Distribution of nitric oxide synthase-containing nerves in the aganglionic intestine of mutant rats: a histochemical study.

We examined the distribution of nerves containing nitric oxide synthase in the intestine of congenitally aganglionic rats, using a reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemical method for whole-mount and cryostat specimens. A constricted intestinal segment extends from the terminal ileum to the anus in this mutant. No nerve elements with the activity were found in the affected terminal ileum, cecum and proximal colon. Although intrinsic ganglionic neurons were absent along the constricted intestine, nerve fibers with the activity were found in both the submucous and intermuscular layers distal to the proximal colon. These fibers increased in density towards the rectum, forming hypertrophic nerve bundles and unusual fiber networks. However, positive fibers were never seen within the circular and longitudinal musculature of the constricted lesion. Some of these hypertrophic nerve bundles were continuous with ectopic ganglia that were situated in the adventitial connective tissue around the lower rectum and in the submucosa near the anus. The hypertrophic nerve bundles seemed to have an extrinsic origin; some of them may have originated from ectopic ganglia. These results suggest that the defective distribution of nerves containing nitric oxide synthase may be involved in the pathogenesis of congenital colonic aganglionosis.

A nigro-rubro-bulbar pathway to the parvicellular reticular formation in the rat.

A possible pathway from the substantia nigra pars reticulata (SNr) to the parvicellular reticular formation (RFp) via the red nucleus (RN) was examined light and electron microscopically by combining anterograde and retrograde tracing techniques. After contralateral injections of biotinylated dextranamine (BDA) into the dorsolateral part of the SNr and cholera toxin B subunit (CTb) into the RFp, many CTb-labelled neurones were distributed contralaterally in the dorsal part of the RN, where numerous BDA-labelled axon terminals originating from the ipsilateral SNr were found. After contralateral injections of BDA into the dorsolateral part of the SNr and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the RFp, ipsilateral axons labelled with BDA were found to make synaptic contacts with the somata and dendrites of contralateral neurones labelled with WGA-HRP in the dorsal part of the RN.

Changes of NADPH-diaphorase activity in the lumbosacral intermediolateral neurons of the rat after pelvic axotomy.

Changes of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the lumbosacral intermediolateral (IML) neurons of the rat were examined for approximately 10 weeks after pelvic nerve transection. Both the number and the staining intensity of NADPH-d-positive neurons in the IML region increased remarkably 1 week after pelvic axotomy; the number of darkly NADPH-d-stained cells on the axotomized side was approximately 2.2-fold greater than on the control side. The number of NADPH-d-positive cells returned to the control level at 5 weeks and decreased significantly below the control level 10-11 weeks postaxotomy. In addition, using a retrograde tracing technique with Fluorogold (FG) combined with NADPH-d histochemistry, approximately 95% of the NADPH-d-positive IML neurons were found to send their axons to the pelvic nerve 1 week after axotomy, whereas nearly 25% of the FG-labeled neurons were found to be negative for NADPH-d. Thus, these results indicate that pelvic axotomy in the rat enhances NADPH-d activity transiently in the IML neurons of the lumbosacral spinal cord, and suggest that the IML region may include different neurons showing different responses in nitric oxide synthase expression after peripheral axotomy.

Characteristics of distribution of peptide-containing nerve fibres in the atrioventricular valves of the rat.

The distribution of vasoactive intestinal polypeptide-, neuropeptide Y-, and calcitonin gene-related peptide-immunoreactive nerve fibres was investigated in the atrioventricular valves of the rat. These nerve fibres were visualized by immunostaining of whole-mount preparations by the avidin-biotin-peroxidase complex method. Vasoactive intestinal polypeptide-immunoreactive nerve fibres were observed mainly in the anterior cusp of the mitral valve and, to a lesser extent, in the medial cusp of the tricuspid valve. Numerous neuropeptide Y-immunoreactive nerve fibres were found covering all of the cusps. Both types of peptidergic nerve fibre formed dense networks that consisted of interlacing and anastomosing nerve fibres. Calcitonin gene-related peptide-immunoreactive nerve fibres were seen in every cusp, but did not form a fine network. These results provide detailed anatomical information for evaluation of the possible roles of each type of peptide-containing nerve fibre in the function of atrioventricular valves.

Origins of nerve fibers containing nitric oxide synthase in the rat celiac-superior mesenteric ganglion.

The origin of nitric oxide synthase-containing nerve fibers in rat celiac-superior mesenteric ganglion was examined using retrograde tracing techniques combined with the immunofluorescence method. Fluoro-Gold was injected into the celiac-superior mesenteric ganglion. Neuronal cell bodies retrogradely labeled with Fluoro-Gold in the thoracic spinal cord, the dorsal root ganglia at the thoracic level, the nodose ganglion, and the intestine from the duodenum to the proximal colon were examined for nitric oxide synthase immunoreactivity. About 60% of sympathetic preganglionic neurons in the intermediolateral nucleus projecting to the celiac-superior mesenteric ganglion were immunoreactive for nitric oxide synthase, as were approximately 27% of nodose ganglion neurons and about 65% of dorsal root ganglion neurons projecting to the celiac-superior mesenteric ganglion. Neurons projecting to the celiac-superior mesenteric ganglion were found in the myenteric plexus of the small and large intestine. In the proximal colon, about 23% of such neurons were immunoreactive for nitric oxide synthase. However, in the small intestine, no immunoreactivity was found in these neurons.

Demonstration of axon collateral projections from the substantia nigra pars reticulata to the superior colliculus and the parvicellular reticular formation in the rat.

It was revealed in the rat that single neurons in the substantia nigra pars reticulata (SNr) innervated both the superior colliculus (SC) and the parvicellular reticular formation (RFp) in the pons and medulla oblongata by way of axon collaterals. After injecting Fluoro-gold into the lateral part of the SC and Fluoro-ruby into the RFp on the same side, some SNr neurons were double-labeled with both tracers. They were localized in the dorsolateral part of the caudal half of the SNr ipsilateral to the injection sites.

Ultrastructural study on NOS-immunoreactive nerve terminals in the rat coeliac ganglion.

Nerve terminals immunoreactive for nitric oxide synthase (NOS) were studied in the rat coeliac-superior mesenteric ganglion by electron microscopy using a pre-embedding immunostaining method. The immunoreactive material was distributed in the axoplasmic matrix and was not specifically associated with any subcellular organelle. In most NOS-immunoreactive axon terminals numerous small clear vesicles (35-50 nm) were seen, and in some terminals a small number of large granular vesicles (70-120 nm) were intermingled with small clear vesicles. Most NOS-immunoreactive axon terminals formed axodendritic as well as axo-somatic synapses with non-immunoreactive ganglion neurones, and axo-axonic contacts were very scarce. These results suggest that NO may be released at the synaptic sites from the axon terminals and may affect ganglion neurones.

Co-localization of nitric oxide synthase and vasoactive intestinal peptide immunoreactivity in neurons of the major pelvic ganglion projecting to the rat rectum and penis.

Nitric oxide synthase (NOS)- and vasoactive intestinal peptide (VIP)-immunoreactive neurons projecting to the upper rectum or penis were examined using retrograde tracing combined with immunohistochemistry in the major pelvic ganglion of male rats. Five days after injection of Fluoro-Gold (FG) into the upper rectum or penis, the major pelvic ganglion was treated with colchicine. FG injected into the upper rectum labelled many ganglion neurons in the major pelvic ganglion. Immunohistochemistry showed that 37% of FG-labelled neurons were immunoreactive for NOS and 33% for VIP. After injection of FG into the penis, 41% of FG-labelled neurons were immunoreactive for NOS and 25% for VIP. Serial cryostat sections stained for NOS and VIP, respectively, showed the co-localization of NOS and VIP in the ganglion cells projecting to the rectum and penis. In the major pelvic ganglion of the colchicine-treated animals, about 17% of the ganglion cells were immunoreactive for NOS and 32% were immunoreactive for VIP. These neurons were small in diameter (less than 30 microns). A histogram showing cell sizes in cross-sectional areas of NOS-immunoreactive neurons coincided with that of VIP-immunoreactive neurons. Most of the NOS- and VIP-immunoreactive neurons were less than 600 microns. These results indicate that small neurons containing both NOS and VIP in the major pelvic ganglion project to the rectum and penis. In the penile erectile tissues and enteric ganglia, NO and VIP may be released from the same axons and may act concomitantly on the target tissue.

Distribution of extrinsic enkephalin-containing nerve fibers in the rat rectum and their origin in the major pelvic ganglion.

The distribution of nerve fibers containing enkephalin (ENK)-like immunoreactivity was examined in the rectum of aganglionosis rats (AGRs) which completely lack the intramural ganglion cells in the large intestine, and was compared with that of their normal littermates. Furthermore, Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL)-like immunoreactive neurons projecting to the rectum were examined using retrograde tracing combined with immunohistochemistry in the major pelvic ganglion of normal male rats. In the intermuscular space of the aganglionic rectum of AGRs, unlike the pattern of the normal intermuscular plexus, moderate numbers of ENK-like-immunoreactive fibers were arranged in an irregular, coarse network; greatly diminished numbers of immunoreactive fibers were found in the submucosa. No ENK-like-immunoreactive fibers were seen in the circular muscle layer and mucosa. In the normal rat rectum, ENK-like-immunoreactive fibers were seen throughout all layers, and immunoreactive nerve cells were found predominantly in the myenteric plexus of colchicine-treated animals. Fluoro-Gold injected into the upper rectum labelled numerous principal ganglion neurons in the major pelvic and inferior mesenteric ganglia. Less than 10% of tracer-labelled neurons were positive for fluorescein immunolabelling of MEAGL in the major pelvic ganglion; no immunoreactive neurons were found in the inferior mesenteric ganglion. In the major pelvic ganglion of the colchicine-treated normal rats, about 5% of principal ganglion neurons were immunoreactive for MEAGL. Comparison of serial paraffin sections of the major pelvic ganglion stained for tyrosine hydroxylase (TH), MEAGL and vasoactive intestinal polypeptide (VIP), respectively, revealed that more than half of MEAGL-like immunoreactive neurons were also positive for TH; there was no case showing co-existence of MEAGL with VIP in the principal neurons. These results indicate that a small number of enkephalin-containing neurons in the major pelvic ganglion project to the rectum, and that more than half of these neurons are postganglionic sympathetic. They may terminate mainly in the myenteric ganglia in the rectum.

Ultrastructural evidence for innervation of the endothelium and interstitial cells in the atrioventricular valves of the Japanese monkey.

BACKGROUND: A rich supply of nerves to the atrioventricular valve has been demonstrated. The role of the valvular nerves is still controversial because the target sites of the nerves have not been confirmed. METHODS: The innervation of the atrioventricular valves of the Japanese monkey (Macaca fuscata) was examined by acetylcholinesterase staining and electron microscopy. Immunoreactivity for neuropeptide Y (NPY) was also investigated by a post-embedding immunogold method. RESULTS: The valvular nerve elements were clearly concentrated between the endothelium and interstitial cells on the atrial side of cusps. Naked axon terminals were observed to make direct contact (20-nm gaps) with interstitial cells and also to be in close proximity (approximately 200-nm cleft) to the endothelium. NPY immunoreactivity was clearly detected on the large granular vesicles in some terminals that were in close proximity to interstitial cells and/or the endothelium. CONCLUSION: The present study suggests that the extensive innervation of the atrioventricular valve, which includes NPY-containing nerves, might affect valvular function via interstitial cells and/or the endothelium.

Descending projections from the superior colliculus to the reticular formation around the motor trigeminal nucleus and the parvicellular reticular formation of the medulla oblongata in the rat.

We observed by the anterograde and retrograde tracing techniques in the rat that the lateral part of the superior colliculus (SC), where the nigrotectal fibers from the dorsolateral part of the substantia nigra pars reticulata (SNr) terminated, sent projection fibers to the reticular region around the motor trigeminal nucleus (RFmt) and parvicellular reticular formation (RFp) of the medulla oblongata, where many premotor neurons for the orofacial motor nuclei were known to be distributed. The SC neurons sending their axons to the RFmt and RFp were mainly located in the stratum griseum intermedium, and additionally in the stratum griseum profundum. Our results suggest that neuronal signals conveyed through the nigro-tecto-bulbar pathway to the RFmt and RFp may exert control influences upon oral behavior.

Coexistence of vasoactive intestinal polypeptide and neuropeptide Y immunoreactivity within axon terminals in the canine and human lower esophageal sphincter: electron microscopy by a double immunogold labeling procedure.

Subcellular localization of vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) immunoreactivity within nerves was investigated in the lower esophageal sphincter of dogs and humans by double immunogold staining of sections prepared for electron microscopy. Coexistence of VIP and NPY immunoreactivity was clearly demonstrated in the large granular vesicles (LGVs) in axon terminals that were closely associated with the smooth muscle cells, as well as in the LGVs within the perikarya of neurons located in the myenteric plexus. Some LGVs appeared immunopositive only for VIP or NPY. This phenomenon might have been partly due to the fact that the double-labeling procedure with immunogold particles of different sizes was performed on both faces of each section. The results obtained in this study suggest that VIP and NPY are synthesized in the same neuron, stored in the same axon terminal, and released together to act on sphincter muscle cells.

Nerve fibres containing neuropeptide Y in the atrioventricular valves of Japanese monkey and rat; a light and electron microscopic study.

Dense distribution of varicose fibres containing neuropeptide Y-like immunoreactivity (NPY-LI) was found in the atrioventricular valves of the Japanese monkey, and moderately in the rat. The immunoelectron microscopy using immunogolds resulted in the localization of NPY-LI within the dense-cored vesicles which existed with the small clear vesicles in the unmyelinated axons near the endocardium. These NPY-LI-containing fibres may participate in regulation of vasomotor role or other functions of the atrioventricular valves.

Heterogenous distribution of peptide-containing nerve fibres within the circular muscle layer of the human pylorus.

The distribution of nerve fibres immunoreactive for vasoactive intestinal polypeptide (VIP), substance P (SP), methionine-enkephalin (ENK), calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) within the circular muscle layer was examined histochemically in the human pylorus, adjacent antrum and duodenum. Longitudinal cryostat sections of the pyloric and surrounding regions were stained by an indirect immunofluorescence method, and the total length of each type of peptide-containing fibre per unit sectional area (micron/mm2) was measured using an image-analysing system. The narrow region of the circular muscle layer bordering the submucosa in the pylorus contained a rich supply of VIP, SP, ENK and CGRP immunoreactive fibres; VIP fibres were most prominent with less SP and ENK fibres and moderate amounts of CGRP. These peptide-containing nerve fibres were more dense than in the pyloric circular muscle, the longitudinal muscle layer and also the adjacent muscle layer. NPY-immunoreactive fibres were sparsely distributed throughout the pyloric region. These results suggest that the inner edge of the circular muscle, lying adjacent to the submucosa and densely innervated with peptide-containing fibres, may be a characteristic feature of the human pyloric sphincter.

Distribution and origin of extrinsic nerve fibers containing calcitonin gene-related peptide, substance P and galanin in the rat upper rectum.

The distributions of nerve fibers containing calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) were examined in the rat rectum of mutants rats, aganglionic rats (AGRs), which completely lack the intramural nerve cells in the large intestine, and of their normal littermates. The origin of extrinsic peptide-containing nerve fibers was examined using retrograde tracing combined with immunohistochemistry in normal rats. In the rectum of normal rats, CGRP-, SP- and GAL-immunoreactive varicose fibers were observed throughout all layers of the rectal wall, and immunoreactive nerve cells were present in the enteric ganglia of colchicine-treated rats. In the aganglionic rectum of AGR, a rich supply of CGRP-immunoreactive fibers was observed in the mucosa, around the blood vessels, and in the submucous and intermuscular spaces. SP- and GAL-immunoreactive fibers in the aganglionic rectum showed a similar distribution to CGRP-immunoreactive fibers but were less dense. These results suggest that most of CGRP-positive fibers in the rectum are extrinsic whereas a large part of SP- or GAL-positive fibers are intrinsic. Fluoro-gold injected into the upper rectum of normal rat labelled nerve cells (less than 10% of total ganglion cells) in the lumbar (L1 and L2) and lumbosacral (L6 and S1) dorsal root ganglia. More than half of nerve cells in the dorsal root ganglia (L6 and S1) projecting to the rectum were immunoreactive for CGRP, and less than 10% were immunoreactive for SP or GAL. Comparison of serial sections of the dorsal root ganglion revealed that about half of the CGRP-immunoreactive cells were also positive for SP or GAL. These results indicate that SP- or GAL-positive neurons projecting to the rectum are scarce in the dorsal root ganglia. The present investigation suggests that CGRP-containing nerves are visceral afferents forming a major component of the sensory innervation of the rat rectum, and SP- and GAL-containing nerves which share their extrinsic origins appear to form a lesser proportion of the sensory innervation.

An in vitro study of the projections of enteric vasoactive intestinal polypeptide-immunoreactive neurons in the human colon.

The anatomical basis of the peptidergic neural control of the human colon is largely unknown. In this study, in vitro retrograde tracing methods have been used on fresh human colon to determine the projection pathways of the enteric nerves and, in particular, those containing vasoactive intestinal polypeptide, one of the most abundant and potent of the gut neuropeptides. Two components of the submucous plexus were identified, the inner one projecting to the lamina propria, and the outer to the circular muscle. The lengths of projections within the submucous plexus were up to 5-14 mm in all directions. Myenteric ganglion cells projected to both longitudinal and circular muscles, for distances of up to only 5 mm. The subpopulation of nerves containing vasoactive intestinal polypeptide arose mainly from the submucous plexus and projected up to 6.5 mm anally, 5 mm orally, and 14 mm within the submucous layer to the mucosa or circular muscle. These findings provide entirely new data on the neuroanatomy of the human colon and may help in the understanding of the neural control of colonic secretion and motility.

The distribution of GAWK-like immunoreactivity in neuroendocrine cells of the human gut, pancreas, adrenal and pituitary glands and its co-localisation with chromogranin B.

GAWK is a recently discovered peptide isolated from extracts of human pituitary gland and subsequently shown to be identical to sequence 420-493 of human chromogranin B. The distribution of this peptide was studied in human gut, pancreas, adrenal and pituitary glands using antisera to two portions of the 74 amino acid peptide (sequences 1-17 and 20-38). In addition, the co-existence of GAWK immunoreactivity with other peptides and chromogranin B was investigated using comparative immunocytochemistry. In the gut, GAWK was localised mainly to serotonin-containing cells of the mucosal epithelium, where electron microscopy showed it to be stored in typical electron-dense (250 nm diameter) granules, and to a moderate population of nerve fibres in the gut wall. Considerable quantities of GAWK-like immunoreactivity were measured in the gut, up to 36.3 +/- 18 pmol GAWK 1-17/g wet weight of tissue (mean +/- SEM) and 12.4 +/- 2.9 pmol GAWK 20-38/g. Chromatography of gut extracts revealed several GAWK-like immunoreactive peaks. GAWK-like immunoreactivity was also detected in endocrine cells of pancreas, pituitary gland and adrenal medulla, where the highest concentrations of GAWK-like immunoreactivity were measured (GAWK 1-17 2071.8 +/- 873.2 and GAWK 20-38 1292.7 +/- 542.7 pmol/g). Endocrine cells containing GAWK-like immunoreactivity were found also to be immunoreactive for chromogranin B. Our results define a discrete distribution of GAWK immunoreactivity in human endocrine cells and nerves and provide morphological support for the postulated precursor-product relationship between chromogranin B and GAWK. Details of the functions of this peptide are awaited.

Specific inhibitors of tyrosine-specific protein kinase, synthetic 4-hydroxycinnamamide derivatives.

Several newly synthesized 4-hydroxycinnamamide derivatives such as 3-(3',5'-di-isopropyl-4'-hydroxybenzylidene)-2-oxindol (ST 280), 3-(3',5'-di-methylthiomethyl-4'-hydroxybenzylidene)-2-oxindole (ST 458), alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638) and 3-(3'-ethoxy-4'-hydroxy-5'-phenylthiomethylbenzylidene)-2-pyrol idinone (ST 642) were found to inhibit tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor with IC50 values of 0.44 microM, 0.44 microM, 0.37 microM and 0.85 microM, respectively. None of them showed inhibitory effect on the enzyme activities of serine- and/or threonine-specific protein kinases such as cAMP-dependent protein kinase, Ca2+/phospholipid-dependent protein kinase C, casein kinase I and casein kinase II. In addition, none of them had effect on Na+/K+-ATPase or 5'-nucleotidase. The results suggest that the compound ST 280, ST 458, ST 638 and ST 642 are potent and specific inhibitors of tyrosine-specific protein kinase.

Distribution of nerves containing vasoactive intestinal polypeptide-like immunoreactivity in rats with congenital aganglionosis of the colon.

The distribution of nerves containing vasoactive intestinal polypeptide-like immunoreactivity was examined immunohistochemically in whole-mount specimens of the colons of mutant rats, which completely lacked intramural nerve cells in the colon, and of their normal littermates. In the aganglionic colon, greatly diminished numbers of vasoactive intestinal polypeptide-like immunoreactive nerve fibers were found in the circular muscle layer, lamina propria of the mucosa, and in the submucosa. In the intermuscular space of the aganglionic colon, unlike the pattern of the normal Auerbach's plexus, vasoactive intestinal polypeptide-like immunoreactive nerve fibers were arranged in an irregular, coarse network. These findings suggest the existence of extrinsic nerves containing vasoactive intestinal polypeptide in the aganglionic colon of the hereditary aganglionic rat.