Department of Pharmacology, School of Medicine and Health Sciences, The George Washington University, Washington, DC 20037
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ABSTRACT |
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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. -Conotoxin GVIA, a selective
antagonist of N-type channels, significantly reduced the nAChR-mediated
enhancement of
-aminobutyric acid (GABA) release in the SpL by 78%
compared with control responses. Nifedipine, an L-type channel blocker,
and
-Agatoxin-TK, a P/Q-type channel blocker, did not inhibit the
enhancement of GABAergic IPSCs. In the LGNv,
-Conotoxin GVIA also
significantly reduced the nAChR-mediated enhancement of GABA release by
71% from control values. Although
-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.
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INTRODUCTION |
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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
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.
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METHODS |
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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.
-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.
-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.
-AgTx-TK and
-CgTx GVIA were applied to the slices by two methods. In the first
method, a carbachol control response was obtained and then
-AgTx-TK
or
-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
-AgTx-TK or
-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 M
. The pipette solution contained (in
mM) 150 KCl, 2 MgCl2, 2 ethylene glycol-bis(
-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.
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RESULTS |
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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
-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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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.
-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
-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
-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
-CgTx GVIA demonstrated that carbachol altered IPSC frequency
without changing IPSC amplitude and that
-CgTx GVIA markedly
inhibited carbachol's effect (Fig. 2B). The nAChR-mediated
postsynaptic inward current, on the other hand, was not significantly
reduced by
-CgTx GVIA (n = 5, P > 0.05). The basal spontaneous GABA release was also not significantly influenced by
-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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The presynaptic effect of carbachol also was reduced by -CgTx GVIA
in the LGNv. However, we found that in the LGNv, 0.5 µM
-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
-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,
-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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Carbachol-induced enhancement of GABAergic IPSCs remained in the
presence of -AgTx-TK
-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
-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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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--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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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).
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DISCUSSION |
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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 -BgTx-binding receptors are present in the LGNv (Sorenson and Chiappinelli
1992
). The carbachol-induced enhancement of GABAergic IPSCs is
not blocked by
-BgTx in either the SpL or LGNv (Guo et al.
1998
; Sorenson and Chiappinelli 1990
;
unpublished observations) implying that
-BgTx-sensitive (
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 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
3
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 -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 -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
-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
-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
-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
-CgTx GVIA with these receptors.
-AgTx-TK produced only a nonsignificant decrease in the
carbachol-induced enhancement of GABAergic IPSCs in both the SpL and
LGNv.
-AgTx-TK is selective for P/Q-type channels, but there is also
evidence that the toxin may bind to
-CgTx GVIA-sensitive N-type
channels in the chick CNS (Maubecin et al. 1995
).
Regardless of where
-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.
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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.
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FOOTNOTES |
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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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REFERENCES |
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