factors influencing the course of neuroplasticity. According to this hypothesis, innervations of the non-junctional variety may regenerate after neurotoxic lesioning, whereas those making numerous synaptic connections are replaced by local sprouting of intact afferents.Presented in part at the Third European Neurosciences Meeting, Rome September 1979.