N-Type Voltage-Dependent Calcium Channels Mediate the Nicotinic Enhancement of GABA Release in Chick Brain

Trevor L. Tredway, Jian-Zhong Guo, and Vincent A. Chiappinelli

Department of Pharmacology, School of Medicine and Health Sciences, The George Washington University, Washington, DC 20037


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Tredway, Trevor L., Jian-Zhong Guo, and Vincent A. Chiappinelli. N-type voltage-dependent calcium channels mediate the nicotinic enhancement of GABA release in chick brain. The role of voltage-dependent calcium channels (VDCCs) in the nicotinic acetylcholine receptor (nAChR)-mediated enhancement of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) was investigated in chick brain slices. Whole cell recordings of neurons in the lateral spiriform (SpL) and ventral lateral geniculate (LGNv) nuclei showed that cadmium chloride (CdCl2) blocked the carbachol-induced increase of spontaneous GABAergic IPSCs, indicating that VDCCs might be involved. To conclusively show a role for VDCCs, the presynaptic effect of carbachol on SpL and LGNv neurons was examined in the presence of selective blockers of VDCC subtypes. omega -Conotoxin GVIA, a selective antagonist of N-type channels, significantly reduced the nAChR-mediated enhancement of gamma -aminobutyric acid (GABA) release in the SpL by 78% compared with control responses. Nifedipine, an L-type channel blocker, and omega -Agatoxin-TK, a P/Q-type channel blocker, did not inhibit the enhancement of GABAergic IPSCs. In the LGNv, omega -Conotoxin GVIA also significantly reduced the nAChR-mediated enhancement of GABA release by 71% from control values. Although omega -Agatoxin-TK did not block the nicotinic enhancement, L-type channel blockers showed complex effects on the nAChR-mediated enhancement. These results indicate that the nAChR-mediated enhancement of spontaneous GABAergic IPSCs requires activation of N-type channels in both the SpL and LGNv.


    INTRODUCTION
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Abstract
Introduction
Methods
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The propagation of an action potential down an axon causes activation of voltage-dependent calcium channels (VDCCs) in nerve terminals, producing a calcium influx that triggers the release of neurotransmitter. Activation of presynaptic nicotinic acetylcholine receptors (nAChRs) in the CNS also can stimulate the release of neurotransmitters. Presynaptic nAChRs have been shown to enhance evoked as well as spontaneous neurotransmitter release (Gray et al. 1996; McMahon et al. 1994a,b; reviewed in Role and Berg 1996; Wonnacott 1997). The mechanism underlying the generation of a calcium influx in nerve terminals sufficient to trigger neurotransmitter release after nAChR activation is not completely understood.

Experimental evidence from studies of nAChRs suggests two potential mechanisms for the generation of a calcium influx. One possibility arises from a unique characteristic of the neuronal nAChRs. When compared with muscle-type nAChRs, the neuronal nAChRs have a high permeability to calcium ions (Mulle et al. 1992; Vernino et al. 1992), thus the calcium influx might be directly through the nAChR channel. Calcium influx through nAChRs has been shown to activate calcium-dependent chloride (Mulle et al. 1992; Seguela et al. 1993; Vernino et al. 1992) and potassium (Fuchs and Murrow 1992) conductances and may activate second-messenger systems. The high calcium permeability of nAChRs, in particular alpha 7-containing subtypes, suggests that they could produce sufficient calcium current to directly stimulate transmitter release in a nerve terminal. This type of direct calcium influx through the nAChR has been shown to enhance synaptic transmission in the hippocampus (Gray et al. 1996).

The alternate explanation is that the calcium influx is not directly through the nAChR channels, but rather it occurs by subsequent activation of VDCCs. In this case, stimulation of the presynaptic nAChRs produces a local depolarization via sodium influx, and this depolarization activates the VDCCs. The calcium flux through VDCCs then would account for the majority of the calcium influx into the nerve terminal and lead to transmitter release. Activation of VDCCs would enable those nAChR subtypes with a lower calcium permeability to modulate neurotransmitter release. Some nAChR subtypes therefore may be more dependent on the VDCCs than others. Studies also indicate that there are several types of VDCCs within the CNS that can potentially participate in triggering neurotransmitter release (reviewed in Bean 1989).

The goal of this study was to investigate the mechanism of the nAChR-mediated enhancement of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs), which we have described within the chick lateral spiriform (SpL) and ventral lateral geniculate (LGNv) (Guo et al. 1998; McMahon et al. 1994a,b). We sought to determine whether VDCCs played a role in this phenomenon and if so to attempt to identify the specific VDCC subtype(s) involved. We now report that in the SpL and LGNv, the nAChR-mediated enhancement of spontaneous GABAergic IPSCs is dependent on the activation of VDCCs. In addition, we found that in both the SpL and LGNv, N-type channels predominantly mediate the nicotinic enhancement. These results provide new information concerning the mechanisms of presynaptic nAChR function in chick brain.


    METHODS
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Methods
Results
Discussion
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Brain slice preparation

Embryonic White Leghorn chicks (18-19 days of incubation) were decapitated rapidly, and their brains were removed and immediately placed in 4°C artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 2.5 KCl, 1.24 NaH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26 NaHCO3, 10 D-glucose, pH 7.3, when bubbled with 95% O2-5% CO2). Atropine sulfate (1.0 µM) was added to all ACSF to block muscarinic responses (Guo and Chiappinelli 1998). The brains were blocked and attached to the stage of a vibrating tissue slicer. Coronal slices (350-400 µm) containing the SpL or LGNv were placed in fresh ACSF at room temperature for >= 1 h before use in experiments. Slices then were placed between two mesh holders in the center of a recording chamber (Warner Instruments, Hamden, CT) on a fixed-stage upright Zeiss microscope fitted with Nomarski optics and a video camera and perfused continuously (2-3 ml/min) in ACSF. Carbachol chloride (Sigma, St. Louis, MO) was applied by bath perfusion for 30-60 s at 20-min intervals. The use of carbachol instead of nicotine and the prolonged interval between agonist applications served to minimize nAChR desensitization (Guo et al. 1998; McMahon et al. 1994a,b).

Calcium channel antagonists

Calcium channel antagonists were prepared and applied as described in the following text. Nifedipine (Sigma, St. Louis, MO) was prepared fresh daily in dimethylsulfoxide (DMSO) to make a 100 mM stock solution. The stock then was diluted to 10 µM in ACSF that yielded a final DMSO concentration of 0.01%. At this concentration DMSO alone had no effect on our whole cell recordings. Nimodipine (RBI, Natick, MA) was prepared in ethanol at a concentration of 100 mM, and verapamil hydrochloride (Sigma) was dissolved in distilled and deionized water at 10 mM. Dilution of these stocks in ACSF produced the final working concentration of 10 µM. Nifedipine, nimodipine, and verapamil were all applied for 10 min before carbachol application. (±)Bay K 8644 (Calbiochem, La Jolla, CA) was dissolved in ethanol at 50 mM and diluted to 1-10 µM in ACSF.

omega -Agatoxin-TK (Peptides International, Osaka, Japan) was dissolved in distilled and deionized water at a concentration of 100 µM. This stock was diluted further in ACSF to a final drug concentration of 0.1 µM for use in experiments. omega -Conotoxin GVIA (Sigma) was dissolved in phosphate-buffered saline (pH 7.4). The stock was diluted to the final concentrations of 0.5 and 1.0 µM in ACSF. omega -AgTx-TK and omega -CgTx GVIA were applied to the slices by two methods. In the first method, a carbachol control response was obtained and then omega -AgTx-TK or omega -CgTx GVIA was applied for 10-15 min, at which point a second carbachol response was recorded. The second method of toxin application was an incubation of the slices in omega -AgTx-TK or omega -CgTx GVIA for 1-2 h. The slices then were placed in the recording chamber and perfused with normal ACSF. Neuronal recordings were made within 10-20 min of removing the slice from the toxin solution.

Electrophysiological methods

Whole cell patch-clamp recordings were performed from slices visualized with Nomarski optics as described in Guo et al. (1998). Patch pipettes were fabricated from borosilicate glass with a two-stage microelectrode puller to produce a tip opening of 1-2 µm with a resistance of 4-8 MOmega . The pipette solution contained (in mM) 150 KCl, 2 MgCl2, 2 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, 2 Mg-ATP, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 5 lidocaine N-ethyl bromide (QX314), pH 7.3, with 1.0 N potassium hydroxide. Signals were amplified with an Axopatch 200B patch clamp amplifier in the voltage-clamp mode (Axon Instruments, Foster City, CA) and low-pass four-pole Bessel filtered at 10 kHz. The amplified output was monitored continuously on an oscilloscope. Filtered data were recorded on a chart recorder and stored on VCR tape. Portions of selected recordings then were transferred through a low-pass eight-pole Bessel filter at 1-2 kHz and digitized by a TL-1 or Digidata 1200 DMA interface and saved to a computer with pClamp 6.03 (Axon Instruments). Neurons were voltage-clamped at a holding potential of -70 mV.

Data analysis

Analysis of spontaneous events was performed on 60 s of continuous data using MINI as described in McMahon et al. (1994a). Our detection threshold for the spontaneous GABAergic IPSCs was set at a di/dt of 20 pA/ms, with a minimal acceptable amplitude for a spontaneous event of 20 pA. To calculate the increase in IPSC frequency, the mean interval for baseline conditions (i.e., before carbachol) was divided by the mean interval during carbachol application. Statistical differences between increases in the presence of the VDCC antagonists and those in ACSF was tested by analysis of variance in Crunch (Version 4.0) statistical software, with a P < 0.05 indicating significance.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Cadmium chloride inhibits the carbachol-induced enhancement of GABAergic IPSCs

In ACSF containing 1.0 µM atropine, carbachol causes a 9.7- ± 1.3-fold increase (mean ± SE, n = 14 cells) in the frequency of spontaneous GABAergic IPSCs in SpL neurons and a 9.8- ± 1.2-fold increase (n = 18 cells) in LGNv neurons. These spontaneous GABAergic IPSCs are blocked completely by the gamma -aminobutyric acid-A (GABAA) receptor antagonist, bicuculline (McMahon et al. 1994a,b). In the SpL, the presynaptic effect of carbachol is accompanied by a pronounced inward current due to activation of postsynaptic nAChRs (McMahon et al. 1994a) (Fig. 1A).



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Fig. 1. Cadmium chloride blocks the carbachol-induced enhancement of spontaneous GABAergic inibitory postsynaptic currents (IPSCs) in the lateral spiriform (SpL) and ventral lateral geniculate (LGNv). Carbachol dramatically increases the frequency of spontaneous GABAergic currents recorded in an SpL neuron (A) and an LGNv neuron (B) in artificial cerebrospinal fluid (ACSF) containing 1.0 µM atropine (top). In the presence of 50 µM CdCl2 (middle), these nicotinic acetylcholine receptor (nAChR)-mediated presynaptic effects are blocked. Responses returned after a 20-min washout of the CdCl2 (bottom). Neurons were voltage-clamped at -70 mV. Bars above the traces indicate 45- (A) and 35-s (B) applications of 30 µM carbachol.

The nAChR-mediated enhancement of spontaneous GABA release seen in SpL neurons was reduced significantly by 82%, (1.7- ± 0.3-fold increase, n = 6 cells; P < 0.05) in the presence of 50-100 µM cadmium chloride (CdCl2), a competitive blocker of all high-threshold voltage-activated VDCCs (Bean 1989) (Fig. 1A). The nAChR-mediated postsynaptic inward current, however, was reduced only slightly by 20% (n = 5, P < 0.02). The basal spontaneous GABA release also was reduced by 0.68-fold (n = 6, P < 0.03) in the presence of CdCl2. In the LGNv, CdCl2 significantly reduced the presynaptic effect of carbachol, by 79% compared with control values (2.1- ± 0.4-fold increase, n = 8 cells; P < 0.05; Fig. 1B), and also decreased the basal spontaneous GABA release by 0.58-fold (n = 6, P < 0.03). The carbachol-induced enhancement of GABAergic IPSCs returned after washout of the CdCl2 in both SpL and LGNv (Fig. 1). These reductions in carbachol's presynaptic effect suggested that the nAChR-mediated enhancement of spontaneous GABA release in the SpL and LGNv was dependent on the activation of VDCCs. Another possible explanation was that CdCl2 was directly blocking the nAChRs because the nAChR-mediated postsynaptic current was blocked slightly in the SpL (Fig. 1A). To determine a definitive role for VDCCs in the nAChR-mediated responses, selective antagonists of VDCC subtypes were used to examine their possible involvement.

omega -CgTx GVIA significantly reduced the carbachol-induced enhancement of spontaneous GABAergic IPSCs

N-type calcium channels appear to be the main VDCCs controlling neurotransmitter release in the chick (Maubecin et al. 1995). In addition, N-type channels have been proposed to be involved in the nAChR-mediated enhancement of neurotransmitter release (Wonnacott 1997). We therefore investigated their role in the SpL and LGNv with omega -CgTx GVIA, a selective and irreversible blocker of N-type calcium channels (Carbone et al. 1990; McCleskey et al. 1987; Nowycky et al. 1985; Olivera et al. 1984; Plummer et al. 1989). In omega -CgTx GVIA (0.5 µM), the nAChR-mediated enhancement of GABAergic IPSCs in the SpL was reduced significantly by 78% (2.1- ± 0.4-fold increase, n = 6 cells; P < 0.05), which was equivalent to that seen with CdCl2 (Fig. 2A). Cumulative distributions of IPSC amplitude and interval in both ACSF and omega -CgTx GVIA demonstrated that carbachol altered IPSC frequency without changing IPSC amplitude and that omega -CgTx GVIA markedly inhibited carbachol's effect (Fig. 2B). The nAChR-mediated postsynaptic inward current, on the other hand, was not significantly reduced by omega -CgTx GVIA (n = 5, P > 0.05). The basal spontaneous GABA release was also not significantly influenced by omega -CgTx GVIA (1.3- ± 0.3-fold increase, n = 5 cells; P > 0.4). These results indicate that in the SpL the nAChR-mediated increase in spontaneous GABA release primarily involves the activation of N-type VDCCs.



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Fig. 2. Effects of omega -CgTx GVIA on the nAChR-mediated enhancement of GABAergic IPSCs in the SpL. A: carbachol-induced increase in spontaneous GABAergic IPSCs (top) is blocked by 0.5 µM CgTx GVIA (bottom). B: effect of carbachol on the cumulative distributions of IPSC amplitude and interval. Plots were constructed from 60 s of continuous data obtained from the same neuron under control conditions and in the presence of carbachol in ACSF (top) or in omega -CgTx GVIA (bottom). Kolmogorov-Smirnov test was used to determine significant differences between cumulative distributions. Interval distributions in ACSF were significantly different (P < 0.01) between control and carbachol. In omega -CgTx GVIA, carbachol's effect was inhibited substantially and the interval distributions were no longer significantly different (P > 0.1). C: in the same cell, averaged spontaneous currents for control and carbachol in ACSF and in the presence of omega -CgTx GVIA are shown superimposed to demonstrate that carbachol as well as omega -CgTx GVIA does not significantly alter the amplitudes, rise times, or decay rates of the individual events. Neuron was voltage-clamped at -70 mV. Bars above the records in A indicate 30-s application of 30 µM carbachol.

The presynaptic effect of carbachol also was reduced by omega -CgTx GVIA in the LGNv. However, we found that in the LGNv, 0.5 µM omega -CgTx GVIA was not sufficient to block carbachol's presynaptic effect. A nonsignificant reduction of 33% (6.6- ± 1.8-fold increase, n = 10 cells) in the carbachol responses was observed at this toxin concentration. Increasing the omega -CgTx GVIA concentration to 1.0 µM caused a significant 71% reduction in the carbachol-induced increase in GABAergic IPSCs (2.8- ± 1.1-fold increase, n = 4 cells; P < 0.05; Fig. 3). As in the SpL, omega -CgTx GVIA had no effect on the IPSC amplitude and basal spontaneous GABA release. The reduction in carbachol's effect was equivalent to the blockade observed with CdCl2, suggesting that in the LGNv N-type channels predominantly mediate the nicotinic enhancement of GABAergic IPSCs.



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Fig. 3. omega -CgTx GVIA blocks the nAChR-mediated enhancement of GABAergic IPSCs in the LGNv. A: enhancement of spontaneous GABAergic IPSCs (top) is blocked by 1.0 µM omega -CgTx GVIA (bottom) in the LGNv. B: portions of the traces in A, for control conditions and in the presence of carbachol, are shown on an extended time scale. Neuron was voltage-clamped at -70 mV. Bars above the records in A indicate a 30-s application of 30 µM carbachol.

Carbachol-induced enhancement of GABAergic IPSCs remained in the presence of omega -AgTx-TK

omega -Agatoxin-TK is a selective antagonist of P/Q-type calcium channels (Adams et al. 1990; Uchitel 1997). Previous studies have shown that P/Q-type channel blockers do not effect calcium influx or transmitter release in chicken brain synaptosomes suggesting that P/Q-type channels do not have a significant role in transmitter release in the chick (Uchitel 1997). In the present study, the carbachol-induced increase of spontaneous GABAergic IPSCs in the presence of omega -AgTx-TK was reduced by 27% (7.1- ± 0.9-fold increase, n = 8 cells) and 39% (6.0- ± 1.7-fold increase, n = 5 cells) in the SpL and LGNv, respectively, when compared with controls (Fig. 4, A and B). In neither case were the observed reductions significantly different from controls, suggesting that P/Q-type channels may be present but that they have at most a minor role in the nAChR-mediated responses.



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Fig. 4. nAChR-mediated increase in spontaneous GABAergic IPSCs is not prevented by omega -AgTx-TK. Carbachol-induced enhancement of spontaneous GABAergic IPSCs in an SpL (A) and an LGNv (B) neuron before (top) and during exposure to 0.1 µM omega -AgTx-TK (bottom). These records show the minor reduction in carbachol's presynaptic effect caused by omega -AgTx-TK. Neurons in both A and B were voltage-clamped at -70 mV. Bars above records indicate a 55- (A) and 60-s (B) application of 30 µM carbachol.

Blockade of L-type channels did not diminish the carbachol-induced increase in spontaneous GABA release in the SpL

Whole cell recordings from SpL neurons showed that nifedipine, a dihydropyridine (DHP) compound and selective blocker of L-type VDCCs, did not by itself affect the frequency of the baseline spontaneous currents or the amplitudes of the IPSCs. In nifedipine (10 µM), the carbachol-induced increase of spontaneous GABAergic IPSCs in the SpL was not significantly different from that observed with carbachol in ACSF (7.8- ± 2.7-fold increase, n = 7 cells). Activation of L-type channels thus does not appear to contribute significantly to the nAChR-mediated effect in the SpL.

Effects of L-type channel antagonists on LGNv neuronal responses

In contrast to the SpL, we found that L-type channel antagonists were difficult to use in the LGNv. To begin with, nifedipine caused a change in the baseline spontaneous events. Perfusion of the slices with nifedipine (10 µM) caused a dramatic increase in the frequency of baseline spontaneous events within 5 min from the start of the drug. The mean interval between spontaneous events decreased from 1.3 ± 0.1 s (mean ± SE; n = 17 cells) for the control group's baseline to 0.32 ± 0.06 s (n = 4 cells) for the baseline of the cells in nifedipine. The baseline activity returned to normal after a 20-min washout of nifedipine. Attempts to block this increase in baseline activity with tetrodotoxin, mecamylamine, dihydro-beta -erythroidine, and CdCl2 failed (data not shown), leading us to conclude that this was likely a nonspecific action of nifedipine in the LGNv. Carbachol still enhanced GABAergic IPSCs in the presence of nifedipine, but the effect was decreased by 65% (3.4- ± 1.4-fold increase, n = 4 cells, P < 0.05; Fig. 5). It was unclear if this reduction was merely the result of the increased baseline or whether it represented a genuine effect of nifedipine on the carbachol responses.



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Fig. 5. Effects of L-type channel antagonists on the nAChR-mediated enhancement of GABAergic IPSCs in the LGNv. This bar graph shows the effects of the L-type antagonists nifedipine, nimodipine, and verapamil as well as the L-type agonist, (±)Bay K 8644 on the presynaptic effect of carbachol in the LGNv. Application of carbachol caused a 9.8- ± 1.2-fold increase (mean ± SE; n = 18 cells) in the frequency of spontaneous GABAergic IPSCs. In the presence of nifedipine (10 µM) and verapamil (10 µM), this x-fold increase was reduced to 3.4 ± 1.4 (n = 4 cells) and 2.3 ± 0.8 (n = 4 cells), respectively. These decreases were significantly different from the controls. In nimodipine (10 µM) the x-fold increase was 5.5 ± 1.8 (n = 5 cells). (±)Bay K 8644 (1-10 µM), an L-type agonist, did not significantly alter the carbachol responses in the neurons tested (6.9- ± 1.0-fold increase; n = 4 cells). *Significant difference from controls (P < 0.05).

Because of these nonspecific actions of nifedipine in the LGNv, two other L-type channel blockers were tested: nimodipine (another DHP) and verapamil (a phenylalkylamine). Nimodipine and verapamil did not alter the frequency of the baseline spontaneous currents as observed with nifedipine. In the presence of nimodipine (10 µM), the carbachol-induced enhancement of GABAergic IPSCs was reduced by 44% (5.5- ± 1.8-fold increase, n = 4 cells; Fig. 5). Verapamil had an even stronger effect on the carbachol responses, which were decreased significantly by 77% (2.3- ± 0.8-fold increase, n = 4 cells, P < 0.05) when compared with control values (Fig. 5).

There are two possible explanations for these results. First, there is the possibility that L-type channels are indeed involved in the nAChR-mediated increase in spontaneous GABAergic IPSCs and that blocking them inhibits the effects of carbachol. The other possibility is that these L-type channel blockers are somehow directly blocking the nAChRs in the LGNv. It has now become evident from other studies that L-type channel antagonists may be very effective at inhibiting the actions of nAChRs (Dolezal et al. 1996; Donnelly-Roberts et al. 1995). It has been suggested that the nAChR may have a specific site at which these L-type antagonists act (Donnelly-Roberts et al. 1995).

In an attempt to distinguish between these two possibilities we used (±)Bay K 8644, an L-type channel agonist, to see if we could enhance further the carbachol-induced increase of spontaneous GABAergic IPSCs. In recordings from four cells, (±)Bay K 8644 (1-10 µM) by itself had no effect on the LGNv neurons and did not potentiate the carbachol-induced increase in GABAergic IPSCs (Fig. 5).


    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

An autoradiographic localization study has demonstrated that most of the nAChRs present in the SpL are of the high-affinity nicotine type, whereas both high-affinity nicotine and alpha -BgTx-binding receptors are present in the LGNv (Sorenson and Chiappinelli 1992). The carbachol-induced enhancement of GABAergic IPSCs is not blocked by alpha -BgTx in either the SpL or LGNv (Guo et al. 1998; Sorenson and Chiappinelli 1990; unpublished observations) implying that alpha -BgTx-sensitive (alpha 7 containing) receptors are not involved. It therefore appears that in both regions the nAChRs enhancing the spontaneous release of GABA are of the high-affinity nicotine type. The specific subunit compositions of these high-affinity nicotine receptors are unknown.

High-affinity nicotine nAChRs are generally not as permeable to calcium as the alpha 7-containing nAChRs (Seguela et al. 1993; Vernino et al. 1992). It is thus less likely that they can carry sufficient calcium current to stimulate neurotransmitter release and would require another mechanism to trigger release. Cadmium's block of the carbachol responses in the SpL and LGNv implicated VDCCs in the enhancement of GABAergic IPSCs. However, an alternative explanation was that the calcium influx needed for transmitter release was in fact coming directly through the nAChRs but that cadmium was blocking the activation of the nAChRs. The slight blockade of postsynaptic nAChR-mediated inward current by cadmium in SpL neurons might support this possibility. However, there is little evidence for cadmium blocking nAChRs at the concentrations (50-100 µM) we used. One study showed that cadmium had no effect on a "ganglionic-like" nAChR (possibly alpha 3beta 4 subunit combination) up to a concentration of 1 mM (Donnelly-Roberts et al. 1995). The fact that the postsynaptic nAChR-mediated inward current is reduced only slightly under conditions where cadmium blocks most of the presynaptic nAChR-mediated spontaneous GABA release also argues against a direct effect by cadmium on nicotinic receptors. However, because we do not know the exact subunit composition of our native nAChRs, we cannot rule out some interaction of cadmium. Our results with the selective VDCC blockers support the conclusion that cadmium's effect was at the VDCCs not the presynaptic nAChRs.

Application of omega -CgTx GVIA to the SpL slices resulted in an irreversible block of the carbachol-induced enhancement of GABAergic IPSCs while having little effect on the postsynaptic nAChR-mediated inward current. This result provided further evidence that VDCCs play a role in the nAChR-mediated presynaptic effects. The amount of the block was similar to that seen with cadmium, suggesting that N-type channels mediate the majority of the response. These results in the SpL are in agreement with those given by others who have shown that in the chick, the predominant VDCC controlling neurotransmitter release is an N-type channel (Maubecin et al. 1995).

In the LGNv, the omega -CgTx GVIA results were somewhat different as initial experiments with the toxin (0.5 µM) suggested that N-type channels were not involved. However, further studies revealed that a higher concentration of omega -CgTx GVIA (1.0 µM) was needed to achieve a significant irreversible block in the LGNv neurons. This might indicate that the number of functional VDCCs required per terminal in the SpL and the LGNv is different. At this higher concentration of toxin, there is also a possibility that if present, some L-type channels would be blocked. Unlike the toxin's actions on N-type channels, however, the L-type channel blockade would be expected to be readily reversible (Aosaki and Kasai 1989; Kasai et al. 1987). In our study, washout of the omega -CgTx GVIA for >= 1 h did not cause our responses to return, again indicating no involvement of L-type channels. The pharmacology of the calcium channel mediating the nicotinic enhancement of GABAergic IPSCs in the LGNv thus is related most closely to that of the N-type channels. There is no direct evidence so far suggesting that omega -CgTx GVIA can bind directly to and block the nAChRs. However, because we do not know the exact subunit composition of our native nAChRs, we cannot completely rule out some interaction of omega -CgTx GVIA with these receptors.

omega -AgTx-TK produced only a nonsignificant decrease in the carbachol-induced enhancement of GABAergic IPSCs in both the SpL and LGNv. omega -AgTx-TK is selective for P/Q-type channels, but there is also evidence that the toxin may bind to omega -CgTx GVIA-sensitive N-type channels in the chick CNS (Maubecin et al. 1995). Regardless of where omega -AgTx-TK was binding, it still did not eliminate the nicotinic enhancement.

L-type channels are not generally thought to participate in neurotransmitter release within the CNS. In a study comparing VDCCs in rat and chicken brain synaptosomes, an L-type channel blocker had no effect on calcium uptake in either preparation (Maubecin et al. 1995). In the present study, the L-type channel antagonist nifedipine showed no significant effect on the nAChR-mediated increase in GABA release in the SpL. Thus in this nucleus, L-type channels do not appear to be important in the nAChR-mediated responses.

The neurons in the LGNv, however, exhibited several responses to nifedipine exposure. The frequency of the baseline spontaneous currents increased in the presence of nifedipine. Interestingly, the same concentration of nifedipine in the SpL did not show this increased baseline activity. As mentioned, we tried to block this nifedipine effect with various pharmacological agents without any success. At this point, we do not know the reason or mechanism for this nifedipine effect, but it appears to be a nonspecific action on the neurons in the LGNv.

Other L-type channel antagonists, nimodipine and verapamil, also demonstrated complex actions within the LGNv. Neither drug altered the frequency of the baseline events, but they caused a reduction in the presynaptic effect of carbachol. Our results in the LGNv are similar to what others have found in chick sympathetic neurons, where (±)Bay K 8644, another DHP compound and an L-type channel antagonist, actually diminished the presynaptic nAChR-mediated release of noradrenaline (Dolezal et al. 1996). The authors concluded that this decrease in noradrenaline release was the result of a direct antagonism of nAChRs. Another study has shown that DHP compounds as well as some non-DHP compounds are effective antagonists of some subtypes of nAChRs (Donnelly-Roberts et al. 1995). Unfortunately, the concentration needed to block the L-type calcium channels is well within the range that would also effectively antagonize the nAChRs.

We used the L-type channel agonist, (±)Bay K 8644, to determine whether L-type channels were present within the LGNv. If L-type channels were present, one might expect a change in baseline spontaneous currents with an L-type channel agonist. In addition, if L-type channels were involved in the nAChR-mediated enhancement of GABA release, the presence of an L-type channel agonist might potentiate the response. We found no effect of (±)Bay K 8644 on the baseline activity of the LGNv neurons. There also was no potentiation of the carbachol responses in the presence of (±)Bay K 8644. These results suggested that L-type channels were not likely to be involved in the nAChR-mediated presynaptic effects.

In summary, we have presented evidence that activation of high-affinity nicotine nAChRs in the SpL and LGNv produces a subsequent activation of VDCCs, leading to an increase in spontaneous GABA release. In both the SpL and LGNv, N-type channels appear to be the predominant subtype of VDCC activated.


    ACKNOWLEDGMENTS

We are grateful to Drs. Terrence Egan, Yi Nong, Eva Sorenson, and Ping-Jun Zhu for helpful comments and valuable discussion.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-17574 to V. A. Chiappinelli.


    FOOTNOTES

Address for reprint requests: J.-Z. Guo, Dept. of Pharmacology, School of Medicine and Health Sciences, The George Washington University, 2300 Eye St., N.W., Washington, DC 20037.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 25 June 1998; accepted in final form 27 October 1998.


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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society