The distribution and colocalization of neuropeptides in perivascular nerves innervating the large arteries and veins of the snake,Elaphe obsoleta

Summary Single- and dual-labelling immunohistochemistry were used to determine the distribution and coexistence of neuropeptides in perivascular nerves of the large arteries and veins of the snake, Elaphe obsoleta, using antibodies for vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, neuropeptide Y, galanin, somatostatin, and leu-enkephalin. Blood vessels were sampled from four regions along the body of the snake: region 1, arteries and veins anterior to the heart; region 2, central vasculature 5 cm anterior and 10 cm posterior to the heart; region 3, arteries and veins in a 30-cm region posterior to the liver; and region 4, dorsal aorta and renal arteries, renal and intestinal veins, 5–30 cm cephalad of the vent. A moderate to dense distribution of vasoactive intestinal polypeptide-like immunoreactive fibres was found in most arteries and veins of regions 1–3, but fibres were absent from the vessels of region 4. The majority of vasoactive intestinal polypeptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were unaffected by either capsaicin or 6-hydroxydopamine (6-OHDA) pretreatment. In the anterior section of the snake, the vagal trunks contained many cell bodies with colocalized vasoactive intestinal polypeptide and substance P-like immunoreactivity. It is suggested that the vasoactive intestinal polypeptide/substance P-like immunoreactive cell bodies and fibres are parasympathetic postganglionic nerves. Neuropeptide Y-like immunoreactive fibres were observed in all arteries and veins, being most dense in regions 3 and 4. The majority of these fibres also contained colocalized galanin-like immunoreactivity, and were absent in tissues from 6-OHDA pretreated snakes, suggesting that neuropeptide Y and galanin are colocalized in adrenergic nerves. A small number of neuropeptide Y-like immunoreactive fibres contained vasoactive intestinal polypeptide but not galanin, and were unaffected by 6-OHDA treatment. All calcitonin gene-related peptide-like immunoreactive fibres contained colocalized substance P-like immunoreactivity, and these fibres were observed in all vessels, being particularly dense in the carotid artery and jugular veins. All calcitonin gene-related peptide/substance P-like immunoreactive fibres appeared damaged after capsaicin treatment suggesting they represent fibres from afferent sensory neurons. A sparse plexus of somatostatin-like immunoreactive fibres was observed in the vessels only from region 4. No enkephalin-like immunoreactive fibres were found in any blood vessels from any region. This study provides morphological evidence to suggest that there is considerable functional specialization within the components of the rat snake peripheral autonomic system controlling the circulation, in particular the regulation of venous capacitance.     

Somatostatin in human enteric nerves

Summary Somatostatin-immunoreactive nerves and endocrine cells were localized by use of immunohistochemistry in human stomach, small and large intestine. The nature of the immunoreactivity in acid extracts of separated layers of intestine was determined with separation by high pressure liquid chromatography followed by detection with radioimmunoassay; authentic somatostatin-14 was found in the external musculature, which contains nerves, and in the submucosa and mucosa, which contain both nerve fibres and endocrine cells.The distribution of somatostatin nerves in the gastric antrum, duodenum, jejunum, ileum, ascending and sigmoid colon, and rectum is described. In the intestine many positive perikarya and fine varicose fibres were seen. Mucosal fibres formed a sub-epithelial plexus and a looser network in the lamina propria; this nerve supply was less dense in the large intestine. Submucous ganglia contained positive perikarya and terminals; many terminals formed pericellular baskets, mainly around non-reactive cells. A small number of nerve fibres were associated with submucosal blood vessels. The innervation of the circular and longitudinal muscle was sparse. Positive nerve terminals were seen in the myenteric plexus, although fewer than in the submucous ganglia; positive perikarya were scarce in myenteric ganglia. Somatostatin-immunoreactive nerves were found in the muscle layers and myenteric plexus of the gastric antrum, but were not detected in the antral mucosa and all layers of the gastric body.The distribution of human enteric somatostatin nerves is compared to that in small laboratory animals, and possible roles for these nerves are discussed.     

Intimate relationship between interstitial cells of Cajal and enteric nerves in the guinea-pig small intestine

Recent studies have suggested that enteric inhibitory neurotransmission is mediated via interstitial cells of Cajal in some gastrointestinal tissues. This study describes the physical relationships between enteric neurons and interstitial cells of Cajal in the deep muscular plexus (IC-DMP) of the guinea-pig small intestine. c-Kit and vimentin were colocalized in the cell bodies and fine cellular processes of interstitial cells of the deep muscular plexus. Anti-vimentin antibodies were subsequently used to examine the relationships of interstitial cells with inhibitory motor neurons (as identified by nitric oxide synthase-like immunoreactivity) and excitatory motor neurons (using substance P-like immunoreactivity). Neurons with nitric oxide synthase- and substance P-like immunoreactivities were closely associated with the cell bodies of interstitial cells and ramified along their processes for distances greater than 300 7m. With transmission electron microscopy, we noted close relationships between interstitial cells and the nitric oxide synthase- and substance P-like immunoreactive axonal varicosities. Varicosities of nitric oxide synthase and substance P neurons were found as close as 20 and 25 nm from interstitial cells, respectively. Specialized junctions with increased electron density of pre- and postsynaptic membranes were observed at close contact points between nitric oxide synthase- and substance P-like immunoreactive neurons and interstitial cells. Close structural relationships (approximately 25 nm) were also occasionally observed between either nitric oxide synthase- and substance P-like immunoreactive varicosities and smooth muscle cells of the outer circular muscle layer. The data suggest that interstitial cells in the deep muscle plexus are heavily innervated by excitatory and inhibitory enteric motor neurons. Thus, these interstitial cells may provide an important, but probably not exclusive, pathway for nerve-muscle communication in the small intestine.     

Distribution and projections of neurons with immunoreactivity for both gastrin-releasing peptide and bombesin in the guinea-pig small intestine

Summary Bombesin-like and gastrin-releasing peptide (GRP)-like immunoreactivities were localized in nerves of the guinea-pig small intestine and celiac ganglion with the use of antibodies raised against the synthetic peptides. The anti-bombesin serum (preincubated to avoid cross reactivity with substance P) and the anti-GRP serum revealed the same population of neurons. Preincubation of the antibombesin serum with bombesin abolished the immunoreactivity in nerves while absorption of the anti-GRP serum with either bombesin or the 14–27 C-terminal of GRP only reduced the immunoreactivity. The immunoreactivity was abolished by incubation with GRP 1–27.Immunoreactive nerves were found in the myenteric plexus, circular muscle, submucous plexus and in the celiac ganglion. Faintly reactive nerve cell bodies were found in the myenteric ganglia (3.2% of all neurons) but not in submucous ganglia. After all ascending and descending pathways in the myenteric plexus had been cut, reactive terminals disappeared in the myenteric plexus, circular muscle (including the deep muscular plexus) and the submucous plexus on the anal side. After the mesenteric nerves were cut no changes were observed in the intestinal wall but the reactive fibres in celiac ganglia disappeared. It is deduced that GRP/bombesin-immunoreactive nerve cell bodies in myenteric ganglia project from the myenteric plexus to other myenteric ganglia situated further anally (average length 12 mm), anally to the circular muscle (average length 9 mm), anally to submucous ganglia (average length 13 mm) and external to the intestine to the celiac ganglia.It is concluded that the GRP/bombesin-reactive neurons in the intestinal wall represent a distinct population of enteric neurons likely to be involved in controlling motility and in the coordination of other intestinal functions.     

Innervation of interstitial cells of Cajal by vasoactive intestinal polypeptide containing nerves in canine colon.

The hypothesis was tested, through structural and functional studies, that interstitial cells of Cajal receive and can respond to direct innervation from nerves containing the vasoactive intestinal polypeptide neuromediator. The submucosal network of interstitial cells of Cajal has been postulated to provide pacemaking activity for the circular muscle and to be involved in neurotransmission from nonadrenergic, noncholinergic nerves for which vasoactive intestinal polypeptide is a putative mediator. The distribution of vasoactive intestinal polypeptide and substance P immunoreactive material in nerve profiles of the enteric nervous system of the canine colon was examined. In addition, electrophysiological studies were done on the interstitial cells bordering the submucosal side of the circular muscle layer after they were electrically isolated using heptanol. The vasoactive intestinal polypeptide immunoreactivity, located exclusively in nerve large granular vesicles, was found throughout the enteric nervous system (myenteric plexus, submucous plexus, and circular muscle--submucosa interface). The highest proportion (38% compared with 22-24%) of profiles of large granular vesicles with vasoactive intestinal polypeptide immunoreactivity was found in nerve profiles of the circular muscle--submucosa interface. In contrast, substance P immunoreactivity was found in nerve profiles of myenteric plexus (33% of large granular vesicles were positive) but not associated with submucosal interstitial cell nerve network. The vasoactive intestinal polypeptide hyperpolarized interstitial cells by 9 mV when electrically isolated by 1 mM heptanol and markedly reduced (about 50%) their input membrane resistance. We conclude that the distribution of vasoactive intestinal polypeptide immunoreactivity and its action are consistent with a postulated role of the interstitial cells as a major site of neurally mediated inhibition of colonic pacemaker activity.     

Distribution of substance p-like immunoreactivity in nerves of the gastrointestinal tract of the frog Rana esculenta L.

Summary Substance P-like immunoreactivity in the alimentary canal of the frogRana esculenta L. was studied by means of the indirect immunoperoxidase method. In all segments of the gastrointestinal tract, immunoreactivity was revealed in both the myenteric and the submucosa plexus. Stained nerve cells were observed in the myenteric plexus but not in the submucous plexus. The proximal part of the oesophagus and hindgut were free of immunoreactive perkarya. The stained nerve cells were of the Dogiel type I category in the foregut, and type II in the midgut. Both the musculature and gastrointestinal glands were supplied with immune-positive fibres. These results indicate that substance P may play similar roles in the frog gut, as described previously in mammals and fish.     

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

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

Peptide-containing nerves in the urinary bladder of the toad,Bufo marinus

The distributions of peptide-containing nerves in the urinary bladder of the toad, Bufo marinus, were studied by means of fluorescence immunohistochemistry of whole-mount preparations. The bundles of smooth muscle in the bladder are well supplied by varicose nerve fibres displaying somatostatin-like immunoreactivity; these fibres probably arise from intrinsic perikarya. The urinary bladder also has a well-developed plexus of nerves containing substance P-like immunoreactive material; these elements probably represent sensory nerves of extrinsic origin. Nerve fibres showing immunoreactivity to vasoactive intestinal polypeptide (VIP) or enkephalin are rare within the urinary bladder of the toad. It is considered unlikely that any of these peptides directly mediates the hyoscine-resistant excitatory response of the smooth muscle to nerve stimulation in the toad bladder.     

Conversion of Neuropeptide K to Neurokinin A and Vesicular Colocalization of Neurokinin A and Substance P in Neurons of the Guinea Pig Small Intestine

The highest concentration of neurokinin A-like immunoreactivity and substance P-like immunoreactivity in the guinea pig small intestine was associated with the myenteric plexus-containing longitudinal muscle layer. Chromatographic analysis of extracts of this tissue demonstrated the presence of neurokinin A and neuropeptide K but the probable absence of neurokinin B. A fraction of synaptic vesicles of density 1.133 +/- 0.003 g/ml was prepared from the myenteric plexus-containing tissue by density gradient centrifugation in a zonal rotor and was enriched 29 +/- 12-fold in the concentration of neurokinin A-like immunoreactivity and 43 +/- 13-fold in the concentration of substance P-like immunoreactivity. This fraction was separated from the fraction of vasoactive intestinal peptide-containing vesicles (density, 1.154 +/- 0.009 g/ml). Chromatographic analysis of lysates of the vesicles indicated the presence of neurokinin A but not neuropeptide K. It is postulated that beta-pre-protachykinin is processed to substance P, neurokinin A, and neuropeptide K in the cell bodies of myenteric plexus neurons but that conversion of neuropeptide K to neurokinin A takes place during packaging into storage vesicles for axonal transport. The data are consistent with the proposal that neurokinin A and substance P are stored in the same synaptic vesicle, but the possibility of cosedimentation of different vesicles of very similar density cannot be excluded.     

VIP-, substance P-, gastrin/CCK-, bombesin-, somatostatin- and glucagon-like immunoreactivities in the gut of the rainbow trout,Salmo gairdneri

Summary The presence of peptides in the gastrointestinal tract of the rainbow trout, Salmo gairdneri, was investigated immunocytochemically. VIP-like immunoreactivity was demonstrated in nerves in all layers of the stomach and the intestine, whereas substance P-like immunoreactivity was localized to endocrine cells, predominantly in the mucosa of the stomach, and to nerves mainly concentrated in the myenteric plexus throughout the gut. Endocrine cells reactive to gastrin/CCK antiserum were demonstrated in the intestinal mucosa, while no immunoreactivity was found in the stomach. Bombesin-immunoreactive and somatostatin-immunoreactive cells were localized in the stomach mucosa, and cells reactive to glucagon antiserum in the intestinal mucosa. Radioimmunoassay of stomach mucosa and muscle confirmed the presence of VIP-like and substance P-like immunoreactivity in these tissues, while gastrin/CCK-like immunoreactivity was low and bombesin-like immuno-reactivity was insignificant. In conclusion, molecules resembling the mammalian brain-gut peptides may be involved in the neuronal and hormonal control of gut function in fish.     

An immunohistochemical study of the innervation of the large intestine of the toad (Bufo marinus)

The distribution of intrinsic enteric neurons and extrinsic autonomic and sensory neurons in the large intestine of the toad, Bufo marinus, was examined using immunohistochemistry and glyoxylic acid-induced fluoresecence. Three populations of extrinsic nerves were found: unipolar neurons with morphology and location typical of parasympathetic postganglionic neurons containing immunoreactivity to galanin, somatostatin and 5-hydroxytryptamine were present in longitudinally running nerve trunks in the posterior large intestine and projected to the muscle layers and myenteric plexus throughout the large intestine. Sympathetic adrenergic fibres supplied a dense innervation to the circular muscle layer, myenteric plexus and blood vessels. Axons containing colocalized calcitonin gene-related peptide immunoractivity and substance P immunoreactivity distributed to all layers of the large intestine and are thought to be axons of primary afferent neurons. Five populations of enteric neurons were found. These contained immunoreactivity to vasoactive intestinal peptide, which distributed to all layers of the large intestine; galanin/vasoactive intestinal peptide, which projected to the submucosa and mucosa; calcitonin gene-related peptide/vasoactive intestinal peptide, which supplied the circular muscle, submucosa and mucosa; galanin, which projected to the submucosa and mucosa; and enkephalin, which supplied the circular muscle layer.     

Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig

The occurrence of tachykinins in sensory neurons of the guinea-pig was studied by means of radioimmunoassay combined with ion-exchange and high-performance liquid chromatography as well as by immunohistochemistry. Antisera raised against kassinin (antiserum K12), neurokinin A (NKA) (antiserum NKA2) and substance P (SP) (antisera SP25 and SP2) were used. Antiserum K12 detected NKA, neuropeptide K (NPK) and a component eluting in the position of eledoisin (ELE) in extracts of the lung and ureter. Neurokinin B (NKB) was, however, not found. Neutral water extraction favored recovery of NKA and of the ELE-like component, while NPK was found only in acid extracts. The SP antisera detected two immunoreactive components of which the major form coeluted with synthetic SP. Capsaicin pretreatment depleted all these various forms of immunoreactivity in several peripheral organs including the ureter and lung. The immunoreactivity detected by antisera K12 or SP25 in radioimmunoassay had a similar regional distribution pattern in peripheral tissues. Immunohistochemical examination revealed that antiserum NKA2 stained the same spinal ganglion cells as the SP2 antiserum. The distribution of capsaicin-sensitive nerve fibers stained by these two antisera was also identical in peripheral organs such as the ureter, inferior mesenteric ganglion, heart and lung. It is concluded that multiple tachykinins, including SP, NKA, NPK and an ELE-like peptide, are present in capsaicin-sensitive sensory nerves in the guinea-pig. This finding can most likely be related to the origin of SP, NKA and NPK from the same precursor molecule, subsequent posttranslational tissue processing and axonal transport to terminal regions.     

Substance P-containing nerve fibres in large peripheral blood vessels of the rat

Substance P-immunoreactive nerve fibres were localized by the indirect immunohistochemical method in the adventitia and the adventitial-medial border of large peripheral arteries and veins of the rat. Arteries showed a richer substance P-containing innervation than veins. The superior mesenteric artery was densely innervated, whereas no substance P-containing fibres were found around the carotid artery. Substance P produced a vasoconstriction of the veins, but was basically without effect on arteries, although with the carotid artery a dose-dependent relaxation was observed. The absence of a correlation between the degree of innervation of the blood vessels and their responsiveness to exogenous substance P suggests that there nerves do not subserve a vasomotor function. The depletion of substance P immunoreactivity from nerves in arteries and veins by capsaicin suggest that substance P-containing vascular nerves are primarily sensory in nature.     

Transient expression of the calcitonin receptor by enteric neurons of the embryonic and early post-natal mouse

Calcitonin receptor-immunoreactivity (CTR-ir) was found in enteric neurons of the mouse gastrointestinal tract from embryonic day 13.5 (E13.5) to post-natal day 28 (P28). CTR-ir occurred in cell bodies in ganglia of the myenteric plexus extending from the esophagus to the colon and in nerve cells of the submucosal ganglia of the small and large intestines. CTR-ir was also found in vagal nerve trunks and mesenteric nerves. Counts in the ileal myenteric plexus revealed CTR-ir in 80% of neurons. CTR-ir was clearly evident in the cell bodies of enteric neurons by E15.5. The immunoreactivity reached maximum intensity between P1.5 and P12 but was weaker at P18 and barely detectable at P28. The receptor was detected in nerve processes in the intestine for only a brief period around E17.5, when it was present in one to two axonal processes per villus in the small intestine. In late gestation and soon after birth, CTR-ir was also evident in the mucosal epithelium. The perinatal expression of CTR within the ENS suggests that the calcitonin/CTR system may have a role in the maturation of enteric neurons. Signals may reach enteric neurons in milk, which contains high levels of calcitonin.     

Neurokinin B- and substance P-like immunoreactivity are co-localized in enteric nerves of rat ileum

The tachykinins (TKs) substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) have conserved C-terminal sequences and mediate similar physiological responses by activating neurokinin receptors found on neural and smooth muscle cells. Many enteric nerves express preprotachykinin A (PPT A) mRNA and synthesize SP and NKA. However, it is unclear if NKB is synthesized in enteric neurons as many antibodies developed against NKB also recognize other TKs. Therefore, the cellular distribution of NKB-like-immunoreactivity (NKB-ir) in rat ileum was examined using selective antisera raised against either synthetic Cys10-NKB or peptide 2 (P2), a non-tachykinergic peptide sequence in NKB precursor protein. NKB-ir and P2-ir had a similar distribution in varicose nerve fibers in submucosal and myenteric ganglia and almost all ganglia contained immunoreactive nerves. Few submucosal or myenteric neuronal somata contained strong immunoreactivity. Preabsorption of NKB or P2 antisera with their respective cognate peptides, but not with other TK peptides, abolished specific immunostaining. Finally, co-localization of NKB-/P2-ir with SP-ir suggested that most NKB-/P2-ir nerve fibers contain SP-ir, but some SP-ir nerves do not contain detectable NKB-/P2-ir. These results indicate that PPT B products P2 and NKB are localized in a subpopulation of enteric nerves containing TKs encoded by PPT A. Stimulation of these nerves may release NKB to activate local neurokinin receptors.     

Innervation of the large arteries and heart of the toad (Bufo marinus) by adrenergic and peptide-containing neurons

Summary The innervation of the major arteries and heart of the toad (Bufo marinus) was examined by use of glyoxylic acid-induced catecholamine fluorescence and peptide immunohistochemistry. All arteries possessed a moderate to dense plexus of adrenergic axons, which also showed neuropeptide Y-like immunoreactivity (NPY-LI). Some adrenergic axons in the intracardiac vagal trunks showed NPY-LI, but the varicose adrenergic axons innervating the cardiac muscle of the atria and ventricle, and the coronary blood vessels did not display NPY-LI. About half of the nerve cell bodies in the anterior sympathetic chain ganglia with dopamine--hydroxylase-LI (DBH-LI) also contained NPY-LI. The nerve cell bodies with DBH-LI alone were generally larger (median diameter 30 m) than those with both DBH-LI and NPY-LI (median diameter 20 m). Some cell bodies showing DBH-LI alone were surrounded by boutons with NPY-LI but not DBH-LI. Axons that displayed simultaneously both substance P-LI (SP-LI) and calcitonin gene-related peptide-LI (CGRP-LI) also formed a plexus around all arteries studied, being particularly dense around the mesenteric and pulmonary arteries. These axons are most likely sensory since SP-LI was reduced by capsaicin treatment, and nerve cell bodies with both SP-LI and CGRP-LI were found in dorsal root ganglia and the vagal ganglion. A dense plexus of axons showing somatostatin-LI was located around the pulmonary artery and its main intrapulmonary branches. A few nerves with vasoactive intestinal polypeptide-LI were found around the dorsal aorta and pulmonary artery. No perivascular nerves with enkephalin-LI were observed. Reversed-phase, high-pressure liquid chromatography of acid extracts of the large arteries showed that the major peaks of NPY-LI and SP-LI coeluted with porcine NPY (1–36) and synthetic SP (1–11), respectively. Thus, the location and structure of these peptides in perivascular nerves has been highly conserved during vertebrate evolution.     

Ontogeny of substance P in the digestive tract, spinal cord and hypothalamus of the human fetus

The time of appearance and tissue concentrations of substance P-like immunoreactivity (SP-LI) were studied in 53 human fetuses aged 8-21 weeks. Detectable amounts were present at 8 weeks of gestation in available fragments of spinal cord and intestine. Thereafter, the tissue concentrations were highest in spinal cord, intermediate in hypothalamus and lowest in digestive tract. Except for a significant increase in the intestinal wall, the concentrations did not vary from the 8-14 to the 15-21 week period. At chromatography, SP-LI in extracts of spinal cord and intestine was essentially eluted in the volume of the synthetic undecapeptide. Using the indirect immunofluorescence technique, the localization of SP-LI positive structures in the digestive tract was studied in 5 fetuses aged 12-18 weeks. Scarce cell bodies were observed in the myenteric plexus. Nerve fibers were recognized in the muscular layer, in the myenteric plexus and in connective tissue of pancreas. The present results demonstrate the early appearance of SP-LI positive structures both in central nervous system and in the enteric nervous system in the human fetus. In the age range tested, SP-LI concentrations were noticeably higher in spinal cord and hypothalamus than in the digestive tract.     

Mapping, quantitative distribution and origin of substance p- and VIP-containing nerves in the uvea of guinea pig eye

VIP- and substance P-like immunoreactivities were found in considerable concentrations (VIP: 17.3 +/- 4.8 pmol/g, mean +/- SEM; substance P:11.1 +/- 1.8 pmol/g) in the uveal portion of the guinea pig eye. Immunocytochemistry localised these two regulatory peptides to nerve fibres found principally in a plexus in the iris (substance P) and in an extensive network surrounding the blood vessels of the choroid (VIP). A remarkable anatomical demarcation of the two types of peptide-containing nerves was established by the staining of substance P-containing nerves, which stops at the level of the ciliary body. This uveal area is known to be involved in the ocular responses to nociceptive stimuli. At the ultrastructural level, immunoreactivity for both peptides was localised to distinct subpopulations of p-type nerves, distinguishable by the size of their large dense-cored vesicles. Those immunoreactive for VIP were significantly larger (p less than 0.0005) than those immunoreactive for substance P (95 +/- 7 nm and 82 +/- 9 nm respectively; mean +/- SD). Interruption of the trigeminal pathway produced a remarkable decrease of substance P immunoreactivity in the anterior portion of the uvea (9.1 +/- 1.5 pmol/g, mean +/- SEM, control; 5.3 +/- 1.3 pmol/g, denervated), but not of VIP immunoreactivity in the choroid. Following colchicine treatment, VIP-immunoreactive neuronal cell bodies were localised in the choroid. The separate anatomical localisations and distributions of the two uveal peptides appear to be related to their different origins and functional roles in the response of the eye to noxious stimuli.     

Perivascular nerves in the feline carotid rete

Summary Numerous nerve fibres containing acetylcholinesterase and noradrenaline, as well as avian pancreatic polypeptide-, vasoactive intestinal peptide-, or substance P-like immunoreactivity are observed around arteries in the external carotid rete of the cat. The nerves are located in the adventitial layer close to the media. It is possible that adrenergic, cholinergic and peptidergic nerve fibres may have a strong neurogenic influence on the rete blood vessels.     

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

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