Localisation of neuropeptide Y in nerves of the rat cardiovascular system and the effect of 6-hydroxydopamine.

The distribution of neuropeptide Y (NPY) in the rat cardiovascular system has been examined by radioimmunoassay, chromatographic analysis of tissue extracts, and immunocytochemistry. High concentrations of NPY were identified throughout the heart and within the major blood vessels of the rat in particular in the renal and superior mesenteric arteries. NPY-immunoreactivity was localised to dense plexuses of nerves in the adventitia of the arteries. Treatment of rats with 6-hydroxydopamine resulted in a significant reduction of NPY concentrations in the major vessels. A depletion of extractable NPY was also seen in the heart of the treated animals. Only few degenerated NPY-containing nerve fibres (swollen and fragmented) were observed in the heart and in the adventitia of the blood vessels. It is concluded that NPY containing nerves of the heart and blood vessels are sensitive to treatment with 6-hydroxydopamine. The pharmacological properties of this peptide suggest that these NPY-containing nerve fibres may act as efferent vasoconstrictor nerves to the blood vessels.

Sympathetic innervation of the rat cornea as demonstrated by the retrograde and anterograde transport of horseradish peroxidase-wheat germ agglutinin.

The sympathetic innervation of the rat cornea was studied by using the method of intraaxonal transport of horseradish peroxidase-wheat germ agglutinin conjugate (HRP-WGA). In the first set of experiments, the relative number of superior cervical ganglion neurons that innervate the rat central cornea was estimated by the method of retrograde HRP-WGA transport. Following tracer application to the scarificed central corneal surface, 49-198 labeled neurons were observed in the ophthalmic region of the ipsilateral trigeminal ganglion and zero to four cells in the rostral pole of the ipsilateral superior cervical ganglion. In the second set of experiments, the three-dimensional distribution and termination sites of the corneal sympathetic nerve fibers were investigated by the technique of HRP-WGA anterograde transport from the superior cervical ganglion. HRP-WGA-labeled axons in corneal whole mounts were identified by the presence within their axoplasm of linear arrays of HRP-TMB reaction product, and their distribution was plotted faithfully onto line drawings made with a drawing tube attachment. Large numbers of HRP-labeled fibers were found in all animals within the corneoscleral limbus where the majority were associated with blood vessels. Fewer fibers (zero to 14 per animal) entered the cornea proper. The latter fibers entered the peripheral cornea in the deep to middle layers of the stroma and ascended into progressively more superficial layers as they coursed centrally. The majority of fibers branched infrequently in the peripheral cornea and increased in branching complexity near the central cornea. HRP-labeled axonal varicosities suggestive of terminal and preterminal expansions were located preferentially in the subepithelial layer of the corneal stroma and in the basal epithelium. Approximately 75% of the axonal varicosities were located in the central half of the cornea. The results of the current investigation reveal that the rat cornea is innervated sparsely by sympathetic nerve fibers derived from the superior cervical ganglion. These data provide additional support to current theories that corneal sympathetic nerve fibers may influence select aspects of corneal physiology, including ion transport and hydration, mitogenesis and wound healing, and sensitivity.

A histochemical study of the innervation of cerebral blood vessels in the turtle.

Specific histochemical techniques for the demonstration of noradrenaline and of acetylcholinesterase have been used to study the distribution of adrenergic and cholinergic nerves to the cerebral blood vessels of turtle, Geoclemys reevesii. The major and medium-sized cerebral arteries were supplied with dense adrenergic nerve plexuses, the plexuses were particularly dense in the medium-sized pial arteries of very thick vascular wall, indicating the functional significance of these arteries in the cerebral circulation. The parenchymal arterioles and capillaries were also supplied with adrenergic nerves. On the other hand, the cholinergic innervation was less dense than the adrenergic one and the acetylcholinesterase activity of the nerve fibres was remarkably weak. By contrast, the parenchymal small arterioles and capillaries exhibited heavy acetylcholinesterase activity on the vascular wall and, in addition, the capillaries were supplied with the well-stained acetylcholinesterase-positive nerve fibres. These fibres and also the adrenergic fibres associating with the capillaries appear to be of central origin. It is suggested that the cholinergic mechanisms in the parenchymal small vessels also play an important role in the cerebral circulation. The basophil leucocytes observed abundantly in the blood of turtle emitted an intensive greenish yellow fluorescence after formaldehyde gas-treatment.

Histochemical evidence for sympathetic innervation of hair receptor afferents in cat skin.

The goal of the present study was to demonstrate histochemically fine catecholamine-containing sympathetic nerve fibers which might be associated with hair receptor afferent fibers in cat skin. Positive results would be consistent with the hypothesis that the sympathetically mediated desensitization of hair afferents, observed in this an earlier studies, is mediated by direct neurotransmitter release onto the afferent fibers. Activity in single primary afferent fibers was recorded electrophysiologically during mechanical stimulation of hairs and electrical stimulation of the lumbar sympathetic trunk in anesthetized cats. The mechanical threshold for activation was seen to increase markedly during low-frequency sympathetic stimulation. Fresh-frozen sections of skin from cat hindleg were treated with glyoxylic acid for detection of fluorescent catecholamines. Fluorescent fibers were observed in association with hair follicles as well as with arterioles and arrector-pili muscles. Subsequent silver staining of the identical skin sections revealed that the fine catecholamine-containing fibers located near hair follicles were closely associated with hair receptor afferent fibers. The results of hair receptor sensitivity is mediated by direct neurotransmitter release onto the afferent fibers.

Localization of neurons in the rat spinal cord which project to the superior cervical ganglion.

Horseradish peroxidase (HRP) was used to determine the location in the spinal cord of neurons projecting to the superior cervical ganglion of the rat. HRP was applied to the proximal cut end of the cervical sympathetic trunk, close to its entry into the superior cervical ganglion. After survival times of 3, 6, or 9 days, the animals were sacrificed and their spinal cords were processed to visualize HRP using diaminobenzidine, benzidine dihydrochloride, or tetramethylbenzidine. Labeled neurons were found only ipsilateral to the site of HRP application and were restricted to spinal segments C8-T5. Ninety percent of these neurons were located in segments T1-T3. Similar numbers of labeled neurons were found at survival times of 3 and 6 days and the mean number +/- S.E.M. for 11 experiments at these two survival times was 1575 +/- 89. Nine days after application of HRP the mean number of labeled cells and the density of label per cell were reduced. Labeled neurons were found in four regions of the spinal cord: the intermediolateral nucleus (75%), the lateral funiculus (23%), the central autonomic area (1%), and the intercalated region (1%). The cells of the intermediolateral nucleus did not form a continuous column along the rostrocaudal axis of the spinal cord, but instead were often found in clusters, several clusters being present per spinal segment.

Segmental organization of sympathetic preganglionic neurons in the mammalian spinal cord.

We have used retrograde transport of horseradish peroxidase to determine the distribution of the preganglionic cell bodies whose axons join particular rami of the thoracic spinal cord in a series of guinea pigs, and in a small number of hamsters and cats. In contrast to other recent studies, our results show that the neurons sending axons to a ramus are confined to a single segment at the corresponding spinal level. This segmental organization supports the idea that the rostro-caudal position of preganglionic cell bodies is one determinant of selective synapse formation between preganglionic axons and sympathetic ganglion cells.

Distribution of phenylethanolamine N-methyltransferase cell bodies, axons, and terminals in monkey brainstem: an immunohistochemical mapping study.

Adrenaline (epinephrine) is an important candidate transmitter in descending spinal control systems. To date intrinsic spinal adrenergic neurons have not been reported; thus adrenergic input is presumably derived from brainstem sites. In this regard, the localization of adrenergic neurons in the brainstem is an important consideration. Maps of adrenergic cell bodies and to a lesser extent axons and terminal fields have been made in various species, but not in monkeys. Thus, the present study concerns the organization of adrenergic systems in the brainstem of a monkey (Macaca fascicularis) immunohistochemically mapped by means of an antibody to the enzyme phenylethanolamine N-methyltransferase (PNMT). PNMT-immunostained cell bodies are distributed throughout the medulla in two principal locations. One concentration of labeled cells is in the dorsomedial medulla and includes the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (X), and an area ventral to X in a region of the reticular formation (RF) known as the central nucleus dorsalis (CnD) of the medulla. A few scattered cells are observed in the periventricular gray just ventral to the IVth ventricle and on midline in the raphe. The second major concentration of PNMT-immunostained cells is located in the ventrolateral RF, lateral and dorsolateral to the inferior olive (IO), including some cells in the rostral part of the lateral reticular nucleus (LRN). Terminal fields are located in the NTS, X, area postrema (AP), and the floor of the IVth ventricle in the medulla and pons. A light terminal field is also observed in the raphe, particularly raphe pallidus (RP). A heavy terminal field is present in locus coeruleus (LC). Fibers labeled for PNMT form two major fiber tracts. One is in the dorsomedial RF extending as a well-organized bundle through the medulla, pons, and midbrain. A second tract is located on the ventrolateral edge of the medulla and caudal pons. Fibers in this tract appear to descend to the spinal cord. A comparison with maps of other catecholamine neurons in primates is discussed, confirming that the distribution of the adrenergic system in monkeys is similar to that described in the human.

Enkephalin-containing sympathetic preganglionic neurons projecting to the inferior mesenteric ganglion: evidence from combined retrograde tracing and immunohistochemistry.

The origin of fibers containing enkephalin immunoreactivity in the inferior mesenteric ganglion of the guinea-pig was studied by combining retrograde axonal tracing and indirect immunofluorescence techniques. Fast Blue was applied into the inferior mesenteric ganglion. Three days later colchicine was administered into the subarachnoid space in order to increase the peptide content of the spinal cord cell bodies. The drug was injected through a catheter which was inserted into the cisterna magna and moved to the appropriate spinal cord levels. After the colchicine injection the animals were perfused with formalin and the L2-L3 spinal cord segments were dissected. Cryostat sections of the spinal cord were analyzed in a fluorescence microscope and subsequently processed for indirect immunohistochemistry using antiserum against enkephalin. Several sympathetic preganglionic neurons containing both Fast Blue and enkephalin-like immunoreactivity were seem mainly in the intermediolateral cell column of the cord. The observations strongly support the view that at least some of the enkephalin-containing fibers in the inferior mesenteric ganglion originate in the sympathetic preganglionic nuclei of the spinal cord. These findings are discussed in view of recent physiological studies which have shown that enkephalin may have a presynaptic inhibitory action on preganglionic neurons as well as on substance P containing primary afferent neurons in the inferior mesenteric ganglion.

Evidence for a sympathetic component in motor branches of the facial nerve: a horseradish peroxidase study in the cat.

The retrograde transport of horseradish peroxidase (HRP) was used to examine the location of sympathetic ganglion cells with axons in facial motor branches of the cat. Large numbers of HRP-labeled neurons were observed in the rostro-anterior part of the superior cervical ganglion. In addition, some labeled neurons were found in the cervical sympathetic trunk, the accessory cervical, middle cervical and stellate ganglia.

Intracranial trajectories of sympathetic nerve fibers originating in the superior cervical ganglion in the rat: WGA-HRP anterograde labeling study.

Intracranial trajectories of sympathetic nerve fibers originating in the superior cervical ganglion (SCG) in the rat were investigated by means of anterograde labeling following the injection of wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the unilateral SCG. The trajectory of the sympathetic fiber innervating the pineal gland and its continuing structures was found advancing along the abducent nerve, through the cavernous plexus, then along the trochlear nerve. Labeled sympathetic fibers showed two patterns of distribution in the blood vessels on the basal surface of the brain. The sympathetic fibers originating in the unilateral SCG were intermingled with those fibers from the contralateral SCG in the pineal gland, its continuing structures and the choroid plexus of the third ventricle as well as in the cerebral blood vessels.

Comparison of 3H-norepinephrine and 3H-metaraminol accumulation as indices of adrenergic nerve density in rabbit blood vessels.

The possibility that accumulation of 3H-metaraminol, which is not metabolized, would provide a better estimate of adrenergic nerve density than 3H-norepinephrine accumulation was examined in a series of rabbit blood vessels. Accumulation of 3H-metaraminol was linear for up to 10 min. Norepinephrine accumulation was correlated with metaraminol acumulation in the vessels studied, but the relationship was not a simple one. The findings suggest that, even with short incubation times, metabolism may significantly alter the amount of norepinephrine retained by the tissue. Nevertheless, accumulation of metaraminol was still not well-correlated with endogenous norepinephrine content. Norepinephrine contents of the basilar artery, mesenteric artery and mesenteric vein were quite similar: 2.7, 2.8 and 2.8 micrograms/g respectively. However, values for metaraminol accumulation in these vessels ranged from 8.7 to 1.78 ml cleared/g. These findings suggest that norepinephrine content of a single adrenergic varicosity may vary from tissue to tissue. The relationship between norepinephrine content, storage capacity, uptake activity and transmitter release needs to be more carefully examined. No single parameter can provide an adequate estimate of adrenergic nerve density.

Terminal course of nerve supply to Müller's muscle in the rhesus monkey and its clinical significance.

Electron microscopic, histochemical fluorescence, and pharmacologic evidence suggested that, in the rhesus monkey, Müller's muscle was not totally denervated either by cutting the levator palpebrae superioris muscle or by a Fasanella-Servat procedure. We examined the terminal course of the sympathetic nerve supply and the potential for preserving Müller's muscle in blepharoptosis surgery.

The structure of the female lower urinary tract and pelvic floor.

This article presents the salient features of the histology of the human female bladder, bladder neck, and urethra. Also described are the structure and relationships of the pelvic floor, with particular emphasis on aspects that concern the functional control of the urethra.

Sympathetic innervation of lymph nodes in mice.

Noradrenergic innervation of popliteal and mesenteric lymph nodes in mice was examined with fluorescence histochemistry. Dense varicose plexuses entered the nodes with the vasculature in the hilar region and continued with the vasculature into the medullary region. Fine, delicate varicosities and small vascular plexuses continued into the cortical and paracortical regions surrounding the germinal centers; some varicosities ended among lymphocytes. A subcapsular plexus contributed fibers into the cortical and paracortical regions. Chemical measurements revealed the presence of norepinephrine in lymph nodes that was depletable with 6-hydroxydopamine. Depletion of norepinephrine from lymph nodes with this agent resulted in a diminished primary immune response in draining lymph nodes following subcutaneous injection of an antigen in two mouse strains, but had no effect in two other strains. These findings suggest that noradrenergic fibers innervate both the vasculature and parenchymal regions of lymph nodes, and may participate in the modulation of immune responses in these organs.

Sympathetic innervation of murine thymus and spleen: evidence for a functional link between the nervous and immune systems.

Sympathetic innervation was demonstrated in both perivascular and parenchymal regions of murine thymus and spleen. Catecholamine varicosities were associated with mast cells in these areas. The antibody response to sheep red blood cells of 7 week old mice that had been sympathectomized with six-hydroxydopamine (6OHDA) at birth was significantly elevated compared with saline treated controls. Alpha-methyl tyrosine (alpha-MT) and 6OHDA treatment of mice, producing a more complete sympathectomy, showed a significantly enhanced anti-SRBC response with respect to mice treated with alpha-MT or 6OHDA alone. Catecholamine levels in thymus, spleen, and adrenals of both experimental and control mice were measured using liquid chromatography with electrochemical detection (LCEC). The present study suggests that the sympathetic nervous system has a functional role in modulating the humoral immune response in vivo.

Noradrenergic sympathetic innervation of lymphoid tissue in the rabbit appendix: further evidence for a link between the nervous and immune systems.

The rabbit appendix, a region of gut with well organized zones of lymphoid tissue, was examined with the glyoxylic acid histofluorescence technique for the localization of noradrenergic fibers, with high performance liquid chromatography with electrochemical detection (LCEC) for the quantitation of norepinephrine and serotonin, with Bielshowski and Giemsa stains for additional information about neural supply, and with acetylcholinesterase histochemistry for the localization of this hydrolytic enzyme. Fluorescent plexuses entered the serosal surface of the appendix associated with blood vessels, traveled longitudinally inside the muscularis interna, mainly in association with blood vessels but adjacent to enteric smooth muscle, and branched into long, linear, varicose plexuses that ran inward in a radial orientation towards the lumen in the internodular septa. As these fibers approached the interdomal regions near the epithelial surface, they passed through thymus-dependent cell zones, and arborized extensively throughout the interdomal region. A high density of varicosities was found in the subepithelial region where immunoglobulin-secreting plasma cells are found. These varicosities were sparse at 21 days of age, but were increased in number at 42 days of age. They were even further increased in number and density in adults. These fibers were further identified with a Bielshowski silver stain, and also demonstrated acetylcholinesterase activity. The noradrenergic varicosities in the interdomal regions of the adult rabbit appendix were closely associated with numerous yellow fluorescent cells of 25-40 microns diameter, which sometimes demonstrated fine varicose processes. The adult rabbit appendix contained a moderate concentration of norepinephrine (163.0 +/- 22.9 ng/g wet weight) and a very high concentration of serotonin (3981 +/- 283 ng/g wet weight). Levels in neonates were considerably lower, suggesting that the yellow fluorescent cells may contain serotonin. Acetylcholinesterase was associated with neural fibers and with non-neural regions of the lymph nodules and the domes, perhaps playing a protective role for these regions of the cellular immune system. The rabbit appendix is a well organized region of lymphoid tissue with specific zones of noradrenergic innervation and possible "paraneuronal" activity, with a readily accessible lumen for the isolation and collections of secretions. We propose this model as an excellent structure for further exploration of interactions between the nervous and immune systems.

Influence of external light conditions on norepinephrine levels in organs innervated by sympathetic nerves from different levels.

Rat tissues receiving their sympathetic innervation from different levels were studied for changes in their norepinephrine level after maintaining the animals for 2 weeks under various lighting conditions. Constant darkness resulted in a doubling of the norepinephrine concentration in iris and a significant increase also in the pia-arachnoid when compared to the tissues from animals kept in alternating light/darkness conditions. Also continuous light gave higher norepinephrine concentrations in the pia-arachnoid. None of the light conditions studied affected cardiac norepinephrine. Norepinephrine levels in vas deferens were reduced after constant light and increased after constant darkness, compared to light/dark conditions. It is concluded that norepinephrine only in tissues innervated from the superior cervical sympathetic ganglia is influenced by external light stimuli, and that the norepinephrine changes in vas deferens are secondary effects of the hormonal alterations produced by continuous light and darkness, respectively, and affecting the entire reproductive tract.

Origin of the adrenergic nerve fibers in the subdiaphragmatic vagus in the dog.

The vagi at the subdiaphragmatic level were studied by the Hillarp-Falck technic in combination with a nerve crush procedure in three normal dogs and in eight dogs that had undergone previous surgical excision of the superior cervical ganglion and/or the stellate ganglia. Unilateral ganglionectomies were performed so that the contralateral vagus served as a control. Based on these results, it is concluded that: (1) the subdiaphragmatic canine vagus contains numerous adrenergic nerve fibers; (2) the main portion of these vagal adrenergic fibers arises from the stellate ganglia; and (3) removal of both the stellate and the superior cervical ganglia results in nearly complete adrenergic denervation of the abdominal vagus.

Changes in ingestive behavior following interruption of a noradrenergic projection to the paraventricular nucleus: histochemical and neurochemical analyses.

Histochemical investigation of the paraventricular nucleus (PVN) identified the tractus filiformis lateralis (TF) as a major noradrenergic preterminal pathway to the PVN. Bilateral knife cuts transecting the TF resulted in a discrete deafferentation of 56% of the noradrenergic input to the PVN region. TF transection also resulted in hypophagia and hypodipsia during the first four days after surgery. A marked hyperdipsia developed between postoperative Days 11 and 13 and persisted until sacrifice on Day 17. The results suggest that the TF contains a noradrenergic system which innervates the PVN and is involved in mediation of ingestive behaviors.

J. Edouard Samson Address: the autonomic nerve supply of bone. An experimental study of the intraosseous adrenergic nervi vasorum in the rabbit.

The anatomy of the autonomic sympathetic vasomotor nerve supply of bone was studied in rabbits by methods of histochemistry, and fluorescent and electron microscopy. Our observations show that the intraosseous vessels are richly supplied by adrenergic nerves. The large primary nerves are located on or about the surface of the vessel; the medium sized secondary nerves spiral around the long axis of vessels lying more deeply in the tunica adventitia; and the fine tertiary nerves form a rich plexus at the outer area of the tunica media. The tertiary nerves have various structures which probably contain neurotransmitter substance--that is, noradrenaline--and function as neuro-vasomuscular synapses. The sympathetic nerve supply of bone originates from the appropriate ganglion, and in the case of the tibial diaphysis it descends through the sciatic nerve and thereafter mainly through the medial popliteal nerve and enters the bone alongside the nutrient artery.