Chromogranin A in neurons of the rat cerebellum and spinal cord: quantification and sites of expression.

Chromogranin A (CGA) is an abundant protein of dense-cored secretory vesicles in endocrine and neuronal cells. The present study, for the first time, compares CGA of neurons of the central nervous system with the CGA of adrenal origin. By S1 nucleus protection assay, we found that the 3' part of the CGA mRNA between exons 5-8 of the cerebellum and the spinal cord of the rat is homologous to that of the adrenal. In situ hybridization histochemistry revealed that CGA mRNA in the cerebellar cortex is present in cell bodies of Purkinje cells and in neurons of the deep cerebellar nuclei. The perikarya of these cells also exhibit CGA-like immunoreactivity. CGA mRNA and CGA-like immunoreactivity are also present in the motoneurons of the ventral, lateral, and dorsal horns of the rat spinal cord. The amounts of CGA, as determined by radioimmunoassay in cerebellum and spinal cord, were about one tenth of the amounts detected in the adrenal, adenohypophysis, or the olfactory bulb. The sites of CGA expression suggest that CGA may be involved in signal transduction in the motor system.     

Structural basis of sympathetic-sensory coupling in rat and human dorsal root ganglia following peripheral nerve injury

Tyrosine hydroxylase immunocytochemistry was used to reveal the sympathetic postganglionic axons that sprout to form basket-like skeins around the somata of some primary sensory neurons in dorsal root ganglia (DRGs) following sciatic nerve injury. Ultrastructural observations in rats revealed that these sprouts grow on the surface of glial lamellae that form on the neurons. Sciatic nerve injury triggers glial cell proliferation in the DRG, and the formation of multilamellar pericellular onion bulb sheaths, primarily around large diameter DRG neurons. We infer that these glia participate in the sprouting process by releasing neurotrophins and expressing growth supportive cell surface molecules. Many DRG cell somata, and their axons in intact nerves and nerve end neuromas, express α2A adrenoreceptors intracytoplasmically and on their membrane surface. However, sympathetic axons never make direct contacts with the soma membrane. The functional coupling known to occur between sympathetic efferents and DRG neurons must therefore be mediated by the diffusion of neurotransmitter molecules in the extracellular space. Sympathetic basket-skeins were observed in DRGs removed from human neuropathic pain patients, but the possibility of a functional relation between these structures and sensory symptoms remains speculative.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetylcholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immunoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholinergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Summary Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetycholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immnoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholingergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.     

Requirement of neuropilin 1-mediated Sema3A signals in patterning of the sympathetic nervous system

Neuropilin 1 is the specific receptor for Sema3A and plays a role in nerve fiber guidance. We report that neuropilin 1 and Sema3A mutant mouse embryos, generated by targeted gene disruption, showed displacement of sympathetic neurons and their precursors and abnormal morphogenesis in the sympathetic trunk. We also show that Sema3A suppressed the cell migration activity of sympathetic neurons from wild-type but not neuropilin 1 mutant embryos in vitro and instead promoted their accumulation into compact cell masses and fasciculation of their neurites. These findings suggest that the neuropilin 1-mediated Sema3A signals regulate arrest and aggregation of sympathetic neuron precursors and sympathetic neurons themselves at defined target sites and axon fasciculation to produce the stereotyped sympathetic nerve pattern.     

Differential responsiveness of RVLM sympathetic premotor neurons to hypoxia in rabbits

To determine whether differential sympathetic nerve responses to hypoxia are explained by opposing effects of hypoxia upon sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM), the cardiac sympathetic nerve and the renal sympathetic nerve were recorded in anesthetized and vagotomized rabbits. Renal sympathetic nerve was activated by the injection of sodium cyanide solution close to the bifurcation of the common carotid artery and/or by inhalation of hypoxic gas (3% oxygen-97% nitrogen). On the other hand, cardiac sympathetic nerve was inhibited by these stimuli. Barosensitive (inhibited by the stimulation of baroreceptor afferents) reticulospinal (antidromically activated by the stimulation of the spinal cord) neurons in the RVLM were divided into three groups according to their responses to hypoxic stimulation: neurons (Type I, n = 25), the activity of which was inhibited by the injection of sodium cyanide solution close to the bifurcation of the common carotid artery and/or by inhalation of hypoxic gas, neurons (Type II, n = 99), the activity of which was facilitated by the same stimulation, and neurons (Type III, n = 11), the activity of which was not changed. These data indicated that the differential responses of cardiac and renal sympathetic nerves might be due to opposing effects of hypoxia on individual RVLM neurons.     

Neonatal treatment with nerve growth factor antiserum eliminates cholinergic sympathetic innervation of rat sweat glands

Most mammalian sympathetic neurons are noradrenergic, and their dependence upon nerve growth factor (NGF) for survival during development is well established. A minor population of sympathetic neurons, including those that innervate sweat glands, is cholinergic. To determine whether cholinergic sympathetic neurons, like their noradrenergic counterparts, require NGF during development, neonatal rats were treated with NGF-antiserum and 3 weeks later their sweat glands were examined for the presence of innervation. Acetylcholinesterase (AChE) staining and vasoactive intestinal polypeptide-like immunoreactivity (VIP-IR) which mark the mature sweat gland innervation were absent from the sweat glands of the anti-NGF treated animals. Further, when the glands were examined with the electron microscope, no axons or nerve terminals were evident. These observations indicate that the elaboration of the sweat gland plexus is NGF-dependent and suggest that at least one population of cholinergic sympathetic neurons in the rat requires NGF for survival. Our findings are consistent with the idea that during development NGF is a required trophic factor not only for noradrenergic sympathetic but also for cholinergic sympathetic neurons.     

Norepinephrine reduces ω-conotoxin-sensitive Ca2+ currents in renal afferent neurons in rats

Sympathetic efferent and peptidergic afferent renal nerves likely influence hypertensive and inflammatory kidney disease. Our recent investigation with confocal microscopy revealed that in the kidney sympathetic nerve endings are colocalized with afferent nerve fibers (Ditting T, Tiegs G, Rodionova K, Reeh PW, Neuhuber W, Freisinger W, Veelken R. Am J Physiol Renal Physiol 297: F1427-F1434, 2009; Veelken R, Vogel EM, Hilgers K, Amman K, Hartner A, Sass G, Neuhuber W, Tiegs G. J Am Soc Nephrol 19: 1371-1378, 2008). However, it is not known whether renal afferent nerves are influenced by sympathetic nerve activity. We tested the hypothesis that norepinephrine (NE) influences voltage-gated Ca(2+) channel currents in cultured renal dorsal root ganglion (DRG) neurons, i.e., the first-order neuron of the renal afferent pathway. DRG neurons (T11-L2) retrogradely labeled from the kidney and subsequently cultured, were investigated by whole-cell patch clamp. Voltage-gated calcium channels (VGCC) were investigated by voltage ramps (-100 to +80 mV, 300 ms, every 20 s). NE and appropriate adrenergic receptor antagonists were administered by microperfusion. NE (20 μM) reduced VGCC-mediated currents by 10.4 ± 3.0% (P < 0.01). This reduction was abolished by the α-adrenoreceptor inhibitor phentolamine and the α(2)-adrenoceptor antagonist yohimbine. The β-adrenoreceptor antagonist propranolol and the α(1)-adrenoceptor antagonist prazosin had no effect. The inhibitory effect of NE was abolished when N-type currents were blocked by ω-conotoxin GVIA, but was unaffected by other specific Ca(2+) channel inhibitors (ω-agatoxin IVA; nimodipine). Confocal microscopy revealed sympathetic innervation of DRGs and confirmed colocalization of afferent and efferent fibers within in the kidney. Hence NE released from intrarenal sympathetic nerve endings, or sympathetic fibers within the DRGs, or even circulating catecholamines, may influence the activity of peptidergic afferent nerve fibers through N-type Ca(2+) channels via an α(2)-adrenoceptor-dependent mechanism. However, the exact site and the functional role of this interaction remains to be elucidated.     

The localization of the beta-subtype of protein kinase C (PKC-beta) in rat sympathetic neurons

The localization of PKC-beta was studied in rat sympathetic neurons using a polyclonal antibody specific for the beta 1- and beta 2-subspecies. The tissues studied included the superior cervical (SCG) and hypogastric (HGG) ganglia and the target tissues of the SCG and HGG neurons: the submandibular gland, iris, prostate and vas deferens. PKC-beta-LI was found in nerve fibers in both ganglia. A proportion of the fibers in the SCG disappeared after decentralization, suggesting that the fibers were of both pre- and postganglionic origin. The somata of the HGG and SCG neurons expressed varying amounts of PKC-beta-LI, the majority of SCG neurons being labelled only after colchicine treatment. In all target tissues there were PKC-beta-immunoreactive nerve fibers in bundles, but the most peripheral branches of the fibers were negatively labelled. The results show that PKC-beta-LI is widely present in sympathetic postganglionic neurons with mainly quantitative differences. The lack of PKC-beta in the most peripheral branches of nerve fibers might be a general feature of sympathetic postganglionic neurons, suggesting that the participation of PKC-beta in neurotransmitter release and in other functions in nerve terminals in sympathetic adrenergic neurons is unlikely.     

Pathomorphology of adrenergic and cholinergic structures of sympathetic nerve ganglia in experimental burn injuries]

Changes in adreno- and cholinergic structures of sympathetic nerve ganglia (superior cervical, stellate, and splanchnic ganglia of the solar plexus) were studied in 15 male white rats aged 5-7 months, b. m. 200-250 g, 3, 7, and 11 days after burn injury (Stages IIIA, IIIB, involving 20-25% of body surface) and in 5 reference animals. The sections were treated in 2% glyoxylic acid solution and by the Karnovsky-Roots technique. Reduced catecholamine concentrations were revealed in sympathetic nerve ganglia neurons in the early periods after burn injury; the mediator reserves are recovered to a certain measure in later periods after thermal injury. The detected shifts in the sympathetic nerve ganglia neurons correlate with the detected shifts in the cardiovascular system.     

Differential effects of oestrogen on developing and mature uterine sympathetic nerves

Oestrogen is a key factor in the remodelling of uterine sympathetic nerves during puberty and the oestrous cycle; these nerves are influenced by changes in their target uterine tissue. The magnitude of oestrogen-induced responses might however be influenced by the maturation stage of sympathetic nerve fibres, the age of the neurons and/or the developmental state of the uterus. We have therefore compared the sympathetic innervation of the uterus following chronic oestrogen treatment of infantile/prepubertal and young adult intact and ovariectomised rats. Treatment of infantile/prepubertal rats resulted in the complete loss of intrauterine noradrenaline (NA)-labelled sympathetic nerves and a marked reduction in the total NA content in the uterine horn. Chronic treatment of young adult rats had little effect. To examine whether the age of the neurons or the degree of development of the uterus determined responsiveness of nerves to oestrogen, we assessed the effects of oestrogen on the sympathetic reinnervation of intraocular transplants of young adult uterine myometrium into ovariectomised adult host rats. Early treatment (10 days post-transplantation) resulted in less sympathetic innervation than late treatment (30 days post-transplantation). Measurements of nerve growth factor (NGF) levels in the uterine horn of control rats before and after puberty and following infantile/prepubertal chronic oestrogen treatment and acute oestrogen treatment of young adult rats revealed a coordinated increase between the growth of the uterus and NGF protein levels. Thus, developing and recently regrown sympathetic nerves are more susceptible to oestrogen-induced changes in the uterus than mature nerves, differential susceptibility is not related to the age of the neurons or the developmental state of the uterus and changes in NGF protein do not account for the differential susceptibility of developing and mature uterine sympathetic nerve fibres to oestrogen. Growing sympathetic fibres are more vulnerable to oestrogen than mature fibres and nerve fibres that have been in contact for longer periods with their target become less susceptible to oestrogen.     

Inhibition of apoptotic signaling cascades causes loss of trophic factor dependence during neuronal maturation

During development, neurons are acutely dependent on target-derived trophic factors for survival. This dependence on trophic support decreases dramatically with maturation in several neuronal populations, including sympathetic neurons. Analyses of nerve growth factor deprivation in immature and mature sympathetic neurons indicate that maturation aborts the cell death pathway at a point that is mechanistically indistinguishable from Bax deletion. However, neither the mRNA nor protein level of BAX changes with neuronal maturation. Therefore, BAX must be regulated posttranslationally in mature neurons.Nerve growth factor deprivation in immature sympathetic neurons induces two parallel processes: (a) a protein synthesis-dependent, caspase-independent translocation of BAX from the cytosol to mitochondria, followed by mitochondrial membrane integration and loss of cytochrome c; and (b) the development of competence-to-die, which requires neither macromolecular synthesis nor BAX expression. Activation of both signaling pathways is required for caspase activation and apoptosis in immature sympathetic neurons. In contrast, nerve growth factor withdrawal in mature sympathetic neurons did not induce the translocation of either BAX or cytochrome c. Moreover, mature neurons did not develop competence-to-die with cytoplasmic accumulation of cytochrome c. Therefore, inhibition of both BAX-dependent cytochrome c release and the development of competence-to-die contributed to the loss of trophic factor dependence associated with neuronal maturation.     

Rapid anterograde and retrograde tracing from mesenteric nerve trunks to the guinea-pig small intestine in vitro

A novel technique for rapid anterograde labelling of cut axons in vitro was used to visualise the peripheral branches of mesenteric nerve trunks supplying the guinea-pig small intestine. Biotinamide, dissolved in an artificial intracellular solution, was applied to the cut ends of the mesenteric nerves and the tissue was maintained in organ culture overnight. Labelled nerve fibres were visualised by fluorescein isothiocyanate (FITC)-conjugated streptavidin. Intense staining of nerve fibres and terminal varicosities in the ganglia and internodal strands of the myenteric plexus was achieved up to 15 mm from the application site. Filled fibres formed baskets around some myenteric nerve cell bodies, suggesting target-specific neurotransmission. When combined with multiple-labelling immunohistochemistry for tyrosine hydroxylase (TH), calcitonin gene-related protein (CGRP) or choline acetyltransferase (ChAT), most anterogradely labelled nerve fibres, and many pericellular baskets, were found to be TH immunoreactive, indicating their postganglionic sympathetic origin. Double-labelling immunohistochemistry revealed that the postganglionic sympathetic pericellular baskets preferentially surrounded 5-hydroxytryptamine (5-HT)-handling myenteric neurons. Some biotinamide-filled fibres were CGRP immunoreactive, and are likely to originate from spinal sensory neurons. We describe for the first time many pericellular baskets labelled from the mesenteric nerves which were ChAT immunoreactive. Retrogradely filled intestinofugal nerve cell bodies were also observed, all of which had a single axon arising from a small nerve cell body with short filamentous or lamellar dendrites. Many of these cells were ChAT immunoreactive. This in vitro technique is effective in identifying the fine arrangement of nerve terminals arising from nerve trunks in the periphery.     

Sympathoinhibition from ventrolateral periaqueductal gray mediated by 5-HT(1A) receptors in the RVLM

The role of 5-hydroxytryptamine 1A (5-HT(1A)) receptors located in the rostral ventrolateral medulla (RVLM) in the mediation of a sympathoinhibitory and depressor response elicited from the ventrolateral periaqueductal gray (vlPAG) matter of the midbrain was examined in pentobarbital sodium-anesthetized rats. Activation of neurons in the vlPAG evoked a decrease in renal and lumbar sympathetic nerve activities and a decrease in arterial blood pressure. After microinjection of the specific 5-HT(1A)-receptor antagonist WAY-100635 into the pressor area of the RVLM, the vlPAG-evoked sympathoinhibition and hypotension was attenuated to control levels (7 of 15 animals) or converted into a sympathoexcitation and pressor response (8 of 15 animals). Baroreflex inhibition of sympathetic nerve activity was not impaired by microinjection of WAY into the sympathoexcitatory region of the RVLM. These data suggest that sympathoinhibition and hypotension elicited by activation of neurons in the vlPAG are mediated by 5-HT(1A) receptors in the RVLM.     

Modified sympathetic nerve system activity with overexpression of the voltage-dependent calcium channel beta3 subunit

N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory beta3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a beta3-overexpressing transgenic (beta3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of beta3-null and CaV2.2 (alpha1B)-null mice. The beta3-Tg mice showed increased expression of the beta3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The beta3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the beta3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the beta3-Tg mice, whereas beta3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the beta3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of beta3-Tg mice revealed increased calcium channel currents, especially N- and L-type currents. These results identify a determining role for the beta3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.     

Activation of slowly conducting medullary raphe-spinal neurons, including serotonergic neurons, increases cutaneous sympathetic vasomotor discharge in rabbit

Neurons in the rostral medullary raphe/parapyramidal region regulate cutaneous sympathetic nerve discharge. Using focal electrical stimulation at different dorsoventral raphe/parapyramidal sites in anesthetized rabbits, we have now demonstrated that increases in ear pinna cutaneous sympathetic nerve discharge can be elicited only from sites within 1 mm of the ventral surface of the medulla. By comparing the latency to sympathetic discharge following stimulation at the ventral raphe site with the corresponding latency following stimulation of the spinal cord [third thoracic (T3) dorsolateral funiculus] we determined that the axonal conduction velocity of raphe-spinal neurons exciting ear pinna sympathetic vasomotor nerves is 0.8 +/- 0.1 m/s (n = 6, range 0.6-1.1 m/s). Applications of the 5-hydroxytryptamine (HT)(2A) antagonist trans-4-((3Z)3-[(2-dimethylaminoethyl)oxyimino]-3-(2-fluorophenyl)propen-1-yl)-phenol, hemifumarate (SR-46349B, 80 microg/kg in 0.8 ml) to the cerebrospinal fluid above thoracic spinal cord (T1-T7), but not the lumbar spinal cord (L2-L4), reduced raphe-evoked increases in ear pinna sympathetic vasomotor discharge from 43 +/- 9 to 16 +/- 6% (P < 0.01, n = 8). Subsequent application of the excitatory amino acid (EAA) antagonist kynurenic acid (25 micromol in 0.5 ml) substantially reduced the remaining evoked discharge (22 +/- 8 to 6 +/- 6%, P < 0.05, n = 5). Our conduction velocity data demonstrate that only slowly conducting raphe-spinal axons, in the unmyelinated range, contribute to sympathetic cutaneous vasomotor discharge evoked by electrical stimulation of the medullary raphe/parapyramidal region. Our pharmacological data provide evidence that raphe-spinal neurons using 5-HT as a neurotransmitter contribute to excitation of sympathetic preganglionic neurons regulating cutaneous vasomotor discharge. Raphe-spinal neurons using an EAA, perhaps glutamate, make a substantial contribution to the ear sympathetic nerve discharge evoked by raphe stimulation.     

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons. These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4). Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6). For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6). Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly. In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons. The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.     

Conducting pathways of the superior cervical sympathetic ganglion of the cat

Individual nerves of the superior cervical sympathetic ganglion were stimulated in acute experiments on cats, and action potentials (AP) were recorded from other nerves of the ganglion in order to clarify whether or not there is transmission of excitation through the ganglion from one nerve to another and to establish whether this transmission is continuous or synaptic. The method of intracellular recording from neurons of the ganglion was also used. It is established that stimulation of the cervical sympathetic nerve evokes AP in all of the peripheral nerves of the ganglion, a circumstance that is the result of synaptic transmission of excitation. There is no transmission of excitation in the reverse direction or between any of the 12 peripheral nerves of the ganglion (including the four branches of the internal carotid nerve). Orthodromic excitation is recorded intracellularly from neurons of the ganglion during stimulation of the cervical sympathetic nerve, and antidromic excitation is recorded during stimulation of a peripheral nerve (the internal carotid nerve). It follows that the pathways through the ganglion which conduct excitation from the cervical sympathetic nerve into all of the remaining nerves of the ganglion are synaptic. Analysis of EPSP latent periods indicated that preganglionic fibers that differ sharply with respect to threshold and conduction rate (groups S₂ and S₄) converge on one and the same neurons of the ganglion.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 2, pp. 216–224, March–April, 1970.