Isolated Rat Superior Cervical Ganglia Research Articles

Two distinct classes of functional 7-containing nicotinic receptor on rat superior cervical ganglion neurons.

Nicotinic acetylcholine receptors (nAChRs) that bind alpha-bungarotoxin (alpha Bgt) were studied on isolated rat superior cervical ganglion (SCG) neurons using whole-cell patch clamp recording techniques. Rapid application of ACh onto the soma of voltage clamped neurons evoked a slowly desensitizing current that was reversibly blocked by alpha Bgt (50 nM). The toxin-sensitive current constituted on average about half of the peak whole-cell response evoked by ACh. Nanomolar concentrations of methyllycaconitine blocked the alpha Bgt-sensitive component of the ACh-evoked current as did intracellular dialysis with an anti-alpha 7 monoclonal antibody. The results indicate that the slowly reversible toxin-sensitive response elicited by ACh arises from activation of an unusual class of alpha 7-containing receptor (alpha 7-nAChR) similar to that reported previously for rat intracardiac ganglion neurons. A second class of functional alpha 7-nAChR was identified on some SCG neurons by using rapid application of choline to elicit responses. In these cases a biphasic response was obtained, which included a rapidly desensitizing component that was blocked by alpha Bgt in a pseudo-irreversible manner. The pharmacology and kinetics of the responses resembled those previously attributed to alpha 7-nAChRs in a number of other neuronal cell types. Experiments measuring the dissociation rate of 125I-labelled alpha Bgt from SCG neurons revealed two classes of toxin-binding site. The times for toxin dissociation were consistent with those required to reverse blockade of the two kinds of alpha Bgt-sensitive response. These results indicate that rat SCG neurons express two types of functional alpha 7-nAChR, differing in pharmacology, desensitization and reversibility of alpha Bgt blockade.

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.

Adenosine selectively depresses muscarinic compared with non-muscarinic receptor mediated depolarisation of the rat superior cervical ganglion

1. 1. A grease gap d.c. recording technique was used to measure electrophysiological responses of the isolated rat superior cervical ganglion. 2. 2. Adenosine at 100 μM depressed depolarisations to the muscarinic agonists carbachol, muscarine and methylfurmethide. In contrast adenosine (100μM) did not alter depolarisations to 1,1-dimethyl-4-phenylpiperazinium, 2-methyl-5-hydroxytryptamine and potassium and enhanced depolarisations to 5-hydroxytryptamine and gamma-aminobutyric acid. 3. 3. Adenosine-induced depressions of the depolarisations to carbachol, muscarine, and methylfurmethide tended to be increased in the presence of 0.3 μM methoctramine (a muscarinic receptor antagonist with slight selectivity for M 2 receptors). The increase was statistically significant ( P < 0.01) for carbachol. 4. 4. Medium containing 0.1 mM Ca 2+ and 0.3 μM pirenzepine augmented the hyperpolarising phase of the response to carbachol. Adenosine (10–300μM) hyperpolarised ganglia and did not significantly alter the hyperpolarisation to 0.3 or 1 μM carbachol but selectively reduced the depolarisation response to 3 μM carbachol. 5. 5. Adenosine-induced hyperpolarisations (100 μM) were enhanced when applied during depolarisations to muscarinic agonists (muscarine, pilocarpine, N-methyl-N-(1-methyl-4-pyrrolidine-2-butynyl)acetamide (BM-5)), and other M-current inhibitors, barium and eledoisin-related-peptide. Adenosine induced hyperpolarisations were not affected by d-Ala 6-luteinizing-hormone-releasing-hormone or uridine 5′-triphosphate which produced small depolarisations. 6. 6. It is concluded that adenosine acts selectively in opposing mechanisms of depolarisation of the rat SCG that are due to the action of muscarinic agonists (acting via M 1-receptors) and by other M-current inhibitors.

Different effects of muscarinic agonssts in rat superior cervical ganglion and hippocampal slices

In this study the effects of muscarinic antagonists and agonists on M 1 muscarinic receptors in the isolated rat superior cervical ganglion and the rat hippocampal slice were investigated. Oxotremorine and APE but not pilocarpine. McN-A-343 or 4-CI-McN-A-343 induced small M 2 muscarinic receptor-mediated hyperpolarizations in the rat superior cervical ganglion. Nevertheless, for all the agonists investigated the pA 2 values of the muscarinic antagonists pirenzepine. AF-DX 116 and p-fluoro-hexahydro-sila-difenidol indicated the presence of only M In1 muscarinic receptors in the rat superior cervical ganglion and hippocampal slice. Full agonistic behaviour with respect to dcpolarization of the rat superior cervical ganglion was observed for pilocarpine. McN-A-343 and 4-CI-McN-A-343. Oxotremorine and arecaidine propargyl ester were partial agonists in this preparation, with maximal effects of 35 and 46% of the maximum obtained with pilocarpine. respectively. Pilocarpine. Oxotremorine and arecaidin propargyl ester displayed full agonistic behaviour on the increase in firing rate of pyramidal cells in rat hippocampal slices. Whereas 4-Cl-McN-A-343 was a partial agonist (maximal effect of 63% of the maximum obtained with pilocarpine). McN-A-343 displayed no agonistic or antagonistic activity in rat hippocampal slices. It remains to be established whether the heterogeneous behaviour of the agonists in both preparations reflects as yet unknown differences in the M 1 receptor protein or results from differences in the coupling of receptor to second messenger.

In vitro effect of thyroxine on cholinergic neurotransmission in rat sympathetic superior cervical ganglion.

This study aimed at examining the effect of thyroid hormones on cholinergic transmission in isolated rat superior cervical ganglia (SCG). In SCG explants incubated with 3H-choline, thyroxine (T4) and 3,3',5-triiodothyronine (T3) added to the medium before a second depolarization stimulus of 60 mM K+ resulted in a dose-dependent increase of S2/S1 ratio for 3H release. The concentration of hormone that produced 50% of maximal increase in K(+)-induced radioactivity release was 8 x 10(-9) M for T4 and 1.6 x 10(-8) M for T3 while 3,3',5,5'-tetraiodothyroacetic acid was almost ineffective. Preincubation of SCG with 10(-7) M iopanoic acid for 30 min before S2, although not affecting by itself S2/S1 ratio, effectively prevented the increase given by T4 or T3. 3H-acetylcholine release by SCG was augmented in a high K+, and the effect was amplified by T4 to a similar extent as that for total 3H release. When added to the incubation medium together with 60 mM K+ for 30 min, T4 (10(-7) M) increased significantly the activity of choline acetyltransferase (ChAT). T4 did not affect ChAT activity in SCG exposed to 4.7 mM K+, nor in SCG homogenates. 3H-choline uptake measured immediately after exposure of SCG to 60 mM K+ decreased by 25%, whereas it increased by 71% after a subsequent 30-min incubation with 4.7 mM K+. Addition of 10(-7) M T4 prevented the changes in choline uptake observed in a high K+ medium. These results indicate that T4 increases SCG cholinergic transmission.

The calcium‐dependent potassium conductance in rat sympathetic neurones.

1. Adult and intact sympathetic neurones of isolated rat superior cervical ganglia were subjected to a two-electrode voltage-clamp analysis at 37 degrees C in order to investigate the Ca2(+)-dependent K+ conductance. 2. At each potential a Ca2(+)-dependent K+ current, IKCa, was determined as the difference between the current that could be attributed to the voltage-dependent K+ current, IKV, following Ca2+ channel blockade by Cd2+ and the total current generated. The final IKCa curves were obtained after correcting the experimental tracings for the underlying ICa current component. 3. IKCa became detectable during commands to -30 mV. About 3.6 x 10(5) Ca2+ ions are required to enter the cell before IKCa is initiated. The current was modelled on the basis of a 0.4-0.6 ms delay followed by an exponential activation of a fast component, IKCaf, simultaneously with a much slower exponential activation, IKCas. Experiments indicate a sigmoidal activation curve for the fast conductance, gKCf, with half-maximal activation at -13.0 mV and a slope factor of 4.7 mV (for 5 mM-Ca2+ in the bath). The associated time constant, tau kcf, ranged from 0.8 to 2.0 ms. The slow conductance exhibited a similar steady-state activation curve but an activation time constant in the 48-280 ms range. The maximum mean gKC was 0.32 microS per neurone for either the fast or slow component. 4. Excess K+ ions accumulate in the perineuronal space during K+ current flow giving rise to rapidly occurring, large K+ reversal potential (EK) modifications (up to -45 mV for the largest currents). The kinetics of K+ extracellular load can be described satisfactorily by a simple exponential function (tau = 0.9-2.8 ms). The characteristics of K+ wash-out appear similar to those of accumulation. 5. The immediate effect of such an extracellular K+ build-up is to make the apparent IKCa activation kinetics faster and to reduce (up to 50%) the true value of the K+ conductance. We simulated the predictions of a K+ diffusion model and generated new functions describing the IKCa steady-state activation, activation rate and maximum conductance values which satisfactorily reconstruct the IKCa current tracings together with the K+ accumulation process near the membrane. 6. A small component of the Ca2(+)-dependent K+ current, IAHP, was observed which survived at membrane potential levels negative to -40 mV. Increasing Ca2+ influx by applying longer pulses enhanced IAHP, which on the other hand was also activated by depolarizations of short duration.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-term potentiation at nicotinic synapses in the rat superior cervical ganglion.

1. Nicotinic fast excitatory postsynaptic potentials (fast EPSPs) were recorded intracellularly from postganglionic neurones in the isolated rat superior cervical ganglion. 2. An hours-long potentiation of the fast EPSP could be induced by brief tetanic stimulation of the preganglionic nerve (5 Hz for 5 s to 20 Hz for 20 s). While long-term potentiation (LTP) can be detected in every ganglion by extracellular techniques, LTP was induced in only two-thirds of the nicotinic synaptic responses. 3. Muscarinic blockade with atropine did not prevent LTP of the fast EPSP. 4. LTP of the fast EPSP did not correlate with changes in input resistance nor cell potential, as recorded in the soma. 5. The formation of nicotinic LTP appeared to depend upon stimulation of the nerve terminals. Non-synaptic tetanic depolarization of the postganglionic neurone, effected by injecting depolarizing current pulses through the intracellular microelectrode, was not sufficient. LTP could be induced by synaptic tetani in two-thirds of the same neurones. 6. The response to exogenous 1,1-dimethyl-4-phenylpiperazinium (DMPP), a selective nicotinic agonist, was not increased during nicotinic synaptic LTP. This was true whether DMPP was applied by pressure-ejection from an extracellular micropipette during intracellular recording, or by brief superfusion during sucrose-gap recording of postganglionic responses. 7. Responses to exogenous acetylcholine and carbachol were increased during nicotinic LTP when these non-selective cholinergic agonists were applied by pressure-ejection during intracellular recording. However, the potentiation of the fast EPSP was always at least twofold greater than the potentiation of the response to these exogenous agonists. 8. Potentiation of the responses to acetylcholine and carbachol may have been due to long-term enhancement of muscarinic responses. Thus, no postsynaptic basis for nicotinic LTP was uncovered in these studies.

Vasoactive Intestinal Polypeptide Increases Inositol Phospholipid Breakdown in the Rat Superior Cervical Ganglion

The effects of VIP and of peptides of the VIP family: secretin, glucagon, the porcine histidine isoleucine containing peptide (PHI) and the rat hypothalamic growth hormone-releasing hormone (rhGRF) on the cyclic AMP and inositol phosphate contents of isolated rat superior cervical ganglia were investigated. We demonstrate that VIP is able to provoke a large inositol lipid breakdown by acting directly on ganglionic cells. This observation suggests the presence in rat superior cervical ganglia of a new type of receptors for VIP or for an unidentified peptide structurally related to VIP.

Vasoactive intestinal polypeptide and carbachol act synergistically to induce the hydrolysis of inositol containing phospholipids in the rat superior cervical ganglion

The effects of vasoactive intestinal polypeptide (VIP) and carbachol on inositol lipid breakdown were assayed in isolated rat superior cervical ganglia. We report here that VIP and carbachol act synergistically to stimulate the formation of inositol phosphates. This synergistic interaction may explain the modulatory effect of VIP on the muscarinic transmission in the sympathetic ganglia.

Effects of synaptic activity on the metabolism and release of purines in the rat superior cervical ganglion.

The release of radioactive metabolites from isolated rat superior cervical ganglia was measured under various conditions following preloading with 3H-adenosine. The 3H label was recovered primarily in the adenosine metabolites, ATP, ADP, AMP, IMP, and inosine, rather than in adenosine itself. Increased release was evoked by preganglionic stimulation or by exposure to a high-K+ medium, whereas in a low-Ca2+-high-Mg2+ medium, both spontaneous release and evoked release of most metabolites were inhibited. Exposure of the ganglion to an atmosphere of N2 also increased the release of most labeled metabolites, but this release was not substantially affected by a low-Ca2+ medium. The fluorescent derivatives of the endogenous adenine-containing compounds present in the ganglion were prepared from homogenates and separated by high-performance liquid chromatography (HPLC). By the end of the testing period (6 hr), levels of ATP in the isolated ganglia had dropped to 10-20% of the initial values, while levels of ADP, AMP, and adenosine increased. There was little difference in these values between nonstimulated ganglia and those exposed to N2 or to a high-K+ medium.

Vasoactive intestinal polypeptide increases inositol phospholipid breakdown in the rat superior cervical ganglion

The effects of VIP and of peptides of the VIP family: secretin, glucagon, the porcine histidine isoleucine containing peptide (PHI) and the rat hypothalamic growth hormone-releasing hormone (rhGRF) on the cyclic AMP and inositol phosphate contents of isolated rat superior cervical ganglia were investigated. We demonstrate that VIP is able to provoke a large inositol lipid breakdown by acting directly on ganglionic cells. This observation suggests the presence in rat superior cervical ganglia of a new type of receptors for VIP or for an unidentified peptide structurally related to VIP.

The pharmacological assessment of RS 86 (2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-decan-1,3-dion hydrobromide). A potent, specific muscarinic acetylcholine receptor agonist

The effects of the compound RS 86 (2-ethyl-8-methyl-2,8-diazaspiro-[4,5]-deecan-1,3-dion hydrobromide) in a number of in vitro and in vivo test systems for muscarinic cholinergic activity were analyzed and compared to those of classical muscarinic receptor agonists. In radioligand binding assays RS 86 presented high nanomolar apparent affinity only for sites labeled by 3H-muscarinic receptor agonists while its apparent affinity for sites labeled by 3H-muscarinic receptor antagonists including [ 3H]QNB, [ 3H]NMS and [ 3H]pirenzepine was in the micromolar range. RS 86 had no or only low affinity (IC 50 > 10 μM) for other neurotransmitter or drug receptor sites. The compound induced scopolamine-sensitive contractions of the isolated guinea-pig ileum showing a pD 2 of 6 in this model. In the isolated rat superior cervical ganglion RS 86 was also an agonist with a pD 2 of 6.7. When given to mice or rats by different routes RS 86 induced central and peripheral effects typical of a muscarinic receptor agonist, such as hypothermia, tremor, mydriasis, salivation, lacrimation, diarrhoea and modification of behavior as observed in an open field. In several of these tests RS 86 was about 10 times less potent than oxotremorine but more potent than arecoline, pilocarpine, aceclidine or the compound (cis) AF-30. The ED 50 values for some central effects, including the induction of hypothermia and alert non-mobile behavior were lower than those for tremor and peripheral effects. Some of the effects lasted for up 6 h, depending on the dose. Finally, RS 86 administration resulted in modifications of brain acetylcholine turnover and high affinity choline uptake typical of a central muscarinic receptor agonist. Taken together these results demonstrate clearly that RS 86 is a potent, centrally acting, selective muscarinic receptor agonist. RS 86 appears to be an adequate tool for the clinical examination of the cholinergic hypothesis of Alzheimer's disease.

Opposite actions of forskolin at pre- and postsynaptic sites in rat sympathetic ganglia.

In isolated rat superior cervical ganglia, forskolin, a powerful activator of adenylate cyclase, augmented the amplitude of fast excitatory postsynaptic potentials. Quantal analysis showed that forskolin acts presynaptically to facilitate the release of the transmitter. The time course of the presynaptic action of forskolin paralleled that of the increase in cyclic AMP level in the ganglia. In addition, forskolin exerted a postsynaptic action on the nicotinic acetylcholine receptor so that the acetylcholine-induced depolarization was depressed. The action of forskolin on the nicotinic acetylcholine receptor seems to be unrelated to the cyclic AMP system.

Membrane currents underlying the cholinergic slow excitatory post‐synaptic potential in the rat sympathetic ganglion.

Non-nicotinic slow synaptic currents were recorded from voltage-clamped neurones in isolated rat superior cervical ganglia bathed in a solution containing d-tubocurarine and (usually) 1 microM-neostigmine. Three components of slow synaptic current could be detected following repetitive preganglionic stimulation: a net inward current resulting from inhibition of the voltage-dependent outward K+ current IM; a net outward current associated with a fall in membrane conductance when IM was deactivated by membrane hyperpolarization or inhibited with external Ba2+ or internal Cs+; and an occasional late inward current associated with an increased membrane conductance. As a result, synaptic current amplitudes showed complex changes with changes in membrane potential. Both the inward current associated with IM inhibition and the outward current were enhanced by neostigmine and blocked by atropine or pirenzepine, and therefore resulted from activation of muscarinic receptors. In unclamped neurones, equivalent stimulation produced a membrane depolarization and induced or facilitated repetitive spike discharges. It is concluded that the principal synaptic response to muscarinic receptor activation is IM inhibition, leading to a net inward current and increased excitability, but that this response may be modified under certain circumstances by other synaptic currents.

Two components of muscarine-sensitive membrane current in rat sympathetic neurones.

Membrane currents induced by muscarine (Imus) were recorded in voltage-clamped neurones in isolated rat superior cervical ganglia. Two components of Imus were regularly recorded: an inward current resulting from inhibition of the outward K+ current, IM; and an outward current attributable to the reduction of a steady inward current. The presence of these two components caused a 'cross-over' in the current-voltage curves at -50 +/- 3 mV in neurones impaled with KCl-filled micro-electrodes or at -63 +/- 4 mV in neurones impaled with K-acetate-filled electrodes. Both components of Imus were prevented by atropine. Both persisted in Krebs solution containing tetrodotoxin (1 microM), Cd2+ (200 microM) or 0 Ca2+. When IM was inhibited by external Ba2+ or internal Cs+ only the outward component of Imus could be detected. This component reversed at +3 +/- 2 mV in cells impaled with CsCl-filled electrodes or at -20 +/- 3 mV in cells impaled with Cs-acetate-filled electrodes. The reversal potentials agreed with those for the currents induced by gamma-aminobutyric acid (+4 +/- 2 mV and -16 +/- 3 mV with CsCl and Cs acetate electrodes respectively). Replacement of external NaCl with Na acetate (so reducing external Cl- concentration ( [Cl-]o) from 155 to 22 mM) shifted the reversal potential for Imus by +25 and +14.5 mV in two cells impaled with CsCl-filled electrodes. A tenfold reduction of external [Na+] (by glucosamine replacement) did not significantly alter the reversal potential for Imus in KCl or CsCl-impaled cells. Under conditions where IM is already inhibited, the residual outward component of Imus can lead to hyperpolarization and inhibition of neuronal activity in unclamped cells. We conclude that both inward and outward components of Imus result from direct activation of muscarinic receptors on the ganglion cells. The inward component results from IM inhibition. We suggest that the outward component results from inhibition of another, voltage-independent current IX which largely comprises a Cl- current. The inward component induces membrane depolarization and an increased excitability; the outward component can lead to hyperpolarization and reduced excitability.

A fast transient outward current in the rat sympathetic neurone studied under voltage-clamp conditions.

Post-ganglionic neurones of the isolated rat superior cervical ganglion were voltage clamped at 37 degrees C using separate intracellular voltage and current micro-electrodes. Control experiments in current clamp suggested that the neurone is electrotonically compact, the soma and the proximal dendritic membranes being under good spatial voltage uniformity. Depolarizing voltage steps from membrane potentials near -50 mV evoked: (i) a voltage-dependent inward Na+ current, (ii) an inward Ca2+ current, (iii) a voltage-dependent outward K+ current, (iv) a Ca2+-activated K+ outward current. Depolarizations from holding potentials more negative than -60 mV elicited, besides the currents mentioned above, a fast transient outward current IA which peaked in 1-2.5 ms and then decayed to zero following an exponential time course. The IA current was shown to be primarily, if not exclusively, carried by K+. It was unaffected by removal of external Ca2+ or addition of Cd2+ and was weakly blocked by tetraethylammonium ions and partially by 4-aminopyridine. The IA current showed a linear instantaneous current-voltage relationship. Its activation ranged from -60 to 0 mV with a mid-point at -30 mV. The A conductance could be described in terms of a simple Boltzmann distribution for a single gating particle with a valency of +3. Both the development and removal of inactivation followed a single exponential time course with a voltage-dependent time constant which was large near the resting potential (42 ms at -70 mV) and small (11 ms) near -100 and -40 mV. Steady-state inactivation h infinity ranged from -100 to -50 mV, with a mid-point at -78 mV, suggesting that approximately 50% of the IA channels are available at the physiological resting potential. Action potentials elicited from various holding potentials showed maximal repolarization rates dependent on the holding potential itself. This voltage dependence was found to be in reasonably good agreement with that of h infinity curve. These data are consistent with the view that in the rat sympathetic neurone, under physiological conditions, it is the IA current rather than the delayed outward current that is responsible for the fast action potential repolarization.

Identification of delayed potassium and calcium currents in the rat sympathetic neurone under voltage clamp.

Post-ganglionic neurones of the isolated rat superior cervical ganglion were studied at 37 degrees C under two-electrode voltage-clamp conditions. Membrane depolarization beyond -40 mV from holding levels between -50 and -100 mV produced a delayed outward current which exhibited no inactivation within this voltage range. The current is carried primarily by K+ ions and its instantaneous I-V relation is linear. The total outward current could be separated into two distinct components on the basis of ion-substitution experiments. A voltage-dependent component of the delayed current, termed IK(V), is activated by membrane depolarization beyond -40 mV when Ca2+ fluxes are selectively blocked by Cd2+ or in Ca2+-free solution. IK(V) develops following first-order kinetics and rises to a peak with a voltage-dependent delay (239 ms at -30 mV and 23 ms at +10 mV). GK(V) attains a saturating value of the order of 17 mS/cm2 at about +20 mV and can be described in terms of a simple Boltzmann distribution for a single gating particle with a valency equal to +2.5. A second component of the delayed outward current, termed IK(Ca), depends on Ca2+ entry for its activation and was isolated as difference current before and after block of Ca2+ movements across the membrane. IK(Ca) is larger and faster than IK(V): it is strictly related to Ca2+ influx and also depends on membrane potential depolarization. A distinct Ca2+ current, ICa, was recorded from the neurone exposed to Na+-free or tetrodotoxin solution. ICa was activated by membrane depolarization beyond -30 mV and reached a maximum value near 0 mV. Its activation agrees with fourth-order kinetics and becomes faster with increasing depolarization. The Ca2+ current developed with a voltage-dependent time to peak of 2.9-1.8 ms and thereafter completely inactivated. The relationship between ICa and IK(Ca) is discussed. The Ca2+-k+ repolarizing system is expected to be mainly associated with action potentials arising from a depolarized neurone, whereas the IA current (Belluzzi, Sacchi & Wanke, 1985) dominates the repolarization mechanism at the normal membrane potential. The effect of muscarine was examined. Muscarine (10-50 microM) produced a fall in conductance with a voltage dependence similar to that exhibited by GK(Ca) and was ineffective when removing extracellular Ca2+ or adding Cd2+. A partial suppression of ICa by muscarine is demonstrated. It is suggested that the decrease of the outward current magnitude in the presence of muscarine may be accounted for qualitatively by the reduction in ICa.

Noncholinesterase actions of an irreversible acetylcholinesterase inhibitor on synaptic transmission and membrane properties in autonomic ganglia.

Superfusion of the organophosphorous acetylcholinesterase inhibitor soman (pinacolyl methylphosphonofluoridate; 0.01-25 microM) produced a dose-dependent reduction of extracellularly and intracellularly recorded synaptic responses in the isolated rat superior cervical ganglia at frequencies of orthodromic stimulation that do not normally produce synaptic depression. The magnitude of depression was dependent upon the frequency of stimulation (0.02-1 Hz), was maintained after the removal of soman from the superfusion solution, and recovered by over 65% during periods of inactivity. The depression of synaptic transmission produced by soman was not dependent upon the inhibition of acetylcholinesterase (AChE) activity by this agent. Transmission was increasingly depressed by doses of soman greater than those needed to inactivate all measurable ganglionic AChE activity. Dose-dependent depression of synaptic transmission in soman also occurred after pretreatment with the irreversible AChE inhibitor diisopropylphosphofluoridate (DFP; 100 microM), which inhibited greater than 98% of the AChE activity in the ganglia. Soman produced a decline in the input resistance, resting potential, spike amplitude, and spike threshold and a reduction in the hyperpolarizing afterpotential. Soman-induced depression of synaptic transmission was not due primarily to a blockade of postsynaptic nicotinic receptors. At concentrations of soman which produced significant depression in transmission, ganglionic depolarization produced by bath-applied carbamylcholine (carbachol) was either slightly depressed or facilitated. In the presence of soman, repetitive focal application of acetylcholine or carbachol did not reveal use-dependent desensitization. Muscarinic antagonists, atropine and pirenzepine, protected against the use-dependent depression of synaptic transmission induced by soman. These results suggest that a principal site of action for soman is at the presynaptic terminal and that this site is sensitive to muscarinic receptor blockade.

Outward currents in voltage-clamped rat sympathetic neurones.

Outward membrane currents were studied in neurones of the isolated rat superior cervical ganglion by using a two-micro-electrode or single-micro-electrode voltage-clamp technique. Under current clamp, depolarization elicited electrotonic potentials that displayed marked outward rectification. From negative resting potentials (-70 mV) a short latency, short duration outward rectification was observed. From more positive potentials (-40 mV) a longer latency persistent outward rectification could be demonstrated. Under voltage clamp, four distinct outward currents were observed: a delayed rectifier (IK); a transient outward current (IA); a Ca2+-activated current (IC) and the M-current (IM). The maximum amplitude of IK, IA and IC was 1-2 orders of magnitude greater than IM. Depolarizing from -40 mV to potentials more positive than -20 mV co-activated IK and IC, producing a characteristic N-shaped current voltage curve with a minimum at about +80 mV. Superfusion with Mn2+-containing solutions reduced outward current at all voltages and abolished the N-characteristic; the remaining current (IK) slowly inactivated (tau greater than 1 s). Raising [K+]o from 6 to 36 mmol/l reversed outward tail currents observed in normal solution. Addition of tetraethylammonium ions (1-3 mmol/l) strongly reduced the amplitude of IK and IC. IA was characterized by very rapid activation at potentials more positive than -60 mV and by fast and complete inactivation at potentials in the activation range. The amplitude of IA was dependent on [K+]o and was reduced by external 4-aminopyridine (1-3 mmol/l). The activation appeared to depend on the nature and concentration of divalent cations present in the superfusate. It is concluded that the soma membrane of rat sympathetic neurones, like many other vertebrate and invertebrate neurones, contains multiple populations of K+ channels. The possible functions of these in the control of ganglion cell excitability are discussed.

A light and electron microscopic study of the rat superior cervical ganglion cells by intracellular HRP-labeling

Horseradish peroxidase (HRP) was injected intracellularly into single neurons of the isolated rat superior cervical ganglia. Intracellular iontophoresis of HRP did not seem to damage the sympathetic neurons or to affect synaptic transmission. Under the light microscope, 9 of the 27 HRP-labeled sympathetic neurons exhibited varicosites in their dendrites, but not in their axons; the varicose dendrites came into close contacts with adjacent non-labeled neurons. With the electron microscope, the varicose dendrites of 3 separate neurons lightly stained with HRP, morphological features of synapses could be identified at the contact site: clusters of vesicles in the varicose dendrites, intercellular space of about 20 nm separating the apposed membranes, and an intermediate density on the postjunctional membrane. These findings suggest at the ultrastructural level the occurrence of dendro-dendritic and dendro-somatic synapses in mammalian sympathetic ganglia.