INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The biophysical and pharmacological diversity of ionotropic GABA receptors (GABA_ARs) is a well-established principle leading to heterogeneous inhibitory synaptic transmission. GABA_ARs are pentameric heteromers assembled from a large multigene family of subunits (_1-6, _1-4, _1-4, , , , and _1-3). Throughout the mammalian brain, GABA_ARs exist as multiple isoforms, generally containing two , two , and one  or  subunit (Farrar et al. 1999; McKernan and Whiting 1996). The subunit-dependent kinetics and pharmacology of GABA_ARs (Macdonald and Olsen 1994), along with developmental- and regional-specific expression patterns of subunits (Laurie et al. 1992; Pirker et al. 2000; Wisden et al. 1992), suggest distinct functional roles for GABA_ARs isoforms in the brain. Recombinant receptors containing the  subunit are characterized by high affinity for GABA and slow desensitization rates (Adkins et al. 2001; Saxena and Macdonald 1994), which may favor signaling by low, persistent amounts of transmitter. The replacement of the 2 with the  subunit in GABA_AR constructs alters the modulation due to endogenous (Mensah-Nyagan et al. 1999) and synthetic neurosteroids such as alphaxalone and pregnenolone sulfate (PS) (Adkins et al. 2001; Wohlfarth et al. 2002; Zhu et al. 1996), which have behavioral, anti-convulsant, and hypnotic actions in rodents (Lambert et al. 1995; Macdonald and Olsen 1994; Vallee et al. 1997). While the above findings obtained with recombinant receptors have advanced our knowledge of the  subunit in general, it is uncertain whether they can be faithfully translated to native GABA_ARs in neurons that express the  subunit.

Native -containing GABA_ARs develop postnatally (Laurie et al. 1992) and are most prominently observed in the cerebellum, followed by forebrain regions of the dentate gyrus and dorsal thalamus (Pirker et al. 2000; Wisden et al. 1992). In cerebellar granule cells,  subunits are co-assembled with 6 subunits into GABA_ARs that are largely restricted to extrasynaptic sites (Nusser et al. 1998). The complete loss of -containing GABA_ARs in cerebellar granule cells, resulting from genetic inactivation of the 6 subunit (6^/) (Jones et al. 1997), is associated with the disruption of a tonic inhibitory current (Brickley et al. 2001) produced by resting GABA levels. While such a  subunit dependent tonic inhibition may also exist in forebrain (Nusser and Mody 2002), there is presently no subcellular anatomical evidence supporting an exclusively extrasynaptic location for -containing GABA_ARs in dentate granule or thalamocortical (TC) relay neurons. A possible synaptic function for -containing GABA_ARs in forebrain has been suggested by results from mice lacking the  subunit itself (^/). Studies of ^/ mice have reported faster decay times for both spontaneous and evoked synaptic GABA_A currents recorded from dentate granule neurons (Li et al. 1998; Mihalek et al. 1999), but this contrasts with no difference in cerebellar granule cells (Vicini et al. 2002). Because dentate granule and TC neurons both assemble -containing GABA_ARs with the 4 subunit (Korpi et al. 2002; Peng et al. 2002; Sur et al. 1999) and undergo a similar reorganization of remaining GABA_AR subunits after the loss of the  subunit (Korpi et al. 2002; Peng et al. 2002), we tested for similar alterations in the synaptic inhibition onto TC neurons from ^/ mice.

GABA-mediated inhibition in TC neurons resulting from activity of the reticular thalamic nucleus (RTN) has been shown to be involved in the generation of synchronous activity including sleep spindles and slow-wave sleep (McCormick and Bal 1997; Steriade et al. 1993). A hypersynchronous state of the intrathalamic circuit, reminiscent of several rodent models of absence, can be experimentally induced through either an enhancement of the inhibitory output from RTN onto TC nuclei (Huguenard and Prince 1994) or a reduction of the intra-RTN GABAergic inhibition (Huntsman et al. 1999). These two opposing roles of GABAergic inhibition within the intrathalamic circuit make it necessary for any potential anti-absence therapy directed against GABA_ARs to differentiate between RTN  TC and intra-RTN connections. With intra-RTN connections devoid of the  subunit (Peng et al. 2002; Wisden et al. 1992; but see Browne et al. 2001), a functional postsynaptic presence of -containing GABA_ARs in TC neurons may provide a novel method to differentiate the two intrathalamic pathways. Additionally, a class of pharmacological compounds, neurosteroids, may be promising in targeting -containing synapses. ^/ mice have attenuated behavioral responses to neurosteroids (Mihalek et al. 1999), which have also been shown to influence specific recombinant GABA_ARs (Brown et al. 2002; Zhu et al. 1996). In this paper, we study the synaptic physiology and neurosteroid sensitivities of TC neurons in ^/ mice, to further understand the above findings.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Thalamic slice preparation

C57BL/6J × Strain 129Sv/SvJ wild-type (^+/+) and knockout (^/) adult mice (Mihalek et al. 1999), of either sex, were anesthetized with 50 mg/kg pentobarbital and decapitated. Brains were blocked, removed, and immediately transferred to ice-cold, oxygen-equilibrated (95% O₂-5% CO₂), sucrose-cutting solution (in mM: 234 sucrose, 11 glucose, 24 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 10 MgSO₄ and 0.5 CaCl₂). After being submerged for 2 min, brains were glued to a petri dish filled with the same cutting solution as above and sectioned into 200-µm-thick horizontal slices on a Vibratome (TPI, St. Louis, MO). Slices were bisected and trimmed to only thalamus and parts of adjacent striatum before being placed into an incubator containing artificial cerebral spinal fluid (ACSF, in mM: 124 NaCl, 5 KCl, 1.25 NaH₂PO₄, 2 CaCl₂, 2 MgSO₄, 10 glucose, 26 NaHCO₃) continuously bubbled with 95% O₂-5% CO₂ at 35°C at least 1 h prior to recording.

Electrophysiology

In the recording chamber, slices were gently weighted down under nylon netting and superfused with a constant flow of ACSF (2 ml/min) equilibrated with 95% O₂-5% CO₂. Glass electrodes (KG-33 borosilicate glass, ID 1.0 mm, OD 1.5 mm; Garner Glass, Claremont, CA) were pulled in multiple stages to a resistance of 2.5-3.3 M using a Flaming-Brown micropipette puller (model P-87, Sutter Instruments, Novato, CA) and filled with a cesium chloride solution [in mM: 135 CsCl, 5 lidocaine-N-ethyl bromide (QX-314, Sigma-RBI, St. Louis, MO), 2 MgCl₂, 10 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, Sigma-RBI), pH = 7.3]. Voltage-clamp recordings were made from identified neurons within the boundaries of either the ventral posterior medial or the ventral posterior lateral thalamic nucleus (referred to as the ventral basal complex, VB) through the use of fixed-stage upright microscope (Axioskop, Carl Zeiss MicroImaging, Thornwood, NY) equipped with an insulated 63× objective, Nomarski optics, and an infrared-sensitive video camera (Cohu, San Diego, CA). All recordings were obtained with either a List-Medical EPC-7 (Darmstadt, Germany) or an Axopatch 200B (Axon Instruments, Foster City, CA) patch-clamp amplifier. Evoked inhibitory postsynaptic potentials (eIPSCs) were elicited via a bipolar tungsten electrode placed into the RTN. Once a minimal response was obtained, the test stimulation consisted of a brief, single pulse, at the same intensity but with a 1.5× duration. An inter-trial interval of at least 5 s was used. All recordings were made at room temperate (23°C) with a holding potential of 60 mV. Using the conditions described above, all GABA_A-mediated currents appeared as inward events in our recordings.

Pharmacology

GABA_A receptor-mediated currents were pharmacologically isolated by bath application of the ionotropic excitatory amino acid receptor antagonists: 6,7-dinitro-quinoxaline-2,3-dione (DNQX, 20 µM final, Sigma-RBI) and 2-amino-5-phosphonopentanoic acid (AP-5, 100 µM final, Sigma-RBI) in physiological saline. GABA_B receptors were blocked internally using Cs⁺ (135 mM) and QX-314 (5 mM) in the pipette solution. PS (Sigma-RBI) was dissolved in distilled H₂O at a 1:1,000 stock concentration and used at a final concentration of 10 µM. 3-Alpha,5-alpha-tetrahydrodeoxycorticosterone (THDOC, Sigma-RBI) was dissolved in DMSO at a 1:4,000 stock concentration and used at a final concentration of 250 nM.

Data collection and analysis

Continuous records of spontaneous inhibitory postsynaptic potentials (sIPCSs) were filtered at 1 kHz and collected with Axotape v.2 or PClamp v.8 (Axon Instruments). sIPSC events were sorted and separated with the customized software Detector v.5.14 (J. R. Huguenard) to obtain raw amplitude, duration, and frequency measurements. Single sIPSC events that decayed completely to baseline before the start of another event (type 1) were hand-sorted, averaged, and curve-fitted with PClamp v.8 (Axon Instruments) to generate decay time constants and amplitudes. eIPSCs were collected with Clampex v.5.5 or PClamp v.8 and curve-fitted with Clampfit v.8. Data were further analyzed with Origin v.6.1 (MicroCal Software, Northhampton, MA) and statistical significance was measured using a Student's t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Spontaneous IPSC comparisons between ^+/+ and ^/ VB neurons

Continuous recordings of sIPSCs were made from VB neurons (^+/+: n = 32, ^/: n = 33) at room temperature (Fig. 1, summary data provided in Table 1). We regularly observed a difference in baseline physiological noise between ^+/+ (Fig. 1A1) and ^/ (Fig. 1A2) recordings. ^/ VB neuron showed a 40% lower baseline noise compared with ^+/+ VB neurons (^+/+: 4.8 ± 0.3 pA; ^/: 3.0 ± 0.3 pA, P 0.001, Fig. 1C). By contrast there were with no differences in sIPSC rise time, frequency, or duration (Fig. 1C). sIPSC decay was quantified by fitting ensemble-averaged type 1 events with a single or more commonly (^+/+: 93%; ^/: 81%, P > 0.05) double-exponential decay function. No significant differences were observed between ^+/+ and ^/ VB neurons (Fig. 1B) in either mean sIPSC amplitude (^+/+: 32.3 ± 3.0 pA; ^/: 31.5 ± 3.3 pA, P = 0.85, Fig. 1C) or decay rate [weighted decay time constant (_d,w), ^+/+: 9.2 ± 0.5 ms; ^/: 8.6 ± 0.4 ms, P = 0.39, Fig. 1C]. For the neurons in which double exponential fits were obtained, there was a modest, but significant, decrease in the percentage of total sIPSC amplitude represented by the slow component in ^/ VB neurons (^+/+: 29.3 ± 4.1%; ^/: 23.0 ± 3.3%, P < 0.05). This slight change in amplitude of the slow component was however not associated with a significant decrease in overall sIPSP duration, as measured by _d,w (see Table 1).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Spontaneous inhibitory postsynaptic potentials (sIPSC) in ventral basal complex (VB) neurons of wild-type (+/+) and knockout animals (/). Representative traces from individual (A1) +/+ and (A2) / VB neurons. B1, B2: corresponding average sIPSCs for the 2 neurons shown in (A). Average sIPSC waveforms contain only events that have fully decayed before another event begins (type 1 events). The mean sIPSC duration from a single neuron (measured by the weighted decay time constant, d,w) was obtained from an exponential fit of the average sIPSC waveform (overlaid gray line, B). C: black bars in the summary histograms refer to +/+ control animals, while white bars indicate / knockout animals. All error bars show ± SE. For this and all subsequent figures: *P < 0.05, **P < 0.01, ***P < 0.005.

View this table: [in this window] [in a new window]   Table 1. sIPSC and eIPSC characteristics for +/+ and / VB neurons

Evoked IPSC comparisons between ^+/+ and ^/ VB neurons

To investigate the population of receptors that might be distal to the synapse, we evoked IPSCs by stimulating GABAergic fibers in RTN (Fig. 2A, summary data provided in Table 1). As with previous studies attempting to assess extrasynaptic GABA_B receptors (Kim et al. 1997; Scanziani 2000), a synchronously evoked response was used to trigger sufficient GABA release and spillover to activate potentially extrasynaptic -containing GABA_A receptors (Roepstorff and Lambert 1994). Robust, monosynaptic eIPSCs were elicited in both ^+/+ and ^/ VB neurons (^+/+: n = 15, ^/: n = 19). Average eIPSC peak amplitude was similar in both groups (^+/+: 1106.2 ± 164.9 pA; ^/: 1045.3 ± 226.9 pA, P = 0.84). Double exponential fits of averaged ^+/+ and ^/ eIPSCs revealed a trend toward faster eIPSCs from ^/ VB neuron recordings compared with ^+/+ controls (Table 1), but this trend was not significant for the eIPSC decay constant of the fast component (₁). However, the slower component (₂) was significantly faster in ^/ VB neurons (^+/+: 55.0 ± 5.5 ms; ^/: 39.9 ± 3.2 ms, P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 2. Evoked inhibitory postsynaptic potentials (eIPSCs) in VB neurons of +/+ and / animals. Representative average traces from individual (A1) +/+ and (A2) / VB neurons. Dots () in this figure, and all subsequent figures containing eIPSCs, indicate the time of RTN stimulation. Average traces from each +/+ and / VB neuron were fit to a double exponential curve to obtain decay kinetics for both the fast (2) and the slow (1) component. B: black bars in the summary histograms refer to +/+ control animals, while white bars indicate / knockout animals. All error bars show SE.

Pregnenolone sulfate modulation of ^+/+ and ^/ VB neurons

Having shown a subtle difference in both sIPSCs and eIPSCs, we next investigated modulation by neurosteroids in ^/ VB neurons. PS is a known negative allosteric modulator of GABA_A receptors lacking the  subunit (Zhu et al. 1996). In wild-type neurons (n = 12), neither amplitude nor decay of averaged eIPSC waveforms were affected by 10 µM PS (Fig. 3, summary data provided in Table 2). In contrast, PS caused a significant transformation of eIPSCs in ^/ VB neurons (Fig. 4, n = 9). Both ₁ (^/: control: 9.7 ± 0.9 ms, PS: 13.1 ± 1.2 ms, P < 0.01) and ₂ (^/: control: 47.9 ± 11.2 ms, PS: 79.4 ± 17.3 ms, P < 0.005) of ^/ average eIPSCs were substantially increased after 10 µM PS. On average, 10 µM PS applications produced a 30% decrease in eIPSC amplitude of ^/ VB neurons (Fig. 4B, ^/: control: 1771.3 ± 490.9 pA, PS: 1142.6 ± 272.6 pA, P < 0.05). These effects were not accompanied by an alteration in sIPSC frequency (^/: control: 13.2 ± 6.4 Hz, PS: 15.2 ± 7.6 Hz, P = 0.38). No effects of PS on sIPSCs were observed in either ^+/+ or ^/ VB neurons (Fig. 5, A and B).

View larger version (19K): [in this window] [in a new window]   Fig. 3. 10 µM pregnenolone sulfate (PS) has no effect on VB eIPSCs in +/+ animals. Average traces from a representative +/+ VB neuron (A1) before and (A2) after 10 µM PS. B: black bars in the summary histograms refer to eIPSPs in control conditions, while gray bars indicate those in 10 µM PS. All error bars show SE.

View this table: [in this window] [in a new window]   Table 2. 10 µM PS modulation of eIPSCs in +/+ and / VB neurons

View larger version (23K): [in this window] [in a new window]   Fig. 4. Loss of the  subunit confers a PS susceptibility on eIPSCs of / VB neurons. Average eIPSC traces from a representative / VB neuron (A1) before and (A2) after 10 µM PS. Inset shows the time course of the eIPSC half-width change due to the 10 µM PS application (gray bar) on the VB neuron from (A). B: black bars in the summary histograms refer to eIPSCs in control conditions, while gray bars indicate those in 10 µM PS. All error bars show SE.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Effects of PS and THDOC on sIPSCs from either +/+ and / VB neurons. Average sIPSC waveforms for representative (A1) +/+ and (B1) / VB neurons in control conditions (black trace) and in 10 µM PS (overlaid gray trace). A2, B2: neither group showed a significant percentage change from control sIPSC amplitude or duration in 10 µM PS. 250 nM 3-alpha,5-alpha-tetrahydrodeoxycorticosterone (THDOC) increased sIPSC duration in both (C1) +/+ and (C2) / VB neurons. All error bars show SE.

PS (10 µM) caused no significant changes in either sIPSC amplitude (^+/+: 97.9 ± 2.7% of control, n = 7, P = 0.33; ^/: 96.5 ± 5.4% of control, n = 12, P = 0.34) or _d,w (^+/+:117.0 ± 10.2% of control, P = 0.22; ^/: 106.0 ± 5.0% of control, P = 0.26). The lack of effect of PS on sIPSCs in ^/ VB neurons is contrasted with the significant effects on eIPSCs in the same cells (Fig. 6) and 10 µM PS also had no effect on baseline noise for either group (^+/+, control: 4.5 ± 0.5 pA vs. PS: 4.6 ± 0.5 pA, P = 0.57; ^/, control: 2.5 ± 0.4 pA vs. PS: 2.3 ± 0.4 pA, P = 0.35).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Time course of PS effects on sIPSCs and eIPSCs simultaneously recorded from a / VB neuron. A1: eIPSC and A2: sIPSC amplitudes () and durations () were averaged together in bins of 30 s and normalized to control values, over a 10-min application of 10 µM PS (gray bars). eIPSC duration was measured as a weighted decay time constant (d,w), while sIPSC duration was obtained from event half-widths. No significant changes in sIPSC rise time were observed for the duration of this experiment, demonstrating stability in recording conditions.

THDOC modulation of ^+/+ and ^/ VB neurons

Unlike PS, THDOC, a neurosteroid shown to potentiate GABA responses, had similar effects in the two animals types tested. In both ^+/+ and ^/ VB neurons, 250 nM THDOC almost doubled the _d,w of average eIPSC waveforms (Fig. 7B2, ^+/+: 180.9 + 8.4% of control, n = 3, P < 0.05; ^/: 211.8 + 35.2% of control, n = 4, P < 0.05), while having no effect on eIPSC amplitude (Fig. 7B1, ^+/+: 87.3 + 1.9% of control, P = 0.07; ^/: 93.8 + 9.0% of control, P = 0.22). In several ^+/+ VB neurons, the lack of PS effect on eIPSCs could be observed preceding the positive modulation of eIPSC duration by THDOC (Fig. 8). Although the increase in eIPSC duration due to THDOC appears to be mirrored in sIPSCs (Fig. 8A2, Fig. 5C), we were unable to show significance due to the high variability of the effect. We observed no change in baseline noise for THDOC applications in either group (^+/+, control: 4.7 ± 0.7 pA vs. THDOC: 4.8 ± 0.5 pA, n = 5, P = 0.52; ^/, control: 2.7 ± 0.2 pA vs. THDOC: 2.9 ± 0.4 pA, n = 5, P = 0.62).

View larger version (20K): [in this window] [in a new window]   Fig. 7. THDOC has a similar effect on eIPSCs from +/+ and / VB neurons. Average eIPSC traces from a representative (A1) / and (A1) +/+ VB neuron before (black traces) and after (overlaid gray traces) 250 nM THDOC. Percentage change from control eIPSC amplitude and duration are shown for both (A1) / and (A1) +/+ VB neurons. Duration was measured as a weighted decay time constant (d,w). All error bars show SE.

View larger version (30K): [in this window] [in a new window]   Fig. 8. Time course of PS and THDOC effects on sIPSCs and eIPSCs simultaneously recorded from a +/+ VB neuron. A1: eIPSC and A2: sIPSC amplitudes () and durations () were averaged together in bins of 30 s and normalized to control values, over a 10-min application of 10 µM PS (first gray bar) followed by 10-min application of 250 nM THDOC (second gray bar). eIPSC duration was measured as a weighted decay time constant (d,w), while sIPSC duration was obtained from event half-widths.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study we have demonstrated subtle changes in the phasic, GABAergic inhibition onto VB neurons, along with an increased sensitivity to PS, in mice lacking the  subunit. Although sIPSCs of ^/ VB recordings showed no significant difference in total amplitude, duration, or frequency compared with ^+/+ controls, we did observe a minor, but significant, decrease in the relative amplitude of the slow decay component of sIPSCs. This change in the late decay of sIPSCs was paralleled by a change in eIPSCs of ^/ VB recordings with a significantly shortened duration of the slow decay component relative to controls. ^/ VB recordings also exhibited a substantial decrease in background physiological noise when compared with ^+/+ recordings. While synaptic ^/ GABAergic inhibition was left relatively intact, neurosteroid modulation was partially affected. PS significantly increased duration and decreased amplitude of eIPSCs recorded from ^/ VB neurons but had no effect on ^+/+ eIPSCs. In contrast, another neurosteroid, THDOC, produced similar changes in the eIPSCs and sIPSCs of ^+/+ and ^/ mice. These differences between ^+/+ and ^/ mice, and a role for the  subunit in the neurosteroidal modulation of native neurons, are discussed in the following text.

sIPSC and eIPSC of ^/ VB neurons are intact

The loss of the  subunit results in significant anatomical reorganization of GABA_ARs in VB neurons. In thalamus, where  subunits are co-assembled with 4 subunits (Sur et al. 1999), the absence of -containing GABA_ARs is accompanied by a significant downregulation of 4 protein (Peng et al. 2002). This decrease in GABA_ARs is partially offset by the increased presence of 42-containing GABA_ARs in ^/ forebrain regions, as supported by an upregulation of the 2 subunit and higher levels of 4 protein immunoprecipitated by 2 antibody (Korpi et al. 2002; Peng et al. 2002). The lack of substantial differences between synaptic GABA_A currents of ^+/+ and ^/ VB neurons suggests that  subunits do not contribute to synaptic GABA receptors, as previously reported in cerebellar granule cells (Nusser et al. 1998). However, if -containing GABA_ARs are functionally active at ^+/+ RTN  VB inhibitory synapses, compensation by the 2 subunit may be a sufficient rescue for the  knockout. The modest amplitude and duration changes we observed in the slow decay components of ^/ sIPSCs and eIPSCs, respectively, could be attributed to the biophysical differences in desensitization rates and channel opening frequencies previously reported for recombinant GABA_ARs containing the  or 2 subunits (Saxena and Macdonald 1994).

Although  subunits co-assemble with different  subunits in thalamus (4) and cerebellum (6) (Nusser et al. 1998; Sur et al. 1999), both recombinant constructs demonstrate the high agonist affinity (Adkins et al. 2001; Saxena and Macdonald 1996) ideal for detecting extrasynaptic signals consisting of low levels of GABA (Attwell et al. 1993). If GABA spillover (Brickley et al. 1999) does occur at RTN  VB inhibitory synapses, the contribution of extrasynaptic -containing GABA_ARs would lead to slow-rising, low-amplitude, and long-lasting sIPSCs and eIPSCs similar to those observed in cerebellar granule cell recordings (Rossi and Hamann 1998). The removal of this slow IPSC component from ^/ recordings, through either a complete loss or a subunit reorganization of extrasynaptic GABA_ARs, may also account for the changes we observed in the slow decay component of mean ^/ sIPSC and eIPSC waveforms (Roepstorff and Lambert 1994). Furthermore, the significant reduction in background noise we observed in ^/ recordings appears to be qualitatively similar to the decrease in noise from 6^/ cerebellar granule cells (Brickley et al. 2001) that lose all -containing GABA_ARs, predominantly from extrasynaptic areas (Nusser et al. 1998, 1999). Although the net loss of GABA_ARs in ^/ VB neurons is significantly less than the 50% decrease observed in 6^/ cerebellar granule cells (Nusser et al. 1999), the compensatory increase of 2-containing GABA_ARs in ^/ thalamus could also contribute to lower background noise because the receptors would have a reduction in overall GABA affinity (Korpi et al. 2002), and they would be less sensitive to resting levels of GABA. These compensatory responses among GABA_ARs subunits may have also been responsible for the alterations in neurosteroid sensitivities we observed in ^/ VB neurons.

Neurosteroid modulation changes in ^/ VB neurons

In contrast to the attenuated neurosteroid sensitivity initially reported for ^/ mice using sleep time assays (Mihalek et al. 1999), here we have reported an elevated PS sensitivity in ^/ VB neurons. PS, a sulfated neurosteroid present in the brain (Wang et al. 1997), is known for its inhibitory actions on GABA_ARs (Majewska et al. 1988) at an EC₅₀ close to 10 µM (Park-Chung et al. 1999; Shen et al. 2000). Believed to reduce GABA_AR channel-opening frequency (Akk et al. 2001; Mienville and Vicini 1989) and increase the rate of desensitization (Shen et al. 2000), PS applications in native neurons can result in reduced peak amplitude and accelerated decay of GABA_A-mediated responses (Shen et al. 2000). In our experiments using PS we observed a similar reduction in eIPSC amplitude, accompanied by a previously undescribed increase in eIPSC duration (Fig. 4). The increased effectiveness of 10 µM PS at reducing ^/ VB eIPSC amplitude is consistent with previous studies reporting a higher PS sensitivity in recombinant GABA_ARs after substituting the  subunit with the 2 subunit (Zhu et al. 1996). However, we cannot completely account for the simultaneous lengthening of ^/ VB eIPSC duration by PS.

Dual modulation by a sulfated neurosteroid has been previous reported with 11-ketopregnenolone sulfate and recombinant GABA_ARs (Park-Chung et al. 1999). PS may also act at distinct positive and negative modulatory sites at ^/ VB GABA_ARs, resulting in the eIPSCs modulation described above. Another explanation for the dual modulation may be a potential inhibitory PS effect on neurotransmitter release (Teschmacher et al. 1997; ffrench-Mullen et al. 1994) at the RTN  VB synapse, coupled with a small potentiation of postsynaptic ^/ VB GABA_ARs. An alteration in presynaptic RTN neurons, which do not express the  subunit, may indicate a change in ^/ mice independent of GABA_ARs. If these changes occurred in a PS-sensitive component of RTN neurons affecting release, such as NMDA receptors (Wu et al. 1991) or high-voltage activated Ca²⁺ channels (ffrench-Mullen et al. 1994), they could have substantial consequences for synaptic VB currents evoked with RTN stimulation.

While a PS effect on neurotransmitter release at ^/ RTN  VB synapses is a distinct possibility, it was not supported by a resultant change in ^/ VB sIPSCs. The lack of effect on ^/ VB sIPSCs suggests the absence of the  subunit cannot be the sole determining factor of PS modulation in GABA_ARs. Therefore eIPSCs and sIPSCs of ^/ VB neurons may originate from different GABA_AR populations based either on subunit composition or on interactions within the native neuronal environment. Given the complex response of ^/ VB neurons to PS, more work is needed to determine the factors involved.

Another neurosteroid, THDOC, showed no difference between effects in ^+/+ and ^/ VB neurons. THDOC is structurally distinct from PS (Park-Chung et al. 1999) and has been shown to prolong GABA_A-mediated currents (Cooper et al. 1999; Zhu and Vicini 1997). As predicted from previous work in native neurons (Cooper et al. 1999), 250 nM THDOC increased sIPSC and eIPSC duration in both ^+/+ and ^/ VB neurons without significantly affecting amplitude (Figs. 7 and 8). The potentiation due to 100-10,000 nM THDOC is substantially increased in recombinant GABA_ARs after the 2 subunit is substituted for the  subunit (Zhu et al. 1996). Interestingly, we observed far less of a difference in THDOC eIPSC potentiation between ^+/+ and ^/ VB neurons (Fig. 7), a finding which agrees with recent results obtained with recombinant receptors (Brown et al. 2002; Wohlfarth et al. 2002). A more extensive screening with THDOC would be required to establish whether differences are statistically significant. It may be possible that a THDOC concentration lower than 250 nM is required to observe any  subunit specific differences in neurons. sIPSCs from cerebellar granule cells show a significantly reduced potentiation due to 100 nM THDOC in ^/ mice compared with wild-type controls (Vicini et al. 2002).

The similar potentiation of both eIPSCs and sIPSCs in ^+/+ and ^/ VB neurons by THDOC contrasts with the effects of PS, which depended on both the presence of  and the response type (eIPSCs vs. sIPSC). These contrasting sensitivities to THDOC and PS support the hypothesis that positive and negative neurosteroid GABA_A modulators act at different binding sites (Park-Chung et al. 1999; Shen et al. 2000).

Future directions

The biophysical and pharmacological differences between ^+/+ and ^/ VB neurons demonstrated in the present experiments including the following: 1) the reduction in background noise; 2) minor changes restricted to the slow decay component of synaptic currents; and 3) altered pharmacological sensitivities in eIPSCs but not sIPSCs with one neurosteroid; suggest -containing GABA_ARs are distant from RTN  VB synaptic areas. Unlike 6^/ mice where there is a clear and complete loss of -containing extrasynaptic GABA_ARs in cerebellar granule cells, ^/ mice undergo a mixture of GABA_ARs loss and compensation with the 2 subunit, which challenges the interpretation of ^/ physiological relevance. A pharmacological block, or inducible genetic manipulation, which selectively renders membrane-inserted, -containing GABA_ARs inactive, might provide a more reliable method for determining the role of the  subunit in thalamus.