J Physiol Volume 514, Number 1, 59-69, January 1, 1999
The Journal of Physiology (1999), 514.1, pp. 59-69
© Copyright 1999 The Physiological Society
Calcium channel subtypes differ at two types of cholinergic synapse in lumbar sympathetic neurones of guinea-pigs
David R. Ireland, Philip J. Davies and Elspeth M. McLachlan
Prince of Wales Medical Research Institute, Randwick, NSW 2031, Australia
Received 27 May 1998; accepted after revision 18 September 1998.
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ABSTRACT |
- The involvement of different presynaptic Ca2+ channels in transmission at 'weak' (subthreshold) and 'strong' (suprathreshold) synapses was investigated in guinea-pig paravertebral ganglia isolated in vitro. Selective Ca2+ channel antagonists were used to block excitatory synaptic currents evoked by stimulating single preganglionic axons.
- The N-type Ca2+ channel blocker,
-conotoxin GVIA (100 nM), reduced peak synaptic conductance by similar amounts at weak synapses (by 39 ± 6 %) and strong synapses (34 ± 6 %).
- The P-type Ca2+ channel blocker, -agatoxin IVA (40 nM), significantly reduced transmitter release at weak synapses (by 42 ± 6 %) but had only a small effect at strong synapses (reduced by 6 ± 2 %).
- Blockers of Q-, L- or T-type Ca2+ channels had no significant effects on peak synaptic conductance at either type of synapse.
- We conclude that the two functionally distinct types of preganglionic terminal in sympathetic ganglia which synapse on the same neurone differ in their expression of particular types of voltage-dependent Ca2+ channels. Both types utilize N-type channels and channels resistant to blockade by specific antagonists, but Ca2+ entry through P-type channels makes a substantial contribution to acetylcholine release only at weak synapses.
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INTRODUCTION |
Depolarization of a presynaptic nerve terminal leads to Ca2+ entry through voltage-dependent Ca2+ channels activating the release of transmitter by initiating exocytosis from vesicles. Functionally, low-voltage-activated (LVA) or high-voltage-activated (HVA) channels, with differing kinetics of inactivation, can be distinguished. These channels can be classified into at least five distinct subtypes (Zhang et al. 1993). The only LVA channel, the T-type channel, is blocked by low concentrations of Ni2+ (100 µM; Fox et al. 1987). Four types of HVA channel have been identified: (i) L-type, blocked by dihydropyridines (e.g. nifedipine) (Fox et al. 1987); (ii) N-type, blocked by -conotoxin GVIA (-CTX GVIA; EC50 = 0·7 nM; Boland et al. 1994); (iii) P-type, blocked by -agatoxin IVA (-Aga IVA EC50 = 2 nM; Mintz et al. 1992; Randall & Tsien, 1995) and by -conotoxin MVIIC with slow binding kinetics (-CTX MVIIC; EC50 = 50 nM; McDonough et al. 1996); and (iv) Q-type, blocked by -Aga IVA (EC50 = 90 nM) and by -CTX MVIIC (fully blocked by 500 nM; Randall & Tsien, 1995). Other Ca2+ channels resistant to all these antagonists have been referred to as 'R-type' (Zhang et al. 1993; Randall & Tsien, 1995) but here they are called 'resistant' channels.
At skeletal neuromuscular junctions, acetylcholine (ACh) release occurs via N-type channels in amphibians (Katz et al. 1995), whereas in mammals most ACh release is mediated via P/Q-type channels (Katz et al. 1996), with a small contribution from resistant channels (Lin & Lin-Shiau, 1997). At mammalian sympathetic neuroeffector junctions (e.g. vas deferens; Smith & Cunnane, 1996) and neuro-neuronal synapses in the chick ciliary ganglion (Yawo & Chuhma, 1994), transmission predominantly depends on Ca2+ influx through N-type channels. The Ca2+ channels mediating synaptic transmission have also been widely studied in the central nervous system, for example, in the hippocampus and cerebellum (see Wu & Saggau, 1997) and spinal cord (Takahashi & Momiyama, 1993; Wall & Dale, 1994). Multiple types of presynaptic Ca2+ channel usually contribute to transmitter release at these central synapses. Most neuro-neuronal synapses have been studied in brain slices using synaptic responses resulting from simultaneous activation of an unknown, but probably large, number of synaptic connections, so that differences between individual synapses cannot be observed. However, in a few cases, the channels involved in transmission have been determined for single synapses (Regehr & Mintz, 1994; Yawo & Chuhma, 1994; Poncer et al. 1997; Wu et al. 1998) where it has been confirmed that multiple types of presynaptic Ca2+ channel are involved.
At the synapses in mammalian sympathetic ganglia, there are two distinct functional types of preganglionic cholinergic input. The majority of inputs give rise to subthreshold or 'weak' synaptic potentials, which must summate to initiate an action potential. The minority evoke suprathreshold or 'strong' synaptic potentials that always bring the postsynaptic membrane to threshold with a large safety margin. Release probability at weak synapses ranges from 0·2 to nearly 1 (McLachlan, 1975). Although it is not possible to determine the release probability for strong synapses, early studies of quantal release imply that the probability of release is 
1 and that the large quantal content reflects a large value of the binomial parameter n. Postganglionic neurones in the paravertebral chain receive one or two strong inputs and several weak ones (McLachlan et al. 1997). In this study, we sought to identify the subtypes of Ca2+ channel responsible for triggering release of ACh from preganglionic terminals in mammalian paravertebral ganglia, and also to determine whether the two functional types of cholinergic synapse depend upon the same or different populations of Ca2+ channels.
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METHODS |
Guinea-pigs (either sex, 150-295 g) were anaesthetized with pentobarbitone sodium (I.P., 100 mg (kg body weight)-1) and exsanguinated by perfusion through the descending thoracic aorta with physiological salt solution (composition (mM): Na+, 151; K+, 4·7; Ca2+, 2·0; Mg2+, 1·2; Cl-, 144·5; H2PO4-, 1·3; HCO3-, 16·3; and glucose, 7·8; pH 7·2-7·4) gassed with 95 % O2-5 % CO2. Ganglia of the lumbar paravertebral chain from L2 to L6, together with attached white rami L2-L4, were dissected free from the animal. The L5 ganglion and associated nerves were pinned in a plastic recording chamber (0·8 ml volume) and superfused at 5 ml min-1 with physiological salt solution of the above composition warmed to 35°C. These procedures were approved by the Animal Care and Ethics Committee of the University of New South Wales.
Intracellular recordings were made from L5 ganglion cells using techniques described previously (Hirst & McLachlan, 1984). Excitatory synaptic potentials (EPSPs) were recorded in single-electrode current clamp and excitatory synaptic currents (EPSCs) in single-electrode voltage clamp (see Hirst & McLachlan, 1984). Passive membrane properties were determined at a holding potential of -60 mV from the electrotonic voltage response (< 10 mV amplitude) to a small hyperpolarizing current step (250 ms duration) passed through the recording microelectrode. Current-voltage (I-V) relationships were also determined at a holding potential of -60 mV. I-V relationships were always linear between -60 and -90 mV (see Fig. 1B).
Lumbar sympathetic ganglion cells receive multiple preganglionic inputs from several levels of the spinal cord. The most caudal white ramus from which they arise in guinea-pigs is L4 (McLachlan et al. 1985). Synaptic responses arising from stimulation of single preganglionic axons were studied by placing separate suction electrodes on the sympathetic trunk above L2 ganglion and below L5 ganglion as well as on individual white rami L3 and L4 in some cases (Fig. 1A). By stimulating these nerve trunks separately (0-20 V, 0·02-1·0 ms), it was possible to activate only a few preganglionic inputs from each source. Experiments were performed when a single input could be distinguished on the basis of its distinct stimulation voltage threshold via one of the electrodes (i.e. the EPSP did not vary in size as stimulus strength was varied over several volts).
Stimulation of a single strong axon always evoked an action potential at resting membrane potential (RMP) and sometimes also when the membrane was hyperpolarized by up to -40 mV, whereas the EPSP in response to stimulation of a single weak axon was subthreshold for an action potential at RMP or when hyperpolarized < 10 mV below RMP in the case of some large weak responses. EPSPs and EPSCs were recorded at -90 mV in an attempt to block active responses. This increased the peak response amplitude by increasing the driving force for the ACh-mediated current. The cell input time constant was also determined at -90 mV using a depolarizing step to mimic the synaptic potential. Large weak inputs were selected so that reductions in amplitude caused by drugs could be quantified more accurately and, whenever possible, relatively small strong inputs (< 2 nA) were selected to improve the quality of the voltage clamp (see also below).
With repetitive stimulation, EPSP amplitude increased with the first few stimuli due to facilitation and then reached a constant value (McLachlan, 1975). A train of 30-40 stimuli were presented at 2 Hz and the last 20 responses were averaged. This train was repeated every 15 min before and after application of a drug until the effect of the drug on the averaged synaptic response had reached a plateau. No stimuli were presented between these trains. Control experiments showed little change in EPSC amplitude (2 ± 3 %, n = 9) with this protocol over the time taken (> 45 min) to perform an experiment.
Inputs that produced EPSCs with amplitudes > 2·0 nA could usually only be poorly voltage clamped (i.e. the residual peak voltage was > 15 % of the unclamped value). In these cases, it was possible to improve the voltage-clamp control by reducing the peak amplitude of strong inputs by application of hexamethonium (10-5-10-6 M; see Results).
Changes in transmitter release were measured as changes in peak synaptic conductance (picosiemens) calculated from the maximum amplitude of the synaptic current as:
g = IEPSC/(Vrev - Vm),
where g is conductance (nanosiemens); IEPSC is the peak EPSC amplitude (nanoamps); Vrev is the ACh reversal potential, taken as 0 V (Selyanko, 1995); and Vm is the membrane potential at which EPSCs were recorded (volts).
All data are expressed as means ± S.E.M. (n, number of cells). Antagonist-induced changes in conductance were normalized with respect to control amplitude in the 15 min prior to addition of drugs. Since weak and strong synapses were selected on the basis of EPSC amplitude, and also because values of conductance were normalized for the purpose of comparison between drug treatments or between weak and strong synapses, non-parametric statistics were usually applied using StatView (Abacus Concepts Inc., Berkeley, CA, USA) (paired comparisons, Wilcoxon signed-rank test; unpaired comparisons, Mann-Whitney U test; comparisons against a given value, one sample sign test). Student's paired and unpaired t tests were used when n < 5, when equivalent non-parametric tests cannot be used. All reported differences have P values < 0·05.
-CTX GVIA and -CTX MVIIC were purchased from Auspep (Parkville, Australia), and FTX-3.3 (synthetic funnel-web spider toxin) from Tocris Cookson (Bristol, UK). -Aga IVA was a kind gift from Pfizer (Groton, CT, USA) and was initially dissolved as a stock solution containing 1 mg ml-1 cytochrome c and kept frozen until immediately before use. Nifedipine (Sigma) was made up fresh for each experiment as a stock solution (10 mM) in ethanol. Care was taken when using nifedipine to minimize its exposure to light. Drugs were added by transferring the inlet of the perfusion system into a recirculating system containing 20 ml of a solution containing the stated concentration of drug. Previous experiments had shown that a steady state of drug concentration is achieved in the organ bath within 2-3 min (see Davies et al. 1996).
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RESULTS |
General properties of ganglion cells
RMP was -52 ± 1 mV (n = 49). Passive membrane properties determined between -60 and -70 mV were: cell input resistance (Rin), 163 ± 9 M
; input time constant (
in), 33 ± 2 ms; cell capacitance, 216 ± 11 pF (n = 43). These values are comparable to those reported previously for neurones in the same ganglion (Cassell et al. 1986).
In current clamp, injection of a suprathreshold depolarizing current pulse through the microelectrode evoked a single action potential or a transient discharge of multiple action potentials at the onset of the step in 48/49 neurones. Therefore, as described previously (Cassell et al. 1986), virtually all of these neurones were of the phasic class.
Weak and strong synaptic inputs
Weak and strong synapses were identified on the basis of the shape and amplitude of the postsynaptic responses when the membrane was hyperpolarized to -90 mV to block action potential discharge (Fig. 1C).
The EPSPs arising at weak synapses (weak EPSPs) had slow rise times (10-90 % of peak amplitude, 4·94 ± 0·22 ms, n = 33), rounded peaks and their decay phases could be fitted with a single exponential (Fig. 1D). The mean EPSP amplitude at weak synapses was 24 ± 2 mV (n = 33) at -90 mV. The decay time constant for weak EPSPs (22 ± 2 ms) was longer than the mean input time constant at -90 mV (15 ± 2 ms, n = 17). The mean EPSC amplitude was 0·75 ± 0·04 nA, which corresponds to a mean peak synaptic conductance of 8·8 ± 0·5 nS at -90 mV (n = 34).
The EPSPs arising at strong synapses (strong EPSPs) typically had rise times (2·7 ± 0·2 ms, n = 10) that were faster than those of weak EPSPs (except in the presence of hexamethonium - see below) and brief sharp peaks. The decay phase of the strong EPSPs could not be fitted with a single exponential except below 30 % of maximum amplitude. The time constant of decay of this latter portion of the EPSP (22 ± 2 ms, n = 21) was longer than the input time constant at -90 mV (16 ± 5 ms, n = 10) but was not different from the decay of weak EPSPs (P = 0·63). At -90 mV, strong synapses had a mean EPSP amplitude of 33 ± 2 mV, a mean EPSC amplitude of 1·30 ± 0·16 nA, and a mean peak synaptic conductance of 18·4 ± 2·9 nS (n = 21). These values include data from 10 strong synapses after addition of hexamethonium (EPSP amplitude, 30 ± 3 mV; EPSC amplitude, 1·00 ± 0·14 nA; conductance, 11·5 ± 1·4 nS).
The decay of the EPSC could be fitted with a single exponential for most of the decay phase in the case of both weak and strong synapses (Fig. 1D). Further, the EPSC decay time constant did not differ between weak (8·3 ± 0·6 ms, n = 26) and strong synapses (7·6 ± 0·6 ms, n = 18) (P = 0·54). In practice, most (78 %) synapses with EPSCs > 1 nA usually had EPSPs characteristic of a strong synapse, and most (93 %) of those with EPSCs < 1 nA had the EPSP shape of a weak synapse. It should be noted that the values for amplitudes and time courses given above are mean values for synapses selected on the basis of size, as described in Methods. They do not represent mean values for all weak and strong synapses in these ganglia.
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Figure 1. Responses evoked at single ganglionic synapses
A, diagram of experimental arrangement for recording from single synapses in L5 paravertebral ganglia. Individual preganglionic axons lying either in the sympathetic chain above L2 ganglion, in L3 or L4 white rami, or projecting beyond L5, were stimulated via suction electrodes. Evoked synaptic events were recorded with an intracellular microelectrode in a postganglionic neurone in L5 ganglion. B, steady-state I-V curve for an L5 neurone showing rectification below -90 mV and above -60 mV. The linear part of curve is indicated by the dotted line. Examples of voltage records in response to depolarizing and hyperpolarizing current steps are shown at the bottom; the dotted line indicates a holding potential of -60 mV. C, responses evoked at a single weak synapse (a) and a single strong synapse (b) in two different ganglion cells. 1, responses at -60 mV consisted of an EPSP in a and an action potential in b. 2, with the membrane held at -90 mV to block action potentials, EPSPs had distinctive shapes. 3, EPSCs recorded under voltage clamp at -90 mV (top) and residual voltage responses (bottom) in the same cells; holding currents were -0·25 nA (a) and -0·32 nA (b). Dotted lines indicate holding potentials and holding currents as appropriate. Calibrations in b also apply to a. The difference in latency of responses between a and b is due to the greater length of axon between the stimulating electrode and the ganglion in a. D, time course of averaged synaptic responses in the same neurones as in B. a, weak EPSPs (digitized points; top) had an exponential decay (fitted curve) with time constant, , slightly longer than the input time constant, in. b, strong EPSPs had a decay phase with an early hump and only the latter part of the voltage trajectory decayed exponentially with a time constant slightly longer than in. The underlying EPSCs (digitized points; bottom) decayed as single exponentials with the time constants shown.
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Effect of hexamethonium on strong synaptic responses
Hexamethonium was used to reduce the size of large strong synaptic responses in 10 of 21 experiments at strong synapses. Because hexamethonium preferentially blocks open channels at hyperpolarized membrane potentials (see Selyanko, 1995), neurones were held close to -90 mV and the strong input was stimulated at 2 Hz. The block progressively increased during the train and maximum block was reached by about the 40th stimulus. Therefore, when hexamethonium was used, 70 stimuli were applied and the last 20 responses were averaged to provide the mean response amplitude. The amount of block varied with concentration; mean conductance was reduced by 49 ± 6 % by 5 × 10-6 M hexamethonium (n = 4), and by 70 ± 3 % by 10-5 M hexamethonium (n = 4). Using our stimulation protocol, the mean peak conductance of strong synapses exposed to hexamethonium did not show evidence of run-down over the time span of an experiment (peak conductance change, 1 ± 6 %; n = 4, P = 0·2).
In the presence of hexamethonium, strong EPSPs were identical in shape to large weak EPSPs. The amplitude of strong EPSPs in hexamethonium was reduced to 30 ± 3 mV (n = 10), which was not different from the amplitude of strong EPSPs not exposed to hexamethonium (35 ± 2 mV; n = 11, P = 0·15) or from the value for weak EPSPs (P = 0·06). The rise time of strong EPSPs in hexamethonium was 4·2 ± 0·3 ms (n = 10) which was longer than the rise time for strong EPSPs in the absence of hexamethonium, but was not different from the rise time of weak EPSPs (P = 0·14).
Blockade of Ca2+ entry by Cd2+ ions
Block of Ca2+ influx by addition of Cd2+ (300 µM) abolished all responses at both weak and strong synapses confirming that transmitter release is entirely dependent on extracellular Ca2+. Addition of Cd2+ did not significantly alter passive membrane properties of the neurones (n = 7) (control RMP, -56 ± 2 mV; Rin, 123 ± 23 M; in, 23 ± 3 ms).
Blockade of N-type Ca2+ channels
Application of the specific N-type Ca2+ channel antagonist, -CTX GVIA (100 nM), reduced peak synaptic conductance at both weak (Fig. 2Aa and Table 1A) and strong (Fig. 2Ab and Table 1B) synapses. Maximal block was achieved in 30-45 min (Fig. 3Aa and Ba). Peak synaptic conductance at weak synapses was reduced by 39 ± 6 % (Table 1A), and at strong synapses by 34 ± 6 % (Table 1B). These changes were not different between the two types of synapse (P = 0·77).
-CTX GVIA did not alter passive membrane properties of impaled neurones (n = 14) (control RMP, -52 ± 1 mV; Rin, 191 ± 24 M; in, 31 ± 2 ms) or the time course of decay of weak (Table 1A) or strong (Table 1B) EPSCs. This latter observation suggests that -CTX GVIA did not have an effect on the opening characteristics of nicotinic receptor channels (see Selyanko, 1995).
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Figure 2. Selective blockade of N- and P-type Ca2+ channels
Effects of 100 nM -CTX GVIA (A) and 40 nM -Aga IVA (B), at examples of weak synapses (a) and strong synapses in 10-5 M hexamethonium (b). 1, averaged EPSPs in current clamp; 2, corresponding averaged EPSCs in voltage clamp (top) and residual voltage (bottom), in control (thin lines) and in the presence of antagonist (thick lines). In Bb, the control and drug traces overlie each other. Dashed lines indicate holding potential (-90 mV) in 1 and 2 (bottom trace) and holding currents in 2 (top trace) of -0·30 nA (Aa), -0·22 nA (Ab), -0·21 nA (Ba) and -0·40 nA (Bb).
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Figure 3. Time course of blockade of transmission by Ca2+ channel antagonists
Peak conductance relative to control is shown at various times after application of the antagonist. -CTX GVIA inhibited transmission at weak (Aa) and strong (Ba) synapses to a similar degree. However, -Aga IVA reduced transmission at weak synapses (Ab) but had little effect at strong synapses (Bb). Error bars indicate S.E.M.
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Blockade of P-type Ca2+ channels
Application of the P-type Ca2+ channel blocker, -Aga IVA (40 nM), reduced peak synaptic conductance evoked by stimulating weak inputs by 42 ± 6 % (Fig. 2B a and Table 1A) in 30-45 min (Fig. 3Ab). In contrast, at strong synapses, this concentration of -Aga IVA reduced peak synaptic conductance by only 6 ± 2 %, a change which was only just significant (P = 0·04) (Fig. 2B b and Table 1B). The effect of -Aga IVA at strong synapses was significantly less than that at weak synapses.
To ensure that a concentration of -Aga IVA of 40 nM was sufficient to block all P-type channels, the concentration was raised to 200 nM, the effects of which were similar to the those of 40 nM. Peak synaptic conductance at weak synapses in 200 nM -Aga IVA was reduced by 40 ± 9 % (n = 3), and at strong synapses was not changed (3 ± 2 %, n = 3, P = 0·20). These data suggest that 40 nM -Aga IVA was effective in producing complete P-type channel blockade. A concentration of -Aga IVA of 200 nM was previously shown to reduce the extracellularly recorded postganglionic nerve spike of the rat superior cervical ganglion (SCG) by 21 % (Gonzalez Burgos et al. 1995). Our data suggest that part of the extracellular signal in their experiments must have arisen from the summation of weak EPSPs to threshold.
Although -Aga IVA produced no significant change in input resistance (control Rin, 174 ± 21 M; n = 8) or input time constant (control in, 33 ± 4 ms; n = 8), there was a small hyperpolarizing shift in RMP (-49 ± 1 to -52 ± 1 mV; n = 8). -Aga IVA did not affect the time course of decay of weak (Table 1A) or strong (Table 1B) EPSCs. This suggests that -Aga IVA also did not have an effect on nicotinic receptor channel properties.
Table 1. Effects of Ca2+ channel antagonists on synaptic conductance at weak (A) and strong (B) synapses
| | Conductance (nS) | EPSC amplitude (nA) | EPSC (ms) | EPSP amplitude (mV) | EPSP (ms) |
| A. Weak synapses |
| Control | 6·8 ± 0·9 (7) | 0·59 ± 0·08 (7) | 7·3 ± 0·9 (7) | 18 ± 3 (4) | 16 ± 3 (4) |
| -CTX GVIA (100 nM) | 4·1 ± 0·6 (7) * | 0·35 ± 0·05 (7) * | 8·0 ± 1·1 (7) | 11 ± 4 (4)  | 15 ± 1 (4) |
| Control | 10·0 ± 0·8 (7) | 0·88 ± 0·07 (7) | 7·3 ± 0·5 (7) | 25 ± 2 (5) | 24 ± 3 (5) |
| -Aga IVA (40 nM) | 5·9 ± 0·7 (7) * | 0·53 ± 0·06 (7) * | 7·7 ± 1·0 (7) | 19 ± 1 (5) * | 23 ± 3 (5) |
| Control | 9·0 ± 0·8 (7) | 0·79 ± 0·06 (7) | 7·2 ± 0·6 (7) | 24 ± 2 (6) | 24 ± 2 (6) |
| -CTX GVIA + -Aga IVA | 2·8 ± 0·6 (7) * | 0·25 ± 0·05 (7) * | 8·9 ± 1·4 (7) | 10 ± 3 (6) * | 21 ± 2 (6) |
| Control | 9·1 ± 1·0 (8) | 0·84 ± 0·09 (8) | 10·3 ± 1·7 (7) | 27 ± 3 (7) | 19 ± 3 (7) |
| -CTX GVIA + -Aga IVA + -CTX MVIIC (500 nM) | 3·7 ± 1·1 (8) * | 0·33 ± 0·08 (8) * | 10·2 ± 1·7 (7) | 9 ± 3 (7) * | 15 ± 4 (7) |
| Control | 8·3 ± 0·9 (5) | 0·73 ± 0·08 (5) | 7·8 ± 0·6 (5) | 17 ± 3 (3) | 18 ± 2 (3) |
| Nifedipine (10 µM) | 7·6 ± 1·0 (5) | 0·67 ± 0·09 (5) | 7·0 ± 0·4 (5) | 17 ± 4 (3) | 15 ± 2 (3) |
| B. Strong synapses |
| Control | 18·0 ± 5·1 (6) | 1·53 ± 0·46 (6) | 6·1 ± 0·4 (6) | 33 ± 5 (6) | 20 ± 5 (6) |
| -CTX GVIA (100 nM) | 12·0 ± 3·5 (6) * | 1·02 ± 0·29 (6) * | 6·3 ± 0·4 (6) | 26 ± 5 (6) * | 20 ± 5 (6) |
| Control | 16·1 ± 3·1 (5) | 1·45 ± 0·27 (5) | 6·8 ± 0·8 (5) | 28 ± 0 (3) | 25 ± 7 (3) |
| -Aga IVA (40 nM) | 15·1 ± 3·0 (5) * | 1·36 ± 0·26 (5) * | 7·3 ± 0·9 (5) | 29 ± 2 (3) | 21 ± 4 (3) |
| Control | 12·2 ± 2·1 (7) | 1·05 ± 0·19 (7) | 6·5 ± 0·6 (7) | 28 ± 3 (7) | 25 ± 4 (7) |
| -CTX GVIA + -Aga IVA | 8·1 ± 1·5 (7) * | 0·70 ± 0·13 (7) * | 6·5 ± 0·7 (7) | 23 ± 4 (7) * | 24 ± 5 (7) |
| Control | 11·2 ± 1·7 (4) | 1·02 ± 0·15 (4) | 11·4 ± 0·4 (4) | 35, 36 (2) | 22, 14 (2) |
| -CTX GVIA + -Aga IVA + -CTX MVIIC (500 nM) | 7·1 ± 1·2 (4) | 0·65 ± 0·11 (4) | 9·5 ± 0·2 (4) | 30, 24 (2) | 18, 12 (2) |
EPSC, EPSC decay time constant; EPSP, EPSP decay time constant. All data are presented as means ± S.E.M.; numbers in parentheses indicate the number of cells. * P < 0·05 vs. Control, Wilcoxon signed-rank test; P < 0·05 vs. Control, Student's t test.
Combined blockade of N- and P-type Ca2+ channels
When -CTX GVIA (100 nM) and -Aga IVA (40 nM) were applied sequentially, it was possible to determine whether or not the effects of blocking N- and P-type channels were additive. In the presence of -Aga IVA, application of -CTX GVIA reduced peak synaptic conductance at weak synapses by a further 32 ± 5 % of control (i.e. to a final amplitude of 0·30 ± 0·09 of control; n = 5) and at strong synapses by a further 22 ± 1 % of control (i.e. to a final amplitude of 0·74 ± 0·05 of control; n = 3). Similarly, in the presence of -CTX GVIA, application of -Aga IVA reduced peak synaptic conductance at two weak synapses by a further 15 and 49 % of control (i.e. to final amplitudes of 0·44 and 0·27 of control, respectively; n = 2). In contrast, at strong synapses, -Aga IVA produced no further change in conductance in the presence of -CTX GVIA (-2 ± 3 % of control; n = 4; P = 0·63). These changes were similar to those that occurred when the antagonists were applied in the absence of other toxins.
At weak synapses, peak synaptic conductance was reduced by 70 ± 6 % in the presence of both toxins (Fig. 4A and Table 1A). This was not different from the sum of the reductions in conductance produced by -CTX GVIA and -Aga IVA individually, which was 81 % of control (P = 0·73; one sample sign test). At strong synapses, application of -CTX GVIA plus -Aga IVA reduced peak synaptic conductance by only 33 ± 6 % (Fig. 4B and Table 1B). This was not different from the block due to -CTX GVIA alone (34 ± 6 %; P = 0·95) or the sum of the blocks produced by -CTX GVIA and -Aga IVA individually (40 %; P = 0·23). However, the degree of block at strong synapses was significantly less than the block produced by the combination of toxins at weak synapses.
The time courses of decay of weak (Table 1A) and strong (Table 1B) EPSCs were not modified by application of both toxins.
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Figure 4. Simultaneous blockade of both N- and P-type presynaptic Ca2+ channels
A, a weak synapse; B, a strong synapse. a, averaged EPSPs in current clamp; b, corresponding averaged EPSCs in voltage clamp (top) with residual voltage (bottom) in control (thin lines) and in the presence of 100 nM -CTX GVIA + 40 nM -Aga IVA (thick lines). Dashed lines indicate holding potential (-90 mV) in a and b (bottom) and holding current in b (top) of -0·41 nA (A) and -0·15 nA (B).
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Blockade of presynaptic Q-type Ca2+ channels
-CTX MVIIC at 500 nM fully blocks the Ca2+ current through Q-type channels in cerebellar granule neurones (Randall & Tsien, 1995). However, this concentration would also be expected to block most N-type channels (half-block, 18 nM in rat SCG neurones; McDonough et al. 1996) and to block P-type channels slowly (half-block, 50 nM in cerebellar Purkinje neurones; McDonough et al. 1996). Thus in order to determine whether or not Q-type channels mediate the component of transmitter release resistant to N- and P-type channel blockade, -CTX MVIIC (500 nM) was added together with -Aga IVA (40 nM) and -CTX GVIA (100 nM) to determine whether the block of transmission achieved was greater than that with a combination of only -CTX GVIA plus -Aga IVA. At weak synapses, application of all three toxins reduced peak synaptic conductance by 63 ± 11 % (Table 1A). This was not different from the combined effects of -CTX GVIA plus -Aga IVA which blocked only N- and P-type channels (70 ± 6 % block; P = 0·56). At strong synapses, the combination of the three toxins applied together blocked peak synaptic conductance by 37 ± 2 % (Table 1B), which was not different from either the block due to the combination of -CTX GVIA plus -Aga IVA (33 ± 6 %; P = 0·40), or that due to 100 nM -CTX GVIA alone (34 ± 6 %; P = 0·72). However, the block by the combination of the three toxins at weak synapses was significantly greater than it was at strong synapses.
In the presence of all three toxins, the time course of decay of weak EPSCs was not modified (Table 1A), although the mean time course of decay of strong EPSCs was slightly decreased (Table 1B).
Blockade of L-type Ca2+ channels
Nifedipine was used to determine the role of L-type channels in transmitter release. Nifedipine (10 µM) did not change mean peak synaptic conductance due to stimulation of weak inputs (-8 ± 7 %; n = 5; P = 0·47) (Table 1A). Transmitter release at strong synapses was also not affected by nifedipine (n = 2) (peak conductance, control/nifedipine: 26·6 nS/25·8 nS, 11·6 nS/13·6 nS).
Blockade of T-type Ca2+ channels
When 100 µM Ni2+ was added to block T-type Ca2+ channels (Fox et al. 1987), peak conductance was not changed at weak synapses (3 ± 3 %; n = 4; P = 0·44), or at strong synapses (-5 ± 1 %; n = 3; P = 0·16). This concentration of Ni2+ would also have blocked resistant or R-type channels in cerebellar granule cells (Tottene et al. 1996).
Effects of FTX-3.3
Both an extract of funnel-web spider venom (FTX) and a synthetic toxin sFTX (Dupere et al. 1996) have been shown to block transmission through the rat SCG (Gonzalez Burgos et al. 1995). We therefore applied 1 µM FTX-3.3, a synthetic analogue of FTX, to three synapses (2 weak, 1 strong) in the presence of steady-state block of N- and P-type channels by 100 nM -CTX GVIA and 40 nM -Aga IVA, and to one strong synapse in the absence of other toxins. FTX-3.3 reduced peak synaptic conductance by 9 ± 1 % of control (n = 4) and the resistant component by 25 ± 3 % (n = 3). Block of ganglionic transmission by 500 µM FTX (Gonzalez Burgos et al. 1995) may have resulted from non-specific block of N- as well as P-type channels (Gonzalez Burgos et al. 1995; Norris et al. 1996) thus reducing the postsynaptic response below threshold without entirely blocking transmitter release.
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DISCUSSION |
This study of synapses in mammalian sympathetic ganglia has shown that the Ca2+ that triggers quantal ACh release in response to an action potential enters the preganglionic nerve terminals through Ca2+ channels of the N- and P-type and also through a channel resistant to selective concentrations of toxins. In this regard, the ganglion synapses bear a closer resemblance to many mammalian central synapses than to peripheral neuromuscular junctions of either the somatic or autonomic type. We have further shown that ganglionic synapses of different effectiveness utilize different combinations of Ca2+ channel types: at weak synapses, N-type (40 %), P-type (40 %), and resistant channels mediate transmitter release whereas, at strong synapses, N-type (35 %) and resistant channels (65 %) are involved with little contribution from P-type channels (Fig. 5). The combination of channels that supports release at weak synapses is the same as that recently reported at the subthreshold cholinergic synapses in submucosal ganglia of guinea-pig caecum (Cunningham et al. 1998). However, in vivo, action potentials are evoked in postganglionic neurones in paravertebral ganglia primarily by activity of strong synapses (McLachlan et al. 1997) where the resistant channels mediate the bulk of transmitter release. This large resistant component at strong ganglionic synapses contrasts with the situation at other suprathreshold synapses, such as the calyx of Held (Wu et al. 1998), the cerebellar climbing fibre synapse (Regehr & Mintz, 1994), and the chick ciliary ganglion (Yawo & Chuhma, 1994), where transmission is predominantly mediated by N- and/or P-type channels.
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Figure 5. Relative contribution of Ca2+ influx through specific Ca2+ channels to transmission in lumbar paravertebral ganglia
Error bars indicate S.E.M. * Significant difference at the 0·01 level between strong and weak synapses. The resistant component (R) is the component resistant to block of N- and P-type channels. Weak and strong synapses differ markedly in the contribution made by P-type and resistant channels to transmission.
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Weak and strong synapses represent two distinct populations characterized by the shape of their synaptic potentials and the average size of their peak synaptic conductance (see Results). It is remarkable that the differences in presynaptic Ca2+ channel types were apparent even though the populations of weak and strong synapses selected for these studies were very similar in terms of their peak synaptic conductance. In addition, large strong EPSPs that had been treated with hexamethonium had amplitudes and time courses that were indistinguishable from those of large weak EPSPs, yet different responses to the Ca2+ channel antagonists were still observed.
Weak and strong preganglionic inputs are both cholinergic, originate in the same group of spinal neurones and are associated with the same kind of target neurones; they have previously been known to differ only in the quantity of transmitter released (Hirst & McLachlan, 1984). Further, as most ganglion cells receive both types of input, we have shown for the first time that the mechanisms of transmitter release at two synapses on the same neurone can differ. It is not clear what the basis of this differentiation is or how it is related to connectivity in the ganglion. Individual preganglionic neurones might form only one type of synapse, that is, one population of preganglionic neurones may make strong synapses at all their connections, like somatic motor units, whereas the other (possibly the majority) forms only weak synapses. Alternatively, each preganglionic neurone may form both strong and weak synapses via different axon collaterals on neurones in the same ganglion or in different segmental ganglia. Most preganglionic axons innervate neurones in more than one paravertebral ganglion but the dominant input to a particular ganglion arises from the equivalent segmental level of the spinal cord (Lichtman et al. 1980). The synaptic inputs arising from the dominant segment must include strong inputs but those that reach more distant ganglia usually do not. Thus it seems likely that the divergent branches of a single preganglionic axon form synapses that utilize different populations of Ca2+ channels. This raises the interesting question of how different Ca2+ channel types are targeted to the appropriate axonal branches of an individual neurone.
An important issue in the interpretation of these experiments is antagonist specificity. To avoid problems of non-specificity described previously (e.g. Wang et al. 1992; Sivaramakrishnan & Laurent, 1995), we used relatively low concentrations of Ca2+ channel antagonists. This meant that steady-state block was achieved rather slowly, over a similar time course to that achieved by the same concentrations of -CTX GVIA or -Aga IVA in brain slices (Nooney & Lodge, 1996). The specificity of blockade by the different antagonists has been determined in patch-clamp studies of neuronal somata. One hundred nanomolar -CTX GVIA is specific for N-channels (Olivera et al. 1994) and is saturating for these channels in mammalian postganglionic neurones (Boland et al. 1994) and for transmitter release from central and peripheral synapses (Wu & Saggau, 1994; Nooney & Lodge, 1996; Smith & Cunnane, 1996). Since we saw no greater effect on transmission at either type of synapse with 100 nM -CTX GVIA plus 500 nM -CTX MVIIC than with -CTX GVIA alone, 100 nM -CTX GVIA is also saturating for preganglionic N-type channels. The results obtained with -Aga IVA are inherently difficult to interpret because of its apparent lack of specificity. Evidence from patch-clamp studies of P-type channels in Purkinje neurones indicates that 40 nM -Aga IVA should fully block these channels (see Introduction). This was confirmed here as 200 nM -Aga IVA had no greater effect than 40 nM -Aga IVA at either weak or strong synapses. In addition, while it is possible that some of the effects of 40 nM -Aga IVA were due to block of a proportion of Q-type channels, this is unlikely given that addition of a further 500 nM -CTX MVIIC to produce a saturating block of all Q-type channels did not increase the amount of inhibition.
The observed differences in the effects of -Aga IVA at weak and strong synapses could be accounted for if toxin access differed, or if strong synapses had presynaptic P-channels which were less sensitive to -Aga IVA than those at weak synapses. However, the time course of block with -CTX GVIA was the same for the two types of synapse which argues that toxin access was similar for weak and strong synapses. In addition, 200 nM -Aga IVA had no greater effect than 40 nM at strong synapses. These observations suggest that the ineffectiveness of P-channel blockade at strong synapses is not due to either limitations to toxin access or to P-channels with low sensitivity to -Aga IVA.
As at other fast synapses (see Dunlap et al. 1995), L-type and T-type channels were found not to be involved in transmitter release in sympathetic paravertebral ganglia. Nifedipine at 10 µM significantly blocks the effects of Ca2+ entry in rat postganglionic neurones (Davies et al. 1996) but had no effect on ACh release from preganglionic terminals in the present experiments. However, nifedipine (10 µM) reduced the amplitude of synaptic potentials evoked by splanchnic nerve stimulation in the guinea-pig coeliac ganglion by about 40 % (Zhai & Ma, 1991). Synapses in the coeliac ganglion may differ from those in the paravertebral chain, but it is also possible that the release of a peptide, such as substance P, which potentiates synaptic responses in this ganglion (Jiang & Dun, 1986), is blocked by L-channel antagonists (Rane et al. 1987).
At most synapses, antagonist-induced changes in postsynaptic responses are unlikely to mirror precisely changes in presynaptic Ca2+ levels, since the relationship between overall Ca2+ influx and the postsynaptic response is a power function 
1 (Wu & Saggau, 1997). Consequently, the sum of the block produced by each of two antagonists may sometimes exceed the block produced by their simultaneous application ('supra-additivity') (see Dunlap et al. 1995). However, the effects of blocking N- and P-type channels at weak synapses were approximately additive, which is consistent with an approximately linear relation between Ca2+ influx and transmitter release in ganglia (n = 1·5; Bennett et al. 1976). Conversely, saturation of the ACh release mechanism by Ca2+ influx through N-type and resistant channels is unlikely to account for the lack of effect of block of P-type channels, given that -Aga IVA has limited effects at strong synapses, whether applied before or after block of N-type channels.
At central synapses, the sensitivity of the release mechanism to Ca2+ entry through P/Q-type channels seems to be greater than that for N-type channels (see Wu & Saggau, 1997; Wu et al. 1998). Thus there might be fewer P-type than N-type channels in the presynaptic membrane of weak synapses despite -Aga IVA and -CTX GVIA blocking transmitter release to similar extents. However, weak synapses utilize P-type channels but strong synapses do not, so that a greater sensitivity of P-type channels to Ca2+ cannot contribute to the difference in synaptic strength. Further, if transmitter release were, for example, more sensitive to the Ca2+ that entered via resistant channels, this might contribute to differences between weak and strong synapses.
What are the functional consequences of the differences in presynaptic Ca2+ channels? Endogenous substances such as adenosine and neuropeptide Y inhibit N-type channels to a greater extent than P/Q-type channels (see Wu & Saggau, 1997). Similarly, the difference in the relative contributions of P-type and resistant channels between weak and strong synapses might underlie their differential regulation by as yet unknown mechanisms. More importantly, since the resistant component at strong synapses in ganglia (67 %) is substantially larger than that at central synapses (see Wu & Saggau, 1997), the identification of an antagonist that specifically blocks resistant presynaptic Ca2+ channels might enable manipulation of sympathetic pathways (such as those controlling vasoconstriction; see McLachlan et al. 1997) without significant effects on the central nervous system.
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Acknowledgements
This work was supported by the National Health and Medical Research Council of Australia and the Motor Accidents Authority of New South Wales. D. R. I. holds an Australian Postgraduate Award. We are very grateful to Pfizer Inc., USA, for providing -agatoxin IVA.
Corresponding author
E. M. McLachlan: Prince of Wales Medical Research Institute, Randwick, NSW 2031, Australia.
Email: e.mclachlan{at}unsw.edu.au
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Introduction
