Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6074
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ABSTRACT |
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Dutar, Patrick, Jeffrey J. Petrozzino, Huan M. Vu, Marc F. Schmidt, and David J. Perkel. Slow Synaptic Inhibition Mediated by Metabotropic Glutamate Receptor Activation of GIRK Channels. J. Neurophysiol. 84: 2284-2290, 2000. Glutamate is the predominant excitatory neurotransmitter in the vertebrate CNS. Ionotropic glutamate receptors mediate fast excitatory actions whereas metabotropic glutamate receptors (mGluRs) mediate a variety of slower effects. For example, mGluRs can mediate presynaptic inhibition, postsynaptic excitation, or, more rarely, postsynaptic inhibition. We previously described an unusually slow form of postsynaptic inhibition in one class of projection neuron in the song-control nucleus HVc of the songbird forebrain. These neurons, which participate in a circuit that is essential for vocal learning, exhibit an inhibitory postsynaptic potential (IPSP) that lasts several seconds. Only a portion of this slow IPSP is mediated by GABAB receptors. Since these cells are strongly hyperpolarized by agonists of mGluRs, we used intracellular recording from brain slices to investigate the mechanism of this hyperpolarization and to determine whether mGluRs contribute to the slow synaptic inhibition. We report that mGluRs hyperpolarize these HVc neurons by activating G protein-coupled, inwardly-rectifying potassium (GIRK) channels. MGluR antagonists blocked this response and the slow synaptic inhibition. Thus, glutamate can combine with GABA to mediate slow synaptic inhibition by activating GIRK channels in the CNS.
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INTRODUCTION |
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Glutamate mediates the vast
majority of excitatory synaptic transmission in the vertebrate CNS
through two classes of receptors. Postsynaptic actions of ionotropic
glutamate receptors are always excitatory. Metabotropic glutamate
receptors (mGluRs), on the other hand, can have diverse effects,
including modulation of second messenger levels, ion-channel activity,
and synaptic efficacy (Conn and Pin 1997;
Nakanishi 1994
; Pin and Duvoisin 1995
).
Effects of mGluRs on membrane potential are almost always depolarizing, either through inhibition of potassium conductances (Charpak and Gahwiler 1991
; Charpak et al. 1990
) or
activation of nonselective cation conductance (Glaum and Miller
1992
; Staub et al. 1992
). In all but two
of the cases in which mGluR activation induces hyperpolarization
(Knoflach and Kemp 1998
; Slaughter and Miller 1981
), the response is mediated by
Ca2+-activated K+ channels
(Fagni et al. 1991
; Fiorillo and Williams
1998
; Holmes et al. 1996
; Rainnie et al.
1994
; Shirasaki et al. 1994
).
In songbirds, the forebrain nucleus HVc (used here as the proper name;
Brenowitz et al. 1997) is essential for
producing learned song and provides input to a circuit that is
essential for learning (Bottjer and Arnold 1997
;
Brenowitz et al. 1997
; Doupe and Kuhl 1999
; Margoliash 1997
; Nottebohm et al.
1976
). HVc has two populations of projection neurons
(Dutar et al. 1998
; Katz and Gurney
1981
). Those projecting to nucleus robustus archistriatalis
(RA) form a primary motor pathway for song production (Nottebohm
et al. 1976
). Those HVc neurons projecting to a basal
ganglia-like region called area X provide input to a circuit that is
essential for song learning but not for song production (Bottjer
et al. 1984
; Scharff and Nottebohm 1991
;
Sohrabji et al. 1990
). The area X-projecting HVc
neurons have physiological and pharmacological properties distinct from
those of the RA-projecting neurons (Dutar et al. 1998
;
Kubota and Taniguchi 1998
). In particular, area
X-projecting HVc neurons exhibit an unusually slow synaptic inhibition
which is only partially mediated by GABAB
receptors (Dutar et al. 1998
; Schmidt and Perkel
1998
). These cells are robustly hyperpolarized by the mGluR
agonist aminocyclopentane dicarboxylic acid (ACPD) (Dutar et al.
1998
) or by the GABAB receptor agonist
baclofen (Dutar et al. 1998
, 1999
; Schmidt and
Perkel 1998
). This hyperpolarization resembles the
GABAB receptor-mediated slow inhibition in
mammalian hippocampus (Dutar and Nicoll 1988
), which
involves activation of a G-protein-coupled inwardly rectifying class
of potassium (GIRK) channels (Lüscher et al.
1997
). mGluRs share substantial sequence homology with
GABAB receptors (Kaupmann et al.
1997
) and, in a heterologous expression system, can activate
GIRK channels (Saugstad et al. 1996
; Sharon et
al. 1997
). However, native coupling of activation of GIRK
channels by mGluR agonists has been observed only in a subtype of
cerebellar interneuron (Knoflach and Kemp 1998
).
To examine whether the coupling of mGluRs to GIRK channels occurs in area X-projecting HVc neurons and contributes to slow inhibition, we made intracellular recordings in brain slices. We report that mGluR agonists hyperpolarize HVc neurons by activating GIRK channels and that mGluR antagonists block slow synaptic inhibition. This action of mGluRs represents a novel synaptic signaling mechanism by which glutamate receptors can inhibit central neurons.
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METHODS |
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Preparation of slices and electrophysiological recording
Adult male zebra finches (Taeniopygia gutatta) at
least 120 days old were obtained from a local supplier or were bred in
our colony. Slices were prepared as described by Schmidt and
Perkel (1998) and the procedures were approved by the
Institutional Animal Care and Use Committee at the University of
Pennsylvania. Briefly, a bird was anesthetized with halothane and
decapitated. The brain was rapidly removed and placed in ice-cold
artificial cerebrospinal fluid (ACSF) that had been pregassed with 95%
O2-5% CO2. The composition of the ACSF was (in mM) 119 NaCl, 2.5 KCl, 1 NaH2PO4, 26.2 NaHCO3, 11 D-glucose, 2.5 CaCl2, and 1.3 MgSO4. Parasagittal slices (300-400 µm thick)
were prepared with a vibrating microtome and stored submerged in
bubbled ACSF. For recording, a slice was transferred to a chamber where
it was submerged and superfused at 1-2 ml/min with pregassed ACSF.
Conventional intracellular recordings were obtained from neurons within
HVc, whose borders could be seen in the unstained, living slice. We
concentrated on those HVc neurons projecting to another song nucleus,
area X, which are distinguished by a number of physiological and
pharmacological properties (Dutar et al. 1998). The
electrodes were filled with 4 M potassium acetate and 0-20 mM KCl and,
when required,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), guanosine 5'-O-(3-thiotriphosphate) (GTP
-S),
guanosine 5'-O-(2-thiodiphosphate) (GDP-
-S), or
N-(2,6-dimethylphenylcarbomoylmethyl)triethyl ammonium bromide (QX-314). Membrane potential was amplified using an
Axoclamp 2B microelectrode amplifier (Axon Instruments, Foster City, CA). Signals were low-pass filtered at 1-5 kHz, digitized at
twice the filter cutoff frequency, and stored on a computer hard disk.
Acquisition and analysis were carried out using a program written in
Labview (National Instruments, Austin, TX). To measure the
current induced by the application of ACPD or baclofen, the membrane
potential was manually clamped to the initial membrane potential.
Average values are given as mean ± SE and the statistical test
used was Student's t-test.
Drugs
Chemicals used in this study included baclofen (30 µM), BAPTA
(100-200 mM in the recording pipette), cesium chloride (10 mM), tetraethylammonium (TEA) (1 mM), TTX (1 µM), bicuculline methiodide (BMI) (40 µM), GTP--S (20 mM in the pipette), and GDP-
-S (20 mM
in the pipette), all obtained from Sigma Chemical Co., St. Louis, MO.
ACPD (100 µM), (2S, 3S, 4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG) (500 µM), and QX-314 (100 mM) were from Tocris Cookson, Ballwin, MO; CGP35348 (0.5 mM) was from Novartis, Basel, Switzerland; and LY307452 (100-200 µM) was from Eli Lilly, Indianapolis, IN). All
drugs were added to the superfusion medium by dilution of a stock
solution made in water, except for BAPTA, GTP-
-S, and QX-314, which
were included in the recording electrolyte and delivered to neurons by
diffusion and by passing current pulses.
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RESULTS |
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Bath application of ACPD (100 µM) caused a robust
hyperpolarization (14.1 ± 0.6 mV, n = 52) (Fig.
1A1) in area X-projecting HVc
neurons, identified by their intrinsic properties (Dutar et al.
1998), accompanied by an increase in membrane conductance (Fig.
1A2). The response was not caused by an action
potential-dependent release of inhibitory neurotransmitters by
neighboring cells because the hyperpolarization was unaffected by the
presence of the Na+ channel blocker TTX (1 µM,
n = 9) (Fig. 1B). The current-voltage relation of
the ACPD-induced response indicated that the conductance had a reversal
potential near
90 mV (Fig.
2A) and was inwardly rectifying (Fig. 2B).
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To investigate the ionic basis of the response, we measured the
reversal potential of the hyperpolarization while the slice was bathed
in different concentrations of K+. The reversal
potential for the ACPD-induced response closely matched that predicted
by the Nernst equation, assuming an internal K+
concentration of 120 mM. This match indicates that ACPD caused an
increase in membrane conductance for K+ ions. In
addition, the nonspecific K+ channel blocker
Cs+ (10 mM) reversibly reduced the ACPD response
from 12.7 ± 3.6 mV to
2.5 ± 1.2 mV (n = 4, P < 0.05).
Use of selective agonists and antagonists indicated that group II or
group III mGluR agonists could evoke this hyperpolarizing response,
which is not caused by cross-reactivity with
GABAB receptors (Dutar et al.
1999). Application of an additional group II-selective mGluR
antagonist, MCCG (Jane et al. 1994
; Knopfel et
al. 1995
), blocked the ACPD response (Fig.
3).
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To test whether mGluRs and GABAB receptors might share effector pathways, we investigated whether the outward current evoked by ACPD and baclofen applied together was different from the sum of the currents evoked by the individual agonists applied separately. In nine cells, ACPD and baclofen induced similar currents. Coapplication of ACPD and baclofen to the same cells induced a current that was significantly less than the current predicted assuming linear addition (P < 0.0005) (Fig. 4). Thus, the currents partially occluded one another, which is consistent with convergence of the two receptors onto the same G protein or channel population.
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MGluRs can activate potassium conductance by raising intracellular
Ca2+ (Rainnie et al. 1994). We
tested for this mechanism in HVc neurons by including the
Ca2+ chelator BAPTA (100-200 mM) in the pipette
solution. In the presence and absence of intracellular BAPTA, ACPD
caused a comparable hyperpolarization (8.9 ± 1.0 mV,
n = 15 with BAPTA; 9.7 ± 1.1 mV,
n = 15 control; P = 0.6) (Fig.
5, A-C). We
verified BAPTA entry into cells by observing broadening of the action
potential, the duration of which is governed by
Ca2+-dependent K+ channels
(Fig. 5B) (Lancaster and Nicoll 1987
).
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As a second test for a role of internal Ca2+, we
bath applied TEA, which blocks one class of
Ca2+-dependent K+ channels
and broadens the action potential in hippocampal neurons (Lancaster and Nicoll 1987). TEA (1 mM), despite
dramatically prolonging action potential duration (Fig. 5E),
had no effect on the hyperpolarization induced by ACPD (11.5 ± 2.6 mV control; 13.1 ± 1.8 mV in the presence of TEA;
n = 4; P > 0.05) (Fig. 5, D
and F). These results indicate that ACPD hyperpolarizes HVc neurons via a mechanism independent of
Ca2+-activated K+ channels.
We hypothesized, based on the results in the preceding paragraphs and
on the high degree of sequence similarity between
GABAB receptors and mGluRs (Kaupmann et
al. 1997), that ACPD activates GIRK channels. We first
investigated the possible role of G proteins by recording HVc neurons
with electrodes containing the hydrolysis-resistant analogues GTP-
-S
and GDP-
-S, which irreversibly activate and block G protein-coupled
responses, respectively (Andrade et al. 1986
). In the
presence of GTP-
-S or GDP-
-S, the response to either ACPD or
baclofen was almost completely blocked (Fig.
6). These data indicate that the effect
of ACPD is mediated by a G protein-dependent mechanism.
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Next, we recorded HVc neurons with electrodes containing the lidocaine
derivative QX-314, which blocks GIRK channels (Alreja and
Aghajanian 1994; Andrade 1991
; Nathan et
al. 1990
) as well as Na+ channels
(Strichartz 1973
). In neurons filled with QX-314, the responses to application of ACPD or baclofen were reduced by 82% (n = 5) and 85% (n = 6), respectively,
compared with those in control cells (Fig.
7, A-C). We
concluded that ACPD hyperpolarizes HVc neurons by activating GIRK
channels.
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Finally, we asked whether synaptically released neurotransmitters can
activate this mechanism. Brief tetanic stimulation of afferents to HVc
neurons causes a slow inhibitory postsynaptic potential (sIPSP) that is
partially mediated by GABAB receptors (Schmidt and Perkel 1998). We isolated the
GABAB receptor-independent component of the
sIPSP by recording in the presence of a cocktail of antagonists to
ionotropic glutamate receptors and GABAA and GABAB receptors. The group II mGluR antagonist
MCCG reduced the peak sIPSP amplitude by 60 ± 11%
(n = 6; P < 0.005) (Fig.
8). In four cases tested, the sIPSP
recovered, following washout of MCCG, to 71 ± 13% of control
values. In addition, the group II mGluR antagonist LY307452
(Wermuth et al. 1996
) reduced the sIPSP measured in the
presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX),
2-amino-5-phosphonovaleric acid (APV), and BMI (but not CGP35348) by
25 ± 6% (n = 8). Thus, group II mGluRs
contribute to the generation of the sIPSP.
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DISCUSSION |
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The main results of the present study are that in X-projecting
cells of nucleus HVc of the adult male zebra finch, group II/III mGluR-induced hyperpolarization (Dutar et al. 1999) is
mediated by activation of GIRK channels, and that this process has a
physiological role in contributing to the slow inhibition observed
after brief tetanic stimulation of afferents. Our conclusion that
mGluRs activate GIRK channels in HVc neurons is based on the following
findings. ACPD, an agonist at mGluRs, activates an inwardly rectifying
K+ conductance. The response requires activation
of G proteins and is independent of intracellular
Ca2+. Occlusion of the ACPD response with that of
baclofen, which is known to involve GIRK channels (Lüscher
et al. 1997
), indicates a shared mechanism. Responses to ACPD
and to baclofen are both blocked by intracellular QX-314, which blocks
GIRK channels (Alreja and Aghajanian 1994
;
Andrade 1991
; Nathan et al. 1990
). Since the GABAB receptor-independent component of the
sIPSP is sensitive to two different group II mGluR antagonists, this
mechanism contributes to slow inhibition.
Presynaptic inhibitory actions of mGluR agonists have been extensively
described in the hippocampus, spinal cord, amygdala, striatum,
neocortex, and cerebellum (Pin and Duvoisin 1995;
Stefani et al. 1996
). In these structures, mGluR
agonists depress the release of neurotransmitters by inhibiting
-conotoxin-sensitive or dihydropyridine-sensitive calcium channels.
Therefore, mGluRs serve as autoreceptors at glutamatergic synapses or
heteroreceptors at GABAergic synapses and contribute to the regulation
of synaptic transmission (Conn and Pin 1997
).
Postsynaptic actions of mGluRs are mainly excitatory in the CNS. For
instance, in hippocampal pyramidal neurons, mGluR activation leads to
neuron depolarization associated with an increase in input resistance
and a decrease in the accommodation of action potential discharge
(Davies et al. 1995). Postsynaptic excitatory effects of mGluR activation also occur in other central structures, many of which are mediated by group I mGluRs, and are caused by the
inhibition of various potassium currents, such as
IM, IAHP, and
ILeak (Pin and Duvoisin 1995
;
Schoepp and Conn 1993
). However, mGluR-induced
depolarization can also occur through a variety of other mechanisms,
including increased cation conductance or electrogenic Na/Ca exchange
current (Conn and Pin 1997
).
Activation of mGluRs by exogenous agonists induces postsynaptic
inhibitory actions in some cases (Fagni et al. 1991;
Fiorillo and Williams 1998
; Hirano and MacLeish
1991
; Holmes et al. 1996
; Knoflach and
Kemp 1998
; Rainnie et al. 1994
; Shirasaki
et al. 1994
; Vranesic et al. 1993
). When the
mechanism is investigated, these effects almost always involve
Ca2+ mobilization and the opening of
Ca2+-activated K+ channels
(Fiorillo and Williams 1998
; Holmes et al.
1996
; Rainnie et al. 1994
) and may be mediated
by group I or group II mGluRs. Application of exogenous group II mGluR
agonists to cerebellar interneurons activates GIRK channels
(Knoflach and Kemp 1998
), but it is not known whether
synaptic activity can evoke this process. Thus, our finding that the
group II/III response observed here is unaffected by manipulations that
prevent activation of Ca2+-activated
K+ channels makes this mechanism in HVc neurons
relatively unusual.
A variety of inhibitory neurotransmitter receptors converge to activate
GIRK channels (Nicoll 1988; North 1989
),
probably via direct activation by G proteins (Clapham
1994
). One of the prototypical receptors known to activate GIRK
channels is the GABAB receptor, the postsynaptic
inhibitory action of which is not observed in knock-out mice lacking
the GIRK2 protein (Lüscher et al. 1997
). Our
results confirm the coupling of GABAB receptors to GIRKs in HVc neurons. In addition, we found that QX-314, which blocks GIRK channel activation, blocks the action of mGluRs, thus adding this receptor type to the family of receptors that activate GIRKs.
GABAB receptors and mGluRs share a high degree of
sequence homology (Kaupmann et al. 1997). In
Xenopus oocytes injected with mRNA coding for mGluRs and
GIRKs, receptor activation causes increased potassium conductance
(Saugstad et al. 1996
; Sharon et al.
1997
), indicating that such coupling is feasible in an
artificial system. But to date, synaptic coupling of these widely
expressed receptors and channels has not been observed.
In some mammalian neurons, such as those of the hippocampus,
GABAB receptors activate GIRK channels to mediate
an sIPSP lasting a few hundred milliseconds (Dutar and Nicoll
1988; Nicoll 1988
). Inhibitory processes of
similar or longer duration may help shape song-selective auditory
responses in songbird nucleus HVc (Lewicki 1996
;
Margoliash 1997
). Moreover, inhibitory processes may
alter or suppress auditory responses during singing or for several
seconds afterward (Lewicki 1996
; Margoliash
1986
; McCasland and Konishi 1981
). Our results
suggest that mGluRs, which activate GIRK channels, contribute to such
slow inhibition in HVc.
More generally, our findings add a new mechanism by which glutamate
receptors can play an inhibitory role in the CNS. An sIPSP mediated by
mGluR activation was observed in dopamine neurons of the rat ventral
tegmental area (Fiorillo and Williams 1998). As in
previously described examples of mGluR-induced hyperpolarization in
mammals, this effect is mediated by the release of
Ca2+ from internal stores and the activation of
Ca2+-activated K+ channels.
Thus, the mGluR action on HVc neurons, not requiring a rise in internal
Ca2+ and depending on GIRK channel activation, is
quite distinct. It will be interesting to determine whether this mode
of synaptic inhibition is specific to avian systems or whether it has
been overlooked or masked in mammals by the wide variety of additional excitatory actions of mGluRs.
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ACKNOWLEDGMENTS |
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We are grateful to Dr. Mark Konishi, in whose lab the initial experiments were performed. We thank Dr. Michael P. Nusbaum for helpful comments on the manuscript. We also thank Dr. D. D. Schoepp (Lilly Corp., Indianapolis, IN) for providing LY307452 and CIBA-Geigy (currently Novartis, Basel, Switzerland) for providing CGP35348. M. Farries and D. Perkel wrote the Labview acquisition and analysis program.
This work was supported by National Institutes of Health Grants R03 DC-02477 and R01 MH-56646 and National Science Foundation Grant IBN 9817889 to D. J. Perkel.
Present addresses: P. Dutar, INSERM U. 159, 2 ter rue d'Alésia,
75014 Paris, France; J. J. Petrozzino, UMDNJRobert Wood Johnson
Medical School, 401 Haddon Ave., Camden, NJ 08103-1506; M. F. Schmidt, Dept. of Biology, University of Pennsylvania, Philadelphia, PA
19104-6018.
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FOOTNOTES |
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Present address and address for reprint requests: D. J. Perkel, Depts. of Zoology and Otolaryngology, University of Washington, Box 356515, Seattle, WA 98195-6515 (E-mail: perkel{at}u.washington.edu).
Received 20 April 2000; accepted in final form 14 July 2000.
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REFERENCES |
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