Activation of GABAA Receptors in Subthalamic Neurons In Vitro: Properties of Native Receptors and Inhibition Mechanisms

Jérôme Baufreton, Maurice Garret, Sandra Dovero, Bernard Dufy, Bernard Bioulac, and Anne Taupignon

Laboratoire de Neurophysiologie Centre National de la Recherche Scientifique Unité Mixte de Recherche 5543, Université de Bordeaux 2, 33076 Bordeaux cedex, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 epsilon  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 epsilon  proteins. Applications of increasing concentrations of muscimol, a selective GABAA agonist, induced Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> currents with an EC50 of 5 µM. Currents induced by muscimol were fully blocked by the GABAA receptor antagonists, bicuculline and picrotoxin. They were strongly potentiated by the barbiturate, pentobarbital (+190%), and by the benzodiazepines, diazepam (+197%) and flunitrazepam (+199%). Spontaneous inhibitory postsynaptic currents were also significantly enhanced by flunitrazepam. Furthermore, immunohistological experiments with an epsilon  subunit-specific antibody showed that the epsilon  protein was not expressed within the STN. Native subthalamic GABAA receptors did not, therefore, display pharmacological or structural properties consistent with receptors comprising epsilon . 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 epsilon  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 alpha beta gamma subunits.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha 1-6, beta 1-3, gamma 1-3, delta , epsilon , and theta ) (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 alpha 1, beta 2, and gamma 2 mRNAs in STN, together with weak signals for beta 3 and gamma 3 (Wisden et al. 1992). Moreover, immunoreactivity for alpha 1 and beta 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. alpha 1beta 2gamma 2 (McKernan and Whiting 1996). Abundant transcripts of epsilon  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 epsilon  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 epsilon  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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega 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 MOmega ) 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-epsilon (1:10,000; raised against the N-terminal sequence unique to the rat epsilon  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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Specific agonist of ionotropic GABA receptors, muscimol, activates a Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> current in subthalamic neurons

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<UP><SUB>3</SUB><SUP>−</SUP></UP> to Cl- permeability ratio of 0.2 (Bormann et al. 1987), assuming an intracellular concentration of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> of 16 mM (Staley et al. 1995). 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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Fig. 1. Muscimol activates a Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> current. A: muscimol response from a subthalamic neuron. Membrane was held at -60 mV, and the pipette contained 120 mM KCl. Muscimol (2 µM) was applied onto the slice by a gravity-feed system, positioned just above the pipette, as indicated by  above the record. B: current-voltage relationship of the response. Membrane voltage was ramped at 0.125 V/s according to the protocol shown in the inset. Currents were first recorded in Krebs' medium, then in Krebs' containing 10 µM muscimol. The difference between the 2 records gave the muscimol response. Emu is the reversal potential of the muscimol current. C: Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> contribute to the reversal potential of the muscimol current. Pipettes with different chloride concentrations ([Cl-]p) yielded reversal potentials closer to those predicted by the Nernst equation for Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (---) than those predicted by the Nernst equation for Cl- (- - -). The same ramp protocol was used as in B. D: cumulative dose-response curve for muscimol. The continuous line is a Hill function with an EC50 of 5.2 µM, a nHill of 1.2, and a maximum current of -1,239 ± 228 pA (n = 8). Each point represents the mean ± SE from 3 to 8 cells.

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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Table 1. Effects of different drugs on muscimol response in STN neurons



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Fig. 2. The benzodiazepine flunitrazepam acts on GABAA receptors from subthalamic neurons. A: flunitrazepam potentiates the whole cell response to muscimol. The current activated by 2 µM muscimol (left) was strongly enhanced by co-application of 1 µM flunitrazepam (middle). Potentiation by flunitrazepam was reversible (right). B: flunitrazepam acts on spontaneous inhibitory postsynaptic currents (IPSCs). Ba: comparison of amplitude and surface distribution of spontaneous IPSCs (left). Average IPSC before (thin trace) and after (1 µM, thick trace) flunitrazepam. Inset: both traces normalized to their peak amplitude (right). Bb: flunitrazepam (1 µM) significantly increased the mean IPSC amplitude, surface, and decay time constant. Histogram bars represent the mean data from 6 cells (*P < 0.05; **P < 0.01). The pipette contained high-chloride solution. The membrane was held at -60 mV (A) and -70 mV (B).

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 alpha , beta , and gamma  subunits. The pharmacological properties of native GABA receptors in subthalamic neurons thus appear to differ from those of recombinant receptors containing the epsilon  subunit. The alpha 1beta 1epsilon 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 epsilon  in STN

A high expression level of the GABAA epsilon  subunit in STN was deduced from Northern blot experiments (Davies et al. 1997). Here, our affinity-purified anti-epsilon subunit antibody was used to test the presence of epsilon -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-epsilon 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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Fig. 3. The GABAA receptor subunit epsilon  is not expressed in STN. Labeling of the STN area in rat adjacent brain sections. A: Hoechst cell nucleus staining revealed the architecture of the STN area. B: anti-neurokinin labeled axons surrounding the STN. C: GABAA epsilon  subunit antiserum only stained cells along the dorsal margin of the STN and in the posterior portion of the lateral hypothalamus. Inset: an enlargement of STN and lateral hypothalamus (with stained cells). The center of the STN is indicated by * in B and C. ZI, zona incerta; cp, cerebral peduncle; STN, subthalamic nucleus. Calibration bar, 100 µm.

Our pharmacological and molecular data do not support the hypothesis that the newly identified GABAA receptor subunit epsilon  is expressed in rat STN. Rather they are consistent with data indicating expression of GABAA receptors formed by alpha , beta , and gamma  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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Fig. 4. Activation of GABA receptors by muscimol inhibits regular single-spike firing. In the cell-attached configuration, the biphasic current waves found corresponded to extracellular recordings of action potentials. The recording pipette contained extracellular solution and was held at 0 mV. A: under basal conditions, the vast majority of neurons regularly fired single spikes. B: muscimol application stopped spontaneous action potential firing. In this and the following figure, A (top trace) is a sample of continuous recording of firing activity with the corresponding instantaneous firing rate below. The discharge density histogram (DH, with in inset, the corresponding interspike interval histogram, IIH) and autocorrelogram (AC) are also shown, all calculated from a continuous sequence of >= 500 action currents. In B, top graph is firing rate (bin width = 3 s) with samples of action currents taken from positions 1-3 below.



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Fig. 5. Muscimol inhibits neurons in burst-firing mode. A: in the cell-attached configuration, ~10% of neurons spontaneously fired bursts of spikes. B: muscimol application stopped firing. Same recording conditions and statistical analysis as Fig. 4.

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 MOmega (n = 10, significant at P < 0.01).



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Fig. 6. Muscimol hyperpolarizes membrane potential and strongly decreases membrane resistance. A displays a representative example of subthalamic neurons that stopped firing when hyperpolarized by injection of an increasingly negative holding current in the whole cell configuration (a). Such neurons responded to an hyperpolarizing current pulse by a low-threshold spike (b) but did not produce plateau potential in response to a depolarizing current step (c). When these neurons were challenged by muscimol at zero current level, the spontaneous activity stopped and the membrane hyperpolarized by ~10 mV (d). B, a-c: the characteristic properties of a neuron which was able to switch from regular, single-spike to burst-firing mode (the burst indicated by * is shown on the right with an expanded time scale for greater detail). The capacity of producing both low-threshold spikes (b) and plateau potentials (c) is the hallmark of this type of neuron. Again, firing stopped and membrane voltage hyperpolarized when muscimol was applied to the slice at zero current level. No evidence for burst firing was registered (d). C: membrane input resistance (calculated off-line as Delta V/Delta I) is plotted in b under the voltage record (a) obtained in a neuron kept at -70 mV by continuous injection of -10 pA. It only returned to control value after 81.5 s. The break of an hyperpolarizing pulse (-80 pA, 1 s) elicited several action potentials on a low-threshold spike in normal Krebs' (trace marked with * is shown in a1 with an expanded time scale). Application of 10 µM muscimol did not induce any change in membrane potential, whereas input resistance first dropped to ~90% and then ~50% of its control value (also compare a1 and a2). Input resistance remained under control value even when a single action potential appeared on the break of the current pulse (a3). Pipette solution contained 2.2 mM chloride throughout. Truncated spikes are displayed.

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 (Delta 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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Fig. 7. In muscimol, subthalamic neurons only fire single spikes at depolarized potentials. In control, intracellular injection of a triangle current into a subthalamic neuron resulted in generation of either single spikes (Aa) or a sequence of a burst of spikes followed by recurrent single spikes (Ba). Insets: the 1st and last few spikes of each control trial with an expanded time scale for detail. In muscimol, injection of the same current triangle did not elicit any voltage response (Ab and Bb). Injection of a much larger current was required to trigger a few single spikes (Ac and Bc). The ability to generate a burst of action potentials was no longer present as shown in Bc. The pipette solution contained 2.2 mM chloride. Truncated spikes are displayed.


    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 epsilon  together with beta 1 and alpha 2 or alpha 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 alpha 1-3/5beta 1-3gamma 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 alpha 1beta 1-3gamma 2 (Hadingham et al. 1995; Herb et al. 1992; Saxena and MacDonald 1996). In situ hybridization experiments showed the expression of alpha 1, beta 2/3 and gamma 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 epsilon  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 epsilon  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 alpha beta gamma . It is thus most improbable that STN could be manipulated using drugs targeted at epsilon . 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. alpha 1beta 2gamma 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 alpha 1beta 2gamma 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 beta 3 and gamma 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 alpha , beta , and gamma  subunits, but STN does not express the newly identified epsilon  subunit. Tonic GABAA receptor activation inhibits spontaneous activity in all subthalamic neurons but does not involve persistent or transient burst firing.


    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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society