Comparative study of the autonomic innervation of the mammalian ovary,with particular regard to the follicular system

Summary The autonomic innervation of the ovary was studied in 12 mammalian species utilizing the cholinesterase method in combination with pseudocholinesterase inhibition for the cholinergic component, and glyoxylic acid histochemistry together with fluorometric determination of noradrenaline for the adrenergic component. Ovaries from cow, sheep, cat, and guinea pig were very richly supplied with adrenergic nerves in the cortical stroma, particularly enclosing follicles in various stages of development. In the follicular wall the nerve terminals were located in the theca externa, where they ran parallel to the follicular surface. Numerous adrenergic terminals also surrounded ovarian blood vessels. The adrenergic innervation was of intermediary density in the human ovary and in the pig, dog, cat, and opossum. Ovaries from rabbit, mouse and hamster had a sparse adrenergic nerve supply. The amount of intraovarian adrenergic nerves agreed well with the tissue concentration of noradrenaline in the various species. The cholinergic innervation was generally less well developed, but had the same distribution as the adrenergic system around blood vessels and in the ovarian stroma, including follicular walls.     

Hepatocyte innervation in primates

The efferent innervation and some characteristics of nerve fibers of the liver lobule in the tree shrew, a primate, are described. Nerve endings on hepatocytes were encountered regularly and were determined to be efferent adrenergic nerves. Transmission electron microscopy revealed nerve endings and varicosities in close apposition to the hepatocytes adjacent to the connective tissue of the triads as well as within the liver lobule in the space of Disse. Fluorescence microscopy indicated the existence of adrenergic nerves with a similar distribution. Autoradiography of the avid uptake of exogenous [3H]norepinephrine indicated that all intralobular nerves are potentially norepinephrinergic (adrenergic). Chemical sympathectomy with 6-OH-dopamine resulted in the degeneration of all intralobular liver nerve fibers as revealed by fluorescence microscopy and electron microscopy. Substantial regeneration occurred after 60-90 days but was not completed by that time. Some nerves were also observed in close association with von Kupffer cells and endothelial cells. The functional significance of the efferent liver innervation is discussed.     

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.     

An improved glyoxylic acid technique for the histochemical localization of catecholamines in brown adipose tissue

Summary A glyoxylic acid method using cryostat sections to demonstrate catecholaminergic fibres of the central nervous system was modified to show the extent of the adrenergic innervation in rat brown adipose tissue. It revealed prominent interlacing fluorescent parenchymal fibres surrounding individual adipocytes. The density of this network of fine fibres was not evident using earlier techniques. The new method also confirmed the dense networks of adrenergic fibres associated with arterial vessels. Its specificity was verified by simultaneously performing radioenzymatic determinations of tissue catecholamine levels and histochemical studies of brown adipose tissue from normal and sympathectomized rats. Chemical sympathectomy with 6-hydroxydopamine resulted in a pronounced decrease in brown adipose tissue and heart catecholamine (noradrenalin and dopamine) levels. Significantly, in brown adipose tissue of sympathectomized animals no fluorescence could be detected in terminal nerves of either the parenchyma or those of vascular smooth muscles. Nevertheless, some intense fluorescence was seen in axon bundles. The findings suggest that catecholamines of the parenchymal innervation form a larger proportion of the total catecholamine content of brown adipose tissue than was previously believed, provide stronger support for direct control of the function of multilocular adipocytes, and also confirm unpublished data reporting considerable dopamine content in brown adipose tissue.     

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.     

A histochemical study on the innervation of the cerebral blood vessels in bats

Summary The adrenergic and cholinergic nerves innervating the cerebral blood vessels of four species of Japanese chiropterids (Rhinolophus ferrumequinum, Murina leucogaster, Vespertilio superans and Miniopterus Schreibersi) have been investigated using specific histochemical techniques. In all these species of bats arteries of the internal carotid system are poorly developed, whereas those of the vertebro-basilar system are well developed. The adrenergic and cholinergic nerves innervating these cerebral arteries, however, all originate from the stem nerve bundles entering the cranial cavity along the internal carotid artery. Both nerve plexuses are among the densest of any vertebrate species so far investigated. Adrenergic nerve plexuses are usually composed of complicated meshworks of fine fibres, while cholinergic ones are composed of rather longitudinally arranging meshworks of both thick and thin fibres, exhibiting a very high acetylcholinesterase activity. Small parenchymal arteries and arterioles are also dually innervated by adrenergic and cholinergic nerve fibres of peripheral origin. Intracerebral capillaries, on the other hand, are in several places directly connected with both adrenergic and cholinergic fibres of parenchymal origin. Capillaries in the cerebral and cerebellar cortices, diencephalon and cochlear nucleus in V. superans exhibit a heavy non-nervous acetylcholinesterase activity in their walls, but in R.ferrumequinum and M. schreibersi, the response is weak or negative, except for that in the cochlear nucleus.     

Phylogenetic and ontogenetic study of neuropeptide Y-containing nerves in the liver

Summary The distribution of neuropeptide Y was investigated by light and electron microscopic immunohistochemistry in the liver of various vertebrates including the eel, carp, bullfrog, turtle, chicken, mouse, rat, guinea-pig, dog, monkey and human. The ontogenetic development of neuropeptide Y was also studied in the mouse liver. In all species examined except the eel, neuropeptide Y-like immunoreactivity was detected in nerve fibres. In the carp, bullfrog, turtle, chicken, mouse and rat, positive fibres were distributed around the wall of hepatic vessels and the bile duct of the Glisson's sheath. The density of the positive fibres increased with evolution. On the other hand, in the guinea-pig, dog, monkey and human, numerous neuropeptide Y-positive fibres were observed not only in the Glisson's sheath but also in the liver parenchyma. Positive fibres formed a dense network to surround hepatocytes. The present immunoelectron microscopic study has confirmed that neuropeptide Y-positive terminals are closely apposing to hepatocytes. Ontogenetically, neuropeptide Y-positive fibres were first found in embryonic liver of 19-day-old mice. Positive fibres increased with age and the highest peak was seen one week after birth. This ontogenetic pattern has suggested that neuropeptide Y plays a certain role in developing liver.     

Organization of the sympathetic innervation in liver tissue from monkey and man

Summary The sympathetic innervation of the liver of monkey and man has been investigated in a combined fluorescence histochemical, chemical and electron microscopical study. By means of the Falck-Hillarp fluorescence method a dense network of monoamine-containing nerve fibers was visualized in liver tissue of monkey and man. The nerve fibers ran in close contact to both hepatocytes and blood vessels. Chemical quantitations showed high concentrations of noradrenaline in both human and monkey liver. Microspectrofluorometry of the intraneuronal monoamine resulted in spectra characteristic of a catecholamine. For the electron microscopical study the dopamine analogue, 5-hydroxydopamine, was used to label the catecholamine terminals in both human and monkey liver. The nerve profiles, identified as catecholamine-containing, were demonstrated in a perivascular location and in close contact to hepatocytes. No synaptic membrane specializations were present between nerve fibers and hepatocytes. The general ultramorphology and intralobular distribution pattern of nerves in the liver of monkey and man were similar. The present results prove the existence of a sympathetic innervation of hepatocytes and blood vessels in the liver of man and monkey.     

Innervation of the bovine naso-labial glands

An investigation was carried out on the innervation of bovine naso-labial glands. Histochemical techniques were used to establish the presence and distribution of cholinergic fibres and peptidergic fibres containing VIP and Substance P were revealed by immunohistochemical methods. Parasympathetic nerve fibres were localized around the secretory units and intralobular excretory ducts. VIPergic nerve fibres, the only peptidergic fibres encountered, were arranged in a similar pattern. Adrenergic fibres were only seen in the periglandular connective tissue around blood vessels of medium size. We are, therefore, of the opinion that the secretory activity is mainly regulated by the parasympathetic nerves and that the VIP acts as a modulator.     

Phylogenic study of calcitonin gene-related peptide-immunoreactive structures in the pancreas

 The immunohistochemical localization of calcitonin gene-related peptide was examined, at both light and electron microscopic levels, in the pancreas of various vertebrates, including the eel, bullfrog, turtle, chicken, mouse, rat, guinea pig, dog, monkey, and human. Immunoreactive staining was observed in nerve fibers in every animal species examined, but positive endocrine cells were limited to the rat, monkey, and human. The density of the positive endocrine cells varied considerably among the three species (monkey > rat > human). Positive nerve fibers were distributed throughout the parenchyma, being particularly rich around pancreatic ducts, and near large or small blood vessels. In four species (eel, mouse, rat, and dog), positive nerve fibers formed a dense network in the islet region. There were positive varicose nerve fibers around exocrine cells. These fibers, varying in density in different species (relatively high in the eel, bullfrog, and rat), were sometimes adjacent to acinar cells. At the electron microscopic level, positive nerve terminals were often demonstrated in close apposition to the outer membrane of acinar cells. The eel pancreas revealed an exceptional pattern of staining in neuronal cell bodies that were scattered in the interlobular connective tissue. Despite these anatomical differences, the omnipresence of this peptide suggests its essential role(s) in the pancreas. Accepted: 12 June 1997     

Adrenergic and cholinergic nerves of bovine,guinea pig and hamster salivary glands

Summary The distribution of formaldehyde-induced fluorescence and acetylcholine-esterase (AChE) activity was histochemically investigated in certain salivary glands of the cow (submandibular gland), guinea pig and hamster (submandibular and sublingual glands). Adrenergic nerves occur around the secretory acini of the bovine, guinea pig and hamster submandibular glands, as well as around those of the hamster sublingual gland. The mucous secretory acini of the guinea pig sublingual gland, however, seem to be devoid of adrenergic nerve supply. Except in the sublingual gland of the hamster, no adrenergic nerves occur in relation to duct cells.The pattern of AChE activity is similar to that of adrenergic nerves. Thus, AChE-positive nerves form a network around secretory acini of all the five glands examined. Furthermore, AChE activity was also observed in nerve fibres in close proximity to striated duct cells.Both adrenergic and AChE-containing fibres were observed around blood vessels of different sizes. Ganglionic cells are occasionally to be seen; they all display AChE-activity. No adrenergic ganglionic cells were observed in any of the glands examined.All glands were also studied in the electron microscope. Interest was focussed on the fine structure of the autonomic nerves with special reference to their contents and type of storage vesicles.The content of noradrenaline was chemically determined in each type of salivary gland studied.This work was supported by grants from the University of Umeå and from the Swedish Society for Medical Research and was also carried out within a research organization supported by the Swedish Medical Research Council (projects B73-04X-712-08C and B73-04X-56-09C). The authors are indebted to Miss Kristina Karlsson and Miss Marianne Borg for valuable technical assistance.     

The origin and distribution of adrenergic nerve fibres in the guinea-pig colon

Summary A detailed study of the origin and distribution of sympathetic fibres in the distal colon of the guinea-pig has been made using the fluorescent histochemical method for localizing catecholamines. The extrinsic adrenergic fibres of the colonie sympathetic nerves follow the inferior mesenteric artery and its branches to the colon. Some of the extrinsic adrenergic fibres are associated with the parasympathetic fibres of the pelvic nerves near the colon. Complete adrenergic denervation follows the removal of the inferior mesenteric ganglion or the destruction of the nerves running with the inferior mesenteric artery.No fluorescent fibres, other than those associated with blood vessels, were observed in air-dried stretch preparations of the isolated longitudinal muscle. However, a substantial number of varicose, terminal fibres, not associated with blood vessels, were observed in the circular muscle. Some varicose fibres, apart from those associated with ganglion cells, were observed in the myenteric plexus. These fibres were seen in the bundles of nerves running between the nodes of the plexus and also as single fibres which branched from the plexus to end in areas free of ganglion cells.Three plexuses of adrenergic nerve fibres have been distinguished in the submucosa: a dense plexus of terminal fibres innervating both the veins and arteries; a plexus consisting of innervated nodes of ganglion cells, connected by bundles of fluorescent and non-fluorescent nerves; and a plexus of varicose and non-varicose fibres, which is not associated with ganglion cells. Some groups of ganglion cells in the submucosa were without adrenergic innervation.A plexus of varicose fibres forms a meshwork in the lamina propria of the mucosa. The muscularis mucosae is sparsely innervated. Most of the blood vessels in the mucosa are not associated with adrenergic fibres.     

Study of the innervation of blood vessels of the digestive organs by fluorescence microscopy]

The blood vessels of the samll intestine and the gallbladder were shown to possess a great amount of adrenergic nerve fibres which, when penetrating the thickness of the wall of the above organs, become thinner and the distributed between the tissue structures of the organs as the thinnest monoaxonal network. The method of Falck--Hillarp--Krokhina was used. Among the vessel nerves there are perivascular nerves accompanying the vessels along their total legnth, juxtavascular and intramural nervous bundles of the sumpathetic nature detected by the fluorescent-microscopy method. Large arteries are disposed in a considerably thicker network of specifically fluorescing fibres than veins and small arteries.     

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.     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.     

Galanin-immunoreactive nerves in the mouse and rat pancreas

Summary Galanin-containing nerve fibers have previously been observed in the human, dog, and pig pancreas. Whether the mouse and rat pancreas also contain galanin nerve fibers has been a matter of debate. Therefore, we examined the distribution of galanin in the mouse and the rat pancreas. Further, the possible localization of galanin to adrenergic nerves was studied using sequential immunostaining for galanin and tyrosine hydroxylase (TH). In the mouse pancreas, numerous galanin-immunoreactive (GIR) nerve fibers occurred around blood vessels. They were less numerous in the exocrine parenchyma and in association with the islets. In contrast, in the rat pancreas, only a few GIR nerves were found. They were located around blood vessels and scattered in the exocrine parenchyma. Occasionally, GIR nerves were also observed in the islets. There was a dense distribution of TH-immunoreactive fibers in both the mouse and the rat pancreas. Sequential immunostaining revealed co-localization of galanin and TH immunoreactivity in nerve fibers in both the mouse and the rat pancreas. Following chemical sympathectomy using 6-hydroxydopamine (6-OHDA), not all GIR nerves disappeared. In the mouse pancreas a remaining population of galanin nerves was found around blood vessels, and occasionally in the islets. In the rat pancreas, a few GIR nerves were seen also after chemical sympathectomy. We conclude that intrapancreatic GIR nerves also occur in the mouse and the rat. These findings suggest that many of the GIR nerves are adrenergic but that non-adrenergic, possibly intrinsic or sensory GIR nerves exist as well in both the mouse and the rat pancreas.     

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.     

Adrenergic innervation of the lower urinary tract in cattle

Studies were carried out on 8 sexually immature male calves. Sections of the ureters, urinary bladder, and urethra were cut with a freezing microtome and the method of Ky?s?la et al. (1980) was used to visualize the adrenergic nerve fibres. It was found that bovine ureters possessed weak innervation; most of the nerves was located in the muscular membrane, and only in the paravesical part, sparse nerve fibres were found in the submucosa of this one. Apex of the urinary bladder was more weakly supplied with the adrenergic nerves than the corpus, whereas bladder's trigonum and cervix possessed numerous nerve fibres in both muscular and submucosal membranes. The distribution pattern of adrenergic nerves in the urethra was similar to that of urinary bladder's cervix. The presence of adregeneric nerve fibres was found in submucosal layer of both the urinary bladder and the urethra. Part of the nerves was connected with blood vessels of the organs under study.     

The adrenergic innervation of the guinea pig ovary during prenatal and postnatal periods

The pattern of adrenergic nerves was visualized and norepinephrine (NE) quantified in guinea pig ovaries from fetal day 50 to term and on days 1, 5, 10, 20 and 28 postnatally. Before birth, there were a few perivascular adrenergic nerves and correspondingly low ovarian NE levels. After birth and through day 20, nerves gradually grew into adjacent stroma. By day 28, there was a proliferation of beaded adrenergic nerves around blood vessels and into adjacent stroma, which was reflected by significantly increased ovarian levels of NE. These findings are discussed in relation to antral follicle development, atresia, and interstitial gland formation in the ovaries of prepubertal guinea pigs.     

Neuropeptide Y-like immunoreactivity in perivascular nerve fibres of the guinea-pig

The distribution of perivascular nerve fibres displaying neuropeptide Y-like immunoreactivity was studied in the guinea-pig. Generally, neuropeptide Y fibres were numerous around arteries and moderate in number around veins. In the heart, immunoreactive fibres were numerous in the auricles and the atria (epi- and endocardium) whereas the ventricles had a more scarce supply. The coronary vessels were richly supplied with fibres. Around large elastic and muscular arteries the fibres formed well developed plexuses. Small arteries in the respiratory tract, the gastrointestinal tract and the genito-urinary tract received a particularly rich supply. In the liver, spleen and kidney only few perivascular fibres were seen. Since immunoreactive fibres around blood vessels disappeared upon surgical or chemical sympathectomy, and sequential immunostaining with antisera against dopamine-beta-hydroxylase (a marker for adrenergic neurons) and against neuropeptide Y revealed their co-existence, it is concluded that neuropeptide Y fibres around blood vessels are sympathetic and adrenergic.