Simultaneous ultrastructural visualization of acetylcholinesterase activity and tritiated norepinephrine uptake in renal nerves

In this investigation we have combined the methods of ultrastructural demonstration of acetylcholinesterase activity with electron microscopic autoradiography for the demonstration of norepinephrine uptake. The results show electron-dense deposits indicative of acetylcholinesterase activity associated with perivascular axons overlaid by concentrations of silver grains representing exogenous tritiated norepinephrine. Forty-five percent of the intervaricose regions and 19% of the varicosities overlaid by autoradiographic grains showed "moderate" amounts of cholinesterase staining. A greater proportion of autoradiographic grains was observed on the varicosities than in the intervaricose regions; however, the amount of acetylcholinesterase activity was greater in the intervaricose regions than in the varicosities. This investigation provides evidence for the presence of periaxonal acetylcholinesterase staining in adrenergic axons in the rat kidney.     

Localization of uptake of tritiated norepinephrine by rat brain in vivo and in vitro using electron microscopic autoradiography

Previous studies have identified granular vesicles in peripheral adrenergic axons, and larger granular vesicles in various brain regions which seem to be correlated with the presence of norepinephrine. In the present study, electron microscopic autoradiography was used to localize ³H-norepinephrine taken up by brain slices in vitro and by brain in vivo after injection via the lateral ventricle. In both preparations, the label was found predominantly in relation to small, unmyelinated axons and nerve endings. Many of these contained granular vesicles in axons which were located in regions of the brain known to contain norepinephrine. It is suggested on the basis of this study and previous work, that exogenous norepinephrine is accumulated by brain axons and nerve endings which contain endogenous norepinephrine, and that the presence of granular vesicles is a common feature of such structures.     

Tissue binding of tritiated norepinephrine in pigmented nuclei of human brain

Regional binding of norepinephrine to tissue structures occurs in sections of brain tissue incubated in vitro with tritiated norepinephrine, followed by radioautography. The binding probably represents one of the mechanisms involved in the uptake and storage of catecholamines by nerve cells. Applying this method to a study of human brains showed excessively strong binding of norepinephrine at the surface membranes of pigmented nerve cells in the substantia nigra, nucleus coeruleus, nucleus dorsalis vagi, and others. Such excessive binding was not found in nonpigmented nuclei in the human brain nor in rat brain. The arrangement of sites of binding at the cell membranes strongly suggested synaptic endings. Melanin pigmentation of nerve cells may be related to the amount of catecholamine-containing synapses at the surface of the neurons.     

Nature of anthopleurin-B-induced release of norepinephrine from adrenergic nerves

Anthopleurin-B (AP-B), a new polypeptide from sea anemone (Anthopleura xanthogrammica), markedly increased the amount of norepinephrine (NA) released from the guinea pig isolated vas deferens. The AP-B-induced release of NA was inhibited or abolished by pretreatment with reserpine and by guanethidine or procaine but remained almost unaffected by mecamylamine. D 600, nifedipine, diltiazem, Mn2+, and Mg2+ markedly inhibited the NA releasing action of AP-B. The AP-B-induced release of NA increased in a linear fashion with increasing Na+ concentrations (85-150 mM). Also the NA release by AP-B increased with an increase in the concentration of external Ca2+ from 0 to 0.8 mM but decreased with an increase in the Ca2+ concentration from 0.8 to 2.0 mM. These results suggest that AP-B increases the permeability across the nerve cell membrane to both Na+ and Ca2+ and that this plays an important role in the NA release from the adrenergic nerve.     

Supraspinal control of reflex activity in renal nerves

1. In anaesthetized cats distension of carotid sinus inhibited renal nerve activity and intercostal-renal nerve reflexes. The degree of inhibition was dependent on the amount of rise in sinus pressure. Intercostal-intercostal reflexes were not inhibited.

2. Inhibition of renal nerve activity and intercostal-renal nerve reflexes could be obtained on stimulating in the dorsal reticular formation but mainly in the ventromedial reticular formation at the level of the obex. The intercostal reflex could also be inhibited at several of these points.

3. The possibility that two inhibitory mechanisms are available is suggested, at least one projecting on to spinal reflex arcs.

4. Other ways of inhibiting renal nerve activity were shown. One was similar to that produced by single shocks to peripheral skin and muscle nerves, and the other was due to a subnormal excitability of vasomotor neurones following intense activation.

     

The gross anatomy of the renal sympathetic nerves revisited

Catheter‐based renal denervation techniques focus on reducing blood pressure in resistant hypertension. This procedure requires exact knowledge of the anatomical interrelation between the renal arteries and the targeted renal nervous plexus. The aim of this work was to build on classical anatomical studies and describe the gross anatomy and anatomical relationships of the renal arteries and nerve supply to the kidneys in a sample of human cadavers. Twelve human cadavers (six males and six females), age range 73 to 94 years, were dissected. The nervous fibers and renal arteries were dissected using a surgical microscope. The renal plexus along the hilar renal artery comprised a fiber‐ganglionic ring surrounding the proximal third of the renal artery, a neural network along the middle and distal thirds, and smaller accessory ganglia along the course of the nerve fibers. The fibers of the neural network were mainly located on the superior (95.83%) and inferior (91.66%) surfaces of the renal artery and they were sparsely interconnected by diagonal fibers. Polar arteries were present in 33.33% of cases and the renal nerve pattern for these was similar to that of the hilar arteries. Effective renal denervation needs to target the superior and inferior surfaces of the hilar and polar arteries, where the fibers of the neural network are present. Clin. Anat. 29:660–664, 2016. © 2016 Wiley Periodicals, Inc.     

The rat renal nerves during development

The prenatal and postnatal development of the innervation of the rat kidney has been investigated using immunocytochemical methods. The efferent innervation was studied using dopamine-beta-hydroxylase and neuropeptide Y antibodies. Calcitonin gene related peptide and substance P antibodies were used to investigate the afferent innervation. Kidneys from embryos of 14 to 20 days, from newborn rats, and from animals of 4, 10, 12, 21, 38, 60, and 90 days of age were studied. Slices of whole kidneys were analyzed, and frozen sections were used to investigate the location of the nerves in more detail. Both afferent and efferent nerves are observed inside the kidney by embryonic day 16. At birth, the afferent nerves are found (1) forming a rich plexus in the renal pelvis; (2) associated with the renal vasculature as far as the interlobular arteries (cortical radial arteries) and (3) in the corticomedullary connective tissue. The efferent innervation appears, at birth, to extend to the interlobular arteries and to the afferent arterioles of the perihilar juxtamedullary nephrons. The efferent innervation increases rapidly during the following days, and by postnatal day 21 a distribution of the innervation similar to that of the adult is observed. While the afferent innervation reaches the major target regions of the kidney by birth, the efferent does most of its expansion into the kidney postnatally. Afferent and efferent fibers are found, extrarenally and intrarenally, in the same nerve bundles. This proximity between afferent and efferent fibers may represent anatomical bases for their interaction in the adult as well as during development.Supported by U.S. Public Health Service Grant Rol 18340 from the National Institute of Health     

Origin of sympathetic efferent axons in the renal nerves of the cat

The origin of efferent axons in the renal nerves of the cat was examined using retrograde transport of horseradish peroxidase (HRP). Nerves on the surface of the left renal blood vessels were dissected 5-7 horseradish mm proximal to the medial margin of the kidney, transected and the central cut ends exposed to HRP. Labeled neurons were typically identified in three locations: (1) centrally along the renal nerve, (2) in the superior mesenteric ganglion, and (3) in the ipsilateral sympathetic chain ganglia (T12-L3). HRP was not detected in preganglionic neurons in the thoracolumbar spinal cord. Labeled cells ranged in size from 15 to 50 micrometers, with those in the renal nerve at the smaller end of the spectrum and those in the superior mesenteric ganglion at the larger end. In the superior mesenteric ganglion labeled cells were typically localized to a small region in the caudal pole of the ganglion around the origin of the renal nerve. The results show that the sympathetic efferent innervation of the kidney is derived from both paravertebral and prevertebral ganglia. In the latter (superior mesenteric ganglion), renal efferent neurons exhibited a topographic distribution.     

Localization of norepinephrine and serotonin transporter in mouse brain capillary endothelial cells

Monoamines function as a vasoactive modulator in the central nervous system (CNS) and are believed to regulate blood-brain barrier (BBB) function. Although monoamine transport is an essential process for regulating the extracellular monoamine concentration, the transport systems for monoamines at the BBB are poorly understood. mRNA expression of norepinephrine transporter (NET) and serotonin transporter (SERT) has been detected in a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4) used as an in vitro model of the BBB, whereas no dopamine transporter (DAT) was detected. Western blot analysis showed the expression of NET and SERT protein in the membrane fraction of mouse brain capillaries and TM-BBB4 cells. Immunohistochemical analysis revealed that NET and SERT are localized at the brain capillaries in the mouse cerebral cortex, and suggests that NET is localized at the abluminal side of brain capillary endothelial cells, and SERT is localized at the luminal and abluminal sides. NET and SERT expressed at the BBB may be involved in the inactivation of monoamines released from neurons around the BBB.     

Influence of renal nerves on renin secretion in the conscious dog

In order to evaluate the influence of renal nerves on renin secretion during changes in blood volume, we studied the mean arterial blood pressure (MAP) and the renal venous plasma renin activity (PRA) in 6 conscious dogs having one intact and one denervated kidney.After a passive head-up tilt PRA increased significantly in the vein of the intact kidney while it remained stable in the denervated one.The intravenous injection of Furosemide (3 mg/kg) induced a rapid elevation of PRA in both renal veins. The kinetics of the variations of renin secretion were similar in the two kidneys, but its magnitude was significantly lower in the denervated side.After a slow hemorrhage of 2, 4 and 6 ml/kg, MAP was unchanged and PRA increased in both renal veins but in a significantly lower degree in the denervated side. When blood loss reached 8 ml/kg, MAP decreased and PRA increased identically in the two renal veins.It was concluded that, in conscious dogs, the renal nerves could participate in the rapid adaptations of renin secretion during small changes in the circulating blood volume.     

Norepinephrine-containing glomus cells in the rabbit carotid body. I. Autoradiographic and morphometric study after tritiated norepinephrine uptake

Summary Rabbit carotid bodies were investigated by autoradiography at both the light and electron microscope levels following tritiated norepinephrine administration eitherin vivo orin vitro. Two kinds of labelled structures were found: nerve fibres (absent in sympathectomized carotid bodies) and some type I glomus cells. Desipramine (a specific norepinephrine uptake inhibitor) prevented labelling. Most of the labelled cells differed from unlabelled ones by the presence of (i) large dense-cored vesicles characterized by a large halo between the membrane and an eccentric dense core; (ii) a nucleus showing a more electron dense chromatin and a more irregular shape; and (iii) relatively abundant glycogen particles. A few weakly-labelled cells were characterized by a pyknotic nucleus and very swollen dense-cored vesicles, and were presumed to be degenerating.Dense core diameters of dense-cored vesicles were distributed according to a unimodal distribution in labelled cells as in unlabelled ones but with an extension towards both large and very small diameters in labelled cells. The mean diameter was higher in labelled cells than in unlabelled ones (127 nm versus 113 nm,P < 0.01). The labelling intensity (as estimated by the number of silver grains per unit of cytoplasmic area) was maximum in cells having dense-cored vesicles whose mean diameter was between 130 and 170 nm, but decreased for cells with mean diameter of dense cores smaller than 130 nm, or larger than 170 nm.Thus, in the rabbit carotid body, some glomus cells differ from others by their ability to take up tritiated norepinephrine and by the presence of larger dense-cored vesicles. However, this distinction is not clearcut and there are many intermediates. The observations suggest a phenomenon of evolution deriving from a unique cell type and typified by both metabolic norepinephrine uptake ability, glycogen accumulation) and morphologic changes (increase in diameter of dense-cored vesicles). It seems, therefore, more appropriate to consider these results in terms of different functional states rather than different types of glomus cells.