Adult Rat Superior Cervical Ganglion Research Articles

G-Protein Modulation of Voltage-Gated Ca2+ Channels from Isolated Adult Rat Superior Cervical Ganglion Neurons.

Sympathetic neurons isolated from adult rat superior cervical ganglia (SCG) are a well-established model to study G-protein modulation of voltage-gated Ca(2+) channels (VGCCs). SCG neurons can be easily dissociated and are amendable to heterologous expression of genes, including genetic tools to study G-protein signaling pathways, within a time frame to maintain good spatial voltage-clamp control of membrane potential during electrophysiological recordings (8-36 h postdissociation). This protocol focuses on examining G-protein modulation of VGCCs; however, the procedures and experimental setup for acute application of agonists can be applied to study modulation of other ion channels (e.g., M-current, G-protein-coupled inwardly rectifying K(+) channels). We also discuss some common sources of artifacts that can arise during acute drug application onto dissociated neurons, which can mislead interpretation of results.

Expression of nestin in superior cervical ganglia of rats is influenced by gender and gonadectomy

Neurons and glia arise from neural progenitor cells that express nestin. Although substantial changes in neuronal development were observed during the postnatal period, data concerning dynamics of nestin expression in the superior cervical ganglia (SCG) of rat during that period are lacking. It is known that gonadectomy and steroid hormones influence the development of neurons in the SCG during the postnatal period, but there are no data on how they influence the persistence of nestin expression in the SCG cells.The dynamics of nestin expression in the SCG in rats of three age groups, as well as the influence of gender and gonadectomy, was investigated.Three groups of male rats were sacrificed at 2, 3 and 6 months of age. Additional groups of male and female Sprague-Dawley rats were gonadectomized at the age of 2 months. After 30 days, they were sacrificed and SCGs were harvested and processed immunohistochemically. Immunoreactivity for nestin in the SCG was observed in satellite glia, based on their expression of s100. The proportion of neurons that were encircled with nestin-immunoreactive satellite cells (nestin encircled neurons, NEN) decreased between second and third month of age (p<0.05). The proportion of NEN was greater in the NPY+ than in the NPY− subpopulation. The proportion of NEN in the SCG of female rats was significantly higher (p<0.05) than that of both, the male rats and ovariectomised groups. The percentage of these neurons was significantly higher (p<0.05) in orchidectomised, in comparison to male rats.Results show the existence of nestin-immunoreactive satellite cells in the SCG of adult rats. A substantial decrease of nestin expression in SCG cells of rats, after the onset of sexual maturation, was observed. This decrease showed significant sex-dependence and was dramatically influenced by gonadal activity.

Ca2+ influx through both L‐ and N‐type Ca2+ channels increases c‐fos expression by electrical stimulation of sympathetic neurons

During direct membrane depolarization, Ca2+ influx primarily through L-type Ca2+ (L-) channels initiates activity-dependent gene transcription. This is surprising given that in most neurons a minority of the total Ca2+ current arises from L-channel activity. However, many studies have stimulated Ca2+ influx with unphysiological stimuli such as chronic membrane depolarization using high K+ medium. Few studies have tested whether other Ca2+ channels stimulate gene transcription in adult neurons as a consequence of direct electrical stimulation. Therefore, we evaluated the role of L- and N-type Ca2+ (N-) channel activity in regulating mRNA levels of c-fos, an activity-dependent transcription factor, in adult rat superior cervical ganglion (SCG) neurons as the majority of Ca2+ channels are N-type, while only a minority are L-type. Changes in c-fos mRNA levels were measured using semi-quantitative and single-cell RT-PCR. Phosphorylation of CREB (pCREB) and changes in c-Fos levels were visualized in dissociated cells by immunocytochemistry. Increases in pCREB, c-fos mRNA and c-Fos protein with either K+ or electrical depolarization required Ca2+ influx. These results support previous findings that elevated c-fos levels result from pCREB stimulating c-fos transcription. Elevation of pCREB, c-fos and c-Fos with K+ depolarization depended on L-channel activity. By contrast, antagonizing either channel at 10-Hz stimulation minimized these increases despite unequal numbers of the two channel types. Transition to exclusive L-channel involvement occurred with increasing frequency of stimulation (from 10 to 20 to 50 Hz). Our results demonstrate that N- and L-channel participation in regulating c-fos expression is encoded in the pattern of electrical stimulation.

The RNA-Binding Protein HuD Binds Acetylcholinesterase mRNA in Neurons and Regulates its Expression after Axotomy

After axotomy, expression of acetylcholinesterase (AChE) is greatly reduced in the superior cervical ganglion (SCG); however, the molecular events involved in this response remain unknown. Here, we first examined AChE mRNA levels in the brain of transgenic mice that overexpress human HuD. Both in situ hybridization and reverse transcription-PCR demonstrated that AChE transcript levels were increased by more than twofold in the hippocampus of HuD transgenic mice. Additionally, direct interaction between the HuD transgene product and AChE mRNA was observed. Next, we examined the role of HuD in regulating AChE expression in intact and axotomized rat SCG neurons. After axotomy of the adult rat SCG neurons, AChE transcript levels decreased by 50 and 85% by the first and fourth day, respectively. In vitro mRNA decay assays indicated that the decrease in AChE mRNA levels resulted from changes in the stability of presynthesized transcripts. A combination of approaches performed using the region that directly encompasses an adenylate and uridylate (AU)-rich element within the AChE 3'-untranslated region demonstrated a decrease in RNA-protein complexes in response to axotomy of the SCG and, specifically, a decrease in HuD binding. After axotomy, HuD transcript and protein levels also decreased. Using a herpes simplex virus construct containing the human HuD sequence to infect SCG neurons in vivo, we found that AChE and GAP-43 mRNA levels were maintained in the SCG after axotomy. Together, the results of this study demonstrate that AChE expression in neurons of the rat SCG is regulated via post-transcriptional mechanisms that involve the AU-rich element and HuD.

Advancing age alters the expression of the ryanodine receptor 3 isoform in adult rat superior cervical ganglia

Sympathetic nerves arising from the superior cervical ganglion (SCG) protect the cerebrovasculature during periods of acute hypertension and may play a role in homeostasis of target organs. The functions of these nerves depend on calcium release triggered by activation of ryanodine receptor (RyR) channels. The function of RyR channels is in part dependent on genetic expression and regulation by numerous protein modulators such as neuronal nitric oxide synthase (nNOS) neurons also found in the SCG. We have shown that release of calcium in SCG cells is altered during late maturation and advancing age. However, the underlying molecular mechanisms that may in part account for these data are elusive. Therefore we used molecular techniques to test the hypothesis that advancing age alters the pattern of genetic expression and/or protein levels of RyRs and their modulation by nNOS in the SCG in F344 rats aged 6, 12, and 24 mo. Surprisingly, ryr1 expression was undetectable in all age groups and ryr2 and ryr3 are the predominantly transcribed isoforms in the adult rat SCG. mRNA and protein levels for RyR2 isoform did not change with advancing age. However, ryr3 mRNA levels increased from 6 to 12 mo and declined from 12 to 24 mo. Similarly, RyR3 receptor protein levels also increased from 6 to 12 mo and declined from 12 to 24 mo. Because nNOS and the phosphorylation of the RyRs have been shown to modulate the function of RyRs, total phosphorylation and nNOS protein levels were analyzed in all age groups. Phosphorylation levels of the RyRs were similar in all age groups. However, nNOS protein levels increased from 6 to 12 mo followed by decline from 12 to 24 mo. These data suggest that advancing age selectively impacts the genetic expression and protein levels of RyR3 as well as modulatory nNOS protein levels. In addition, these data may part provide some insight into the possible changes in the function of RyRs that may occur with the normal aging process.

Comparative effects of high glucose on different adult sympathetic neurons in culture

In vivo, diabetes causes neuropathy in the sympathetic celiac/superior mesenteric ganglion (CG/SMG) but not the superior cervical ganglion (SCG). Therefore, our aim was to compare the effects of high glucose in vitro on adult rat SCG and CG/SMG neurons. High glucose decreased viability and neurite outgrowth and increased TUNEL staining in freshly dissociated neurons from both ganglia. In cultures established for 24 h before exposure to high glucose, a significant (p < 0.01) decrease in neurite-bearing neurons from CG/SMG but not SCG occurred. CG/SMG neurons took longer (p < 0.05) to initiate neurite outgrowth and had lower (p < 0.002) anti-oxidant defence enzyme activity. These properties may contribute to the selective development of neuropathy in CG/SMG in diabetes in vivo.

Novel changes in gene expression following axotomy of a sympathetic ganglion: a microarray analysis.

Neurons of the peripheral nervous system are capable of extensive regeneration following axonal injury. This regenerative response is accompanied by changes in gene expression in axotomized neurons and associated nonneuronal cells. In the sympathetic nervous system, a few of the genes affected by axonal injury have been identified; however, a broad sampling of genes that could reveal additional and unexpected changes in expression has been lacking. We have used DNA microarray technology to study changes in gene expression within 48 h of transecting the postganglionic trunks of the adult rat superior cervical ganglion (SCG). The expression of more than 200 known genes changed in the ganglion, most of these being genes not previously associated with the response to injury. In contrast, only 10 genes changed following transection of the preganglionic cervical sympathetic trunk. Real-time RT-PCR analysis verified the upregulation of a number of the axotomy-induced genes, including activating transcription factor-3 (ATF-3), arginase I (arg I), cardiac ankyrin repeat protein, galanin, osteopontin, pituitary adenylate cyclase-activating polypeptide (PACAP), parathyroid hormone-related peptide, and UDP-glucoronosyltransferase. Arg I mRNA and protein were shown to increase within neurons of the axotomized SCG. Furthermore, increases in the levels of putrescine and spermidine, a diamine and polyamine produced downstream of arg I activity, were also detected in the axotomized SCG. Our results identified many candidate genes to be studied in the context of peripheral nerve regeneration. In addition, the data suggest a potential role for putrescine and spermidine, acting downstream of arg I, in the regenerative process.

Differential regulation of survival and growth in adult sympathetic neurons: an in vitro study of neurotrophin responsiveness.

The survival and growth of embryonic and postnatal sympathetic neurons is dependent on both NGF and NT3. While it has been established that adult sensory neurons survive independently of neurotrophins, the case is less clear for adult sympathetic neurons, where the studies of survival responses to neurotrophins have relied upon using long-term cultures of embryonic neurons. We have previously established a method to culture purified young (7 day) and adult (12 week) sympathetic neurons isolated from adult rat superior cervical ganglia (SCG) in order to examine their survival and growth responses to neurotrophins. We now show that by 12 weeks after birth virtually all neurons (90%) survive for 24 h in the absence of neurotrophins. Neuron survival is unaffected by treatment with anti-NGF antibodies (anti-NGF) or with the tyrosine kinase inhibitor, K252a, confirming the lack of dependence on extrinsic neurotrophins. Duration of neuron survival in culture increases significantly between E19 and day 7 and week 12 posnatally, and is similarly unaffected by the presence of anti-NGF or K252a. Saturating concentrations of NGF and NT3 are equipotent in promoting neurite extension and branching. However, we find that NGF is more potent than NT3 in promoting neurite growth, irrespective of postnatal age. The growth-promoting effects of NGF and NT3 are almost entirely blocked by K252a, demonstrating that these effects are mediated via activation of Trk receptors, which therefore appear to remain crucial to plasticity of adult neurons. Our results indicate that maturing neurons acquire protection against cell death, induced in the absence of neurotrophin, while retaining their growth responsiveness to these factors.

Plasticity in the phenotypic expression of catecholamines and vasoactive intestinal peptide in adult rat superior cervical and stellate ganglia after long-term hypoxia in vivo

Sympathetic ganglia in the adult rat contain various populations of nerve cells which demonstrate plasticity with respect to their transmitter phenotype. The plasticity of the neuronal cell bodies and of the small intensely fluorescent cells in the superior cervical and stellate ganglia in response to hypoxia in vivo (10% O 2 for seven days) was assessed by studying the expression of catecholamines and vasoactive intestinal peptide. The levels of norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid and vasoactive intestinal peptide immunoreactivity were determined. In addition, the density of the immunohistochemical staining of cells for tyrosine hydroxylase and vasoactive intestinal peptide was evaluated. In the intact superior cervical ganglion, hypoxia increased the dopamine level as well as the density of small intensely fluorescent cells immunolabelled for tyrosine hydroxylase and vasoactive intestinal peptide. In the axotomized ganglion, hypoxia elicited a twofold rise in the level of the vasoactive intestinal peptide as well as enhancing the density of neuronal cell bodies immunostained for this peptide. Thus, the effect of hypoxia on the expression of vasoactive intestinal peptide expression in neurons was dependent on neural interactions. In the intact stellate ganglion, hypoxia alone induced a 1.5-fold increase in the density of neuronal cell bodies immunostained for vasoactive intestinal peptide. Thus, ganglia-specific factors appeared to play a role in determining changes in neuronal phenotype in response to hypoxia. The present study provides evidence for the involvement of dopamine and vasoactive intestinal peptide in ganglionic responses to long-term hypoxia as well as for differential responses by the two ganglionic cell populations, i.e. neuronal cell bodies and small intensely fluorescent cells. Changes in the expression of the vasoactive intestinal peptide during long-term hypoxia may be of energetic, trophic and/or synaptic significance. Hypoxia may be considered to be a vasoactive intestinal peptide-inducing factor in sympathetic ganglia.

Galanin expression is decreased by cAMP-elevating agents in cultured sympathetic ganglia.

Galanin expression is co-regulated in peripheral neurons with that of vasoactive intestinal peptide (VIP) under a variety of conditions. For example, the expression of both increase after explantation of adult rat superior cervical ganglia (SCG). Because VIP participates in a positive feedback loop regulating its own expression, we examined whether VIP also increases galanin expression. Galanin mRNA and peptide are nearly undetectable in the SCG in vivo, but increase dramatically after 24-48 h in organ culture. Addition of VIP or forskolin to the culture medium reduced galanin mRNA expression by 75% and 77%, respectively, and reduced galanin peptide expression by 76% and 82%, respectively, compared with ganglia cultured in control medium. In contrast, isoproterenol stimulation did not significantly alter levels of galanin mRNA or peptide, consistent with previous observations that isoproterenol exerts its effect on SCG non-neuronal cells, but not on neurons. The results indicate that galanin and VIP are differentially regulated in sympathetic neurons by cAMP- elevating agents.

Vasoactive Intestinal Peptide Enhances Its Own Expression in Sympathetic Neurons after Injury

Neurons in the adult rat superior cervical sympathetic ganglion (SCG) dramatically increase their content of vasoactive intestinal peptide (VIP) and its mRNA after axotomy in vivo and after explantation. Because the VIP gene contains a functional cAMP response element, the effects of cAMP-elevating agents on VIP expression were examined. VIP, forskolin, or isoproterenol increased cAMP accumulation in explanted ganglia. Secretin, a peptide chemically related to VIP, or forskolin increased VIP levels above those seen in ganglia cultured in control medium, whereas treatment with VIP or secretin increased the level of peptide histidine isoleucine (PHI), a peptide coded for by the same mRNA that encodes VIP. VIP or forskolin also increased VIP-PHI mRNA. In contrast, isoproterenol did not alter levels of VIP, PHI, or VIP-PHI mRNA. Although VIP or forskolin increased cAMP levels in both dissociated neurons and in non-neuronal cells, isoproterenol significantly stimulated cAMP accumulation only in the latter. VIP6-28 was an effective antagonist of the actions of exogenous VIP on cAMP and VIP-PHI mRNA in neuron-enriched cultures. When adult SCG explants were cultured in defined medium, endogenous VIP immunoreactivity was released. When VIP6-28 was added to such cultures, it significantly inhibited the increase in VIP-PHI mRNA that normally occurs. These data indicate that VIP, or a closely related molecule, produced by adult neurons after injury can enhance the expression of VIP. Such a mechanism may prolong the period during which VIP is elevated after axonal damage. The possibility is also discussed that, because VIP is present in preganglionic neurons in normal animals, its release during periods of increased sympathetic nerve activity could alter VIP expression in the SCG.

Pituitary adenylate cyclase-activating peptide (PACAP) and PACAP type 1 receptor expression in regenerating adult mouse and rat superior cervical ganglia in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP), a regulatory peptide belonging to the vasoactive intestinal peptide (VIP) family, is widely distributed in the central and peripheral nervous system. Recent studies have shown that PACAP expression is upregulated in sensory neurons in response to axonal injury. Here we report that PACAP and PACAP type 1 receptors are located in rat and mouse superior cervical ganglia (SCG). PACAP-immunoreactivity (-IR) was demonstrated in preganglionic fibers, whereas only occasional PACAP-IR cell bodies could be observed. In situ hybridization histochemistry using 35S-labeled deoxyribonucleotide probes confirmed that PACAP mRNA was present only in occasional cell bodies. In contrast, PACAP type 1 receptor mRNA was expressed in virtually all cell bodies within the ganglia. After removal and culturing of the SCG for 24 h, there was a marked increase in PACAP mRNA, whilst PACAP type 1 receptor mRNA expression appeared to be downregulated in most nerve cell bodies except for a few scattered neurons displaying a strong upregulation. The total specific binding of PACAP to isolated SCG membranes as assayed by [125I]PACAP-27 binding showed an increase in SCG cultured for 48 h. PACAP-27 neither affected axonal outgrowth from the cultured SCG nor the survival of cells within the SCG. We conclude that PACAP and PACAP receptors are rapidly upregulated in sympathetic ganglia in response to axonal injury and that PACAP may play a role during nerve regeneration.

Regulation of galanin in rat sympathetic neurons in vitro

The neuropeptide galanin is induced in sensory and autonomic neurons after peripheral nerve lesion. Leukemia inhibitory factor (LIF) has been suggested to be involved in the up-regulation of galanin. A direct effect of LIF on galanin content in pure sympathetic neuron cultures dissociated from newborn rat superior cervical ganglia was investigated by radioimmunoassay and immunohistochemistry. Galanin increases in sympathetic neurons during a 12 day culture period in the presence of NGF (10 ng/ml). Five days after addition of LIF (10 ng/ml) a 7-fold elevation is observed when compared to control cultures. Furthermore, galanin increases significantly in the presence of non-neuronal cells and in response to potassium-induced depolarization. The proportion of galanin-immunoreactive neurons in mixed cultures is similar to that found in adult rat superior cervical ganglia after transection of the major postganglionic branches. The results corroborate the hypothesis that LIF, presumably released from ganglionic satellite cells, induces galanin in a subpopulation of sympathetic neurons in vivo and in vitro.

Speed of Ca2+ channel modulation by neurotransmitters in rat sympathetic neurons.

We have measured the onset and recovery speed of inhibition of N-type Ca2+ channels in adult rat superior cervical ganglion neurons by somatostatin (SS), norepinephrine (NE), and oxotremorine-M (oxo-M, a muscarinic agonist), using the whole cell configuration of the patch-clamp method with 5 mM external Ca2+. With a local perfusion pipette system that changed the solution surrounding the cell within 50 ms, we applied agonists at various times before a brief depolarization from -80 mV that elicited I(Ca). At concentrations that produced maximal inhibition, the onset time constants for membrane-delimited inhibition by SS (0.5 microM), NE (10 microM), and oxo-M (20 microM) were 2.1, 0.7, and 1.0 s, respectively. The time constants for NE inhibition depended only weakly on the concentration, ranging from 1.2 to 0.4 s in the concentration range from 0.5 to 100 microM. Inhibition by oxo-M (20 microM) through a different G-protein pathway that uses a diffusible cytoplasmic messenger had a time constant near 9 s. The recovery rate constant from membrane-delimited inhibition was between 0.09 and 0.18 s(-1), significantly higher than the intrinsic GTPase rate of purified G protein Go, suggesting that Ca2+ channels or other proteins in the plasma membrane act as GTPase activating proteins. We also measured the rate of channel reinhibition after relief by strong depolarizing prepulses, which should reflect the kinetics of final steps in the inhibition process. In the presence of different concentrations of NE, reinhibition was four to seven times faster than the onset of inhibition, indicating that the slowest step of inhibition must precede the binding of G protein to the channel. We propose a kinetic model for the membrane-delimited NE inhibition of Ca2+ channels. It postulates two populations of receptors with different affinities for NE, a single population of G proteins, and a single population of Ca2+ channels. This model closely simulated the time courses of onset and recovery of inhibition and reinhibition, as well as the dose-response curve for inhibition of Ca2+ channels by NE.

Inhibition of M-type K+ and N-type Ca2+ channels by the human gonadotropin-releasing-hormone receptor heterologously expressed in adult neurons.

Gonadotropin-releasing hormone (GnRH) controls all aspects of reproductive function. GnRH is secreted by hypothalamic neurons and exerts its effects on the endocrine system through pituitary gonadotropes, while its effects on sexual receptivity are mediated by the central nervous system. The electrophysiological responses of central neurons to GnRH have shown both excitatory and inhibitory responses, but little is known about the mechanisms by which GnRH can change neuronal excitability. The present study addresses the mechanisms whereby stimulation of the human GnRH receptor changes neuronal excitability by using a combination of electrophysiological and heterologous expression techniques. Microinjection of in vitro transcribed cRNA coding for the human GnRH receptor into enzymatically dissociated adult rat superior cervical ganglion neurons resulted in GnRH receptor expression. Activation of the GnRH receptor inhibited both M-type K+ and N-type Ca2+ channels. Inhibition of M-type K+ channels was insensitive to pertussis toxin pretreatment and blocked by intracellular GDPbetaS. Inhibition of Ca2+ channels was slow in onset, voltage independent and insensitive to pertussis toxin. Wash-out of GnRH resulted in an unusual transient reversal of tonic G-protein-mediated Ca2+ channel inhibition. Block of the N-type Ca2+ channel with omega-conotoxin GVIA decreased Ca2+ current inhibition from 43 to 15%, indicating that the N-type Ca2+ channel is an effector target. Ca2+ channel inhibition was completely abolished by including a Ca2+ chelator in the patch pipette. Cell-attached macropatch experiments indicated that Ca2+ channel inhibition is mediated by a diffusible second messenger. These results demonstrate that the human GnRH receptor can inhibit M-type K+ and N-type Ca2+ channels when heterologously expressed in adult rat neurons. Modulation of M-type K+ and N-type Ca2+ channels in central neurons which contain GnRH receptors is likely to contribute to the changes in neuronal excitability elicited by GnRH.

PACAP immunoreactivity in the rat superior cervical ganglion in comparison to VIP.

The presence and regulation of pituitary adenylate cyclase activating peptide (PACAP) was investigated in the adult rat superior cervical ganglion. Double-labelling immunofluorescence using specific antibodies against PACAP and vasoactive intestinal peptide (VIP) revealed a dense innervation of postganglionic sympathetic neurones by PACAP-immunoreactive but VIP-negative nerve endings that mainly were of preganglionic origin since they disappeared after transection of the cervical sympathetic trunk. Since PACAP regulates transmitter synthesis and release in sympathetic neurones in vitro, this finding provides a morphological correlate for modulatory effects of PACAP in vivo. Few neurones contained PACAP under normal conditions. However, the number of PACAP-positive neurones increased in response to axotomy of the postganglionic nerves. Many of the newly PACAP-positive neurones also contained VIP, suggesting that both peptides are regulated by similar mechanisms.

Decreased developmental cell death in sympathetic and spinal sensory nervous systems of the Kyoto spontaneously hypertensive rat.

To investigate the hypotheses that Kyoto spontaneously hypertensive rats (SHR) possess more sympathetic neurons than do normotensive Wistar-Kyoto (WKY) animals due to reduced perinatal cell death and that this is due to increased availability of the sympathetic survival neurotrophin, nerve growth factor. Total cell counts of neuron numbers were performed in neonatal and adult SHR and WKY rat superior cervical ganglia and correlated with counts of apoptotic cells. The values for sympathetic neuron numbers were compared with those for a spinal sensory ganglion. Immunocytochemistry was used to obtain more information about the phenotypes of neurons counted. Adult SHR sympathetic ganglia contained about 25% more sympathetic neurons than did those of WKY animals. Similar elevation of numbers was found both for neurons containing and for those devoid of neuropeptide Y. In neonatal animals, in contrast, there was no strain difference in sympathetic cell numbers but the number of apoptotic cells was reduced in SHR. Spinal sensory neuron numbers in adult SHR were elevated to a similar extent as were sympathetic neurons, but biochemical and morphometric data suggested that this change does not involve cells that are sensitive to nerve growth factor. Although our results support the view that there is reduced developmental cell death both in sympathetic and in sensory systems, they also suggest that this is unlikely to be due to a simple excess of nerve growth factor during development.

Effects of benzodiazepine receptor ligands on isolated rat superior cervical ganglia neurons.

We studied the effects of diazepam, CL 218,872, Ro 15-1788, beta-CCM and Ro 15-4513 on the gamma-aminobutyric acid-activated current in adult and newborn rat superior cervical ganglion neurons. Diazepam (10-1,000 nmol/l) potentiated the current in a concentration-dependent manner. CL 218,872 was less effective and weaker than diazepam. The other ligands reduced the amplitude of the current. These peripheral receptors might be involved in some of the side effects of benzodiazepines.

Neurotransmitters acting via different G proteins inhibit N-type calcium current by an identical mechanism in rat sympathetic neurons.

1. We studied the mechanism of voltage-dependent inhibition of N-type calcium current by norepinephrine (NE) and vasoactive intestinal peptide (VIP) in adult rat superior cervical ganglion (SCG) neurons using the whole cell patch-clamp technique. 2. The voltage dependence of inhibition is manifest in the reversal of inhibition by strong depolarization. We tested the hypothesis that this voltage dependence results from disruption of G proteins binding to calcium channels. According to this hypothesis, the kinetics of calcium current reinhibition following a strong depolarization should become faster for higher concentrations of active G proteins. 3. Assuming that larger inhibitions result from higher concentrations of active G proteins, we used different concentrations of NE to alter the amplitude of inhibition and, thus, the active G protein concentration. We found that the kinetics of reinhibition at -80 mV following a depolarizing pulse to +80 mV were faster for larger inhibitions. 4. VIP induces voltage-dependent inhibition of N-current via a different G protein (Gs) than that of NE (Go). We found that the effect of VIP on reinhibition kinetics was identical to that produced by NE. 5. Combined application of NE and VIP did not greatly increase the amplitude of the inhibition but significantly increased the rate of reinhibition. Thus NE plus VIP appear to greatly increase the concentration of the molecule binding to the channel (G protein according to the hypothesis). 6. The kinetics of calcium current disinhibition during strong depolarization (step to +80 mV) did not change with the size of the inhibition induced by NE, VIP or application of NE and VIP together. 7. Both the concentration-dependent reinhibition kinetics and concentration-independent disinhibition kinetics are consistent with the hypothesis that active G proteins bind directly to N-type calcium channels to modulate their activity in rat sympathetic neurons.

Multiple mechanisms regulate sympathetic neuronal phenotype.

Adult rat sympathetic neurons can possess specific neuropeptides utilized as cotransmitters along with norepinephrine, but the factors that regulate their expression remain unknown. 60% of adult rat superior cervical ganglion (SCG) neurons express neuropeptide Y (NPY) in vivo. To determine whether the restricted expression was an intrinsic property of sympathetic ganglia, we examined if embryonic sympathetic precursors gave rise to NPY immunoreactive (-IR) neurons in vitro. After one week in culture, 60% of neurons derived from the E14.5 rat SCG were NPY-IR. Thus, ganglia isolated before peripheral target contact or preganglionic innervation were capable of regulating NPY expression both in the number of neurons with NPY and in the developmental timing of NPY expression. To determine if the restricted expression of NPY was a reflection of neuroblasts committed to an NPY fate, SCG precursors were labeled with a replication incompetent retrovirus carrying lacZ, and NPY expression in lacZ-labeled clones examined after one week. Two thirds of neuronal clones obtained were uniformly NPY-IR; that is, all neurons in a clone either possessed or lacked NPY. One-third of the neuronal clones were mixed and contained both neurons with and without NPY. We provide a novel demonstration that both lineage and environmental cues contribute to neuropeptide phenotype.