Adrenergic pattern of aorta and superior mesenteric arteries in young and senescent rats

The noradrenergic innervation of two portions of the aorta (thoracic and abdominal) was studied by the fluorescence method in rats belonging to two different age groups (3 and 28 months). Adrenergic fibres were clearly visible only in the abdominal tract of the aorta, and appeared to be more distinct and more numerous in young rats compared with the older ones. By contrast, the superior mesenteric artery was regularly found to possess a rich adrenergic plexus, even if catecholamines were more densely present in younger animals.     

Ultrastructure of the red pulp in spleen innervation in horse and pig.

The innervation of the red pulp in the spleen of horse and pig was investigated by electron microscopy. In addition, the neurilemma was studied by immunohistochemistry specific for S-100 protein. In the pig, a large population of smooth-muscle cells extending from the smooth-muscle trabeculae was present in the red pulp. The cytoplasmic processes of reticular cells enwrapped the smooth-muscle cells, and nerve fibres were distributed between the smooth-muscle cells and the reticular cells. The nerve terminals clustered toward the facing of the smooth-muscle cells. Nerve fibres and terminals were not disclosed within the sheathed artery. Immunohistochemically the neurilemma showed a reaction positive for S-100 protein. In the horse, no smooth-muscle cells were noted in the red pulp. The nerve fibres terminated around the cytoplasmic processes of the reticular cells. Nerve fibres and terminals were disclosed within the sheathed artery, and the terminals contained both large and small dense-cored vesicles. Immunohistochemically the neurilemma showed a reaction negative for S-100 protein. These findings support the presence of the axon-bearing reticular cells described earlier in the horse spleen.     

Distribution of dopamine-beta-hydroxylase-like immunoreactivity in the rat pineal organ

The aim of the present investigation was to study the distribution in the rat pineal gland of dopamine-beta-hydroxylase (DBH) which is essential for the formation of the melatonin synthesis-regulating substance noradrenaline (NA). In 5- and 8-month-old male Sprague-Dawley rats DBH-like immunoreactivity (DBH-LI) was studied using polyclonal antibodies against DBH and the indirect immunofluorescent technique. DBH-LI was mainly located in pineal nerve fibres coming from the superior cervical ganglia. The intensity of the staining reaction was considerably lower than in non-pineal noradrenergic nerve fibres and the impression was gained by comparison of DBH-LI specimens with glyoxylic acid-treated sections that only approximately one third of the NA-containing intrapineal nerve fibres exhibited DBH-LI. There were no detectable differences in DBH-LI with regard to time of day and age of the animals. These results suggest that NA synthesis may be relatively low in intrapineal sympathetic nerve fibers and that the NA required for the regulation of pineal melatonin synthesis may, to a large degree, stem from the circulation. In addition to nerve fibres, some rare intrapineal cell bodies exhibited DBH-LI; in 5-month-old rats their numbers did not reveal significant differences between day and night. These cells do not appear to represent pinealocytes. They may be a special population of noradrenergic nerve cells perhaps belonging to an as yet unknown intrapineal regulatory system.     

An investigation of a sympathetic innervation of the vertebral artery in primates: is there a neurogenic substrate for vasoconstriction?

Vasoconstriction of the vertebral artery may be neurogenic in origin. Although the existence of a perivascular sympathetic plexus of the vertebral artery is not in doubt, no method used to date has conclusively demonstrated a direct sympathetic innervation of the vascular smooth muscle cells and, hence, vasomotor function. It was the aim of this study, therefore, to visualise and localise noradrenergic fibres in the wall of the vertebral artery. Intracranial vertebral artery specimens (10 vervet monkeys and 10 baboon vessels) were sectioned (40 mm serial sections) and treated with anti-tyrosine hydroxylase, anti-dopamine b-hydroxylase, and anti-chromogranin-A antibodies. Some evidence of catecholaminergic fibres in the tunica adventitia but not penetrating the external elastic lamina or tunica media of the vertebral artery wall was seen. These findings were confirmed by electron microscopy. It was concluded that although a perivascular sympathetic plexus exists, the vertebral artery of primates was not shown to have a direct sympathetic innervation and a neurogenic vasoconstrictor function is unlikely.     

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.     

Noradrenergic innervation of the heart in untreated and daunomycin-pretreated animals

Summary The innervation of guinea-pig and rat cardiac tissue consists of thin noradrenergic axons running parallel to the myocardial fibres and producing well-developed fluorescent networks which are denser in guinea-pig tissue. Transverse sections of arterial vessels exhibit a marked polar-like disposition of adrenergic nerves, which mainly appear to be concentrated in two opposite areas. Tissue sections from daunomycin-treated animals fluoresced a striking organge-red. This drug-specific fluorescence emanated from all parts of the ventricular tissue, with particular intensity in the nuclei. Neither the density nor the shape of the adrenergic nerves were affected by treatment with the drug. Despite penetration and storage of the antibiotic into the ventricular tissue, cardiotoxicity was not found in the present study.     

Elimination of cobalt from the frog brain introduced into the optic centres through the optic nerve.

One optic nerve in several frogs was filled with cobaltous-lysine complex, and the animals were left to survive from 1 day to 52 days. Degenerated cobalt-filled retinal fibres were phagocytosed by ependymo-glial, and microglial cells. The cobalt appeared in the ependymo-glial cells in the 4th postoperative day, and its amount was greatly reduced by the 52nd day. Within 12 days the labelled axons were replaced by cobalt-loaded microglial cells in the termination sites of optic fibres. By the end of the experimental period, the number of labelled cells increased in the periventricular layers, and decreased in places where retinal fibres had terminated. These processes were accompanied by the appearance of cobalt in the choroid plexus. It is supposed that glial cells dischargd the cobalt into brain ventricles, and the metal left the nervous tissue via the cerebrospinal fluid.     

Exhaustive physical exercise and acid hydrolase activity in mouse skeletal muscle. A histochemical study.

Adult, untrained NMRI mice were exhausted on a motor-driven treadmill by an intermittent-type running programme. Serial cryostate sections for the staining of NADH-tetrazolium reductase, beta-glucuronidase, beta-N-acetylglucosaminidase, and beta-glycerophosphatase activities and for making hematoxylin-eosin staining were cut from m. quadriceps femoris 1, 2, 3, 5, 7, and 15 days after physical exhaustion. A strong increase in the activities of beta-glucuronidase and beta-N-acetylglucosaminidase was observed 7 days after exhaustion and the activity changes, which were similar for the both glycosidases, were more prominent in the highly oxidative red compared to less oxidative white fibres. Activity granules were more numerous in the perinuclear than the interfibrillar area of red fibres. Spots were arranged like longitudinal chains between myofibrils. Activity in connective tissue was usually observed only in animals exhausted 3--7 days earlier. Simultaneous activity in fibres exceeded that in connective tissue. beta-Glycerophosphatase activity was not, by the method used, seen in histologically "healthy" or normal-looking fibres. In samples taken 2--5 days after exhaustion some degenerating and necrotic fibres were observed. Inflammatory reaction was also observed being at its strongest five days after loading when mononuclear cells were seen inside necrotic fibres. The number of regenerating muscle cells was most abundant 7 days after exhaustion. It is suggested that temporary hypoxia, which accompanies exhaustive physical exercise in skeletal muscle, upsets the energy metabolism and homeostasis of fibres and causes the observed histological and histochemical alterations, which possess features typical of both lethal and sublethal acute cell injury.     

Autonomic innervation of receptors and muscle fibres in cat skeletal muscle

Cat hindlimb muscles, deprived of their somatic innervation, have been examined with fluorescence and electron microscopy and in teased, silver preparations; normal diaphragm muscles have been examined with electron microscopy only. An autonomic innervation was found to be supplied to both intra- and extrafusal muscle fibres. It is not present in all muscle spindles and is not supplied at all to tendon organs. Fluorescence microscopy revealed a noradrenergic innervation distributed to extrafusal muscle fibres and some spindles. On the basis of the vesicle content of varicosities the extrafusal innervation was identified as noradrenergic (32 axons traced), and the spindle innervation as involving noradrenergic, cholinergic and non-adrenergic axons (14 traced). Some of the noradrenergic axons that innervate spindles and extrafusal muscle fibres are branches of axons that also innervate blood vessels. We cannot say whether there are any noradrenergic axons that are exclusively distributed to intra- or extrafusal muscle fibres. The varicosities themselves may be in neuroeffective association with striated muscle fibres only, or with both striated fibres and the smooth muscle cells in the walls of blood vessels. The functional implications of this direct autonomic innervation of muscle spindles and skeletal muscle fibres are discussed and past work on the subject is evaluated.     

Immunocytochemical localization of a growth-associated protein (GAP-43) in rat adrenal gland

We have localized at light and electron-microscopic level the growth-associated protein GAP-43 in adrenal gland using single and double labelling immunocytochemistry. Clusters of GAP-43-immunofluorescent chromaffin cells and many immunofluorescent fibres were observed in the medulla. GAP-43-immunoreactive fibres also formed a plexus under the capsule, crossed the cortex and ramified in the zona reticulata. Double labelled sections showed the coexpression of GAP-43 with a subpopulation of tyrosine hydroxylase-and of dopamine--hydroxylase-immunoreactive chromaffin cells. Dual colour immunofluorescence for GAP-43 and calcitonin gene-related peptide (CGRP) revealed that some of the GAP-43-immunoreactive fibres also express CGRP. Pre-embedding electron microscopy showed GAP-43 immunoreactivity associated with the plasma membranes and cytoplasm of noradrenaline-producing chromaffin cells, and with processes of nonmyelin-forming Schwann cells. Immunoreactive unmyelinated axons and terminals were also observed. The immunostained terminals made symmetrical synaptic contacts with chromaffin cells. Immunoreactive unmyelinated fibres and small terminals were present in the cortex. Our results show that GAP-43 is expressed in noradrenergic chromaffin cells and in various types of nerve fibres that innervate the adrenal. Likely origins for these fibres include preganglionic sympathetic fibres which innervate chromaffin cells, postganglionic sympathetic fibres in the cortex, and CGRP containing sensory fibres.     

Noradrenergic and acetylcholinesterase-positive nerve fibres of the uterus in sexually immature and cycling rats

Summary The distribution and density of the noradrenergic and acetylcholinesterase-positive nerve fibres were histochemically studied in different uterine regions of prepubertal and cycling rats in dioestrus and oestrus. Besides the rich and double innervation of blood vessels, both types of nerve fibre were found in the myometrium and cervical musculature. The non-vascular noradrenergic network looked denser at the tubal end of the horns and at the cervix, whereas the acetylcholinesterase-positive innervation was poor at the tubal end, increasing toward the cervix. Contrasting with the middle third of the uterine horn, at the tubal end, the myometrial longitudinal layer was much more innervated than the circular one, especially by the noradrenergic nerve fibres. The prepubertal rats presented an adult pattern of uterine autonomic innervation. In the cycling animals, this innervation was nearly the same during oestrus and dioestrus regarding both the density of nerve fibres and intensity of the histochemical reactions.     

Scanning electron microscope study of the splenic red pulp in relation to the sequestration of immature and abnormal red cells

Spleens from normal, healthy cats, dogs and rabbits were perfused with Ringer solution until only a few red cells remained. After fixation of the intact organ, small pieces of tissue were dried by a camphene method and examined under the scanning electron microscope. In all three species the red cells remaining in the spleen were either reticulocytes, spiculated cells, or cells of tear-drop shape and they were found adhering to macrophages and reticulum cells throughout the red pulp. Elongated masses were found on the sinusal surface of fenestrated endothelium (only in dog and rabbit); some of these appeared to be cells of tear-drop shape emerging from the cords into the sinus. This may perhaps denote a pitting process, as suggested by others, but it cannot be a unique function of fenestrated endothelium for red cells of similar shape were found elsewhere in the pulp. In all three species the network of reticulum fibres presents a very large contact surface area for blood cells and it seems likely that increased cell stickiness, rather than decreased deformability, leads to the trapping of immature red cells in the spleen.     

Histochemistry and ultrastructure of nerve fibres and contractile cells in the tunica albuginea of the rat testis

Whole-mounted preparations of the tunica albuginea of the rat testis were studied using light microscopy techniques for demonstration of cholinergic nerve fibres (Karnovski-Root method), catecholaminergic nerve fibres (De la Torre's method) and actin filaments (avidin-biotin-peroxidase method). An ultrastructural study of different regions of the albuginea was also performed. Cholinergic fibres were seen to penetrate into the albuginea with the testicular artery to form a broad network in the mediastinum testis, many fibres ending beneath the rete testis epithelium. Catecholaminergic fibres penetrated through the middle part of the cauda epididymis and formed a plexus in the albuginea covering the inferior testicular pole. This plexus gave rise to straight fibres which formed varicosities, some of them appeared related with mast cells. Actin-containing cells were only found beneath the rete testis epithelium. These cells were similar to myofibroblasts. The location of both cholinergic fibres and contractile cells among the rete testis channels suggests that these cells may be involved in the pumping of semen towards the ductuli efferentes and that their contractility may be regulated by cholinergic fibres.     

Monoamines and acetyl-cholinesterase in the pineal gland and habenula of the ferret

Summary A histochemical method for demonstrating amines by fluorescence showed that the pinealocytes of the ferret contained a high concentration of a yellow fluorophore (probably 5-HT). Numerous green-fluorescent (noradrenaline-containing) nerve fibres occurred around intrapineal blood vessels, between pinealocytes and in the N. conarii (which entered the gland caudally). A collection of neuron-like cells (the pineal ganglion) lay, surrounded by a meshwork of nerve fibres, in the posterior part of the pineal. Neither the cells nor the fibres of the pineal ganglion contained monoamines, but both showed the presence of acetyl-cholinesterase which otherwise was found in the pineal only in fibres which stretched from the ganglion towards the cranial pole of the gland. The medial habenular nucleus showed a remarkable perivascular green fluorescence not seen in the lateral habenular nucleus nor anywhere else in the adjacent diencephalon and brain stem. The cells and fibres of this nucleus also contained much acetyl-cholinesterase.Bilateral superior cervical ganglionectomy, or treating animals with reserpine, removed the green fluorescence from both pineal nerve fibres and the habenula. Ganglionectomy also resulted in a progressive alteration in the colour of the parenchymal fluorescence from yellow to green; the original yellow colour was restored by treating ganglionectomised animals with nialamide (a monoamine oxidase inhibitor). L-Dopa, 5-hydroxytryptophan or nialamide, alone or in combination, had no effect on the fluorescence of the nerve fibres or cells of the pineal, or on the habenula.These results are related to previous findings that pinealectomy or ganglionectomy prevents the acceleration by artificial light of oestrus in ferrets.     

Fluorescence microscopical and chemical characterization of the adrenergic innervation in mammalian liver tissue

Liver tissue from 12 different mammalian species was studied with a fluorescence histochemical technique for the cellular localization of amines (Falck-Hillarp technique) and with a chemical method for the determination of norepinephrine (HPLC-technique). Adrenergic nerve plexus were found in interlobular blood vessels derived from the portal vein and hepatic artery. Varicose adrenergic nerve fibres were, generally, seen to branch from the fibres around the blood vessels and to enter the liver parenchyma, where they formed a randomly distributed intralobular network. The density of these intralobular fibres showed marked species variation. Human liver and liver from the rhesus monkey, baboon, cynomolgus monkey and guinea pig showed a high density of parenchymal adrenergic nerves. Rabbit, cat, pig, cow and horse liver formed an intermediate group, having fewer varicose adrenergic nerve fibres but an unequivocal distribution of these nerves to the liver parenchyma. In rat and mouse liver no parenchymal innervation could be demonstrated. The density of the parenchymal innervation generally correlated with the concentration of norepinephrine in the liver tissue.     

Evidence for exclusive adrenergic innervation of feather muscles (mm. pennati) in the chicken

Summary Feather follicles in the avian skin are interconnected by well-defined bundles of smooth muscle cells, which are responsible for the erection and depression of feathers and thus play an important role in thermoregulation. The depressing and erecting muscle bundles were found to receive a very dense supply of unmyelinated nerve fibres that displayed ultrastructural and histochemical characteristics of noradrenergic axons (formaldehyde- and glyoxylic acid-induced catecholamine fluorescence; uptake to 5-hydroxydopamine). No nerve fibres were encountered showing histochemical acetylcholinesterase activity. There was no indication of the presence of peptidergic or purinergic nerve endings.The neuromuscular space usually ranged from 40–60 nm in width and contained a basal lamina. Occasionally, this space was reduced to approximately 20 nm. At such close neuromuscular contacts a basal lamina was lacking, and focal densities beneath the pre- and postsynaptic plasma membrane were observed. Since no gap junctions between muscle cells were detected, the dense supply with noradrenergic nerve fibres indicates a high amount of directly innervated smooth muscle cells.An additional finding of the present study was the observation that high local concentrations of 5-hydroxydopamine led to degeneration of noradrenergic nerve endings.Supported by a grant from the Deutsche Forschungsgemeinschaft (Dr. 91)     

Coexistence of nitrergic and acetylcholinesterase (ACHE)-positive nerve structures of the phaesant spleen

The coexistence of neuronal NADPH-diaphorase and ACHE activities were investigated in the phaesant spleen by successive double histochemical staining of the same sections. Two types of nerve structures were found in pheasant the spleen: nerve cells and nerve fibres. NADPH-d and ACHE-positive nerve fibres in colocalization enter the spleen in its hilum in the vicinity of splenic artery branches and are gradually distributed in periarterial topography in the white pulp. Only NADPH-d positive nerve cells were seen around the splenic vessels. In the red pulp and splenic capsule, only ACHE-positive nerve fibres were present.     

Effect of castration and testosterone on norepinephrine storage and on the release of [3H]norepinephrine from rat vas deferens

Norepinephrine and dopamine-β-hydroxylase, used as noradrenergic vesicle markers, were found to be decreased in the rat vas deferens 10 days after castration. Five days of testosterone administration to castrated animals increased the enzyme activity over that of controls but did not modify norepinephrine content. In tissue fractions obtained by differential centrifugation, the highest activities of the noradrenergic markers appeared in the vesicular fraction of controls and in the soluble fraction of castrated animals. Testosterone reversed the effect of castration: it increased dopamine-β-hydroxylase activity in the vesicular and soluble fractions, while norepinephrine increased only in the vesicular fraction. Results obtained after continuous sucrose gradient centrifugation of vesicular fractions suggested that these changes principally affected the number of light noradrenergic vesicles while testosterone increased the number of vesicles reduced by castration. Hormonal manipulations also modified some functional properties of nerve endings: norepinephrine depletion after transmural stimulation in the presence of tetraethylammonium, as well as the release of the neurotransmitter, were decreased after castration. These effects were reversed by testosterone. The results suggest a modulatory effect of testosterone on the norepinephrine storage system and on the functional properties of the adrenergic innervation of vas deferens.     

The Effects of Neonatal Pedunclectomy on [3H]Noradrenaline Uptake and the Development of β-Adrenergic Receptors in the Rat Cerebellum

A newly developed method for cutting the cerebellar peduncles in neonatal rats has allowed the study of the development of cerebellar beta-adrenergic receptors in the absence of noradrenergic afferents. Cutting the cerebellar peduncles of neonatal animals did not affect the pattern of development of the beta-adrenergic receptors, nor their final numbers. Pedunclectomy induced a decline in the ability of slices of cerebellar cortex to accumulate [3H]noradrenaline although high-affinity noradrenaline uptake, was never completely abolished. It is suggested that the remaining high-affinity noradrenaline uptake cannot be attributed to noradrenergic fibres from the locus coeruleus.