Decentralisation of neurones in the pelvic ganglion of the guinea-pig: Reinnervation by adrenergic nerves

An electron-microscopic study has been made of adrenergic and cholinergic nerve fibres and synapses in the pelvic ganglion of the guinea-pig at intervals of up to 60 days following section of the hypogastric and pelvic nerves. Transection of the hypogastric nerves led to degeneration of 80-90% of the cholinergic nerve profiles and synapses in the ganglion. The small number of adrenergic nerves and synapses did not change, but 30-60 days after section, this number increased 8-10 times. Transection of the pelvic nerves led to degeneration of about 15% of the cholinergic nerve terminals, but no change in adrenergic terminals. After transection of both hypogastric and pelvic nerves, only about 1% of cholinergic nerves survived, but after 30-60 days, the number of adrenergic nerves increased 8-10 times. It is concluded that following cholinergic nerve degeneration in the ganglion, adrenergic nerves, probably originating as collateral sprouts from postganglionic neurones and granule-containing cells, can replace them to some extent.     

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.     

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.     

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.     

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.     

Cholinergic nerves in the human liver

Summary The cholinergic innervation of the human liver was studied. Slices (150–200m thick) of human liver and of the greater hepatic blood vessels (hepatic artery and vein, portal vein) were incubated in a solution of 6-hydroxydopamine (6-HDA) in order to obtain a selective degeneration of adrenergic nerves. Controls were prepared from samples incubated with buffer alone. The slices were cut on a cryostat into 15–20m thick sections and processed for the histochemical detection of cholinesterases.Cholinergic nerve fibres innervate the extra hepatic and the intrahepatic branches of the hepatic artery, the portal vein as well as the hepatic vein. Fewer cholinergic fibres innervate the hepatocytes and the hepatic sinusoids. The 6-HDA treatment does not seem to alter the pattern of the cholinergic innervation of the liver. The findings indicate the presence of a cholinergic parasympathetic innervation in the human liver.     

Possible axo-axonal synapses between peripheral adrenergic and cholinergic nerve terminals

Summary The relations between adrenergic and cholinergic terminals were studied in rat iris and rat heart with the electron microscope. Adrenergic terminals were identified by treating the animals with 5-hydroxydopamine, which produces dense-cored synaptic vesicles in adrenergic terminals in tissues fixed in glutaraldehyde and osmium. The specificity of this observation was verified. It was found that adrenergic and cholinergic nerve terminals often come in close contact with one another, the distance between the adjoining membranes being about 250 Å. At times, faint membrane thickenings could be observed in these places. The available pharmacological, physiological, and morphological evidence leaves little room for doubt that cholinergic terminal fibres can influence the adrenergic fibres. From mainly morphological evidence, it is also postulated that adrenergic terminals influence cholinergic ones.This work was supported by grants from the United States Public Health Service (06701-04), the Faculty of Medicine, University of Lund, Lund, Sweden and the Swedish Medical Research Council (B70-14X-2321-03 and B70-14X-712-05). Svenska sällskapet för medicinsk forskning.     

Cholin- and adrenergic components of the vegetative innervation of the dura mater]

Cholin- and adrenergic nerves in the fornix and base of the dura mater of rats, cats and dogs have been studied by methods of Kelle, Falck and Glenner. It has been established that the dura mater has a developed cholin- and adrenergic nervous apparatus innervating arteries, veins and the connective tissue of the mater. The concentration of nerve fibres is always greater on a meningea media and its daughter branches. The statistical processing of the data obtained has shown that the maximum quantity of nerve fibres is in cats, less in dogs and still less in rats. It has been established that in the dura fornix of cats and dogs there are more cholinergic nerve fibres than in the base. In rats there is no such difference. The amount of fibres with monoaminoxidase approximately corresponds to the amount of conductors with noradrenaline.     

Perivascular nerves in the feline carotid rete

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

Innervation of the ureterovesical junction musculature of man

The ultrastructure of the innervation of the human ureterovesical junction was studied. Three different nerve terminals were distinguished among the smooth muscle cells. 1. Nerve processes containing predominantly small granular vesicles (40--60 nm in diameter). 2. Other nerve fibres contained predominantly small round agranular vesicles (30--50 nm in diameter). 3. Processes with large granulated vesicles (80--120 nm in diameter). The first type may be adrenergic, the second cholinergic and the third may originate from the local nerve cells. The gap between the nerve fibres and muscle cells was 300 to 500 nm wide and no synaptic thickenings were observed. This suggests that the transmitter may influence several muscle cells, and the different nerve fibres may directly innervate the smooth muscle cells.     

Nerves containing substance P,vasoactive intestinal polypeptide,enkephalin or somatostatin in the guinea-pig taenia coli

Summary The guinea-pig taenia coli is rich in peptide-containing nerves. Nerve fibres containing substance P (SP), vasoactive intestinal peptide (VIP), or enkephalin, were numerous in the smooth muscle while somatostatin fibres were very few. Nerve fibres displaying SP or VIP immunoreactivity were numerous in the myenteric plexus. Enkephalin nerve fibres were fairly numerous in the plexus while somatostatin nerve fibres were sparse. Nerve cell bodies containing immunoreactive SP or VIP were regularly seen in the plexus. Delicate varicose elements of the different types of nerve fibres were found to ramify around nerve cell bodies in a manner suggestive of innervation.In the electron microscope the various peptide-storing nerve fibres (i.e., elements containing SP, VIP or enkephalin) were found to contain a varying number of fairly large, electron-opaque vesicles in the varicose swellings. These vesicles represent the storage site of the neuropeptides.The isolated taenia coli responded to electrical nerve stimulation with a contraction. After cholinergic and adrenergic blockade the contractile response was replaced by a relaxation followed by a contraction upon cessation of stimulation. SP contracted the taenia while VIP caused a relaxation. The enkephalins raised the resting tension slightly while somatostatin had no effect. These observations are compatible with a role for SP as an excitatory neurotransmitter and for VIP as an inhibitory one, and with the view that both SP neurones and VIP neurones act as motor neurones. In preparations contracted by SP the electrically induced contractions were reduced in amplitude while the electrically induced relaxations seen after adrenergic and cholinergic blockade were enhanced in amplitude. In preparations relaxed by VIP there was an increased contractile response to electrical stimulation, while in the atropine + guanethidine-treated preparation the electrically induce relaxations were reduced in amplitude. The enkephalins reduced the contractile response to electrical stimulation, while somatostatin induced a very small reduction in the amplitude of such responses. These observations suggest that SP neurones and VIP neurones may play additional roles as interneurones. Somatostatin neurones probably act as interneurones. Enkephalin-containing fibres may serve to modify the release of transmitter from other nerves in the smooth muscle, perhaps through axo-axonal arrangements. Alternatively, the enkephalin nerve fibres in the smooth muscle are afferent elements involved in mediating sensory impulses to the myenteric plexus.     

Differences in the chemical coding of nerve fibres supplying major populations of neurons between the caudal mesenteric ganglion and anterior pelvic ganglion in the male pig

The present study was designed to investigate and to compare the chemical coding of nerve fibres supplying major populations of neurons in the caudal mesenteric (CaMG) and anterior pelvic (APG) ganglion in juvenile male pigs (n=5) using double-labelling immunofluorescence. The co-existence patterns of some biologically active substances including tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT) as well as vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), Leu5-enkephalin (LENK) and serotonin (5-HT) were analysed under a confocal laser scanning microscope. Profound differences in the neurochemical features of the nerve terminals between the ganglia were observed. Moreover, there were also distinct differences in the chemical coding of nerve fibres associated with the particular populations and subpopulations of neurons within the ganglia. In both ganglia, nearly all adrenergic and cholinergic neurons were supplied with VAChT-positive nerve fibres (putative preganglionic fibres). However, in the CaMG, they were more numerous and, in contrast to the APG, many of them also stained for VIP. In the APG, a great number of nerve terminals expressed immunoreactivity to SP and CGRP (putative collaterals of sensory neurons). Interestingly, they densely supplied almost exclusively adrenergic neurons. SP-positive nerve fibres were moderate in number in the CaMG, but, in addition to VAChT-IR nerve terminals, the most numerous populations of nerve fibres in this ganglion were those expressing highly colocalized immunoreactivities to CGRP and LENK, and those which stained for 5-HT (putative processes of enteric neurons). However, these fibres supplied almost exclusively larger, intensely stained for TH and clustered adrenergic neurons. This diversity of the nerve terminals reflects the complexity of nerve circuits involved in the innervation of structures supplied by neurons in the porcine CaMG and APG. It also demonstrates the importance of nerve inputs for the proper function of autonomic neurons and thus their target tissues.     

A comparative characteristic of effector innervation of cerebral arteries in mammals and humans.

The neural fibres of cerebral arteries in humans, rats, guinea-pigs, rabbits, cats, dogs, swine, cows and horses have been studied using the methods of Flack-Hillarp and Koelle. The large arteries of the carotid and vertebral systems bear dense cholinergic and adrenergic plexuses formed by transversal and longitudial neural fibres, which are located in the superficial and deep adventitial layers. The highest density of cholinergic and adrenergic fibres was observed on the arteries of humans and large mammals. Depending on the density of cholinergic and adrenergic fibres in the said arteries, the animals may be listed in the following order: rats, guinea-pigs, rabbits, cats, dogs, swine, cows and horses. Basic differences in the structure of neural plexuses of humans and animals were not observed.     

Fine structural and cytochemical study of the innervation of smooth muscle in an amphibian (Bufo marinus) lung before and after denervation

Summary The innervation of the toad (Bufo marinus) lung was studied with transmission electron microscopy and fluorescence techniques, both before and after 12 or 20 days close vagosympathetic denervation. Four cytologically distinct types of neuronal processes were recognised, in relation to the visceral muscles of the lung. These were described as cholinergic, adrenergic, nonadrenergic/non-cholinergic (NANC) and sensory on the basis of the characteristics of their vesicular content and cytochemical reactions. An apparent efferent innervation of visceral smooth muscle was achieved by NANC (50%), cholinergic (25%) and adrenergic (25%) fibres. A few sensory fibres were also present. After denervation only NANC fibres persisted, showing that the cell bodies of these fibres were intrapulmonary. The vascular smooth muscle was supplied by cholinergic, adrenergic and sensory fibres. In the walls of the proximal branches of the pulmonary artery were fibres containing large dense-cored vesicles. These profiles, which were associated with the vasa vasorum, were similar to neurosecretory fibres. After denervation all neural profiles associated with the vasculature had degenerated. The observations suggest that vagal vasodepressor effects in the toad lung are mediated indirectly through relaxation of visceral muscle strands which in their contracted state compress vascular channels.The authors would like to thank Dr. J.R. McLean for technical advice on fluorescence microscopy. This work was supported by a grant from the Australian Research Grants Committee     

Neurogenic inflammation of gingivomucosal tissue in streptozotocin-diabetic rat

Previously it was assumed that nerve fibres are involved in the neurogenic inflammation induced by mechanical or chemical irriations. It has been also suggested that in diabetes mellitus the unmyelinated small diameter fibers are impaired as a result of diabetic neuropathy. Therefore, our aim was to study the alterations of the nerve processes in the gingivomucosal tissue in streptozotocin (STZ)-diabetic rats. Light- and electronmicroscopical examinations were made to analyze the changes in nerve fibres. After one week of steptozotocin treatment, the gingivomucosal tissue had inflammatory cell infiltration and some degenerated nerve fibres were also observed. Dense mitochondria, disorganization of cell organelles, and appearance of myelin-like dense bodies were found in the axons of degenerared nerve fibres. Semiquantitative analysis showed that 14 +/- 4% of the unmyelinated nerve fibres degenerated after one week of STZ treatment. However, degeneration of the myelinated nerve fibers was not observed. Two weeks after STZ treatment, most of the unmyelinated and myelinated nerve fibers showed degeneration (86 +/- 5%) and the placement of the ligature revealed a non-inflammatory connective tissue adjacent to a normal epithelium. The myelin sheath was disrupted and dark axoplasm with cytolysosomes became manifest. These findings demonstrated that both unmyelinated and myelinated nerve fibers are altered and inflammatory reaction exists in the gingivomucosal tissue only in the early stage of diabetes mellitus.     

Simultaneous demonstration of adrenergic and non-adrenergic nerve fibres

Summary A technique for simultaneous demonstration of adrenergic and non-adrenergic nerve fibres is described, using methylene blue staining and fluorescence microscopy after formaldehyde treatment. The procedure is applicable to whole mounts as well as to microtome sections.     

The state of adrenergic and cholinergic fibers in the heart of rabbits subjected to sensitization and anaphylactic shock

It was determined by neurohistochemical methods that after sensitization and anaphylactic shock the adrenergic and cholinergic nervous structures of the heart change. The lowered fluorescence intensity, alterations in density of the adrenergic nervous structures of the heart are observed following the fourth sensitizing injection of normal horse serum. These changes are more obvious on day 7 after anaphylactic shock. The changes in the structure of cholinergic nerve fibers were noted only on day 7 after anaphylactic shock. They manifested in lowered activity of histochemical reaction of nerve elements to acetylcholine in all the regions of the heart.     

Increased Activity of Choline Acetyl-transferase in Sympathetic Ganglia after Prolonged Administration of Nerve Growth Factor

A prolonged increase in the activity of preganglionic sympathetic nerves produces characteristic changes in the enzyme pattern of the postganglionic adrenergic neurones in adult rats^1–4. In newborn mice the normal development of adrenergic neurones depends materially on the intactness of the preganglionic cholinergic nerves⁵. These two findings contribute to the definition of the so far vague term “trophic response to neuronal activity” and the question arises whether these trophic actions are unidirectional only, or whether there is also a retrograde effect, that is, a dependence of the preganglionic cholinergic nerves on the function of the postjunctional adrenergic neurones. Transection experiments in the brain have shown that after lesioning of particular areas, for example, optic nerve tract, cingulate or visual cortex, there occurs not only orthograde but also retrograde trans-synaptic degeneration⁶. It seemed of interest to study this possible retrograde trophic effect in a less complex system and to investigate whether there are not only negative (degeneration) but also positive effects. The administration of nerve growth factor (NGF) to newborn rats produces a marked growth and enhanced differentiation of the adrenergic neurones⁷. The biochemical correlate to these morphological changes is a selective induction of tyrosine hydroxylase and dopamine β-hydroxylase⁸. We have investigated whether these effects are also reflected by changes in the preganglionic nerves.     

Interneuronal synapses in the local ganglia of the cat urinary bladder.

The interneuronal synapses of the urinary bladder in the cat were studied by electron microscopy. The great majority of the fibres containing vesicles are found within the ganglia occurring in the trigonum area. Morphologically differentiated synaptic contacts could be observed on the surface of the local neurons and between the different nerve processes. The presynaptic terminals can be divided into three types based on a combination of synaptic vesicles. Type I terminals, presumably cholinergic synaptic terminals, contain only small clear vesicles of 40-50 nm in diameter. Type II terminals, presumably adrenergic terminals, are characterized by small granulated vesicles of 40-60 nm in diameter. Type III terminals, probably of local origin, contain a variable number of large granulated vesicles of 80-140 nm in diameter. Occasionally, a single nerve fibre contacted several (two or four) other nerve processes forming a typical synapse. In other cases, on one nerve cell soma or on other nerve processes there are two or three different-type nerve terminals establishing synapses. It might be inferred from these observations that convergence and divergence can occur in the local ganglia and that cholinergic and adrenergic synaptic terminals can modulate the ganglionic activity. However, a local circuit also can play an important role in coordinating the function of the bladder.