Innervation of intestinal arteries by axons with immunoreactivity for the vesicular acetylcholine transporter (VAChT)

The presence of a cholinergic innervation of arterioles within the gut wall is suggested by pharmacological studies of nerve mediated vasodilatation, but attempts to identify nerve cells that give rise to cholinergic vasodilator fibres have yielded discrepant results. In the present work, antibodies to the vesicular acetylcholine transporter protein (VAChT) were used to investigate the relationships of immunoreactive nerve fibres to submucosal arterioles. Comparison was made with cerebral arteries, which are known to be cholinergically innervated. Double labelling immunohistochemical techniques revealed separate VAChT and tyrosine hydroxylase (TH) immunoreactive (IR) fibres innervating all sizes of arteries of the submucosa of the stomach, ileum, proximal colon, distal colon and rectum as well as the cerebral arteries. Arterioles of all digestive tract regions had greater densities of TH-IR innervation than VAChT-IR innervation. In the ileum, double labelling for VAChT-IR and VIP-IR or calretinin-IR showed more VAChT-IR than either VIP-IR or calretinin-IR fibres. Calretinin-IR and VAChT-IR were colocalised in a majority of calretinin-IR axons, but VIP-IR and VAChT-IR were not colocalised. All calretinin-IR nerve cells in submucous ganglia were immunoreactive for choline acetyltransferase, but only 1–2% of VIP-IR nerve cells were immunoreactive. Extrinsic denervation of the ileum did not alter the distribution of VAChT-IR fibres, but it eliminated TH-IR fibres. Removal of myenteric ganglia (myectomy) did not alter the distribution of fibres with VAChT or TH-IR. This work thus provides evidence for cholinergic innervation of intrinsic arterioles throughout the digestive tract and indicates that the fibres in the small intestine originate from submucosal nerve cells.     

Distribution of NADPH-diaphorase activity in the embryonic chicken gut

The appearance and distribution of NADPH-diaphorase activity in neuronal cells and fibres in different regions of the embryonic chicken gut was studied histochemically using whole mount preparations and cryostat sections. NADPH-diaphorase activity was detected in neuronal cell bodies as early as embryonic day 5.5 (E5.5 — the earliest age examined), mainly in the foregut, although some positive cells were also seen in the hindgut at this stage. NADPH-diaphorase-positive fibres were first detected in the developing nerve tracts which connect the ganglia at E5.5. The complexity of the network was maximal in the proventriculus-gizzard junction. By E9.5, NADPH-diaphorase-positive fibres were found in the circular muscle layer. NADPH-diaphorasepositive submucosal neurons were first detected at E11.5. The density of innervation was maximal at E15.5 and declined later in development. The expression of neuronal NADPH-diaphorase activity progressed in a craniocaudal direction and followed a developmental pattern similar to that previously described for several neuropeptides in the avian gut.     

家兔回肠淋巴管三维结构的扫描电镜观察

目的　探讨回肠淋巴管的三维结构及流注关系。方法　将Mercox注入家兔回肠壁内制成淋巴管铸型 ,在扫描电镜下观察回肠各层淋巴管的三维结构。结果　回肠的粘膜层、粘膜下层、肌层和浆膜层都有毛细淋巴管网及淋巴管。在小肠绒毛内存有中央乳糜管 ,铸型样品上可见其立体结构 ,可见中央乳糜管注入粘膜层和粘膜下层的毛细淋巴管网 ,该网汇合而成的淋巴管穿过肌层进入浆膜层 ,之后以淋巴集合管的形式入肠系膜淋巴管而离开回肠。铸型标本可见淋巴管呈串珠状外观 ,管壁表面有双凹切迹 ,相当于淋巴管瓣膜的部位。铸型表面还可见淋巴管内皮细胞的椭圆形压迹 ,光镜下可见粘膜下与肌层之间存有三角形间隔 ,其内动脉与两条淋巴管并行。结论　中央乳糜管注入粘膜下毛细淋巴管网 ,该网与粘膜下淋巴管网相连 ,此淋巴管再穿肌层入浆膜层后离开回肠 ,并见淋巴通道存在于中央糜管周围。     

Histochemical localization of nitric oxide-synthesizing neurons and vascular sites in the guinea-pig intestine.

Laminar preparations of fixed segments of the guinea-pig intestine were examined for nitric oxide synthase activity using reduced nicotinamide adenine dinucleotide phosphate and nitroblue tetrazolium salt as substrates. Under conditions specific for detecting nitric oxide synthase-related diaphorase activity, a subpopulation of neural elements in the myenteric plexus, deep muscular plexus and submucosa were intensely stained. Intensely stained nerve fibres were distributed throughout the meshworks of the myenteric plexus and its innervation of the circular muscle, and in the submucosa within Henle's plexus. Intensely stained nerve cells and their processes were evident in most myenteric ganglia but were rare in ganglia of Henle's plexus. Stained ganglion cells comprised types I, II and VI of the morphologically defined enteric nerve cells. Stained neural elements were increasingly prevalent within successively more caudal segments of the intestine. In addition to neuronal staining, arterioles of the submucosal vascular network displayed distinct, punctate patches of staining distributed over their surface. Perivascular nerve fibre staining was absent. These results show nitric oxide synthase activity to be present within neurons and fibres of the major enteric nerve layers and within submucosal blood vessels throughout the guinea-pig small and large intestine.     

Projections of substance P, vasoactive intestinal peptide and tyrosine hydroxylase immunoreactive nerve fibres in the canine intestine, with special reference to the innervation of the circular muscle.

Antisera raised against neuron specific enolase (NSE), substance P, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase (TH) were used to reveal nerve fibres in the wall of the canine small and large intestine. The circular muscle of the colon was innervated by nerve fibre bundles that ran parallel to the muscle throughout its thickness. A plexus of fibre bundles was found against the inner (submucosal) surface of the circular muscle. Fibres with substance P, VIP and TH immunoreactivity all contributed to this innervation. The circular muscle of the small intestine was distinctly separated into outer and inner layers by a dense plexus of nerve fibres, the deep muscular plexus. The outer and inner circular muscle were innervated by substance P, VIP and TH fibres. Extrinsic denervation through the severing of nerve fibres in the mesentery caused TH fibres in the intestine to degenerate, but had no detectable effect on the fibres with substance P or VIP immunoreactivity. Myectomy (the removal of the myenteric plexus from the full circumference of the intestine over a distance of 2-3 cm), performed 7-13 days before tissue was taken, resulted in an almost complete loss of substance P fibres from the circular muscle of the colon and the outer circular muscle of the small intestine. However, many fibres persisted in the deep muscular plexus of the small intestine, and most fibres remained in its inner circular muscle. The changes in distribution of VIP fibres were almost identical, except that a small proportion of reactive fibres remained in the circular muscle of the colon and the outer circular muscle of the small intestine. It is concluded that the circular muscle layers of the small intestine and colon have dual sources of intrinsic nerve supply: the myenteric ganglia supply fibres primarily to the outer part of the muscle and the submucous ganglia supply fibres to the inner muscle. The present study further demonstrated that VIP fibres ran anally in the myenteric plexus of both the small and large intestine, whereas substance P fibres ran orally in the large intestine and both orally and anally in the small intestine. The innervation of the muscularis mucosae and mucosa by substance P and VIP fibres was not affected by myectomy or extrinsic denervation, and these structures are therefore likely to be innervated by nerve cells in the submucous ganglia.     

回肠淋巴管三维结构的扫描电镜观察

目的:探讨回肠淋巴管的三维结构及流注关系;方法:使用16只家免,将Mercox注入动物回肠壁内,制成淋巴管铸型后,在扫描电镜下观察了回肠各层淋巴管的三维结构。为配合铸型标本,同时制作了半薄切片;结果:通过对回肠淋巴管的二维及三维结构的观察,可见回肠的粘膜层、粘膜下层、肌层和浆膜层都有毛细淋巴管网及淋巴管。在小肠绒毛内存有中央乳糜管,铸型样品上可见其立体结构。又观察到中央乳糜管注入粘膜层和粘膜下层的毛细淋巴管网。该网汇合而成的淋巴管穿过肌层,进入浆膜层,之后以淋巴集合管的形式入肠系膜淋巴管而离开回肠。铸型标本可见淋巴管呈串珠状外观,管壁表面有双凹切迹,此相当于淋巴管瓣膜的部位。铸型表面还可见淋巴管内皮细胞的椭圆形压迹。光镜下可见粘膜下与肌层之间存有三角形间隔,其内动脉与两条淋巴管并行;结论:中央乳糜管注入粘膜下毛细淋巴管网,该网与粘膜下淋巴管网相连。此淋巴管再穿肌层入浆膜层后离开回肠。本文见到淋巴通道存于中央乳糜管周围。淋巴液的形成、运输与淋巴管周围的动脉搏动有关。     

Immunohistochemical localisation of cholinergic muscarinic receptor subtype 1 (M1r) in the guinea pig and human enteric nervous system

Little is known regarding the location of cholinergic muscarinic receptor 1 (M1r) in the ENS, even though physiological data suggest that M1rs are central to cholinergic neurotransmission. This study localised M1rs in the ENS of the guinea pig ileum and human colon using fluorescence immunohistochemistry and RT-PCR in human colon. Double labelling using antibodies against neurochemical markers was used to identify neuron subytpes bearing M1r.

M1r immunoreactivity (IR) was present on neurons in the myenteric and submucosal ganglia. The two antibodies gave similar M1r-IR patterns and M1r-IR was abolished upon antibody preabsorption. M1r-IR was present on cholinergic and nNOS-IR nerve cell bodies in both guinea pig and human myenteric neurons. Presynaptic M1r-IR was present on NOS-IR and VAChT-IR nerve fibres in the circular muscle in the human colon. In the submucosal ganglia, M1r-IR was present on a population of neurons that contained cChAT-IR, but did not contain NPY-IR or calretinin-IR. M1r-IR was present on endothelial cells of blood vessels in the submucosal plexus.

The localisation of M1r-IR in the guinea pig and human ENS shown in this study agrees with physiological studies. M1r-IR in cholinergic and nitrergic neurons and nerve fibres indicate that M1rs have a role in both cholinergic and nitrergic transmission. M1r-IR present in submucosal neurons suggests a role in mediating acetylcholine's effect on submucosal sensory and secretomotor/vasodilator neurons. M1r-IR present on blood vessel endothelial cells suggests that M1rs may also mediate acetylcholine's direct effect on vasoactivation.     

Ultrastructural observations in the rat ileal mucosa of possible epithelial “taste cells” and submucosal sensory neurons

Electronmicroscopical studies of the rat ileum have demonstrated the presence of submucosal neuronlike cells located just under the basal lamina of intestinal crypts. These cells had dendrite-like processes and frequently made contacts with adjacent submucosal nerve terminals. Furthermore, within the mucosa epithelial ‘clear’ cells with apical cytoplasmic processes into the gut lumen and basal cytoplasmic processes extending underneath adjacent epithelial cells were demonstrated. The ‘clear’ cells were devoid of secretory granules, as e.g. endocrine cells. and had a morphological resembiance to taste cells. Occasionally the ‘clear’ cells made contacts with submucosal nerve tcrminals. The possibility that the ‘clear’ cells and submucosal neuronlike cells represent a receptive function within the intestine is discussed.     

Glomerular handling of immune complex in the acute phase of active in situ immune complex glomerulonephritis employing cationized ferritin in rats

The distribution and co-localization of nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) were examined by means of immunohistochemistry and NADPH diaphorase (NADPH-d) histochemistry in the gut of patients with Hirschsprung's disease. In the normoganglionic segment, many nitrergic nerve cells were localized in Auerbach's plexus and nerve fibres were observed preferentially in the circular muscle. The submucosal nitrergic nerve cells were mainly situated in Schabadasch's plexus with occasional cells demonstrable in Meissner's plexus. NOS and VIP were co-localized in most ganglion cells of Auerbach's plexus. In the oligoganglionic segment, a marked reduction of NOS- and VIP- positive nerve cells and fibres was noticed in both the myenteric and submucosal plexuses, and nitrergic fibres had disappeared in the inner layer of the circular muscle. In the aganglionic segment, NOS and VIP were revealed only in extrinsic nerve fasciculi and rami and co-localized in a few fibres. From these observations, the inner layer of the circular muscle of the oligoganglionic segment and the whole of the muscularis propria of the aganglionic segment were considered to be totally lacking in nitrergic innervation. Nitrergic nerves of the human colon comprise both intrinsic and extrinsic elements and the majority of intrinsic nitrergic nerve cells contain VIP. Very low numbers of extrinsic nitrergic fibres contain VIP.     

血管周肾上腺素能神经密度与管壁组成成分的关系

本文应用组织化学技术和电镜方法对家兔、豚鼠和大鼠血管周的肾上腺素能神经密度与管壁组成成分的关系进行了观察。实验结果证明,血管周神经分布于外膜层,中膜内未见有神经分布,肌性动脉(以肠系膜动脉为代表)较弹性动脉(以颈总动脉为代表)的血管周神经密度和含膨体数都较高。神经肌肉间隔近(0.05—3微米),最近者神经与肌肉间除基板外无其它组织成分。弹性动脉神经肌肉间隔较远(1—12微米),神经肌肉间隔以外弹力膜、成纤维细胞和板层状的结缔组织。股动脉和肾动脉周的神经分布特点介于上二者之间。静脉较相应动脉神经分布稀疏。但也存在部位的特殊性和种属差异性。如脐动脉虽属肌性动脉,但动脉周并无神经分布,豚鼠肾动脉周的神经密度远较兔及大鼠稀疏。作者认为血管周的神经密度与血管壁中平滑肌的含量有关。本文并对肌性动脉周神经分布致密的原因,不同类型动脉的神经分布特点与生理功能的关系进行了讨论。     

Occurrence and developmental pattern of neuromedin U-immunoreactive nerves in the gastrointestinal tract and brain of the rat

Neuromedin U is a newly described regulatory peptide, found by radioimmunoassay in significant concentrations in both the brain and gut of the rat. The aim of the present study was to localize this peptide immunoreactivity to discrete structures of the gut and brain and to map its distribution using immunocytochemistry. In the gut, neuromedin U was confined to nerve fibres mainly in the myenteric and submucous plexuses and the mucosa of all areas except stomach. Immunoreactive ganglion cells were seen in both ganglionated plexuses and their number did not increase following colchicine administration. This observation and the finding that the population of neuromedin U-immunoreactive nerves in the ileum was not affected by complete extrinsic denervation indicated that the nerves are mostly intrinsic in origin. Colocalization studies revealed neuromedin U and calcitonin gene-related peptide were present in the same myenteric and submucosal ganglion cells. Transection experiments showed that, like calcitonin gene-related peptide-immunoreactive nerves, fibres containing neuromedin U project for very short distances in both an oral and anal direction. At the electron microscopic level, neuromedin U immunoreactivity, demonstrated using the immunogold technique, was localized to large granular vesicles. In the central nervous system, neuromedin U immunoreactivity was localized to fibres which were widespread throughout the brain, except in the cerebellum. The presence of neuromedin U-immunoreactive cell bodies was restricted to the rostrocaudal part of the arcuate nucleus. Colocalization studies showed that a proportion of the neuromedin U-immunoreactive cell bodies in the arcuate nucleus also contained pro-opiomelanocortin. Neuromedin U-immunoreactive fibres were first detected in the rat intestinal mucosa at day 1 after birth. In the brain, the arcuate nucleus showed neuromedin U-immunoreactive neuronal cell bodies at E16 but not at E14. In conclusion, neuromedin U is a new member of the group of molecules known as brain-gut peptides.     

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

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

Innervation and Neurotransmitter Localization in the Lung of the Nile bichir Polypterus bichir bichir

Anatomical and functional studies of the autonomic innervation in the lung of dipnoan fishes and the bichirs are lacking. The present immunohistochemical studies demonstrated the presence of nerve fibers in the muscle layers of the lung of the bichir, Polypterus bichir bichir, and identified the immunoreactive elements of this innervation. Tyrosine hydroxylase, acetylcholinesterase, and peptide immunoreactivity was detected in the intramural nerve fibers. Extensive innervation was present in the submucosa where adenylatecyclase/activating polypeptide 38, substance P, P₂X₂, and 5‐hydroxytryptamine (5‐HT)–immunoreactive nerve fibers mainly supplied blood vessels. A collection of monopolar neurons located in the submucosal and the muscular layers of the glottis expressed a variety of various transmitters. These neurons may be homologous to ganglion cells in the branchial and pharyngeal rami of the vagus in fishes. Nerves containing 5‐HT and P₂X₂ receptor immunoreactivity projected to the lung epithelium. Associated with neuroepithelial cells in mucociliated epithelium, were neuronal nitric oxide synthase–immunopositive axons. The physiological function of this innervation is not known. The present study shows that the pattern of autonomic innervation of the bichir lung may by similar in its elements to that in tetrapods. Anat Rec, 290:1166–1177, 2007. © 2007 Wiley‐Liss, Inc.     

An immunohistochemical study of the ontogeny of the neuroendocrine system in the chicken oesophagus

 The ontogenesis and distribution of serotonin-, chromogranin A-, chromogranin B-, galanin-, neurotensin-, bombesin- and neuropeptide Y-immunoreactive elements were studied in the chicken oesophagus during pre- and post-hatching life. Unlike positive nerve elements, that were present in pre- and post-hatching life, positive endocrine cells were observed only during embryonic life in the oesophageal epithelium. The first endocrine cells, immunoreactive for serotonin and chromogranins, appeared on day 12, in the cervical and thoracic portions of the oesophagus. At the same age, but only in its distal portion, a few bombesin- and neurotensin-immunoreactive cells also appeared. The number of the endocrine cells progressively increased, reaching a maximum on day 15. They then decreased, with a cranio-caudal progression, until they disappeared a few days after hatching. Almost all the serotonin-immunoreactive cells but only a subpopulation of bombesin- and neurotensin-immunoreactive cells colocalized chromogranins. About half of this subpopulation also colocalized serotonin. All these cells reacted positively with Grimelius argyrophile stain. The mucosa of the crop never contained positive endocrine cells. Positive nervous elements appeared first in the wall of the terminal oesophagus and only one or two days later in the proximal oesophagus including the crop. Nervous elements immunoreactive for galanin first appeared from days 6 to 7, for neurotensin from days 7 to 8, for neuropeptide Y from 13 to 15 and for bombesin from 15 to 18. At day 15 galanin-immunoreactive ganglionic cells and fibres occupied both the myenteric and submucous plexus and galanin-positive nerve fibres could be seen throughout the oesophageal wall from the adventitia to a thin subepithelial network. Neurotensin- and neuropeptide Y-immunopositive ganglionic cells and fibres, by contrast, invariably occupied the muscular and submucous layers. Scattered bombesin-immunoreactive ganglionic cells were observed only in the myenteric plexus. The number of positive nerve elements progressively increased until some weeks after birth. Density and intensity were always much higher for galanin and neurotensin than for neuropeptide Y and bombesin. Accepted: 5 November 1997     

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

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

Size of neurons and glial cells in the enteric ganglia of mice, guinea-pigs, rabbits and sheep

Summary A quantitative light microscopic study has been carried out on the myenteric and submucosal ganglia of the stomach, duodenum, ileum, proximal colon and rectum of the guinea-pig; the enteric ganglia of the ileum were studied also in the mouse, rabbit and sheep. The area of the profiles of nerve cells, of nerve cell nuclei and of glial nuclei, and the proportion of the area of ganglia occupied by neuropil were measured, and the relative numbers of neurons and glial cells were estimated. The myenteric ganglia were found to be firmly anchored to the stroma of the muscle coat; their shape and the shape of their component cells varied with contraction and distension of the musculature. The range of neuronal sizes in the myenteric ganglia was extremely wide. In the guinea-pig, the myenteric neurons were on average largest in the stomach and duodenum and smallest in the ileum, with intermediate values in the colon and rectum; the submucosal neurons showed little variation in average size along the length of the gut. The average size of ganglion neurons in the ileum was greatest in the sheep and smallest in the mouse, and had intermediate values in the guinea-pig and rabbit. The percentage volume of neuropil in the myenteric ganglia was 51% in the mouse, 65% in the guinea-pig, 70% in the rabbit, and 74% in the sheep. The number of glial cells relative to the number of neurons was also ranked in the same order. In all the species examined the submucosal ganglia, when compared with the corresponding myenteric ganglia, had a smaller percentage volume of neuropil, a much smaller number of glial cells and (except in the mouse ileum) neurons of smaller average size. In all the ganglia there was a positive correlation between size of neurons and size of glial cells. The results are discussed in the light of possible relations between body size (and length of the intestine), numerical density of ganglion neurons, average size of neurons, amount of musculature, average distance between neurons, and amount of neuropil.     

Evaluation of PGP9.5 in the diagnosis of Hirschsprung's disease.

The ability of an acetylcholinesterase-stained frozen section to detect an increase in large cholinergic nerve fibres within the muscularis mucosae and extending into the lamina propria was a significant step forward in the diagnosis of Hirschsprung's disease (HD). However, such frozen section diagnosis is not always possible. The purpose of this study was to assess the ability of PGP9.5 to detect this pattern of mucosal nerve fibre staining immunohistochemically. Sixty-four specimens were included in the study. Twenty-six of these had been diagnosed as HD by conventional means. All cases were stained immunohistochemically with PGP9.5, S100, and anti-neurofilaments (NF). Twenty-four cases of HD were also stained with neurone-specific enolase (NSE). PGP9.5 reliably stained fibres in the mucosal and submucosal plexuses, and ganglion cells, when the latter were present. This positive staining of ganglion cells was more intense than that seen with NSE, and the positive fibre staining was more intense than that seen with NF. Increased lamina propria fibres were detected with PGP9.5 in only 37 per cent of HD cases compared with S100 positive staining in 60 per cent of cases. However, when S100 staining was assessed alone, it gave a higher false-negative rate in diagnosing HD than PGP9.5 used alone. Therefore we would recommend the use of PGP9.5 and S100 together for the immunohistochemical diagnosis of HD in formalin-fixed biopsies.     

Immunocytochemical identification of luteinizing hormone-releasing hormone-positive fibres and terminals in the olfactory system of the rat

Luteinizing hormone-releasing hormone immunoreactivity was studied in the olfactory system of the rat in combination with acetylcholinesterase histochemistry. Neuronal perikarya containing luteinizing hormone-releasing hormone lie in the medial septal nucleus, the vertical limb of the diagonal band of Broca, the olfactory tubercule and the ganglionated plexus of the terminal nerve. Labelled fibres spread in the superficial layers of the main and accessory olfactory bulbs, some encompassing the strongly acetylcholinesterase-positive atypical glomeruli. Others are observed on the medial side of the bulb, running along the terminal nerve bundles and ganglia. These fibres join the vomeronasal nerve branches and proceed distally towards the nasal cavity. In the septal submucosa, immunoreactive fibres are partly associated with the terminal nerve network. Conspicuous endings filled with luteinizing hormone-releasing hormone are observed on blood vessels of the olfactory mucosa. Such well-differentiated terminals might be the neurosecretory afferents of a new neurohemal area. Immunoreactive terminals are also observed around the excretory ducts of the anterior medial glands. We have failed to observe any labelled fibres in the olfactory and vomeronasal epithelia. The results of the present study are discussed with respect to possible functional interpretations. It is suggested that significant amounts of luteinizing hormone-releasing hormone could be released in the submucosal capillaries in spite of the scarcity of immunoreactive fibres. Similar afferents could also modulate the secretory activity of some nasal glands. Synaptic events involving the neuropeptide might occur in the olfactory bulb, particularly in atypical glomerular areas previously characterized by their high acetylcholinesterase content. Finally, no anatomical support for a chemosensory function of fibres containing luteinizing hormone-releasing hormone has been brought out by our work.     

Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut.

The pattern of distribution and colocalization of nitric oxide-synthase (NOS) and NADPH-diaphorase in the myenteric plexus of whole-mount preparations of the antrum, duodenum, ileum, caecum, proximal colon and distal colon of the rat were investigated using immunohistochemical and histochemical staining techniques. Almost all the myenteric neurons that were NOS-positive in all regions of the gut examined were also stained for NADPH-diaphorase. However, in the stomach, duodenum and ileum, only a few of the NOS-positive nerve fibres in the tertiary and secondary plexuses and circular muscle layer were also stained for NADPH-diaphorase, whereas in the caecum and distal colon almost all the NOS-positive nerve fibres were also stained for NADPH-diaphorase. The results in the present study are consistent with the view that nitric oxide (NO) has a mediating role in gastrointestinal neurotransmission.