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The actions of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, on G-protein-mediated inhibition of N-type Ca2+ channels in adult rat superior cervical ganglion (SCG) neurons were studied using whole-cell voltage clamp. In SCG neurons, inhibition of ICa occurs by at least three separable pathways: one pertussis toxin (PTX) sensitive and voltage dependent, and two PTX insensitive and voltage independent. NEM blocked PTX-sensitive inhibition nearly completely, with only small effects on PTX-insensitive inhibition. Somatostatin inhibition is completely PTX sensitive and was wholly blocked by a 120 sec exposure to 50 microM NEM, with shorter exposure times producing a less complete block. Inhibition of ICa by norepinephrine (NE) is approximately half PTX sensitive and was also approximately half NEM sensitive. One component of muscarinic inhibition is PTX insensitive, voltage independent, and mediated by a diffusible cytoplasmic messenger; this pathway was largely spared by NEM treatment. Another pathway is also PTX insensitive and voltage independent, used by substance P, and was also largely NEM insensitive. Hence, in SCG neurons, NEM selectively inactivates PTX-sensitive G-proteins. We also find evidence that the PTX-insensitive action of NE is distinct from the other PTX-insensitive pathways, and therefore assign it to a fourth signaling pathway.

1. Modulation of Ca(2+)-channel currents by phorbol-12-myristate-13-acetate (PMA) was investigated in acutely dissociated adult rat superior cervical ganglion neurons using the whole cell variant of the patch-clamp technique. 2. PMA (500 nM) increased the current amplitudes, accelerated the inactivation of step currents, retarded the deactivation of tail currents, and shifted the tail current activation to more negative potentials. 3. The effects of PMA were concentration and voltage dependent and mediated through activation of protein kinase C (PKC). PMA also increased Ca2+ currents recorded with the perforated patch technique. 4. PMA affected the N-type Ca2+ channels and an omega-conotoxin GVIA-resistant current component. Ca2+ currents affected by PMA were not sensitive to omega-agatoxin IVA or nimodipine. 5. PMA not only attenuated Ca(2+)-channel inhibition induced by alpha 2-adrenoceptor agonist, which modulates Ca2+ channels via a pertussis toxin (PTX)-sensitive pathway, but also attenuated current inhibition by vasoactive intestinal polypeptide, which modulates Ca2+ channels via a PTX-insensitive but cholera toxin-sensitive pathway. 6. PMA reversed Ca(2+)-channel inhibition induced by tonic activation of G-protein in the absence of neurotransmitter (even in neurons pretreated with PTX) or induced by activation of G-proteins with guanosine 5'-O-(3-thiotriphosphate) (GTP)-gamma-S. 7. Inhibition of phosphatase by okadaic acid or substitution of Ba2+ for Ca2+ in the external solutions accelerated the PMA effect. 8. Our results suggest that activation of PKC antagonizes G-protein mediated inhibition of Ca2+ channels by shifting Ca2+ channels from the "reluctant" state to the "willing" state. The G-proteins and, more likely, the N-type Ca2+ channels may be the target of PKC phosphorylation. Protein phosphatases may be involved in counteracting the PKC phosphorylation in rat sympathetic neurons.

1. The reserpine-like agent, Ro4-1284 (2-hydroxy-2ethyl-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro- 11b-[H] benzo (a)quinolizine) releases [3H]noradrenaline ([3H]NA) from prelabelled superior cervical ganglion (cell bodies) and nictitating membrane (nerve endings) of the cat. 2. The potency of Ro 4-1284 29.0 microM was higher in the cell bodies than in the nerve endings. 3. In both tissues, exposure to the reserpine-like agent Ro 4-1284 induced a selective increase in the spontaneous outflow of [3H]DOPEG, while the [3H]OMDA metabolites to the release induced by Ro 4-1284 was very small. 4. The desamination is the preferential way of the metabolic inactivation of the [3H]NA released by the reserpine-like agent in both parts of the noradrenergic neuron.

1. The release and the metabolism of [3H]noradrenaline ([3H]NA) induced by tyramine was studied in the superior cervical ganglion (cell bodies) and in the nictitating membrane (nerve endings) of the cat. 2. Exposure of the ganglia to 58.0 and 174.0 microM tyramine resulted in the release of 13.7 and 11.8% respectively of the total tissue radioactivity. In the nictitating membrane, the fractional release of radioactivity was directly proportional to the concentration of tyramine (5.8, 58.0 and 174.0 microM). 3. In ganglia [3H]DOPEG accounted for 55.8% of the radioactivity released by 58.0 microM tyramine and only 10.5% of the radioactivity was unmetabolized NA. In presence of 174.0 microM tyramine, [3H]NA increased to 28.0% of the total radioactivity and [3H]DOPEG and [3H]OMDA decreased to 45.3 and 18.9% respectively. 4. In the nerve endings, the contribution of [3H]NA, [3H]DOMA and [3H]NMN increased with the concentration of tyramine while [3H]DOPEG decreased. 5. The deamination is the first step of the metabolic inactivation of [3H]NA induced by tyramine in the cell body of the postganglionic adrenergic neuron while in the nerve endings [3H]NA is preferentially O-methylated.

Large dense-cored vesicles are transported centrifugally in the cervical sympathetic trunk and are depleted in a calcium-dependent manner from synaptic boutons of the cat superior cervical ganglion during orthodromic stimulation at 20-40 Hz [P. Weldon et al. (1993) Neuroscience 55, 1045-1054]. In the present study, we tested in awake cats whether the normal tonic firing of the sympathetic preganglionic neuron contributes to the turnover of large dense-cored vesicles in synaptic boutons of the superior cervical ganglion. Tetrodotoxin was applied with a mini-osmotic pump to one cervical sympathetic trunk, while vehicle alone was applied to the contralateral cervical sympathetic trunk, for two, four or seven days. The appearance of Horner syndrome ipsilateral to the tetrodotoxin application demonstrated block of action potential propagation. Both superior cervical ganglia were excised and processed for electron microscopy. The number of large dense-cored vesicles per bouton cross-section was higher in the ganglion with tetrodotoxin-treated input than in the control. The content at four days was higher than at two days; the content at seven days was similar to that at four days. The number of lysosomes per bouton profile also increased in the ganglion with tetrodotoxin-treated input. No changes were observed in size of bouton profiles, number of boutons or of synapses per grid square and length of the presynaptic densities in the ganglion with tetrodotoxin-treated input.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous experiments showed that exposure of sympathetic ganglia to exogenous adenosine increased acetylcholine (ACh) content and its subsequent release. This effect was not mediated through extracellular adenosine receptors, but at an intracellular site following its uptake through nitrobenzylthioinosine (NBTI)-resistant nucleoside transporters. We postulated that endogenous adenosine may play a role in modulating synaptic transmission in the superior cervical ganglion. The present study tested whether adenosine is involved in the activation of ACh synthesis that occurs during a rest period following prolonged presynaptic tetanic activity. Conditioning of ganglia with high-frequency stimulation (15 Hz) for 45 min followed by a 15 min rest increased their ACh content by 45%. The appearance of this "rebound ACh" showed sensitivity to nucleoside transport inhibitors; it was prevented by dipyridamole, but not by NBTI or meclonazepam, and it was reduced in the presence of RO 11-3624, suggesting an involvement of NBTI-resistant transporters. The effect of dipyridamole was specific for the synthesis of rebound ACh in that it did not inhibit ACh release or ACh synthesis during stimulation. The inhibitory action of dipyridamole on the synthesis of rebound ACh was not evident if it was present only during the tetanic stimulation but it was if dipyridamole was present during the rest period following it, suggesting that adenosine's presence after tetanic stimulation is of importance. This conclusion was strengthened by experiments showing that the presence of cyclopentyltheophylline, an antagonist at inhibitory adenosine receptors, increased ACh output evoked by test stimulation immediately following tetanic activity, as if endogenous adenosine was available at that time to activate the adenosine receptors that inhibit transmitter release. ACh release from conditioned ganglia was 44% greater than that from the controls. However, the rebound ACh was not mobilized in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a vesicular ACh transporter inhibitor. These results suggest that endogenous adenosine released after tetanic stimulation activates ACh synthesis, which results in an increase of ganglionic ACh that is available for subsequent mobilization and release.

The endogenous taurine content, and the uptake and release of [3H]taurine were examined using the rat superior cervical ganglion. Taurine was found to be one of the most abundant amino acids in the superior cervical ganglion, and the superior cervical ganglion took up [3H]taurine from the incubation medium. Carbachol stimulated the release of [3H]taurine in a concentration-dependent manner with an EC50 of 26 microM and maximal stimulation at 100 microM. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium stimulated release with the same potency but with greater efficacy than carbachol. The nicotinic receptor antagonist hexamethonium (1 mM) inhibited carbachol-stimulated release by 74%. (+/-)-Muscarine stimulated release with an EC50 of 8 microM but with a maximal effect of only 32% of that produced by 100 microM carbachol. Oxotremorine, another muscarinic receptor agonist, was ineffective, even at 1 mM. The muscarinic receptor antagonist atropine inhibited carbachol-stimulated release by 30% at 10 microM. These results show that [3H]taurine release from rat superior cervical ganglion can be stimulated by cholinergic receptor agonists. Release is mediated predominantly by a nicotinic receptor and partially by a muscarinic receptor.

Treatment of rat superior cervical ganglion cells in culture for 4 days with an antisense oligodeoxynucleotide corresponding to a partial sequence of the mRNA coding the m1 muscarinic receptor decreased m1 receptor mRNA by 64% without affecting m2 receptor mRNA. [3H]Pirenzepine binding to M1 receptors was decreased by 43%. Intraventricular infusion of this antisense oligodeoxynucleotide reduced muscarinic receptor density in M1 but not M2 receptor-rich brain regions.

The effects of parathyroid hormone (PTH) and calcitonin on acetylcholine release by rat superior cervical ganglion (SCG) were evaluated in vitro. SCG labeled with [3H]choline were exposed to four 5 min-long pulses of 40 mM K+, 35 min apart. PTH increased, and calcitonin inhibited, in a dose-dependent way, K(+)-elicited [3H]acetylcholine release, with apparent effective doses 50 of about 10(-9) M. The effect of PTH was inhibited by co-incubation with the PTH receptor antagonist NLe [8-18]-PTH (3-34) amide. Incubation of SCG for 120 min with PTH or calcitonin resulted in dose-dependent augmentation or inhibition of K(+)-induced increase of high affinity [3H]choline uptake, respectively, with a maximal effect at 10(-8) M concentration (PTH) and 10(-9) M concentration (calcitonin) and declining at higher concentrations. The increase in SCG [3H]choline uptake induced by PTH was blunted by preincubation with the PTH antagonist NLe [8-18]-PTH (3-34) amide. At 10(-7) M concentrations, PTH increased significantly the in vitro conversion of [3H]choline to [3H]acetylcholine, an effect inhibited by PTH receptor antagonist. Calcitonin did not modify SCG [3H]acetylcholine synthesis by rat SCG. The results indicate that, in vitro, PTH increases, and calcitonin inhibits, acetylcholine release in rat SCG.

Age-differences in the sensitivity of peripheral sympathetic neurons to chronic ethanol exposure and ethanol withdrawal were studied in male Wistar rats aged 4 months, 12 months, or 24 to 25 months. The superior cervical ganglia (SCG) of the young (4 months) and the 2-year-old rats responded to a 12-day or 4-week ethanol exposure with significantly increased catecholamine turnover, while the ganglia of the middle-aged rats (12 months) showed only a minor increase in the intensity of catecholamine fluorescence and tyrosine hydroxylase immunoreactivity. Extensive neuronal vacuolation was found in the 4 months ethanol-exposed SCG, probably as a reaction of a subpopulation of neurons to increased stimulation. Ethanol-induced neuronal loss was most prominent in the SCG of the oldest age group. Contrary to the marked changes in SCG functional and morphometric parameters, the pelvic sympathetic neurons in the hypogastric ganglion showed no significant changes after ethanol exposure. The pattern of ethanol-induced morphological alterations found in the present study did not provide unambiquous support for either the "accelerated aging" or the "increased vulnerability" concept regarding ethanol-aging interactions in the nervous system.

The cholinergic differentiation factor/leukaemia inhibitory factor (CDF/LIF) and retinoic acid (RA) induce in sympathetic neurones, a switch from the noradrenergic to the cholinergic neurotransmitter phenotype. In particular, these molecules alter the activities of the biosynthetic enzymes choline acetyltransferase (ChAT), tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). Recently, five rat ChAT mRNA species have been identified although no data have yet been reported concerning their production and regulation in sympathetic neurones. By use of the reverse transcription polymerase chain reaction technique we analysed the effects of CDF/LIF and RA on the levels of ChAT, TH and DBH mRNAs. Each ChAT mRNA was produced in sympathetic neurones and was induced by both molecules, whereas the mRNAs encoding TH and DBH enzymes were down-regulated.

Prolonged contact of the rat superior cervical ganglia (SCG) with the purine alpha,beta-methylene-ATP (alpha,beta-Me-ATP) selectively depressed responses to ATP and alpha,beta-Me-ATP but not responses to uridine 5'-triphosphate (UTP), potassium or adenosine. Prolonged contact with the pyrimidine UTP selectively depressed responses to UTP but not responses evoked by alpha,beta-Me-ATP, potassium or adenosine. These results are consistent with the presence of P2-purinoceptors and pyrimidinoceptors on the rat SCG and the hypothesis that pyrimidinoceptors exist within the nervous system.

The effect of interleukin (IL)-6 on cell survival, and the expression of choline acetyltransferase (ChAT) and neuropeptide mRNAs was examined in cultured rat sympathetic neurons. Reverse transcription-polymerase chain reaction analysis indicated that IL-6 treatment led to six-fold increase in ChAT mRNA without a concomitant increase in ChAT immunoreactivity. In contrast, treatment with ciliary neurotrophic factor or leukemia inhibitory factor/cholinergic differentiation factor increased ChAT mRNA levels 25-fold and resulted in intense ChAT immunoreactivity. Moreover, the latter factors increased somatostatin and preprotachykinin mRNA levels 10-fold whereas IL-6 had no effect. Maximal doses of IL-6 had no effect on cell survival. These findings provide a basis for the overlapping and yet divergent actions of these three factors which share some components of their signal transduction machinery.

Background and Purpose: Studies showing efficacy with free radical scavengers have been conflicting, and when protection was demonstrated it was attributed to action at the level of the vascular endothelium. The purpose of this study was to test the hypotheses that neuronal free radical formation plays a role in the ischemic cascade and occurs intracellularly and that free radical scavengers, if taken up intracellularly, will protect against hypoxic damage., Methods: A tissue culture model of hypoxia followed by restoration of oxygen was employed, using both superior cervical ganglia and hippocampal neurons. Cells were exposed to an anoxic atmosphere of 95% N2 and 5% CO2 and examined 2 to 24 hours later after restoration of oxygen. Determination of survival was measured by trypan blue exclusion. Nitroblue tetrazolium stain was used to assess free radical formation., Results: Pretreatment with free superoxide dismutase did not decrease cell death after hypoxia as measured by trypan blue exclusion. However, when superoxide dismutase was taken up intracellularly under depolarizing conditions (55 mmol/L KCl in the medium), cell death was decreased significantly compared with hypoxic controls (28.7 +/- 4.34 versus 40.3 +/- 4.33; P < .03). During hypoxia neurons reduced nitroblue tetrazolium to form the blue precipitate formazan, and the color change was blocked in neurons pretreated with superoxide dismutase in depolarizing medium. Similar findings occurred in both superior cervical ganglia and hippocampal neurons., Conclusions: These findings provide evidence to support the role of neuronal free radical formation in cell death secondary to hypoxia. In addition, free radical scavengers, if taken up intracellularly, may partially ameliorate their deleterious effect.

To understand the role of low-affinity neurotrophin receptor p75 in neural development, we previously generated mice carrying a null mutation in the p75 locus (Lee, K. F., Li, E., Huber, L. J., Landis, S. C., Sharpe, A. H., Chao, M. V. and Jaenisch, R. (1992) Cell 69, 737-749). To elucidate the mechanisms leading to deficits in the peripheral nervous system in p75 mutant mice, we have employed dissociated cultures to examine the responses of p75-deficient dorsal root ganglion (DRG) and superior cervical ganglion (SCG) neurons to different neurotrophins. We found that p75-deficient DRG and SCG neurons displayed a 2- to 3-fold decreased sensitivity to NGF at embryonic day 15 (E15) and postnatal day 3 (P3), respectively, ages that coincide with the peak of naturally occurring cell death. Furthermore, while p75-deficient E15 DRG neurons did not change their response specificity to BDNF, NT-3, and NT-4/5, P3 SCG neurons became more responsive to NT-3 at higher concentrations (nanomolar ranges). These results may help explain the deficits in the peripheral nervous system in p75 mutant mice and provide evidence that p75 can modulate neurotrophin sensitivity in some neurons.

1. A single administration of the ganglion blocker, chlorisondamine (10 mg kg-1, s.c.) is known to produce a quasi-irreversible blockade of the central actions of nicotine in the rat. The mechanism of this persistent action is not known. It is also unclear whether chlorisondamine can block neuronal responses to excitatory amino acids and whether chronic blockade of nicotinic responses also occurs in the periphery. 2. Acute administration of chlorisondamine (10 mg kg-1, s.c.) to rats resulted in a blockade of central nicotinic effects (ataxia and prostration) when tested 1 to 14 days later, but caused no detectable cell death in tissue sections sampled throughout the rostrocaudal extent of the brain which were stained in order to reveal neuronal degeneration. 3. Long-term blockade of central nicotinic effects by chlorisondamine was not associated with significant alterations in the density (Bmax) of high-affinity [3H]-nicotine binding to forebrain cryostat-cut sections. 4. In cultured dissociated mesencephalic cells of the foetal rat, chlorisondamine and mecamylamine inhibited [3H]-dopamine release evoked by N-methyl-D-aspartate (NMDA, 10(-4) M), but only at high concentrations (IC50 approx. 600 and 70 microM, respectively). A high concentration of chlorisondamine (10(-3) M) had no effect on responses to quisqualate (10(-5) M) and only slightly reduced responses to kainate (10(-4) M). Mecamylamine (10(-3) M) was ineffective against both agonists. 5. In adult rat hippocampal slices, chlorisondamine depressed NMDA receptor-mediated synaptically-evoked field potentials, but again only at high concentrations (10(-4)-10(-3) M). Synaptic responses that were mediated by non-NMDA excitatory amino acid receptors were less affected. 6. In rat isolated superior cervical ganglion, electrically-evoked synaptic transmission was reduced 1 h after acute in vivo administration of chlorisondamine (0.1 mg kg-1, s.c.). However, in vivo administration of a higher dose (10 mg kg-1, s.c.) did not significantly affect ganglionic transmission when tested two weeks later, despite the continued presence of central nicotinic blockade.7. These results indicate that the persistent CNS nicotinic blockade by chlorisondamine is not accompanied by changes in nicotinic [3H]-nicotine binding site density or by neuronal degeneration in the brain; that at doses sufficient to produce nicotinic receptor blockade, chlorisondamine acts in a pharmacologically selective manner; and that chronic central blockade is not accompanied by long-term peripheral ganglionic blockade.

1. The actions of adenosine 5'-triphosphate (ATP) and related purine analogues on neurones of isolated superior cervical ganglia in guinea-pig were studied using intracellular recording techniques. 2. Application of ATP (0.1-1 mM) by superfusion evoked a response in half (26 out of 52) of the neurones studied. The response consisted of a slow depolarization (in 23 of the cells) or a hyperpolarization (in 3 cells). The amplitude of depolarization was concentration-dependent and was accompanied by a decrease in input resistance (in 12 of 23 responses). 3. Superfusion of the P2 chi-purinoceptor selective agonist alpha, beta-methylene ATP (1-100 microM), evoked a response in the majority (42 of 58) of neurones studied. The response consisted of a depolarization (in 39 cells) or a hyperpolarization (in 3 cells). The depolarization was also concentration-dependent and was accompanied by a decrease in input resistance (36 of 39 responses). Superfusion of another P2 chi-purinoceptor agonist, beta, gamma-methylene ATP (10 microns), also produced a small depolarization (5 of 8 cells) which was accompanied by a decrease in input resistance. 4. Application of the P2 gamma-purinoceptor selective agonist, 2-methylthio ATP (1-100 microM) evoked a response in approximately half (28 of 55) of the neurones studied. The response consisted of a depolarization (in 23 cells) or a hyperpolarization (in 5 cells). The amplitude of the depolarization was concentration-dependent and accompanied by a decrease in input resistance (13 of 21 responses). 5. Superfusion of the P1-purinoceptor selective agonist 2-chloroadenosine (100 microM) evoked a response in only a few (3 of 21) of the neurones tested.(ABSTRACT TRUNCATED AT 250 WORDS)

The development and maturation of cells depends not only on their genetic history, but also on sequences and combinations of environmental signals appropriate to their developmental age. Early postnatal rat sympathetic neurons are dependent on nerve growth factor (NGF) for survival in vivo and in vitro, but earlier sympathetic neuroblasts may not require NGF. Ciliary neurotrophic factor (CNTF) provides short-term in vitro trophic support to embryonic and neonatal sympathetic neurons, but its role in vivo is not understood. In this study we examined further the capability of CNTF to support neonatal rat superior cervical ganglion (SCG) sympathetic neurons in vitro and the effect of CNTF on the trophic activities of NGF. SCG neurons cultured with either CNTF or NGF survived for 24 hr in low-density cultures depleted of nonneuronal cells, in contrast to neurons with neither factor. However, with CNTF only a fraction of the NGF-maintained number of neurons survived for 6 days. CNTF given in combination with NGF in these nonneuron-depleted cultures produced a significant decrease in the number of neurons surviving for 6 days, compared to the number supported by NGF alone. If such cultures were supplemented with Schwann cells, very different results were obtained: CNTF alone supported the 6-day survival of 80% as many neurons as did NGF, and the combination of CNTF with NGF produced no decrease in neuronal survival. Antibody to NGF did not block the support provided by CNTF and Schwann cells. Immunostaining for the low-affinity NGF receptor (LNGFR), intense in the NGF-supported neurons, was absent in the CNTF-supported neurons and reduced in the neurons exposed to the combination of NGF and CNTF. These results show that CNTF can act synergistically with a Schwann cell-derived agent to provide trophic support to neonatal sympathetic neurons, and that it can down-regulate the responsiveness of those neurons to NGF.

This study describes the antagonistic properties of himbacine, in comparison with those of pirenzepine, at muscarinic receptors mediating the depolarization of rat superior cervical ganglion, the inhibition of electrically-induced twitch contractions of rabbit vas deferens and the contraction of dog saphenous vein, currently classified as putative muscarinic M1 sites. The affinity of himbacine for the vas deferens site (pA2 8.08) was nearly ten times higher than those for the M1 receptors of rat ganglion and dog saphenous vein (pA2 7.14 and 7.16, respectively); affinity estimates for pirenzepine were similar throughout the different preparations. The present data are consistent with the allocation of ganglion and saphenous vein receptors into the M1 subclass; the profile of the vas deferens site, conversely, appears to be different, and possibly more closely related to that reported for the M4/m4 receptor.

Exposure of neuronal terminals to target-derived NGF has been hypothesized to regulate cell body responses at a distance. To test this hypothesis and, more specifically, to determine whether NGF distally regulates the synthesis of its two characterized receptors, we developed the following paradigm. Sympathetic neurons of the superior cervical ganglion (SCG) that project either to the eye or to the ear were labeled, in adult animals, with the retrograde tracers Fast Blue and Fluoro-Gold, respectively. NGF was then injected daily into the anterior chamber of one eye, exposing the terminals of the ipsilateral eye neurons to increased NGF. To control for systemic and/or localized injury effects, cytochrome C or PBS were injected into the contralateral eye of the same animals. In situ hybridization and image analysis were then used to determine neuronal levels of p75 NGF receptor, trkA, and T alpha 1 alpha-tubulin mRNAs, with the latter providing a correlative measure of neuronal sprouting. Morphological measurements revealed that exogenous, terminally-derived NGF increased the mean cross-sectional area of eye neurons by 37%. Grain counts for p75 NGF receptor mRNA increased from 2- to 6-fold in the NGF-treated neurons, and grain densities, which accounted for neuronal hypertrophy, also increased significantly. In contrast, grain counts for trkA mRNA were not significantly increased by this treatment, while T alpha 1 alpha-tubulin mRNA levels increased only 1.5- to 2-fold. No increase in grain density was detected for either of these mRNAs. The NGF-induced increased in p75 NGF receptor mRNA levels was accompanied by terminal sprouting and by an increase in the density of p75 NGF receptors on terminal neurites, as indicated by IgG-192 immunostaining of the NGF-treated iris. These data therefore suggest that, in addition to promoting local sprouting, increased target-derived NGF increases the levels of p75 NGF receptor relative to trkA on terminal neurites, by differentially regulating receptor synthesis. Such a direct regulatory feedback loop may well play an integral role in precisely modulating neuronal responses as a function of the amount of available trophic support and/or target tissue.

1. Using cell-attached patch pipettes, sustained activity of single potassium M-channels was recorded from dissociated rat superior cervical ganglion neurones. Previous results indicated that this activity, consisting of three main levels of open-channel conductance (congruent to 7, congruent to 12 and congruent to 19 pS) was activated by membrane depolarization and inhibited by muscarine added outside the patch. Consequently, a kinetic analysis was undertaken in order to identify M-channel states sensitive to muscarine and membrane potential. 2. Channel activity recorded at 30 mV positive to the resting membrane potential level (congruent to -60 mV) showed three shut and two open times. Mean shut times were: tau s1 = 8.0 +/- 2.2 ms; tau s2 = 71.3 +/- 8.6 ms and tau s3 = 740 +/- 220 ms. Mean open times were: tau o1 = 10.6 +/- 1.9 ms and tau o2 = 59.3 +/- 8.7 ms. When bursts of channel openings were determined as those including tau s1, two exponential components were evident in burst duration distributions (tau b1 = 11.0 +/- 0.9 ms and tau b2 = 80.4 +/- 11.0 ms). 3. Membrane hyperpolarization significantly lengthened all three shut times and shortened both open times. It also slightly enhanced the relative contribution of high-conductance channels and decreased the relative contribution of low-conductance channels to overall activity. 4. All three shut times of the M-channels were lengthened by 10 microM muscarine without significantly affecting their open times. 5. It is concluded that both open and shut states of the M-channel are voltage sensitive while only shut states are sensitive to muscarine.

1. Using a grease-gap technique, we have investigated the effects of purine and pyrimidine nucleotides on the d.c. potential of the rat isolated superior cervical ganglion (SCG). 2. Of the purines tested, adenosine, adenosine 5'-triphosphate (ATP), beta,gamma-methylene-adenosine 5'-triphosphate (beta,gamma-MeATP) at up to 300 microM produced concentration-dependent hyperpolarizations, whereas 2-methyl-thio-ATP (2-Me.S.ATP) and alpha,beta-methylene-ATP (alpha,beta-MeATP) depolarized ganglia. Of the pyrimidines tested, uridine 5'-triphosphate (UTP) produced concentration-dependent depolarizations and cytosine 5'-triphosphate (CTP) at 1000 microM produced considerably smaller but significant depolarizations. In contrast uridine 5'-monophosphate (UMP) at 1000 microM hyperpolarized ganglia. The relative order of potency of purines and pyrimidines to depolarize ganglia was: UTP > alpha,beta-MeATP >> CTP > 2-Me.S.ATP and to hyperpolarize ganglia was: adenosine = beta,gamma-MeATP > ATP > UMP. 3. The ability of purines and pyrimidines to alter the depolarizing response caused by muscarine and of purines to alter depolarization induced by gamma-aminobutyric acid (GABA) was determined. The relative order of potency of nucleotides in depressing submaximal depolarization caused by muscarine (100 nM) was: adenosine = ATP > beta,gamma-MeATP whereas 2-Me.S.ATP, alpha,beta-MeATP and UTP did not significantly alter depolarization caused by muscarine. At 100 microM beta,gamma-MeATP and adenosine but not ATP potentiated GABA-induced depolarizations. 4. Hyperpolarizations caused by adenosine, ATP, beta,gamma-MeATP and UMP and depolarizations caused by alpha,beta-MeATP were enhanced in medium containing reduced concentrations of calcium (0.1 mM) and potassium (2 mM). In this medium 8-phenyltheophylline abolished hyperpolarizations caused by adenosine and reversed hyperpolarizations caused by ATP into depolarizations. Suramin (300 microM), a P2-purinoceptor antagonist, significantly reduced the depolarizing response caused by alpha,beta-MeATP and significantly increased hyperpolarizations caused by ATP and Beta,gamma-MeATP. Suramin (300 microM) did not significantly alter depolarizations caused by l,l-dimethyl-4-phenylpiperazinium (10 microM), potassium(3 mM) or muscarine (100 nM) and significantly potentiated depolarizations caused by UTP (100 microM).5.It is concluded that the rat SCG contains PI-purinoceptors that hyperpolarize the ganglion and diminish sensitivity to muscarine, and P2X-purinoceptors that depolarize the SCG. There is also some evidence to suggest the presence of receptors for UTP, i.e., pyrimidinoceptors, which depolarize SCG neurones.

We have examined the effects of GABA and several GABAA receptor modulators on isolated rat (1-160 days) superior cervical ganglion (SCG) neurons with whole-cell recordings. The neurons were sensitive to GABA within 12 h after birth, and the relationship between the current amplitude and GABA concentration (GABA50 = 19 microM) in these newborn neurons was fitted with a Hill coefficient close to 1.0. Adult neurons, on the other hand, had a GABA50 value of 30 microM, and the slope was steeper and fitted with a Hill coefficient of 1.6. Unlike the results of previous studies in cultured SCG neurons, we found that the concentration of Zn++ that reduced the response by 50% was 37 +/- 12 microM in newborns and 43 +/- 6 microM in adults. Bicuculline (10 microM) inhibited the current by 41 +/- 6% and 36 +/- 11% in newborn and adult neurons, respectively. With 5 microM GABA, diazepam and pentobarbital potentiated the response in newborn and adult neurons. No changes were found in the sensitivity of the receptor for these ligands, but their efficacy was enhanced 2-fold during development. Diazepam shifted the relationship between GABA and the response to the left in all the neurons studied. Ethanol (40 mM) reduced the amplitude of the GABA current to 64 +/- 12% of control in newborn neurons and to 85 +/- 4% in adult neurons. The inhibitory effect of 100 mM ethanol on the GABA current was noncompetitive. Addition of a very large concentration of ethanol (850 mM) to newborn neurons potentiated the response to 164 +/- 13% of control.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of three tachykinin NK1 receptor antagonists (L-668,169, (+/-)-RP 67580, and (+/-)-CP 96.345) were examined for their ability to antagonise responses evoked by substance P O-methyl ester (a selective NK1 receptor agonist) in isolated neuronal tissue (rat superior cervical ganglia and guinea-pig locus coeruleus) and smooth muscle tissues (rat urinary bladder and guinea-pig ileum longitudinal muscle/myenteric plexus). (+/-)-RP 67580 was similarly effective in antagonising responses in both rat superior cervical ganglia and urinary bladder (estimated pKa value = 7.4 for both tissues); however, (+/-)-CP 96,345 was 50-fold less effective in antagonising responses in guinea-pig locus coeruleus than in ileum longitudinal muscle/myenteric plexus (estimated pKa values = 7.6 and 9.3 respectively). It is suggested that the differential effects of (+/-)-CP 96,345 may reflect the existence of a population of NK1 receptors within guinea-pig locus coeruleus that are less sensitive to the effects of this NK1 receptor antagonist.

1. Differences in the neuromodulation of Ca2+ currents between superior cervical ganglion (SCG) and more caudal paravertebral ganglion (PVG) neurons acutely isolated from the same rats were investigated using the whole-cell patch-clamp technique. 2. Norepinephrine (NE) induced a concentration-dependent inhibition of Ca2+ currents in both SCG and PVG neurons. The concentration producing 50% inhibition (IC50) for NE estimated from concentration-response curves was similar between SCG and PVG neurons but the maximal inhibition estimated from the concentration-response curve for PVG neurons was decreased compared with that of SCG neurons. 3. Tail current activation curves of both SCG and PVG neurons in the absence and presence of NE (5 microM) could be fitted to a double Boltzmann equation. In the presence of NE, the activation curves for both SCG and PVG neurons were shifted toward more depolarized potentials. The magnitude of the shift was greater in SCG than in PVG neurons, which could be accounted for by a greater decrease (P < 0.05) in the fractional amplitude of the first current component of SCG neurons (delta 1.4 +/- 0.4 nA, mean +/- SE, 39%) compared with that of PVG neurons (delta 0.9 +/- 0.1 nA, 16%). 4. Ca2+ current density, expressed as maximal tail current amplitude normalized to cell capacitance, was greater in PVG neurons than that in SCG neurons. 5. In SCG neurons, a saturating concentration of omega-conotoxin GVIA (omega-CgTx) produced a greater decrease of Ca2+ current amplitude at +20 mV (77.4 +/- 1.9%) than in PVG neurons (71.2 +/- 1.5%, P < 0.05). 6. After pretreatment with 15 microM omega-CgTx, NE still decreased the Ca2+ currents in both populations of neurons; however, the inhibition was greater in SCG neurons (31.1 +/- 3.4%) than in PVG neurons (12.8 +/- 3.6%, P < 0.01). 7. The dihydropyridine Ca2+ channel "agonist" Bay K 8644 (10 microM) prolonged Ca2+ tail currents in both SCG and PVG neurons. After normalizing to cell capacitance, there was no significant difference in Bay K 8644-induced tail current amplitude between the two populations of neurons. Moreover, NE (5 microM) increased the prolonged Ca2+ tail current amplitude induced by Bay K 8644 (10 microM) by 44.7 +/- 13.5% in SCG and 41.9 +/- 11.9% in PVG neurons. 8. Under control conditions, Ca2+ currents were facilitated by a depolarizing conditioning pulse (50 ms to +100 mV) in both PVG neurons (29.2 +/- 5.1%) and SCG neurons (20.1 +/- 4.0%).(ABSTRACT TRUNCATED AT 400 WORDS)

We examined the effects of single and multiple systemic injections of nicotine on the expression of five immediate early genes in the rat superior cervical ganglion by in situ hybridization histochemistry. A single nicotine injection resulted in a rapid and transient activation phase of nerve growth factor I-A, c-fos and jun-B at 20 min, and a later and less prominent activation of c-jun, which stayed high from 20 to 60 min. there was a parallel slow and long-lasting activation of jun-D, which remained high 4 h after nicotine treatment. Tyrosine hydroxylase mRNA, but not neuropeptide Y mRNA, was also induced by nicotine. Denervation of the ganglion did not prevent the induction of immediate early genes, but the nicotine antagonists hexamethonium and mecamylamine completely blocked the induction of immediate early genes, indicating that nicotine acted directly on receptors present on ganglion cells. When repeated nicotine injections were given, there was a refractory period of 1-2 h for c-fos, nerve growth factor I-A and jun-B induction. Repeated nicotine injections at 1-h intervals prevented about 80% of c-fos, nerve growth factor I-A and jun-B mRNA induction seen after a single injection. Because nicotine is known to induce immediate early genes in the adrenal glands as well, we examined whether similar kinetics of the gene induction could be seen in the adrenal medulla. However, no refractory period for repeated nicotine treatment or down regulation of the induction of the immediate early genes could be demonstrated in the adrenal medulla. The results show that sympathetic neurons respond to nicotine with altered expression of immediate early genes. Nicotine-induced expression of immediate early genes may be mediated and regulated by different factors in neuronal and endocrine noradrenergic cells.

1. Adenosine analogues caused hyperpolarization and inhibition of the depolarizing response to muscarine of the rat isolated superior cervical ganglion (SCG) measured by a 'grease gap' recording technique. The receptors mediating these responses have been characterized by use of a range of selective adenosine analogues and adenosine receptor antagonists. 2. In decreasing order of potency N6-cyclopentyladenosine (CPA), 2-chloroadenosine (2CA), adenosine, 2-phenylaminoadenosine (PAA), caused concentration-dependent hyperpolarizations whilst N6-(9-fluorenylmethyl)adenosine (PD 117,413) was inactive at up to 100 microM. 3. The order of potency of adenosine analogues in depressing depolarization caused by a submaximal concentration of muscarine (100 nM) was: CPA > R-PIA = 2CA > NECA > S-PIA > BZA > adenosine > PAA, where R- and S-PIA = R(-)- and S(+)-N6-(2-phenylisopropyl)adenosine, NECA = 5'N-ethylcarboxamidoadenosine and BZA = N6-benzyladenosine. PD 117,413 was inactive at concentrations up to 100 microM. The maximum inhibitions of the muscarine-induced depolarization by CPA, 2CA, NECA and BZA were similar. R-PIA, S-PIA and PAA produced similar maximal inhibitions which were significantly smaller than those produced by CPA. 4. Hyperpolarizations caused by adenosine were antagonized by the P1-purinoceptor selective antagonist 1,3-dimethyl-8-phenylxanthine (8PT) and by the selective A1-adenosine receptor antagonist, 1,3-dipropyl-8-(4-((2-aminoethyl)amino)carbonylmethyloxyphenyl++ +)xanthine (XAC). Hyperpolarizations caused by CPA, adenosine and PAA were antagonized by the A1-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) but not by the A2-selective antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX). 5. Inhibition of the muscarinic-induced depolarization by CPA was antagonized by 8PT and DPCPXbut not by DMPX.6. It is concluded that the neurones of the rat SCG possess P1-purinoceptors of the Al-adenosine receptor subtype which mediate hyperpolarization and inhibition of depolarization caused by muscarine.

Nicotinic transmission in the superior cervical ganglion of the cat was studied in vivo and in vitro by recording the postganglionic compound action potential evoked, under partial block with hexamethonium, by supramaximal 0.2 Hz stimulation of the cervical sympathetic trunk. The compound action potential was depressed following a stimulus train (5 Hz, 40 s) applied to the same set of axons. The inhibition was antagonized by naloxone, suggesting mediation by endogenous opioids. Agonists selective for mu-, delta- and kappa-opioid receptors, injected into the arterial supply of the in situ ganglion or added to the perfusion fluid in the in vitro ganglion, also produced a naloxone-sensitive inhibition of the compound action potential. The synaptic inhibition was antagonized by the delta-selective antagonist ICI 174,864 and by the mu-selective antagonist CTAP, but not by the kappa-selective antagonist Nor-BNI. These results suggest that all three main types of opioid receptors are present in the ganglion but only mu- and delta-receptors are involved in the inhibition of ganglionic transmission mediated by the endogenous opioids.

1. Sympathetic neurones derived from superior cervical ganglia of neonatal rats and maintained in tissue culture were used to investigate the modulation of neurotransmitter release by presynaptic receptors. Three week old cultures of neurones were loaded with [3H]-noradrenaline to label endogenous neurotransmitter stores. Release of noradrenaline was evoked by depolarization with raised extracellular K+ in the presence of desipramine and corticosterone to prevent uptake of released catecholamine. 2. Potassium (55 mmol l-1) depolarization for 30 s caused more than a four fold increase in 3H overflow from basal levels but this increase was reduced by up to 40% in the presence of exogenous noradrenaline (1 mumol l-1). The inhibition by noradrenaline of depolarization-evoked overflow was blocked by the alpha 1/alpha 2-adrenoceptor antagonist, phentolamine. Phentolamine alone did not increase K(+)-evoked 3H overflow. 3. The alpha 2-adrenoceptor antagonist, yohimbine, produced a concentration-dependent block of the inhibition by noradrenaline of K(+)-evoked overflow, while the alpha 1-adrenoceptor antagonist, prazosin, was without effect at concentrations up to 0.1 mumol l-1. 4. The beta-adrenoceptor antagonist, propranolol, neither reduced K(+)-evoked overflow nor increased the degree of inhibition caused by the addition of 1 mumol l-1 noradrenaline. 5. The alpha 2-adrenoceptor agonist, clonidine (1 mumol l-1) was less effective than noradrenaline at inhibiting K(+)-evoked overflow, while the alpha 1-adrenoceptor agonist, phenylephrine (1 mumol l-1) had no significant effect. 6. The L-channel calcium blocker, nicardipine (1 mumol l-1) significantly inhibited 3H overflow evoked by K+. In the presence of L-channel block, however, noradrenaline still inhibited residual evoked overflow.7. In the presence or absence of nicardipine, pertussis toxin pretreatment (1 nmol 1-1) reduced, but did not prevent, the effect of noradrenaline (1 micromol 1-1). Pertussis toxin alone caused a significant enhancement of K+-evoked 3H overflow.8. The data indicate that on postganglionic neurones of cultured rat sympathetic ganglia there are alpha 2-adrenoceptors that modulate neurotransmitter release, but no functional beta-adrenoceptors that mediate an enhancement of transmitter release. The data suggest further that in this preparation the mechanism of alpha2-adrenoceptor modulation may involve pertussis toxin sensitive and insensitive G-proteins and effects on calcium channels other than L-type.

A grease-gap extracellular recording technique was used to detect 5-HT1A receptor-mediated hyperpolarizing responses to 5-hydroxytryptamine (5-HT) in the rat isolated superior cervical ganglion. In the presence of the novel 5-HT1A receptor antagonist, WAY-100135 [N-tert-butyl-3-(4-(2-methoxyphenyl)piperazin-1-yl)-2-phenylpropan amide], the responses to 5-HT were antagonised in a competitive manner with a pA2 value of 7.2 (6.9-8.5) and Schild plot slope of 1.0 (0.4-1.6), n = 20. The antagonist activity was greater in the (+) than the (-)enantiomer of WAY-100135. The pA2 value of the (+)enantiomer was 7.5 (7.2-8.0), Schild plot slope 1.2 (0.8-1.6), n = 17. In contrast the (-)enantiomer had weak antagonist activity (pA2 6.3 +/- 0.25, n = 3). No agonist activity of WAY-100135 or its enantiomers were observed in this study.

1. Patch-clamp techniques in conjunction with indo-1 fluorescent measurements were used to measure increases in intracellular free calcium concentration and membrane conductance induced by the activation of nicotinic and calcium channels in cultured rat sympathetic neurons. 2. Under voltage-clamp conditions, pressure application of the nicotinic agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide, 100 microM, 100 ms) increased [Ca2+]i by 193 +/- 26 nM at a clamp potential of -60 mV. This was accompanied by an inward current of -4.53 +/- 0.89 nA, giving a mean ratio of the delta (Ca2+]i to the total inward charge transfer of 42.7 nmoles per litre of free calcium per nanocoulomb of charge (M/q ratio). 3. The DMPP-induced current and associated delta [Ca2+]i were reduced by mecamylamine (100 nM-10 microM) but were unaffected by alpha-bungarotoxin (100 nM) or cadmium (100 microM). 4. The M/q ratio was not affected by the holding potential (from -80 to -40 mV) but was a function of the external calcium concentration. 5. The M/q ratio was reduced by increasing the intracellular calcium buffering capacity and increased by heparin but not affected by ryanodine or by depletion of the caffeine-sensitive calcium store. 6. Under the same recording conditions, we quantified the increase in [Ca2+]i associated with activation of the voltage-dependent calcium current. On average at -60 mV, the M/q ratio of this highly calcium-selective permeability was 1961 mM nC-1, which is 46 times that obtained for the nicotinic channel. 7. Assuming constant-field theory, ion-substitution experiments suggest that in 2.5 mM external calcium, the permeability sequence for the nicotinic conductance was Cs+ < Li+ < Na+ < K+ < Ca2+. 8. We conclude that the nicotinic channels in rat sympathetic neurones are significantly permeant to Ca2+ and that the influx of Ca2+ through these channels is the principal cause of the rise in [Ca2+]i seen under voltage clamp.

1. An unanticipated inward tail current was recorded from freshly isolated adult rat superior cervical ganglion (SCG) neurones using the whole-cell variant of the patch-clamp technique. The tail current was present when Na+ was substituted for tetraethylammonium (TEA) as the primary monovalent cation in external solutions designed to isolate Ca2+ channel currents (0.5 microM tetrodotoxin present and K+ omitted). 2. The tail current was observed following step potentials positive to -30 mV and reached half-activation near -9.0 mV. The decay of the tail current was voltage dependent and could be described with two time constants. Between potentials of -120 and -70 mV, tau f, the fast component, varied from 3 to 8 ms and tau s, the slow component, changed from 12 to 30 ms, respectively. 3. The tail current was not carried by Ca2+, and did not appear to flow through a voltage-gated Ca2+ channel or a Ca(2+)-dependent channel as it persisted in the absence of external Ca2+ or in the presence of the Ca2+ channel blocker, Cd2+ (0.1 mM). 4. Varying the external [Cl-] did not alter the reversal potential of the tail current indicating that Cl- was not the charge carrier. 5. The reversal potential of the tail current changed in accordance with the Nernst relationship when [Na+]i/[Na]o was altered. Our results suggested that this 'unusual or unanticipated current' (Iu) was carried primarily by Na+. 6. Iu was inhibited by the K+ channel-blocking agents quinidine (0.1 mM), external Ba2+ (5 mM) and internal Cs+ (145 mM). TEA (20 mM either internally or externally) and dendrotoxin (10 microM) were not effective inhibitors of Iu. 7. The decay time constants of the tail current and parameters of activation and inactivation of Iu were similar to those of TEA-insensitive delayed rectifier-type K+ channel currents observed in the presence of 145 mM external K+. 8. Iu was reduced in the presence of either external or internal K+. The interaction of external K+ with Na+ on the Iu tail amplitude was reminiscent of anomalous mole-fraction behaviour. 9. Ion permeability studies revealed that the channel producing Iu had a permeability sequence to monovalent cations of 3.5:2.5:2:1:0.5 for Rb+, K+, Cs+, Na+ and Li+, respectively. 10. These data suggest that in the absence of external K+, the ion selectivity of a TEA-insensitive K+ channel in sympathetic neurones is profoundly diminished. Under these conditions, Na+ traversing a K+ channel can generate an unanticipated inward current.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The ionic mechanism responsible for hyperpolarization of the rat superior cervical ganglion (SCG) and depression of the depolarizing response to muscarine by adenosine was studied using an extracellular grease-gap recording technique. 2. Both the hyperpolarizations to adenosine and 2-chloroadenosine and the depression of the response to muscarine by adenosine were potentiated in reduced external calcium (Ca2+). Hyperpolarizations to adenosine were either unaltered or potentiated in the presence of the dihydropyridine Ca2+ channel antagonists, nitrendipine or (+)PN200 110 respectively. Hyperpolarizations to adenosine were unaltered by inorganic Ca2+ channel antagonists except for cobalt, which also antagonized hyperpolarizations to carbachol and depolarizations to muscarine. 3. Hyperpolarizations to adenosine were unaltered in nominally magnesium (Mg2+)-free or in reduced external chloride (Cl-) media. When sodium ions (Na+) were replaced by lithium ions (Li+) maximal responses to adenosine were initially enhanced, returning to pretreatment levels and subsequently reduced in their duration. In contrast, responses to adenosine were significantly enhanced in nominally potassium (K+)-free medium and reduced upon doubling the extracellular K+. 4. Hyperpolarizations were enhanced in the presence of the K+ channel antagonists, 4-aminopyridine and 3,4-diaminopyridine, and reduced by a low concentration (2 mM) of tetraethylammonium (TEA), but not in 10 mM TEA. 5. The results support the hypothesis that adenosine-mediated hyperpolarization of postganglionic neurones of the rat SCG is by a Ca(2+)-independent mechanism and is probably mediated via an increase of a K+ current. The results also indicate that adenosine-induced hyperpolarizations of the rat SCG are independent of the presence of extracellular magnesium.

The ATP-activated inward current (IATP) in cultured rat superior cervical ganglion neurons and its modulation by extracellular Zn2+ were examined. ATP activated a non-specific cation conductance and caused a transient rise in intracellular Ca2+. The current response was specifically activated by ATP and was blocked by the P2-purinoceptor antagonist, suramin. Low concentrations of extracellular Zn2+ rapidly and reversibly potentiated both IATP and the intracellular Ca2+ rise. The potentiation by 10 microM Zn2+ was dependent on the concentration of agonist; Zn2+ increased the sensitivity of activation without potentiating the maximum response. Higher concentrations of Zn2+ reduced and prolonged the current, consistent with open-channel block. We hypothesize that there exist two sites of action for Zn2+: a positively acting allosteric site that enhances current amplitude and a site, possibly within the pore, that blocks conductance through the channel.

The activity of transglutaminase (TG), a Ca(2+)-dependent enzyme contributing to cross-linkage formation of intracellular polypeptide chains decreased rapidly to ca. 25% of control level in superior cervical ganglia (SCG) within 0.5 h following denervation. The reduced level was maintained for at least 24 h. By contrast, following axotomy, ganglionic TG activity increased by ca. 50% within 1 h, maintained the increase to 4 h, and returned to control level by 24 h. When SCG were transferred to aerobic in vitro incubation conditions 3 h following denervation, the addition of the protein kinase C (PKC) inhibitor, trifluoperazine (TFP, 10 micrograms/ml), to the medium partially reversed the denervation-induced reduction in ganglionic TG activity. Addition of a PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA, 1 microM), had no effect on the TG activity. These findings suggest that the pathway resulting in the rapid, denervation-induced inhibition of TG activity may involve the transsynaptic activation of PKC. When SCG were placed in vitro 3 h following axotomy, addition of nerve growth factor (NGF, 0.25 micrograms/ml) to the medium reversed approximately one-half of the axotomy-induced increase in TG activity. Thus, following axotomy, the reduction in delivery to the SCG of NGF, which can be transported retrogradely within the axon and is indispensable for morphological and functional survival of sympathetic neurons, may trigger the transient, axotomy-induced TG activation in the SCG.

1. Using grease-gap recordings from the isolated superior cervical ganglion of mouse, rat and guinea-pig, we have compared the depolarization evoked by 5-hydroxytryptamine (5-HT) with that evoked by the selective 5-HT3 receptor agonist 2-methyl-5-HT (2-Me-5-HT). 2. The maximum depolarization induced by 2-Me-5-HT was smaller than that induced by 5-HT in all three species, and particularly in the guinea-pig. 3. The 5-HT2 receptor antagonist ketanserin (1 microM) caused a clear rightward shift of the dose-response curve to 5-HT on the guinea-pig ganglion, but not on the mouse or rat ganglion. Spiperone (0.03 microM) had a quantitatively similar action to ketanserin (0.1 microM) on the 5-HT dose-response curve of the guinea-pig ganglion. Ketanserin had no significant effect on the dose-response curve to 2-Me-5-HT on any of these ganglia. 4. Using 2-Me-5-HT as the agonist, we determined the pA2 values for two 5-HT3 receptor antagonists. The potency of ICS 205-930 varied by approximately 100 fold between the species and that of (+)-tubocurarine varied by over 1000 fold. The differences in the pA2 values of these compounds varied independently among the species. 5. We conclude that 5-HT3 receptors are present on the superior cervical ganglion from the rat, mouse and guinea-pig, but these receptors may be pharmacologically distinct from each other. In addition, the depolarization of the guinea-pig superior cervical ganglion by low concentrations of 5-HT is largely mediated by ketanserin-sensitive receptors.

Dissections of five human adult cadavers revealed that the C2 spinal ganglion bears a constant relationship to the dorsal aspect of the lateral atlanto-axial joint. Radiologically, the ganglion lies extradurally opposite the midpoint of the silhouette of the lateral atlanto-axial joint space. Needles can be introduced onto this target point using fluoroscopic control and used to perform selective local anesthetic blocks of the C2 spinal nerve. This technique is applicable in cases where it is difficult to decide on clinical grounds whether occipital headaches are due to an upper cervical abnormality or are a symptom of tension headache or common migraine. In particular the technique anesthetizes the otherwise inaccessible articular branches of the median and lateral atlanto-axial joints which may be an occult source of headache.

Superior cervical ganglia from 2-day-old and 3-week-old rats were maintained in vitro for up to 2 weeks in the presence of a range of concentrations of nerve growth factor up to 100 micrograms/ml. Nerve fibre length and density were measured and tyrosine hydroxylase activity of these cultures assayed after various times. Ganglia were also examined for catecholamines and neuronal numbers using fluorescence histochemistry and histology respectively. In cultures maintained without nerve growth factor, or in those containing low concentrations of nerve growth factor (3 ng/ml), tyrosine hydroxylase decreased to 5-10% of the initial levels by 14 days in vitro. The presence of the high concentration of 1 microgram/ml nerve growth factor in the culture medium or the addition of such a concentration during the culture period did not prevent an initial decrease in tyrosine hydroxylase but subsequently increased the enzyme activity. The maximal effect of nerve growth factor on nerve fibre density was at low concentrations whereas its maximal effect on neuronal survival, tyrosine hydroxylase activity or nerve fibre elongation was at high concentrations. After 2 days in culture, maximum neurite production occurred in cultures containing 10 ng/ml, while maximum nerve fibre elongation and tyrosine hydroxylase activity occurred in cultures containing 100 micrograms/ml nerve growth factor. We conclude that low concentrations of nerve growth factor, as occur in plasma, cause maximum axon formation while high concentrations of nerve growth factor, as occur in effector organs, induce maximum tyrosine hydroxylase activity and cell survival. The former process may be mediated via cell surface receptors and the latter via retrograde axonal transport of nerve growth factor to the cell body, following uptake by the terminal regions of the axons.

The synthesis, subcellular distribution and turnover of dopamine beta-hydroxylase was studied in organ cultures of rat adrenal medullae and superior cervical ganglia. After exposure to [3H]leucine for 1 or 3 h, the tissues were homogenized at various time intervals and the amount of labelled dopamine beta-hydroxylase in different subcellular fractions (cytosol, soluble and membrane-bound fraction of catecholamine storage vesicles) was determined by immunoprecipitation and subsequent electrophoresis. In cultured adrenal medullae, induction of dopamine beta-hydroxylase initiated in vivo by administration of reserpine affected both soluble and membrane-bound pools of dopamine beta-hydroxylase to a similar extent after pulse-labelling for 1 or 3 h. The half-lives of dopamine beta-hydroxylase, which amounted to 6 h for the cytosol, 7.5 h for the soluble vesicular and 32 h for the membrane-bound vesicular pools were not altered by pretreatment with reserpine. In superior cervical ganglia the half-lives of the soluble pools were 2-3 times longer than in the adrenal medulla, whereas the half-life of the membrane-bound fraction was the same as in the adrenal medulla. In both organs the most heavily labelled fraction (both after a pulse of 1 or 3 h) was always that of the vesicular membrane, suggesting that newly-synthesized dopamine beta-hydroxylase is immediately incorporated into the storage vesicles and not via release into the cytosol from the site of synthesis. The fact that the half-life of membrane-bound dopamine beta-hydroxylase is markedly longer than that of the two soluble pools suggests that the single pools are not only independently supplied by newly-synthesized DBH but there is also no appreciable subsequent exchange between soluble and membrane-bound pools.