An immunohistochemical study of serotonin-containing nerves in the colon of rats

The localization of the serotonin-like immunoreactive nerves of the rat colon was investigated by means of immunohistochemistry, utilizing an antibody against serotonin. In non-treated colons, serotonin-positive neuropils were consistently detected around the myenteric plexus. In pargyline-treated colons, serotonin-like fibres were demonstrated in association with either the small intramural blood vessels of the submucosa or the extramural nerve bundles. Treatment with 5-hydroxytryptophan (5-HTP) permitted the visualization of additional serotonin-immunoreactive fibres around the large extramural blood vessels. Immunoreactive nerve cell bodies were demonstrated in the myenteric plexus of colons treated with 5-HTP or colchicine. From these observations, it is suggested that the serotoninergic nerves of the rat colon comprise both intrinsic and extrinsic elements.     

An immunohistochemical study of serotonin-containing nerves in the colon of rats

Summary The localization of the serotonin-like immunoreactive nerves of the rat colon was investigated by means of immunohistochemistry, utilizing an antibody against serotonin. In non-treated colons, serotonin-positive neuropils were consistently detected around the myenteric plexus. In pargyline-treated colons, serotonin-like fibres were demonstrated in association with either the small intramural blood vessels of the submucosa or the extramural nerve bundles. Treatment with 5-hydroxytryptophan (5-HTP) permitted the visualization of additional serotonin-immunoreactive fibres around the large extramural blood vessels. Immunoreactive nerve cell bodies were demonstrated in the myenteric plexus of colons treated with 5-HTP or colchicine. From these observations, it is suggested that the serotoninergic nerves of the rat colon comprise both intrinsic and extrinsic elements.     

Adrenergic Nerves in the Avian Eye and Ciliary Ganglion

Summary The distribution of adrenergic fibres to the eye and to the ciliary ganglion was studied in pigeons, chicken and ducks with the aid of the sensitive and highly specific fluorescence method of Falck and Hillarp. In some animals the intensity of the fluorescence was increased by treating the animals with Nialamide and 1-DOPA. The cornea contained no adrenergic fibres except at the limbus, where a plexus of adrenergic varicose fibres was seen, partly associated with vessels. In the chamber angle, adrenergic varicose fibres were common in the loose connective tissue covering the canal of Schlemm. The canal of Schlemm was supplied by only few adrenergic fibres, but such fibres appeared along the intrascleral aqueous drainage vessels. In the iris, adrenergic varicose fibres appeared immediately in front of the posterior layer of pigment cells, strongly indicating the presence of a dilator homologous with that seen in mammals. The frontal third of the stroma contained several adrenergic varicose fibres, many of which seemed to lack association with any vessel. Varicose adrenergic fibres were also sparsely seen in the striated muscle of the iris. The ciliary processes contained many adrenergic varicose fibres, at least part of which seemed to be associated with the ciliary epithelium. The striated muscles of the ciliary body contained adrenergic varicose fibres along the vessels only. The retina contained adrenergic varicose fibres in three layers in the inner plexiform layer. Adrenergic ganglion cells of two sizes were detected in the inner nuclear layer. The retinal vessels had no adrenergic nerve fibres. The pecten was also devoid of adrenergic nerve fibres, except along the vessels close to the papilla. The optic nerve contained adrenergic varicose nerve fibres along vessels only. In the ciliary ganglion, varicose adrenergic fibres appeared at the small ganglion cells, often forming baskets of synaptic character.Acknowledgements. The work has been supported by the United States Public Health Service (grant NB 06701-01), by the Swedish Medical Research Council (project B 67-12 X-712-02 A) and by the Faculty of Medicine, University of Lund, Sweden.     

Avian enteric nerve plexuses

Summary The enteric nerve plexuses of the domestic fowl (Gallus domesticus) were investigated in sections and stretch preparations by means of the cholinesterase and glyoxylic acid fluorescence histochemical techniques. Cholinesterase-positive and varicose and non-varicose fluorescent nerve fibres were distributed at all levels of the gut in myenteric, submucosal, muscle and mucosal plexuses, and in a perivascular plexus. The density of the innervation and the detailed distribution of the nerves varied in different parts of the intestinal tract. All nerve plexuses appeared to be best developed in the rectum. Whereas the circular muscle coat contained a substantial number of nerves at all levels of the gut, the longitudinal coat was well innervated only in the rectum. The major portion of the mucosal plexus appeared to be associated with the intestinal glands. The nerve cell bodies were restricted to the myenteric and submucosal plexuses and were mainly cholinesterase-positive. Fluorescent ganglion cells were not observed. Pretreatment of stretch preparations with NADH: Nitro BT to stain ganglion cells showed that the majority of the cells were surrounded by a meshwork of fluorescent varicose fibres, although none of the fibres appeared to be associated with individual cells. The perivascular plexus was mainly associated with the arteries. The functional significance of the innervation is discussed.We would like to thank the British Council for financial support for Mr. H.A. Ali     

The origin and possible significance of substance P immunoreactive networks in the prevertebral ganglia and related structures in the guinea-pig

The distribution and origin of substance P immunoreactive nerve elements have been studied in the guinea-pig prevertebral ganglia by the indirect immunohistochemical technique, using a monoclonal antibody to substance P. Non-varicose substance P immunoreactive nerve fibres enter or leave the ganglia in all nerves associated with them, traversing the ganglia in larger or smaller bundles. Networks, mainly single-stranded, of varicose substance P immunoreactive nerve fibres also permeate the ganglia, forming a loose meshwork among the neurons. Similar networks are present in the lumbar paravertebral ganglia. In all these ganglia, neuronal somata do not in general show substance P immunoreactivity. The various nerves connected with the inferior mesenteric ganglion have been cut, in single categories and in various combinations, and the ganglion examined, after intervals of up to six days. Cutting the colonic or hypogastric nerves, which connect the ganglion with the hindgut and pelvic organs, leads to accumulation of substance P immunoreactive material in their ganglionic stumps, extending retrogradely to intraganglionic non-varicose fibres traceable through into the intermesenteric and lumbar splanchnic nerves. There is some local depletion of intraganglionic varicose networks. Cutting the intermesenteric nerve, which connects the coeliac-superior mesenteric ganglion complex with the ganglion, leads to accumulation of substance P immunoreactive material in its cranial stump and depletion of its distal stump; a minimal depletion is detectable in the inferior mesenteric ganglion itself. Cutting the lumbar splanchnic nerves, which connect the ganglion with the upper lumbar spinal cord and dorsal root ganglia, leads to accumulation of substance P immunoreactive material in their proximal stumps and total depletion of their distal, ganglionic stumps; in the ganglion there is subtotal loss of non-varicose substance P immunoreactive fibres and of varicose nerve networks, and the few surviving non-varicose fibres are traceable across the ganglion from the intermesenteric nerve to the colonic and hypogastric nerves. Cutting the intermesenteric and lumbar splanchnic nerves virtually abolishes substance P immunoreactive elements from the ganglion within three days postoperatively. It is concluded that these arise centrally to the ganglion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of the blood-brain barrier to horseradish peroxidase in the chick embryo

Summary The development of the adrenergic sympathetic innervation of the rabbit choroid plexus system was studied prenatally and up to two months after birth by a combination of fluorescence histochemistry (formaldehyde and glyoxylic acid methods) and quantitative enzymatic determinations of noradrenaline. The first signs of adrenergic nerves are found in the plexus of the third ventricle within the first day after birth. Fluorescent fibres subsequently appear in the choroid plexuses of the lateral ventricles (five days post partum) and the fourth ventricle (two weeks post partum). During the following development nerve fibres grow along blood vessels to form a plexus located between small vessels and the overlying epithelium. The nerve plexus, with varicose axon terminals, is fully developed at three weeks post partum, and maturation is then established by an increase in the number of terminals within the network of axons. There is a good agreement between (a) the development of the fluorescent nerves and histochemically visible adrenergic innervation, and (b) the tissue level of noradrenaline in the various choroid plexuses. Against the background of available information on the development of the secretory functions in choroid plexus, it is concluded that possibilities for a sympathetic neurogenic influence on the formation of cerebrospinal fluid exist already a few weeks after birth.     

Sympathetic innervation of the reproductive organs of the male opossum, Didelphis albiventris (Lund, 1841)

The dissection of nerves and ganglia anatomically related to the pelvic organs revealed one inferior mesenteric ganglion, two testicular ganglia, two hypogastric nerves, two pelvic ganglia and two pelvic nerves. The histochemical demonstration of catecholamines by a glyoxylic acid fluorescence method revealed a rich sympathetic innervation in the ductus deferens, in the three segments of the prostate and in the convoluted ductuli efferentes. The testis, epididymis and all three pairs of bulbourethral glands presented fluorescent nerve fibers only around blood vessels. Removal of the inferior mesenteric and testicular ganglia, and hypogastric neurectomy with our without ligature and sectioning of testicular arteries, had no effect on the density of the nonvascular fluorescent fibers. Removal of the periprostatic tissue caused complete denervation of the prostate and marked denervation of the ductuli efferentes and ductus deferens. Small ganglia containing fluorescent nerve cell bodies were found close to the capsule of the prostate. The results indicate that short adrenergic neurons are responsible for the sympathetic innervation of the reproductive organs of the male opossum.     

Quantitative analysis of the density and pattern of adrenergic innervation of blood vessels

Summary Automated quantitative image analysis (QIAF) was used to measure and compare the adrenergic nerve plexuses of 4 blood vessels from the guinea pig, demonstrated by glyoxylic acid fluorescence (GAF). The results showed considerable quantitative variation of plexus density, size of bundles, and numbers of varicosities. A range of alternative procedural and anatomical sources of variability were investigated and assessed. The carotid artery was found to have a dense plexus with more nerves than that of the mesenteric artery; the mesenteric vein and abdominal aorta had sparse plexuses. The carotid artery plexus, despite the density of its nerves, possessed only half the number of varicosities of the mesenteric artery plexus. This sparse varicosity population was shown to have a similar density to the varicosities demonstrated by QIAF in the scattered nerves of the mesenteric vein and abdominal aorta. QIAF confirmed visual estimates of adrenergic plexus density, and was able to demonstrate less obvious differences of nerve density and size, and varicosity populations, between the different plexuses studied. The method is applicable to stretch preparations and transverse sections of many adrenergically innervated tissues.     

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.     

Effects of pregnancy on the extrinsic innervation of the guinea pig uterus. A histochemical, immunohistochemical and ultrastructural study

Summary The extrinsic innervation of the guinea pig uterus was studied by immunohistochemical, ultrastructural and enzyme histochemical methods.The extrinsic innervation was organized in two major ways. One consisted of nerve trunks and non-varicose nerve fibres running in the suspensory ligament, and the other of a plexus of varicose nerve fibres surrounding vessels, and non-vessel-related non-varicose nerve fibres in the mesouterus. The use of different neuronal and Schwann cell markers showed that the extrinsic innervation was predominantly adrenergic and contained only few peptidergic nerves. Acetylcholinesterase-positive (cholinergic) nerves were only found around the uterine artery.In late pregnancy, the extrinsic nerves of the mesouterus adjacent to foetus-containing uterine horns underwent pronounced degenerative changes comprising both Schwann cell and axonal structures. In comparison, no changes were found in extrinsic nerves of mesouteri adjacent to non-foetus-bearing uterine horns or in extrinsic nerves in the suspensory ligaments. Further, chemical sympathectomy produced axonal degeneration but no changes in the Schwann cells.In conclusion, the pregnancy-induced nerve degeneration is of a very special type different from that following chemical sympathectomy and represents a local phenomenon related to the conceptus. Hypothetically, this could be of importance for counteracting disturbances in placental blood flow.     

Fluorescent Histochemistry and Cholinesterase Staining of Sympathetic Ganglia in a Teleost,Gadus morrhua

The anatomy and histochemistry of the sympathetic nervous system in the cod were studied by osmic acid staining, cholinesterase staining and fluorescent histochemistry of ganglia and nerve fibres. Large bundles of fluorescent fibres from the sympathetic ganglia in the head enter the cranial nerves and run with these. These bundles are exceptionally large to the vagi, and the cod vagi may therefore be regarded as vago-sympathetic trunks. All the sympathetic ganglion cells contain specific (acetyl-) cholinesterase, although the degree of staining was variable. The vast majority of cells in the ganglion coeliacum and other anterior ganglia show specific fluorescence of variable intensity. Ganglion cells completely devoid of specific fluorescence are scarce in the anterior ganglia, but abundant in the posterior ganglia associated with the vesicular nerve. A separate and distinct bundle of medullated fibres leaves the sympathetic chain on the left side and spreads in the wall of the left posterior cardinal vein, presumably innervating the chromaffin tissue. Similar fibres on the right side are also present, but do not form a distinct nerve.     

Innervation of the renal vasculature of the toad (Bufo marinus)

The innervation of the dorsal aorta and renal vasculature in the toad (Bufo marinus) has been studied with both fluorescence and ultrastructural histochemistry. The innervation consists primarily of a dense plexus of adrenergic nerves associated with all levels of the preglomerular vasculature. Non-adrenergic nerves are occasionally found in the renal artery, and even more rarely near the afferent arterioles. Many of the adrenergic nerve profiles in the dorsal aorta and renal vasculature are distinguished by high proportions of chromaffin-negative, large, filled vesicles. Close neuromuscular contacts are common in both the renal arteries and afferent arterioles. Possibly every smooth muscle cell in the afferent arterioles is multiply innervated. The glomerular capillaries and peritubular vessels are not innervated, and only 3-5% of efferent arterioles are accompanied by single adrenergic nerve fibres. Thus, nervous control of glomerular blood flow must be exerted primarily by adrenergic nerves acting on the preglomerular vasculature. The adrenergic innervation of the renal portal veins and efferent renal veins may play a role in regulating peritubular blood flow. In addition, glomerular and postglomerular control of renal blood flow could be achieved by circulating agents acting via contractile elements in the glomerular mesangial cells, and in the endothelial cells and pericytes of the efferent arterioles. Some adrenergic nerve profiles near afferent arterioles are as close as 70 nm to distal tubule cells, indicating that tubular function may be directly controlled by adrenergic nerves.     

Adrenergic Innervation of Bowel in Hirschsprung's Disease

The adrenergic innervation of normal and aganglionic regions of bowel from patients with Hirschsprung''s disease was investigated by a fluorescent histochemical technique. In normal bowel the adrenergic nerves end about intramural ganglion cells. In aganglionic bowel the adrenergic nerves form a dense varicose plexus in both muscularis externa and muscularis mucosae. It is suggested that the cause of megacolon in Hirschsprung''s disease is due to a lack of nervous pathways controlling the intrinsic reflexes, which is probably congenital in origin.     

A histochemical study of the innervation of the cerebral blood vessels in the domestic fowl

Summary Adrenergic and cholinergic nerves innervating the cerebral arteries of the domestic fowl were examined by specific histochemical techniques.The adrenergic nerve plexuses of the cerebral carotid system are markedly denser than those of other vertebrates observed by similar techniques. They form longitudinally elongated meshworks of fine fibres in the vascular wall of the arterial branches. Those innervating the vertebro-basilar system are less dense and more elongated, and, as the size of the artery diminishes, the fibres of the plexus become coarser. In the small pial and parenchymal arteries they are reduced to a few fibres running parallel to, or spiralling around the vascular axis.The cholinergic nerve plexuses are not as dense as the adrenergic system. The acetylcholinesterase activity is very weak, except in the plexuses innervating the cerebral carotid artery and the proximal portion of the anterior and posterior rami. In the vertebro-basilar system, a few thick nerve bundles run alongside the blood vessels of the vertebral and basilar arteries. Cholinergic nerves enter the cranial cavity along the internal carotid, the vertebral and possibly the cerebro-ethmoidal arteries.Intracerebral capillaries and some arterioles are not innervated with cholinergic and adrenergic fibres of peripheral origin, but with ones arising from parenchymal nerve cells.     

The neuronal and extra-neuronal localisations of biogenic amines in the cervical region of the domestic fowl (Gallus Gallus domesticus L.)

Summary The Falck-Hillarp technique has been used to demonstrate the neuronal and extra-neuronal localisations of biogenic amines in the cervical region of the domestic fowl. Adrenergic cell bodies were found in the superior cervical ganglion and in the ganglia of the cervical paravertebral chain. The axons of the latter ran into the corresponding spinal nerves and thus to the periphery. Very few adrenergic fibres were found in the interganglionic portions of the cervical paravertebral chain. The precarotid branch of the glossopharyngeal nerve, and the vagus nerve, below its junction with the former, contained numbers of adrenergic fibres. The retrocarotid nerve-trunk from the superior cervical ganglion was composed of adrenergic fibres. With the exception of the parathyroid gland, the adrenergic nerves seen in the branchial derivatives (thymus, thyroid and ultimobranchials) appeared to be associated with blood vessels. Under normal conditions the cells of the ultimobranchial body were nonfluorescent, but after injection of 6-hydroxydopamine the cells were brightly fluorescent. The carotid body was devoid of adrenergic nerves other than those with blood vessels, but the cells of the carotid body were brightly fluorescent. Various fluorescent cell types were found throughout the cervical region, particularly in association with the vasculature. I should like to thank Prof. G. Burnstock (Department of Zoology, Melbourne University) in whose department this work was carried out and Dr. R. D. Hodges (Wye College, London University) for his indispensable advice on the disposition of the avian ultimobranchial body. The author held a Postdoctoral Research Fellowship of the National Heart Foundation of Australia during part of this study.     

A histochemical study of the innervation of cerebral blood vessels in the snake

Summary Dual innervation of snake cerebral blood vessels by adrenergic and cholinergic fibres was demonstrated with the use of histochemical methods. Although the nerve plexuses are somewhat less dense, the essential features of innervation of the blood vessels are similar to those of mammals with the exception that the adrenergic plexuses are more prominent than the cholinergic plexuses. The major arteries of the cerebral carotid system have a rich nerve supply. However, the innervation is less rich in the basilar and poor in the spinal (vertebral) arteries. Although the arteries supplying the right side of head are poorly developed, three pairs of arteries, cerebral carotids, ophthalmics and spinals, supply the snake brain. The carotids and ophthalmics are densely innervated and are accompanied by thick nerve bundles, suggesting that the nerves preferentially enter the skull along those arteries. Some parenchymal arterioles are also dually innervated. Connection between the brain parenchyma and intracerebral capillaries via both cholinergic and adrenergic fibres was observed. In addition cholinergic nerve fibres, connecting capillaries and the intramedullary nerve fibre bundles, were noticed. Capillary blood flow may be influenced by both adrenergic and cholinergic central neurons. The walls of capillaries also exhibit heavy acetylcholinesterase activity. This may indicate an important role for the capillary in the regulation of intracerebral blood flow.     

Neuropeptide Y: presence in sympathetic and parasympathetic innervation of the nasal mucosa

Summary The occurrence of neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) in the sympathetic and parasympathetic innervation of the nasal mucosa was studied in various species including man. A dense network of NPY-immunoreactive (IR) fibres was present around arteries and arterioles in the nasal mucosa of all species studied. NPY was also located in nerves around seromucous glands in pig and guinea-pig, but not in rat, cat and man. The NPY-IR glandular innervation corresponded to about 20% of the NPY content of the nasal mucosa as revealed by remaining NPY content determined by radioimmunoassay after sympathectomy. These periglandular NPY-positive fibres had a distribution similar to the VIP-IR and PHI-IR nerves but not to the noradrenergic markers tyrosine hydroxylase (TH) or dopamine--hydroxylase (DBH). The NPY nerves around glands and some perivascular fibres were not influenced by sympathectomy and probably originated in the sphenopalatine ganglion where NPY-IR and VIP-IR ganglion cells were present. The venous sinusoids were innervated by NPY-positive fibres in all species except the cat. Dense NPY and DBH-positive innervation was seen around thick-walled vessels in the pig nasal mucosa; the latter may represent arterio-venous shunts. Double-labelling experiments using TH and DBH, and surgical sympathectomy revealed that the majority of NPY-IR fibres around blood vessels were probably noradrenergic. The NPY-positive perivascular nerves that remained after sympathectomy in the pig nasal mucosa also contained VIP/PHI-IR. The major nasal blood vessels, i.e. sphenopalatine artery and vein, were also densely innervated by NPY-IR fibres of sympathetic origin. Perivascular VIP-IR fibres were present around small arteries, arterioles, venous sinusoids and arterio-venous shunt vessels of the nasal mucosa whereas major nasal vessels received only single VIP-positive nerves. The trigeminal ganglion of the species studied contained only single TH-IR or VIP-IR but no NPY-positive ganglion cells. It is concluded that NPY in the nasal mucosa is mainly present in perivascular nerves of sympathetic origin. In some species, such as pig, glandular and perivascular parasympathetic nerves, probably of VIP/PHI nature, also contain NPY.     

Catecholamine containing nerve cells in the mammalian myenteric plexus

Summary Previous fluorescence histochemical studies have shown that extrinsic denervation causes a disappearance of adrenergic fibres from the gut wall. However, in the present work, adrenergic terminals persisted in the myenteric plexus of the guinea-pig proximal colon following interruption of paravascular nerves. Fluorescent cell bodies are found in the myenteric plexus. The fluorescence reaction of the cells does not appear after reserpine treatment and is restored by -methyl-noradrenaline.     

Degeneration of adrenergic nerves in the urinary bladder during pregnancy

The adrenergic innervation of the urinary bladder of normal female and pregnant rats has been studied using a fluorescence histochemical method. The bladder is richly innervated by adrenergic nerve fibres as is evidenced by the presence of numerous adrenergic nerves in the adventitia, musculosa and submucosa. However, adrenergic nerve cells could not be observed. During pregnancy, adrenergic nerve fibres showed signs of degeneration, as most of the nerve fibres disappeared and the surviving fibres were much swollen. 10 days after parturition the pattern and density of adrenergic innervation became almost similar to those of the control animals.