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The Journal of Neuroscience, March 15, 1998, 18(6):1963-1969
Glutamate and GABA Release Are Enhanced by Different Subtypes
of Presynaptic Nicotinic Receptors in the Lateral Geniculate
Nucleus
Jian-Zhong
Guo,
Trevor L.
Tredway, and
Vincent A.
Chiappinelli
Department of Pharmacology, The George Washington University,
School of Medicine and Health Sciences, Washington, D.C. 20037
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ABSTRACT |
The functional role of nicotinic acetylcholine receptors (nAChRs)
in the ventral lateral geniculate nucleus (LGNv) was examined in chick
brain slices. Whole-cell patch-clamp recordings of neurons in the LGNv
revealed the presence of bicuculline-resistant spontaneous postsynaptic
currents (PSCs), which were subsequently blocked by
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA receptor antagonist. Carbachol and other nicotinic agonists produced marked increases in the frequency of the glutamatergic spontaneous PSCs in the
presence of tetrodotoxin, whereas they had little or no effect on
current amplitude. The nicotinic receptor antagonist dihydro- -erythroidine (DHE) blocked the carbachol-induced
enhancement of spontaneous glutamatergic PSCs.  -bungarotoxin
(-BgTx) selectively blocked the nAChR-mediated enhancement of
spontaneous glutamatergic PSCs but did not prevent nAChR-mediated
enhancement of spontaneous GABAergic PSCs in the LGNv.
Methyllycaconitine and strychnine, other blockers of nAChRs containing
the 7 subunit, failed to inhibit carbachol's increase of
spontaneous glutamatergic and GABAergic PSCs. These results demonstrate
that the LGNv neurons receive both glutamatergic and GABAergic inputs
and that the release of these transmitters can be modulated by
different presynaptic nAChRs. Thus, the regulation of synaptic efficacy
in the brain by presynaptic nAChRs can be complex, involving multiple
neurotransmitters acting on the same neuron.
Key words:
neuronal nicotinic acetylcholine receptors; presynaptic; glutamate; GABA; modulation; -bungarotoxin; methyllycaconitine; ventral lateral geniculate nucleus
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INTRODUCTION |
An important function of neuronal
nicotinic acetylcholine receptors (nAChRs) in the CNS appears to be
modulation of neurotransmitter release (Wonnacott et al., 1990 ; Vidal
and Changeux, 1996; Wonnacott, 1997). Presynaptic nAChRs have been
implicated in the enhanced release of a number of transmitters,
including norepinephrine, dopamine, serotonin, acetylcholine, GABA, and
glutamate (for review, see Role and Berg, 1996). These studies have
focused predominantly on nAChR modulation of a single transmitter in a
diverse group of synaptosomal and brain slice preparations.
The presynaptic nAChR subtypes, their location, and their mechanisms
for enhancing transmitter release vary with each experimental model.
Evidence indicates that both -bungarotoxin (-BgTx) binding nAChRs
and high-affinity nicotine binding nAChRs have the ability to modulate
transmitter release. In addition, sensitivity to blockade by TTX
suggests various locations for these presynaptic nAChRs (Lena et al.,
1993). Consequently, it becomes difficult to uniformly define the
nature and function of presynaptic nAChRs.
The chick ventral lateral geniculate nucleus (LGNv) is a large
retinorecipient region that has a high density of nAChRs (Ehrlich and
Mark, 1984; Morris et al., 1990; Sorenson and Chiappinelli, 1992).
Information that a majority of these receptors are presynaptic comes
from a study in which nicotinic agonists caused a marked increase in
spontaneous GABAergic postsynaptic currents (PSCs), whereas they
produced no significant direct postsynaptic response (McMahon et al.,
1994b). GABA release was enhanced even when axonal sodium channels were
blocked by tetrodotoxin (TTX), indicating that the nAChRs that were
responsible were likely to be located near transmitter release sites on
GABAergic nerve terminals. During our studies in the LGNv, we observed
spontaneous postsynaptic currents in neurons that were not blocked by
the GABAA-receptor antagonist bicuculline, suggesting that
other transmitters were being released. Therefore, the LGNv presented
us with the possibility to study concurrent presynaptic nAChR actions
on multiple transmitters in one region.
The goal of the present study was to identify the other transmitters
released in the LGNv, determine whether they too are subject to
modulation by nAChRs, and further characterize any nAChR-mediated
enhancement. We now report that nAChRs located on presynaptic nerve
terminals in the LGNv enhance the release of glutamate. The
pharmacological properties of the nAChRs enhancing glutamate release
are uniquely distinguishable from those responsible for GABA release,
indicating different receptor subtypes. In chick LGNv neurons,
nicotinic agonists increase the frequencies of both spontaneous
glutamatergic and GABAergic postsynaptic currents, demonstrating a
potentially complex regulatory function for acetylcholine.
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MATERIALS AND METHODS |
Brain slice preparation. Embryonic White Leghorn
chicks (18- to 20-d-old) were decapitated rapidly, and their brains
were removed and immediately placed in 4°C artificial cerebrospinal fluid (ACSF) (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). The brains were
blocked and attached to the stage of a vibrating tissue slicer. Coronal
slices (350-400 µm) containing the LGNv were placed in fresh ACSF at
room temperature for at least 1 hr before use in experiments. Slices
were then 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. Slices were
perfused continuously (2-3 ml/min) in ACSF with 1 µM
atropine sulfate to block muscarinic receptor responses. Bicuculline
(10 µM) was included in most experiments to eliminate the
GABAergic component. Agonists were applied by bath perfusion for 30 sec at 15-30 min intervals to minimize receptor desensitization.
Because binding of -BgTx to nAChRs is slow, we used the following
method to test its action. Slices were first incubated in ACSF
containing -BgTx (0.1 µM) for 2 hr. The slices were
then placed in the recording chamber and continuously perfused with -BgTx. Because of the considerable amounts of -BgTx required, continuous perfusion was maintained by recirculation.
Electrophysiological methods. Whole-cell patch-clamp
recordings were performed from slices visualized with Nomarski optics. 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 potassium gluconate, 2 MgCl2, 2 EGTA, 2 Mg-ATP, 10 HEPES, 5 QX314, pH 7.3, with 1.0N potassium
hydroxide. In some experiments the potassium gluconate was replaced
with 150 mM potassium chloride. Signals were amplified with
an Axopatch 1-D patch-clamp amplifier in the voltage-clamp mode (Axon
Instruments, Foster City, CA) and a low-pass four-pole Bessel filtered
at 10 kHz. Amplified output was monitored continuously on an
oscilloscope. Filtered data were recorded on a chart recorder and
stored on VCR tape using a Vetter Model 200 PCM data recorder. Portions of selected recordings were then transferred through a low-pass eight-pole Bessel filter at 1-2 kHz and digitized by a TL-1 or Digidata 1200 DMA interface. Data were acquired and analyzed with pClamp 6.03 (Axon Instruments). Spontaneous events were analyzed as
described previously by McMahon et al. (1994a), using MINI Ver.1.2
software package. Our detection threshold was set at a di/dt of 5 pA/msec, with minimum and maximum rise
times set at 0.1 and 10 msec, respectively. The minimal acceptable
amplitude for a spontaneous event was 7 pA. Significant difference
between two distributions was determined using the Kolmogorov-Smirnov test, within Crunch Version 4.0, with a p value < 0.01 indicating significance.
Materials. Drugs used were obtained from the following
sources: carbachol chloride, acetylcholine chloride, ( )-nicotine
bitartrate, 1,1-dimethyl-4-phenyl-piperzinium, cytisine, and
bicuculline methiodide from Sigma (St. Louis, MO); lidocaine
N-ethyl bromide (QX314), and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) from RBI (Natick, MA); and
tetrodotoxin (TTX) from Calbiochem (San Diego, CA). DHE was a gift
from Merck, Sharp & Dohme Research Labs (Rahway, NJ). Methyllycaconitine was kindly provided by Dr. M. H. Benn,
University of Calgary (Aiyar et al., 1979). -BgTx was purified from
the crude venom of Bungarus multicinctus as described
(Chiappinelli, 1983).
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RESULTS |
Characterization of glutamatergic spontaneous postsynaptic currents
in LGNv neurons
Whole-cell patch-clamp recordings from LGNv neurons
(n = 150) in brain slices were used to characterize the
properties of spontaneous postsynaptic currents (PSCs). GABAergic IPSCs
were inward-going at 70 mV when recorded with KCl in the internal recording solution (McMahon et al., 1994a). To separate these chloride-mediated inward currents from cationic-mediated inward currents, we replaced the KCl with potassium gluconate to shift the
chloride ion reversal potential from 0 mV to 70 mV. Under these
recording conditions with the membrane potential clamped at 50 mV,
the chloride-mediated GABAergic IPSCs were seen as outward-going
currents and could be distinguished from the cationic-mediated EPSCs,
which were inward-going currents. Initial recordings showed spontaneous
PSCs in both outward and inward directions (Fig.
1A). The amplitude of the
inward currents ranged from 6 pA to 200 pA, with a mean of 13.7 ± 0.7 pA (n = 11 cells). The AMPA receptor antagonist
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 µM) selectively blocked the inward-going currents without affecting the
outward currents, indicating that these inward spontaneous PSCs are
caused by release of glutamate (Fig. 1B). The
GABAA receptor antagonist bicuculline (10-20
µM) selectively blocked the outward-going currents. (Fig.
1C). Recovery of both the CNQX-sensitive and
bicuculline-sensitive currents was seen after a 10-20 min washout of
each drug. These results suggest that LGNv neurons receive excitatory
glutamatergic input in addition to inhibitory GABAergic input.
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Figure 1.
Glutamatergic and GABAergic spontaneous
postsynaptic currents are simultaneously presented in LGNv neurons.
A, Both inward- and outward-directed spontaneous
currents were recorded in normal ACSF with atropine (1 µM). In B the inward-going spontaneous
currents were completely blocked by CNQX (20 µM).
C, The CNQX-insensitive outward-going spontaneous
currents were completely blocked by bicuculline (10 µM).
Potassium gluconate was in the recording pipette, and the neuron was
clamped at 40 mV.
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Pharmacology of the glutamate receptors on LGNv neurons
To characterize the pharmacological properties of the glutamate
receptors on LGNv neurons, the effects of CNQX and
2-amino-5-phosphovalerate (AP5), an NMDA receptor antagonist, were
examined on exogenous L-glutamate-induced inward currents.
A brief (1-5 sec) pulse of L-glutamate (100-200
µM) applied via a fast superfusion system (DAD-12, ALA
Scientific Instruments, Westbury, NY) produced a large inward current
(n = 3 cells) (Fig.
2A, left trace).
CNQX (20 µM) blocked 90% of the glutamate-evoked current
(Fig. 2A, middle trace), which recovered
completely after washout of CNQX (Fig. 2A,
right trace). The addition of AP5 (50 µM) to
the bath solution with CNQX blocked all of the L-glutamate
current (data not shown). The current-voltage relationship of the
L-glutamate-evoked current showed the AP5-sensitive
component at holding potentials from 50 to 10 mV (Fig.
2B). These results indicate that both AMPA and NMDA
glutamate receptors are present on LGNv neurons and are potential sites
of action for spontaneously released glutamate.
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Figure 2.
AMPA and NMDA glutamate receptors participate in
LGNv neuronal responses. A, Exogenous
L-glutamate (L-Glu; 200 µM) applied by fast
perfusion (1 sec) evoked a large postsynaptic inward current (left trace). CNQX (20 µM) blocked the
90% of the postsynaptic inward current induced by L-Glu (middle
trace), which recovered after washout of CNQX (right
trace). The neuron was clamped at 50 mV and perfused with
ACSF containing TTX (0.5 µM) and bicuculline (10 µM). B, L-Glu (500 µM) was
applied by bath perfusion (90 sec) during which time the neuron holding
at 50 mV in ACSF with TTX (0.5 µM) and bicuculline (10 µM) was ramped from 130 to +50 mV. AP5 (50 µM) selectively blocks the additional inward current
induced by L-Glu at membrane potentials of 50 to 10 mV.
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Nicotinic receptors mediate an enhancement of glutamatergic
spontaneous PSCs
Our lab has shown previously that spontaneous GABA release in the
LGNv is enhanced by nAChRs (McMahon et al., 1994b). Therefore in the
present study we investigated whether glutamate release could also be
modulated by nAChRs. Bath-application of nAChR agonists carbachol
(10-100 µM), acetylcholine (100 µM),
nicotine (100 µM), 1,1-dimethyl-4-phenyl-piperzinium (100 µM), and cytisine (100 µM) produced a
dramatic increase in spontaneous glutamatergic EPSCs (Figs.
3A,4). Carbachol at 100 µM caused a significant 2- to 12-fold [mean 6.0 ± 1.1, (n = 10 cells); p < 0.0001]
increase in the frequency of glutamatergic EPSCs, whereas it had little or no effect on current amplitude [mean 15.6 ± 0.6 pA,
(n = 9 cells); p > 0.25 compared with
control], indicating that the effect was presynaptic. The cumulative
distributions of EPSC interval and amplitude for an LGNv neuron are
shown in Figure 5, A and B, respectively. In Figure 5C, averaged
spontaneous EPSCs for control and carbachol are superimposed to show
that carbachol did not affect the amplitudes, rise times, or decay
rates of the EPSCs. CNQX (20 µM) completely eliminated
the basal spontaneous activity as well as the carbachol-induced
enhancement, both of which returned after washout of CNQX (Figs.
3B,C). Dihydro--erythroidine (DHE; 30 µM), a competitive nAChR antagonist, had no
effect on basal spontaneous activity, whereas it completely blocked the effect of carbachol, demonstrating that nAChRs mediated the enhanced glutamate release (Fig. 6).
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Figure 3.
Carbachol-induced enhancement of spontaneous EPSCs
is blocked by CNQX. A, Basal spontaneous EPSCs
(left) and carbachol (100 µM) induced
enhancement of EPSCs (right) recorded in normal ACSF with TTX (0.5 µM) and bicuculline (10 µM).
B, CNQX (20 µM) blocked the basal
spontaneous and the carbachol enhancement of EPSCs, both of which
returned after washout of CNQX (C). The neuron was clamped at 50 mV.
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Figure 4.
Four other nAChR agonists enhance the frequency of
spontaneous EPSCs. ACh (A),
Nicotine (B), DMPP
(C), and Cytisine
(D) all showed similar effects on the spontaneous
EPSCs. These responses were recorded in normal ACSF with TTX (0.5 µM) and bicuculline (10 µM), and the neuron
was clamped at 50 mV.
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Figure 5.
Influence of carbachol on the cumulative
distributions of EPSC interval and amplitude. The neuron was clamped at
50 mV in ACSF with TTX (0.5 µM) and bicuculline (10 µM). Carbachol (100 µM) was bath-applied
for 30 sec. Plots of data were constructed from 45 sec of continuous
data under control conditions (101 events) or in the presence of 100 µM carbachol (585 events). The Kolmogorov-Smirnov test
was used to determine significant differences between cumulative distributions. The amplitude distributions (B)
were not significantly different (p > 0.1)
in control versus carbachol (mean amplitude in Control,
16.8 pA; in Carbachol, 16.6 pA). However, the interval distributions (A) were significantly different
(p < 0.00001) between control and carbachol
(mean interval in Control, 493 msec; in Carbachol, 91 msec). In this cell, carbachol increased
the EPSC frequency 5.4-fold. C, In the same cell,
averaged spontaneous EPSCs for control (81 events) and carbachol (507 events) are shown superimposed to demonstrate that carbachol does not
significantly alter the amplitudes, rise times, or decay rates of the
events.
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Figure 6.
Carbachol-induced enhancement of spontaneous EPSCs
is blocked by dihydro--erythroidine (DHE). A,
Basal spontaneous EPSCs (left) and carbachol (100 µM) induced enhancement of EPSCs (right) recorded in normal ACSF with TTX (0.5 µM) and bicuculline
(10 µM); B, DHE (30 µM)
blocked the carbachol-induced enhancement of spontaneous EPSCs but did
not influence the basal spontaneous activity. C, The
carbachol-induced enhancement recovered after washout of DHE. The
neuron was clamped at 50 mV.
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Carbachol enhancement of spontaneous glutamatergic EPSCs was observed
in 90% of the LGNv neuronal recordings. In 60% of these neurons,
enhancement of glutamatergic EPSCs was still observed in the presence
of 0.5 µM TTX. This result suggests that these nAChRs are
likely to have two distinct locations: (1) near release sites on
presynaptic terminals, which on activation directly cause neurotransmitter release, and (2) some distance from release sites, requiring voltage-dependent sodium channels for their action. To
concentrate on those nAChRs near release sites, only the
TTX-insensitive responses are included in the present study.
Pharmacological profiles of presynaptic nAChRs
A previous binding study has shown that there are -BgTx,
 -BgTx, and nicotine binding sites in the chick LGNv (Sorenson and Chiappinelli, 1992). The -BgTx binding sites, presumably 7 and 8 subunit-containing nAChRs, and the high-affinity nicotine binding sites (likely 4/2-containing) have been reported to function presynaptically to enhance neurotransmitter release (Role and Berg,
1996). We therefore used methyllycaconitine (MLA) and -BgTx, both
known blockers of 7-containing nAChRs, to examine whether these
receptors are involved in modulation of transmitter release in LGNv
(Couturier et al., 1990; Ward et al., 1990; Alkondon et al., 1992;
Alkondon and Albuquerque, 1993; Gray et al., 1996). The
carbachol-induced enhancement of spontaneous glutamatergic EPSCs
remained in the presence of MLA (0.1 µM) (Fig.
7A), which is sufficient to block
the -BgTx-sensitive subclass of neuronal nAChRs in hippocampal
neurons (Alkondon et al., 1992). However, incubation with -BgTx (0.1 µM) for 2 hr eliminated the carbachol-induced response,
which gradually returned during an extended (90 min) washout of the
toxin (Fig. 8A,B).
Alternatively, in the same slices the carbachol-induced enhancement of
spontaneous GABAergic IPSCs continued in the presence of both MLA and
-BgTx, indicating that 7 subunit-containing nAChRs are not likely
involved (Figs. 7B, 8A). The effect of
strychnine (STR) on the carbachol-induced increase in transmitter
release was also examined. STR, a high-affinity antagonist of the
glycine-gated chloride channel, has also been shown to antagonize
-BgTx-sensitive homomeric 7-channels and 8-channels expressed
in oocytes (Seguela et al., 1993; Gerzanich et al., 1994). The
enhancement of spontaneous glutamatergic EPSCs or GABAergic IPSCs by
carbachol (30-100 µM) was not altered by STR (1.0-3.0
µM) (Fig. 9). These results
suggest that the presynaptic nAChRs mediating the enhancement of
glutamate release in the LGNv represent a previously undefined nAChR
subtype and that two pharmacologically distinct subtypes of nAChRs
modulate glutamate and GABA release within the LGNv.
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Figure 7.
Carbachol-induced enhancement of spontaneous PSCs
persisted in the presence of MLA. A, Carbachol continued
to enhance spontaneous glutamatergic EPSCs in (0.1 µM)
MLA. Potassium gluconate was used as the internal recording solution to
observe the glutamatergic EPSCs. B, In a separate
neuron, potassium chloride was used in the electrode to record
GABAergic IPSCs. Carbachol-induced enhancement of GABAergic IPSCs also
remained in the presence of MLA. The neuron in A was
clamped at 50 mV in ACSF with TTX (0.5 µM) and
bicuculline (10 µM), whereas the neuron in
B was held at 70 mV in ACSF with TTX. MLA was applied
by bath perfusion for 15 min before carbachol challenge in both
neurons. Bars above records indicate 30 sec application
of 100 µM carbachol.
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Figure 8.
The effects of -BgTx on the carbachol-induced
enhancement of spontaneous PSCs. A, The
carbachol-induced enhancement of spontaneous glutamatergic EPSCs is
blocked by a 2 hr incubation with -BgTx (0.1 µM)
(top left), whereas in a separate neuron, -BgTx did not block the carbachol-induced increase in GABAergic IPSCs
(bottom left). Cumulative distributions of EPSC and IPSC
intervals are shown on the right. B, In
another neuron from the same slice, the enhancement of spontaneous
glutamatergic EPSCs slightly recovered after 35 and 75 min of washout
of -BgTx. The neurons were clamped at 50 mV in ACSF with TTX (0.5 µM) and bicuculline (10 µM) and potassium
gluconate in the recording electrode for glutamate and 70 mV in ACSF
with TTX and potassium chloride in the recording electrode for GABA.
Bars above records indicate 30 sec application of 100 µM carbachol.
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Figure 9.
Strychnine (STR) failed to inhibit the
carbachol-induced increase of either glutamate or GABA release.
A, Carbachol continued to enhance spontaneous
glutamatergic EPSCs in (1.0 µM) STR. Potassium gluconate
was used as the internal recording solution to observe the
glutamatergic EPSCs. B, In a separate neuron, potassium
chloride was used in the electrode to record GABAergic IPSCs.
Carbachol-induced enhancement of GABAergic IPSCs also remained in the
presence of STR. The neuron in A was clamped at 60 mV
in ACSF with TTX (0.5 µM) and bicuculline (10 µM), whereas the neuron in B was held at
70 mV in ACSF with TTX. STR was applied by bath perfusion for 15 min
before carbachol challenge in both neurons. Bars above records indicate 30 sec application of 100 µM
carbachol.
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DISCUSSION |
Depending on recording conditions, in all neurons we
observed either glutamate-mediated or GABA-mediated spontaneous
currents, leading us to conclude that LGNv neurons receive both
glutamatergic and GABAergic inputs. Previously it has been shown that
presynaptic nAChRs in the LGNv can enhance the release of GABA in a
TTX-insensitive manner (McMahon et al., 1994b). We report here that
presynaptic nAChRs within the LGNv also enhance the spontaneous release
of glutamate by a TTX-insensitive mechanism. The failure of TTX to inhibit the nAChR-mediated enhancement indicates that the location of
these nAChRs, like those for GABA, are near release sites. This
modulation of glutamate release in the LGNv is significant because it
shows conclusively that within one region, presynaptic nAChRs can
regulate the release of multiple transmitters. In understanding the
complex regulatory function of presynaptic nAChRs in the brain, our
data suggest that modulation of excitatory and inhibitory inputs can
occur simultaneously to fine tune synaptic transmission.
As indicated previously, the majority of the nAChRs in the LGNv appear
to be presynaptic, because very little postsynaptic current is seen in
the neuronal recordings (McMahon et al., 1994b). Immunohistochemical
studies have indicated that a number of nAChR subunits are present
within the LGNv (Britto et al., 1992); these include the 2,
3, 4, 7, 8, and 2 subunits. The stoichiometric arrangement of these subunits within native nAChRs is unknown. However,
various combinations of subunits can be distinguished pharmacologically
(Luetje et al., 1990; Luetje and Patrick, 1991). After finding that
glutamate and GABA release are both modulated by presynaptic nAChRs in
the LGNv, we then set forth to determine whether the nAChR subtypes
that were responsible were similar.
Nicotinic AChR antagonists, such as MLA and -BgTx, have proven to be
useful in distinguishing neuronal nAChRs and determining their
involvement in specific responses. MLA and -BgTx were used in the
present study to determine the role, if any, of 7/8
subunit-containing nAChRs in enhancing glutamate and/or GABA release in
the LGNv. Carbachol-induced increases of both glutamate and GABA
release were observed in the presence of 0.1 µM MLA,
suggesting that 7-containing nAChRs are not likely involved in these
LGNv responses. A higher dose of MLA (10 µM), which is
nonselective for nAChR subtypes (Yum et al., 1996), blocked both
responses, confirming again that nAChRs mediated the increased
transmitter release (data not shown).
The 7-containing nAChRs are known to desensitize rapidly, making it
difficult to observe their actions (Couturier et al., 1990; Gerzanich
et al., 1994; Zhang et al., 1994). Our method of carbachol application
in these studies was bath perfusion for a period of 30 sec. The
nAChR-mediated enhancement of neurotransmitter release that we observed
showed little desensitization over the 30 sec carbachol perfusion. One
would expect under this method that any 7-containing
receptor-mediated response would have been desensitized. The MLA
results therefore are consistent with the notion of 7-containing
nAChRs having little involvement in the enhancement of glutamate or
GABA release in the LGNv.
Our results from experiments with -BgTx, however, did not coincide
with our MLA findings. Surprisingly, we found that -BgTx blocked the
carbachol-induced increase in spontaneous glutamate release. In
contrast, the nAChR-mediated enhancement of GABA release was not
inhibited by the 2 hr incubation in -BgTx. Alternately recording
carbachol's enhancement of spontaneous glutamate and GABA release
within the same -BgTx-treated slice allowed us to verify that only
the carbachol-induced increase of spontaneous glutamate release was
blocked. STR was used in an attempt to further characterize the
receptor subtypes involved and, similar to MLA, did not inhibit the
carbachol-induced increase of glutamate or GABA release. Thus, it
appears that the presynaptic nAChRs mediating the carbachol enhancement
of glutamate release have unique pharmacological properties.
As indicated by these results, the presynaptic nAChR subtypes
modulating glutamate and GABA release within the LGNv are different. Because these excitatory and inhibitory inputs are likely to have distinct neuronal origins, it is not surprising that we find a differential expression of nAChR subtypes. The pharmacological profile
of the nAChRs mediating GABA release suggests that they are
high-affinity nicotine sites, presumably an 4/2-like subtype of
nAChR. Conversely the enhancement of glutamate release seems to be
mediated by an nAChR subtype that is not like any described previously.
The fact that -BgTx blocked carbachol's enhancement of glutamate
release argues that an 7/8 subunit may be involved. However, the
lack of desensitization of the response and the failure of MLA and STR
to block it implies otherwise. The receptor appears to exhibit
characteristics of 7-containing and non-7-containing nAChRs. At
this time the involvement of an 7/8 subunit or any other specific
subunit cannot be determined conclusively, because we are dealing
with native receptors located on presynaptic nerve terminals.
Although our results may indicate a unique arrangement of the 7/8
subunit with other nAChR subunits, an alternative explanation is
provided by the work of Pugh et al. (1995), who describe
-BgTx-binding nAChRs in the chick ciliary ganglion that are devoid
of any known nicotinic receptor subunits, including 7/8.
We have no direct evidence of endogenous ACh being released at these
presynaptic sites, but a significant number of cholinergic fibers have
been shown to terminate within the LGNv (Sorenson et al., 1989). One
explanation as to how these receptors may be activated in
vivo is through direct axo-axonic synapses between cholinergic
fibers and the glutamatergic and GABAergic inputs. Stimulation of the
cholinergic fibers concurrently with the glutamate or GABA inputs would
enhance the release of that transmitter into the synapse. Such a direct
interaction would presumably result in the selective modulation of one
specific transmitter.
Another explanation takes into account the idea that the presynaptic
nAChRs might participate in a "volume" transmission phenomenon (Agnati et al., 1995). Acetylcholine released in a sufficient amount
could diffuse through the LGNv and activate the presynaptic nAChRs. In
this instance the release of both transmitters would be affected
simultaneously. Activation of presynaptic nAChRs by this method would
appear to go against the premise of selectively modulating transmitter
release. However, a selective modulation of the transmitters might be
achieved if the presynaptic nAChRs were of different subtypes and
displayed different sensitivities to ACh. Additionally, in terms of
receptor activation, it should be considered that exogenous agonists,
such as nicotine, have the ability to reach these presynaptic nAChRs
and thereby cause activation. Regardless of the process by which the
presynaptic nAChRs are activated, the end result is a direct modulation
of synaptic transmission in the LGNv.
| |
FOOTNOTES |
Received Oct. 24, 1997; revised Dec. 29, 1997; accepted Dec. 31, 1997.
This work was supported by National Institutes of Health Grants NS17574
and NS33135 to V.A.C. The software program KyPlot Version 1.0 that was
used to make the figures in this paper was a gift from Dr. K. Yoshioka,
Tokyo Medical and Dental University, Tokyo, Japan.
Correspondence should be addressed to Dr. Jian-Zhong Guo, Department of
Pharmacology, The George Washington University, School of Medicine and
Health Sciences, 2300 Eye Street, N.W., Washington, D.C.
20037.
| |
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Top
Abstract
Introduction
