Laboratoire de Neurophysiologie Centre National de la Recherche Scientifique Unité Mixte de Recherche 5543, Université de Bordeaux 2, 33076 Bordeaux cedex, France
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ABSTRACT |
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Baufreton, Jérôme,
Maurice Garret,
Sandra Dovero,
Bernard Dufy,
Bernard Bioulac, and
Anne Taupignon.
Activation of GABAA Receptors in Subthalamic Neurons
In Vitro: Properties of Native Receptors and Inhibition Mechanisms.
J. Neurophysiol. 86: 75-85, 2001.
The subthalamic nucleus (STN) influences the output
of the basal ganglia, thereby interfering with motor behavior. The main inputs to the STN are
GABAergic. We characterized the GABAA receptors expressed in the STN and investigated the response of subthalamic neurons to the activation of GABAA receptors.
Cell-attached and whole cell recordings were made from rat brain slices
using the patch-clamp technique. The newly identified subunit
confers atypical pharmacological properties on recombinant receptors, which are insensitive to barbiturates and benzodiazepines. We tested
the hypothesis that native subthalamic GABAA
receptors contain
proteins. Applications of increasing
concentrations of muscimol, a selective GABAA
agonist, induced Cl
and HCO
subunit-specific antibody showed that the
protein was
not expressed within the STN. Native subthalamic
GABAA receptors did not, therefore, display
pharmacological or structural properties consistent with receptors
comprising
. Burst firing is a hallmark of Parkinson's disease.
Half of the subthalamic neurons have the intrinsic capacity of
switching from regular-firing to burst-firing mode when hyperpolarized
by current injection. This raises the possibility that activation of
GABAA receptors might trigger the switch.
Statistical analysis of spiking activity established that 90% of
intact neurons in vitro were in single-spike firing mode, whereas 10%
were in burst-firing mode. Muscimol reversibly stopped recurrent
electrical activity in all intact neurons. In neurons held in whole
cell configuration, membrane potential hyperpolarized by
10 mV whilst
input resistance decreased by 50%, indicating powerful membrane
shunting. Muscimol never induced burst firing, even in neurons that
exhibited the capacity of switching from regular- to burst-firing mode.
These molecular and functional data indicate that native subthalamic
GABAA receptors do not contain the
protein
and activation of GABAA receptors induces
membrane shunting, which is essential for firing inhibition but
prevents switching to burst-firing. They suggest that the STN, like
many other parts of the brain, has the physiological and structural features of the widely expressed GABAA receptors
consisting of
subunits.
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INTRODUCTION |
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The precise role(s) of the subthalamic nucleus (STN) in the
operation of basal ganglia remain(s) unclear. It forms part of what has
recently been called the indirect "network" (instead of indirect
pathway), to underscore the complexity of its microanatomy (Smith et al. 1998). The indirect network comprises the
STN, the globus pallidus (globus pallidus externalis in
primates), and their afferences and efferences. In brain slice
preparations, STN neurons spontaneously fire regular, single spikes at
low frequency (Beurrier et al. 1999
; Bevan and
Wilson 1999
; Overton and Greenfield 1995
).
Firing persists in the absence of both excitatory and inhibitory synaptic transmission (Beurrier et al. 2000
;
Bevan and Wilson 1999
). Furthermore, half of the neurons
switch from single-spike to burst-firing mode when continuously
hyperpolarized (Beurrier et al. 1999
). In contrast, the
STN-globus pallidus network reconstituted in organotypic cultures
spontaneously produces bursts. Bursts occur during longer periods of
either silence or irregular firing and depend on the reciprocal
connections between STN and globus pallidus (Plenz and Kitai
1999
). Pallidal inputs to STN use GABA as a neurotransmitter
(Bevan et al. 1997
; Shink et al. 1996
). Stimulation of pallido-subthalamic fibers has been shown to evoke monosynaptic inhibitory postsynaptic potentials (IPSPs) involving chloride ions that were abolished by bicuculline, the antagonist of
GABAA receptors (Kita et al. 1983
;
Nakanishi et al. 1987
).
GABAA receptors are primarily chloride ionophores
(Bormann et al. 1987). Pharmacologically different
receptors are formed by differential assembly of multiple subunits
(
1-6,
1-3,
1-3,
,
, and
) (Bonnert et al.
1999
). Indeed, studies have demonstrated that the subunit
composition determines the specific effects of allosteric modulators,
such as benzodiazepines, barbiturates, and steroids (Hevers and
Luddens 1998
). Strong hybridization signals have been found for
1,
2, and
2 mRNAs in STN, together with weak signals for
3
and
3 (Wisden et al. 1992
). Moreover,
immunoreactivity for
1 and
2/3 has been detected on postsynaptic
membranes within STN (Charara and Smith 1998
). This
suggested that STN expressed the major subtype of
GABAA receptors found throughout the brain, i.e.
1
2
2 (McKernan and Whiting 1996
). Abundant
transcripts of
were reported in human STN (Davies et al.
1997
), but this finding was not confirmed by another study of
monkey brains (Whiting et al. 1997
). Unambiguous
demonstration of
expression in STN is important since its presence
may confer atypical pharmacological properties on
GABAA receptors. In view of its restricted
expression in the brain, it has been suggested that
could allow
selective manipulation of STN (Davies et al. 1997
). This
could have opened up substantial prospects for the treatment of basal
ganglia dysfunctions such as Parkinson's disease.
To understand the GABAergic input to STN neurons, we investigated the effects of activation of GABAA receptors in slices of rat brain. Our aims were to characterize the GABAA receptors expressed in subthalamic neurons and to test the possibility that activation of GABAA receptors might switch neurons to burst-firing mode.
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METHODS |
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Slice preparation
Experiments were performed on STN neurons in 400-µm-thick coronal slices obtained from 18- to 22-day-old Wistar rats. After cervical dislocation, the animals were killed by fast decapitation, the brain was rapidly removed, and a block of cerebral matter containing the STN was isolated in an ice-cold solution containing (in mM) 250 sucrose, 26 NaHCO3, 7 MgCl2, 2 KCl, 1.15 NaH2PO4, 0.5 CaCl2, and 11 glucose, bubbled with 95% O2-5% CO2 (pH 7.4). Three coronal slices containing STN neurons were prepared from one brain in the same solution using a vibroslicer (Campden Instruments, Loughborrough, UK). They were incubated at room temperature in a Krebs' solution containing (in mM) 124 NaCl, 26 NaHCO3, 3.6 KCl, 1.3 MgCl2, 2.4 CaCl2, 1.25 HEPES, and 10 glucose (pH 7.4), bubbled with 95% O2-5% CO2, for a 2-h recovery period.
Electrophysiological recordings
One slice was transferred to an immersion-type recording chamber
(Guerineau et al. 1997) and continuously superfused (3.5 ml/min) with the oxygenated Krebs' solution maintained at 25°C. The
slice was examined under a dissecting microscope; STN was readily
identified as ovoid gray matter immediately dorsal to the cerebral
peduncle. Recordings were made using the blind patch-clamp technique in
cell-attached or whole cell configurations. In the cell-attached
experiments, the pipette contained Krebs' solution and the patch was
held at bath potential (Fenwick et al. 1982
). In the
whole-cell configuration, both current- and voltage-clamp modes were
used. Four pipette solutions were used for whole cell recordings.
Solution 1 contained (in mM) 120 K-gluconate, 10 KCl, 10 NaCl, 11 EGTA,
10 HEPES, 1 CaCl2, 2 ATP-Mg, and 0.4 Na-GTP (Beurrier et al. 1999
). Solution 2 was a low-chloride
pipette medium in which KCl and NaCl were removed and K-gluconate
raised to 140 mM. Solutions 3 and 4 were high-chloride pipette
solutions, otherwise similar to solution 2. Solution 3 contained 140 mM
KCl. In solution 4, CsCl was used instead of KCl. In all cases, the osmolarity of intrapipette solutions for whole cell recordings was
between 280 and 300 mOsm and pH adjusted to 7.25. Spontaneous synaptic
activity was recorded using a pipette solution 3 in presence of 50 µM
DL-2-amino-5-phosphonovaleric acid (AP5) and 40 µM
6-cyano-7-nitroquinoxalene-2,3-dione (CNQX). Electrodes were pulled
from borosilicate thin-glass capillaries (GC150F-15, Harvard Apparatus,
Edenbridge, UK) on a vertical puller (PP-830, Narishige, Japan) and had
resistances of ~7 and ~12 M
W when filled with high- and
low-chloride pipette media, respectively. Signals were recorded using
an Axopatch-1D amplifier (Axon Instruments, Foster City, CA) with the
amplifier filter set at 5 kHz, stored on a video tape, and/or digitized
at 2.5-20 kHz using a Digidata 1200B. Access resistance (~20 M
)
was regularly monitored. Junction potentials were measured according to
the method described by Neher (1992)
and voltage errors
corrected off-line (
5,
13,
2, and
5 mV for solutions 1-4, respectively).
Drugs
All drugs were purchased from Sigma (St. Louis, MO) except
bicuculline, CNQX, and AP5, which were purchased from Tocris (Bristol, UK), and TTX, which was purchased from Latoxan (Valence, France). They
were prepared as stock solutions and stored at 80°C. The solvent
was water except in the case of furosemide (diluted in ethanol) and
diazepam, flunitrazepam, pentobarbital, and CNQX (prepared in DMSO).
Drugs, diluted in the oxygenated Krebs' solution, were delivered
either by bath perfusion or, more frequently, by means of a
multi-barrel gravity-feed system (HSSE-2, ALA Scientific Instruments,
Sega Electronique, France) composed of two capillaries positioned just
above the patch pipette allowing up to seven solutions to be tested
successively. In the latter case, the minimal duration of a single
application was 2 s. Shorter applications produced inconsistent
results presumably because of the time taken by drugs to reach the
recorded neuron in the slice. Final dilution of the solvent was always
kept <0.002.
Data analysis
Records were analyzed using pClamp 6.01 software (Axon
Instruments). One hundred to 300 synaptic events were detected and acquired (at 25 kHz) using the Fetchex subroutine. The acquired segments were then visually inspected to remove electrical artifacts and superimposed inhibitory postsynaptic currents (IPSCs). Peak amplitude, surface, and decay time constant were calculated for each
IPSC using programmable software (Acquis 1, Biologic, France). Decay
kinetics of IPSCs were usually well described by a single exponential.
The cell-attached experiments were carried out in the voltage-clamp
mode. Action potentials were therefore seen as action currents. On-cell
activity was continuously recorded on video tape. Two sequences from
the continuous recording, containing 500-1,500 action currents (i.e.,
lasting ~3 min), were digitized at 10 kHz and analyzed off-line. One
sequence was taken from the continuous recording before application of
muscimol, the other following the return of the first action current
after application of muscimol. The interspike intervals were obtained
from the intervals between the action currents detected by the Fetchan
subroutine of pClamp. The firing frequency was calculated from the
interspike intervals by the pStat subroutine of pClamp. The interspike
interval distribution was analyzed and plotted according to the Kaneoke and Vitek method (Kaneoke and Vitek 1996) using in-house
software (courtesy of Dr. C. Gross). In the Kaneoke and Vitek method,
discharge density histograms are used to determine the firing pattern
statistically. Autocorrelograms were built to assess the regularity of
any periodic discharge feature in the sequences. In the text and
figures, values are given as means ± SE. Statistical comparisons
were made using paired, Student's t-test. Unless otherwise
stated, values of P < 0.05 were considered significant.
Immunohistochemistry
Rats were overdosed with pentobarbital and immediately perfused
transcardially with 100 ml saline followed by 200 ml of a fixative
solution containing 2% paraformaldehyde and 0.2% picric acid in 0.1 M
phosphate buffer (pH 7.4). Brains were dissected out and soaked
overnight at 4°C in phosphate buffer containing 20% sucrose. The
brains were then stored at 80°C, and 50-µm sections were cut in a
cryostat and processed for immunohistochemistry. Sections were
incubated with affinity-purified anti-
(1:10,000; raised against the
N-terminal sequence unique to the rat
subunit) (Moragues et
al. 2000
) or anti-neurokinin A (1:15,000) (Magoul et al.
1994
) antibodies for 48 h at 4°C in a 0.01 M veronal
buffer, containing 0.2% Triton X-100 and 0.4% casein. Sections were
then incubated for 2 h at room temperature in Fc fragment specific biotinylated goat anti-rabbit IgGs (1:2,000; Jackson Immunoresearch Laboratories), and 2 h in peroxidase-conjugated streptavidin
(1:2,000; Jackson Immunoresearch Laboratories) in veronal buffer with
Triton X-100 (0.2%). The peroxidatic activity was revealed for 20 min using the glucose oxidase/nickel diaminobenzidine method (Shu et
al. 1998
).
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RESULTS |
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Specific agonist of ionotropic GABA receptors, muscimol, activates
a Cl and HCO
Muscimol is a powerful exogenous agonist of ionotropic GABA
receptors. Figure 1A shows a
typical inward current recorded from a subthalamic neuron on
application of muscimol for 20 s. Similar responses were observed
in all 83 neurons tested. The current-voltage relationship for the
muscimol response was examined (Fig. 1B). The positive slope
of the line indicated that a membrane conductance increase occurred
during the muscimol response. Reversal potentials determined with
various pipette solutions were examined (Fig. 1C). Solutions
1-4 were used. The mean experimental value obtained with the
low-chloride pipette solution clearly diverged from the Nernst
equilibrium potential for chloride (- - -); the solid line plots the
values for the reversal potentials calculated from the Nernst equation
with a HCO
permeability ratio of 0.2 (Bormann et al. 1987
),
assuming an intracellular concentration of HCO
). The correspondence of the
data points to the solid line suggests that the response may be
mediated by chloride and bicarbonate.
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The dose-response curve for muscimol is shown in Fig. 1D.
The EC50 (concentration producing 50% of the
maximum response) for muscimol was 5.2 µM. Hence, concentrations of 2 or 10 µM were used in the next set of experiments designed to further
characterize the GABAA receptors in subthalamic
neurons. Mean currents induced by 2 and 10 µM muscimol were
301 ± 50 pA (n = 22) and
936 ± 112 pA
(n = 24), respectively.
Conductance density correlates with receptor density. An estimate of
conductance density was obtained as reported by Zhang and
Jackson (1995). A mean conductance of 27.75 ± 2.61 nS
(n = 5) was calculated from maximal currents by
dividing by the driving force. Dividing by the mean area, estimated
from mean capacitance (7.96 ± 1.27 pF, n = 5),
and assuming a spherical structure (which would only be true of a cell
body) with 1 µF.cm
2,
gave a conductance density of ~3.5 mS.cm2.
Pharmacology of ionotropic GABA receptors in subthalamic neurons
GABAA receptors are defined pharmacologically: in addition to being potently activated by muscimol, they are sensitive to antagonism by the convulsant, bicuculline, but are insensitive to baclofen. Modulators in the picrotoxin, barbiturate, and benzodiazepine families contribute to further delineate GABAA receptor properties.
We investigated the effects of bicuculline, picrotoxinin,
pentobarbital, diazepam, and flunitrazepam on the properties of GABA
receptors in subthalamic neurons. First, we activated both synaptic and
extra-synaptic receptors using muscimol. Table
1 summarizes our results. Bicuculline and
picrotoxinin strongly inhibited muscimol-induced currents
(n = 6 and n = 4, respectively), whereas 10 µM 2-hydroxysaclofen (a GABAB
receptor antagonist) did not alter the response to muscimol
(n = 2, not shown). Pentobarbital at 25 µM
significantly potentiated the currents activated by muscimol (Table 1).
Diazepam (100 nM), a physiologically relevant concentration (Zhang et al. 1993), and flunitrazepam (1 µM, Fig.
2A) also significantly potentiated the currents activated by muscimol. Currents obtained at
EC20 for muscimol were doubled by the two
benzodiazepines.
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We further tested the action of benzodiazepines on spontaneous GABAergic synaptic activity. Spontaneous IPSCs were recorded in the presence of glutamatergic receptor inhibitors (50 µM AP5 and 40 µM CNQX). The IPSCs were fully blocked by bicuculline (20 µM, n = 4), indicating GABAA receptors. Flunitrazepam significantly enhanced both the peak amplitude and the time constant of IPSC decay (Fig. 2B). It shifted the peak amplitude and surface distribution to higher values, resulting in an enhanced average IPSC.
Taken together, our pharmacological data show that extrasynaptic and
synaptic GABA receptors in subthalamic neurons are sensitive to the
positive allosteric modulators, diazepam, flunitrazepam and
pentobarbital, in the same way as GABAA receptors
composed of ,
, and
subunits. The pharmacological properties
of native GABA receptors in subthalamic neurons thus appear to differ
from those of recombinant receptors containing the
subunit. The
1
1
receptor has been reported to be insensitive to the
benzodiazepine, flunitrazepam (Whiting et al. 1997
).
Absence of immunoreactivity for the newly identified
GABAA receptor subunit in STN
A high expression level of the GABAA subunit in STN was deduced from Northern blot experiments
(Davies et al. 1997
). Here, our affinity-purified
anti-
subunit antibody was used to test the presence of
-immunoreactive structures in STN. Delineation of STN boundaries
(Fig. 3, A and B)
was established on the basis of both cell nucleus fluorescent labeling
and the distribution of neurokinin A immunoreactivity in rat midbrain
sections. Using this latter material, STN only displayed a few
neurokinin-A-immunoreactive axons but was bordered by many such axons,
as previously shown (Halliday et al. 1995
). In adjacent
sections incubated with the anti-
antibody (Fig. 3C),
labeled neuronal profiles were detected along the dorsal margin of the
STN but not within the STN proper (see also the enlarged view in the
inset).
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Our pharmacological and molecular data do not support the hypothesis
that the newly identified GABAA receptor subunit
is expressed in rat STN. Rather they are consistent with data
indicating expression of GABAA receptors formed
by
,
, and
subunits (Wisden et al. 1992
).
Physiological effect of muscimol on firing of subthalamic neurons
The physiological intracellular chloride concentration
([Cl]i) of subthalamic
neurons is unknown. This makes the whole cell configuration inadequate
for evaluating the physiological consequences of activation of type-A
GABA receptors. However, firing can be recorded on-cell
(Beurrier et al. 1999
; Bevan and Wilson
1999
). In the voltage-clamp mode, the biphasic current waves
recorded correspond to action potentials (Fenwick et al.
1982
). The cell-attached configuration therefore provided a
physiological test, while leaving [Cl
]i unaltered. Under
basal conditions, in the cell-attached configuration, spontaneous
activity in the STN only displayed two distinctive, stereotypic
patterns of action currents: regular, single-spike firing mode and
periodic, high-frequency (burst) discharge mode. This was confirmed by
analyzing discharge patterns using the method of Kaneoke and
Vitek (1996)
. We never detected any irregular, random discharge
patterns under basal conditions. The vast majority of neurons (38/43)
were found to be regularly firing single spikes. This seemed an
invariable firing mode since all neurons (n = 38) remained in the single-spike mode for the duration of the recording (between 10 and 20 min). An example of a spike train and
instantaneous firing rate of a neuron in the single-spike firing mode
is shown in Fig. 4A, together
with its interspike interval distribution and discharge density
histogram. The mean firing frequency of neurons in the single-spike
firing mode was 8.44 ± 0.78 Hz (n = 38; range:
2.2-19.5). As evidenced by the autocorrelograms, the single-spike
firing mode showed a high degree of regularity. Only 10% of the
neurons (5/43) were in the burst-firing mode. A representative example
is shown in Fig. 5A. The mean
firing frequency of these neurons was 6.45 ± 2 Hz
(n = 5), whereas their mean burst frequency was
0.43 ± 0.07 Hz (n = 5). Again, the
autocorrelograms indicated a high degree of regularity. The mean spike
frequency within bursts was 33.6 ± 2.7 Hz (n = 5); instantaneous spike frequency within bursts was successively
crescendo and decrescendo, with peaks up to 100 Hz. Burst firing appeared to be less stable than single-spike
firing since two neurons (2/5) switched to single-spike firing mode in
the course of the recording.
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Muscimol (0.1 µM to 1 mM) stopped action currents in all the 24 neurons tested. The inhibition duration was a linear function (slope = 3.94 ± 0.72 s/s, n = 13) of application duration in the range of 4-110 s. Firing frequency fell to zero whether the neurons were in the single-spike (Fig. 4B, see insets) or burst-firing mode (Fig. 5B). The inhibiting effect of muscimol was clear-cut. On some occasions, at low concentrations, it was preceded by a gradual decrease in firing frequency. It never caused changes in the firing pattern of the neurons in single-spike firing mode. Conversely, it did not produce any change in the firing pattern of neurons in burst-firing mode prior to arrest of firing.
Inhibition of firing activity by muscimol was reversible in all neurons
tested. Discharge patterns were again analyzed using the Kaneoke
and Vitek method (1996). This analysis indicated that most
neurons (10/14) resumed firing without changing their firing pattern
(data not shown). A minority of neurons (4/14) recovered slowly from
inhibition, entering an irregular firing mode. This change was
presumably due to a slow wash of muscimol around neurons deeper in the
slice than the main set of neurons.
These results clearly indicate that activation of GABAA receptors does not switch the firing activity of intact subthalamic neurons to burst-firing mode.
Modes of action of muscimol
Inhibition could result from several distinct mechanisms: membrane
shunting could lessen the effects of excitatory currents; voltage
changes could either move the membrane away from the action potential
threshold or alter the readiness of voltage-dependent channels.
Experiments were conducted in the whole cell configuration to determine
how activation of GABAA receptors influenced
membrane potential and resistance as well as action potentials. Two
populations of subthalamic neurons can be distinguished on the basis of
their membrane properties (Beurrier et al. 1999). The
neurons that are able to burst in the current-clamp mode also generate
low-threshold spikes (LTS) and plateau potentials when stimulated by
current pulses. The neurons that only fire in rhythmic single spikes do not show plateau potentials and only produce LTS. Neurons of both populations were recorded in the current-clamp mode at resting membrane
potential and were challenged with muscimol. The pipette solution
contained 2.2 mM chloride. Figure
6A shows a representative example of neurons that fired single-spikes (a) and only
generated LTS (b) but no plateau potential (c).
When muscimol was applied, activity stopped as long as the membrane
potential remained hyperpolarized (Fig. 6Ad). No switch to
burst-firing mode, even transient, was ever encountered during muscimol
action. Single-spike activity resumed with a few spare spikes and then
returned to control rhythm. In the same way, neurons that had the
capacity of switching to burst-firing when hyperpolarized (Fig.
6Ba) and responded to current pulses by both LTS and plateau
potential (Fig. 6B, b and c), also were fully
inhibited (Fig. 6Bd). The most hyperpolarized level achieved
by membrane potential on application of muscimol was
72 ± 1.8 mV (n = 18), a value significantly different from the mean lowest membrane potential measured prior to muscimol (
62.5 ± 1.6 mV, n = 18). In the same way, in the presence of
2-hydroxy-saclofen (20 µM), 10 µM GABA induced an hyperpolarization
of
9 ± 4 mV (n = 6). Muscimol action was also
investigated at a membrane potential held at about
70 mV by current
injection (Fig. 6C). This membrane potential is close to the
value of the measured reversal potential for muscimol current with 2.2 mM Cl
in the pipette (as shown in Fig.
1C). By minimizing changes in voltage, this procedure
isolated the shunting action of muscimol. A plot of membrane input
resistance against time (Fig. 6Cb) showed that it dropped
by ~50% with muscimol. Application of 10 µM GABA induced a
similar drop in input resistance (44 ± 6%, n = 6). Furthermore, before muscimol, an LTS with ~10 action potentials
was evoked by the break of a negative current pulse. Both of them
disappeared on muscimol application. On return from inhibition, only a
few spikes on the top of an LTS were evoked by the same current pulses. It should be noted that, during muscimol application, a much increased current pulse (range
800 to
1,000 pA) could still evoke an LTS but
only a single spike. In the same way, experiments at resting potential
using the pipette medium containing 22 mM chloride (reversal potential
of muscimol current about
45 mV) showed no significant changes in
resting membrane potential, whereas spontaneous activity was arrested
in all cells tested (n = 3, data not shown). Finally, experiments were performed in the voltage-clamp mode at
60 mV. Input
resistance, measured from the current response to repetitive small
voltage steps, dropped from 347 ± 84 to 154 ± 29 M
(n = 10, significant at P < 0.01).
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A triangle current paradigm (Sanchez-Vives et al. 1996)
was used to determine the effect of muscimol on action potentials (Fig.
7). Again, under basal conditions,
subthalamic neurons exhibited two behaviors. The triangle current
either evoked recurrent single spikes (Fig. 7Aa) or a
sequence composed of one burst of spikes followed by recurrent single
spikes (Fig. 7Ba). This was in agreement with both the
limited ability of some neurons to only fire in the single-spike mode
and the dual capacity of the other neurons to fire regular, single
spikes as well as bursts of spikes (data not shown). However,
qualitatively similar responses were obtained in both cases with
muscimol. When the same current intensity was used, no voltage response
was evoked (Fig. 7, Ab and Bb). In most cases,
however, the triangle protocol could still generate a few single
spikes, provided that its intensity was sufficient (Fig. 7,
Ac and Bc). The first spike was found at
61.9 ± 1.5 mV (n = 8) in normal Krebs',
whereas it was found at
35.1 ± 4.7 mV (n = 6)
in muscimol; no action potential could be evoked, even with strong
(
I = 1,000 pA) triangle-current stimulation in the two remaining cells. This experiment shows that muscimol does not
prevent spike firing but does inhibit recurrent spiking such as
regular, single-spike firing or burst firing.
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DISCUSSION |
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All the subthalamic neurons tested in our experiments (in
cell-attached as well as whole cell configuration) were sensitive to
muscimol. Muscimol-evoked currents were potently inhibited by
bicuculline but unaffected by 2-hydroxysaclofen. This clearly indicated
that muscimol activated GABAA receptors but not
metabotropic (GABAB) receptors. Our results
therefore support earlier studies that suggested that subthalamic
neurons expressed GABAA receptors (Kita et
al. 1983; Nakanishi et al. 1987
;
Rouzaire-Dubois 1980
).
Pharmacology and subunit composition of GABAA receptors in STN
To the best of our knowledge, the present study is the first to
investigate the pharmacology of GABAA receptors
in STN. It is also the first to collate data correlating the properties
of the native receptors in subthalamic neurons with their structure. Our recordings showed that GABAA receptors were
strongly potentiated by classical benzodiazepine agonists (diazepam and
flunitrazepam) and the typical barbiturate, pentobarbital.
GABAA receptors are thought to be composed of
five subunits. Combinations of the various subunits produces receptor
subtypes with specific properties (Sieghart 1995).
Indeed, expression of
together with
1 and
2 or
1 formed recombinant receptors lacking sensitivity to pentobarbital and flunitrazepam, respectively (Davies et al. 1997
;
Whiting et al. 1997
). The pharmacological profile found
in STN matched that of receptor subtypes composed of
1-3/5
1-3
1-3 (Barnard et al. 1998
). The
potentiation found with the two benzodiazepines was of the same order
as that measured for recombinant receptors formed by heterologous
expression of rat
1
1-3
2 (Hadingham et al. 1995
; Herb et al. 1992
; Saxena and MacDonald
1996
). In situ hybridization experiments showed the expression
of
1,
2/3 and
2/3 transcripts in adult rat STN (Wisden
et al. 1992
). However, it has been suggested on the basis of
Northern blot data that the human STN contained
transcripts
(Davies et al. 1997
). This was not confirmed by in situ
hybridization on primate (Whiting et al. 1997
) or rat brains (Moragues et al. 2000
). Using a specific
antibody, we clearly demonstrated that the
protein was not
expressed in the rat STN. Overall, pharmacological and molecular data
are consistent with the presence in STN of GABAA
receptors mainly composed of
. It is thus most improbable that
STN could be manipulated using drugs targeted at
. STN is a
glutamatergic, excitatory nucleus. It is hyperactive in animal models
of Parkinson's disease (Bergman et al. 1994
; see for
review Blandini et al. 2000
). Drugs that selectively
enhance GABAergic inhibition in the STN may prove beneficial for
patients suffering from Parkinson's disease.
1
2
2 subunits
form the major subtype of GABAA receptors in the
brain present particularly on hippocampal and cortical interneurons (McKernan and Whiting 1996
). A GABA receptor composed of
1
2
2 is not, therefore likely to be a valuable target for
pharmacological treatment of the symptoms of Parkinson's disease.
However, a minor expression of
3 and
3 in STN has been reported
(Wisden et al. 1992
). Furthermore changes in subunit
expression have been described in an animal model of Parkinson's
disease (Chadha et al. 2000
). Thus there is a potential
for receptor heterogeneity in subthalamic neurons that may help, in the
future, to define new pharmacological targets, aimed at decreasing STN
neuronal activity in Parkinson's disease and improving patients'
motor function.
Inhibition of firing activity by GABAA receptors in STN
Activation of GABAA receptors generally
inhibits neuronal excitability (Curtis at al. 1970). In
some cases, however, it can be excitatory (Leinekugel et al.
1995
; Reichling et al. 1994
; Staley and
Mody 1992
; Staley et al. 1995
; Wagner et
al. 1997
). Excitation is not thought to depend on the structure
of the GABAA receptors but to arise from a flow
of anions driven by a depolarizing electrochemical gradient.
Experiments to assess the physiological activation of
GABAA receptors should therefore be carried out on intact neurons, with unperturbed ion gradients. Our cell-attached experiments met this criterion. In all cases, muscimol reversibly stopped spontaneous activity, i.e., activation of
GABAA receptors was always inhibitory.
Our cell-attached results do not indicate whether muscimol was
hyperpolarizing or depolarizing. Experiments using the perforated-patch method with gramicidin (which avoids artifactual changes in
intracellular chloride concentration) showed that subthalamic neurons
were hyperpolarized on activation of GABAA
receptors, with a reversal potential of 77 mV (Bevan et al.
2000
). These findings indicate that our whole cell experiments
(using low-chloride pipette medium that yielded a reversal potential of
75 mV for muscimol-evoked currents) faithfully mimicked the voltage
change induced by activation of GABAA receptors in intact subthalamic neurons. The 10-mV hyperpolarization measured on
activation of GABAA receptors was exactly the
voltage change required to switch half of the subthalamic neurons from
regular, single-spike to burst-firing mode (Beurrier et al.
1999
). Moreover, intermittent bursts of action potentials,
relying on pallido-subthalamic afferences, were found in a
reconstituted network of basal ganglia and cortex neurons (Plenz
and Kitai 1999
). It is, therefore surprising that no burst
firing was ever found in the presence of muscimol. In rats, the globus
pallidus is frontal to STN. Thus globus pallidus and STN are most
likely to be disconnected in our coronal brain slices. The feedback
response necessary for recurrent bursting (McCormick and Bal
1997
; Plenz and Kitai 1999
) is therefore
expected to be lacking. Nevertheless, intact neurons in coronal slices do show persistent burst-firing activity (Beurrier et al.
1999
; present study). Two pieces of evidence revealed by our
whole cell recordings may explain the unexpected finding that
applications of muscimol did not induce burst-firing mode. The first is
that single spikes could still be evoked by appropriately scaled
stimulation, whereas no recurrent firing activity (regular, single
spike, or burst firing) was present. The second is that a major
conductance change occurred in the presence of muscimol. Large
conductance changes (of several
mS.cm
2, as was our
estimate) were produced by GABAA receptor
agonists in soma and dendrites, whereas small changes (0.1 mS.cm
2) were found in
nerve terminals (Zhang and Jackson 1993
). The fine
interplay of several time-, voltage-, and/or calcium-dependent conductances underlying the regular, single-spike and burst-firing modes has been revealed (Beurrier et al. 1999
,
2000
; Bevan and Wilson 1999
). Shunting
these conductances is expected to drastically reduce their ability to
change the membrane potential.
Inhibition processing in the indirect network
Tonic applications of muscimol as in the present work presumably
result in massive, sustained activation of all synaptic as well as
extrasynaptic GABAA receptors. By contrast,
activation of GABAA receptors by afferent fiber
stimulation is assumed to induce membrane shunting and voltage changes
with spatial specificity and speed. GABA receptors are present on both
perikarya and dendrites of subthalamic neurons (Smith et al.
1998). Our results suggest that massive activation of somatic
GABAergic synapses will halt the capacity to generate action potentials
and bring the somatic membrane potential close to
ECl. This may then lead to rebound burst firing, as postulated by Bevan et al. (2000)
.
Alternatively, activation of only a few somatic synapses may switch the
neurons that have this intrinsic capacity to burst firing, provided
that shunting of somatic conductances does not prevail and membrane hyperpolarization is sufficient. However, the computational properties of subthalamic neurons may be influenced in a more subtle way. Specific
excitatory inputs may be shunted, depending on the localization of the
GABAergic synapses on the dendritic tree. This is likely to occur for
cortico-subthalamic inputs since the same postsynaptic structures that
receive glutamatergic, cortical afferents also receive GABAergic
afferents (Bevan et al. 1995
). Obviously, studies of
synaptic activation of GABAA receptors by
stimulation of defined sets of afferents are now required to delineate
the interplay of IPSPs and intrinsic membrane properties of subthalamic neurons.
In summary, GABAA receptors in subthalamic
neurons display pharmacological properties consistent with predominant
expression of ,
, and
subunits, but STN does not express the
newly identified
subunit. Tonic GABAA
receptor activation inhibits spontaneous activity in all subthalamic
neurons but does not involve persistent or transient burst firing.
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ACKNOWLEDGMENTS |
---|
We gratefully acknowledge Dr. C. Gross for help with the algorithms for analysis of firing rhythms. We thank Prof. G. Tramu and Dr. P. Cioffi for help with the immunohistochemical experiments, Drs. M. D. Bevan, E. Bézard, and T. Boraud for comments and discussions, and J. M. Calvinhac, G. Gaurier, and D. Varoqueaux for technical assistance.
This work was supported by grants from Université of Bordeaux 2, Centre National de la Recherche Scientifique and Conseil Régional d'Aquitaine (to M. Garret). J. Baufreton received a doctoral fellowship from Conseil Régional d'Aquitaine.
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FOOTNOTES |
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Address for reprint requests: A. Taupignon (E-mail: anne.taupignon{at}umr5543.u-bordeaux2.fr).
Received 12 December 2000; accepted in final form 13 March 2001.
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REFERENCES |
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