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High-Affinity, Slowly Desensitizing GABA_A Receptors Mediate Tonic Inhibition in Hippocampal Dentate Granule Cells

Department of Neurology, University of Virginia-Health Sciences Center, Charlottesville, Virginia

Received July 11, 2005; accepted November 10, 2005

	Abstract

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The tonic form of GABA-mediated inhibition requires the presence of slowly desensitizing GABA_A receptors with high affinity, which has not yet been directly demonstrated in hippocampal neurons. Low concentration of GABA (1 µM) persistently increased baseline noise, increased membrane slope conductance, but did not affect spontaneous inhibitory postsynaptic currents (sIPSCs) in dentate granule cells (DGCs). Higher concentrations of GABA (10–100 µM) desensitized synaptic currents quickly, and there was a large residual current. Saturating concentration of GABA (1 mM) completely desensitized synaptic currents and revealed a slowly desensitizing, persistent current. Penicillin (300 µM) inhibited baseline noise without affecting mean current and inhibited decay time of sIPSCs. GABA_A receptors mediating baseline noise in DGCs were sensitive to allopregnanolone, furosemide, and loreclezole and insensitive to diazepam and zolpidem. These studies demonstrate persistently open GABA_A receptors on DGCs with high affinity for GABA, slow desensitization rate, and pharmacological properties similar to those of recombinant receptors containing ₄, ₁, and the subunits.

In keeping with this hypothesis, previous studies on cerebellar and hippocampal granule cells demonstrated that tonic inhibition has pharmacological properties similar to those of recombinant receptors containing the subunit. For example, zolpidem does not affect holding (mean) current, whereas it enhances synaptic currents in DGCs in vivo (Nusser and Mody, 2002) and in cultured hippocampal neurons (Yeung et al., 2003). In cerebellar granule cells, receptors mediating tonic inhibition are sensitive to the neurosteroid THDOC (Stell et al., 2003) and furosemide (Hamann et al., 2002; Wall, 2002) but insensitive to diazepam (Hamann et al., 2002). These pharmacological properties are shared with recombinant receptors containing the ₆ and or ₄ and subunits. Postembedding electron microscopic studies further demonstrated that subunit-containing GABA_A receptors are present on extrasynaptic membranes in cerebellar granule cells (Nusser et al., 1998). Based on these studies it has been proposed that subunit-containing receptors mediate tonic inhibition. However, earlier studies do not provide direct evidence of the presence of high-affinity, slowly desensitizing GABA_A receptors in neurons that are necessary to mediate tonic inhibition.

We directly demonstrate that DGCs express persistently open GABA_A receptors with high GABA affinity. Furthermore, these cells express GABA_A receptors with a slow rate and limited extent of desensitization, which remain open when synaptic receptors have desensitized. We also characterized pharmacological properties of tonic currents, which were similar to those of recombinant receptors containing ₄, ₁, and subunits.

	Materials and Methods

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Adult male Sprague-Dawley rats (150–200 g) were anesthetized with halothane before decapitation according to the University of Virginia Animal Care and Use Committee guidelines. Brains were dissected free and immersed in ice-cold (2–4°C) artificial cerebrospinal fluid (ACSF) saturated with 95% O₂, 5% CO₂. The ACSF consisted of 127 mM NaCl, 2 mM KCl, 1.5 mM CaCl₂, 1.5 mM MgSO₄, 25.7 mM NaHCO₃, 1.1 mM KH₂PO₄, and 10 mM glucose (osmolarity, 300–305 mOsM). After cooling, the brains were blocked and mounted on a vibratome stage (Camden Instruments Ltd., Leicester, UK), and 300-µm-thick horizontal sections containing the ventral hippocampus were cut. Slices were maintained in continuously oxygenated ASCF, at 32°C in a holding chamber for 30 to 45 min, and then at room temperature in a recording chamber mounted on the stage of an Olympus BX51 microscope equipped with a 40x water-immersion objective, infrared-differential interference contrast optics, and video. DGCs were identified in dentate granule layer as small- and medium-sized neurons with typical oval-shaped soma and single process. Patch electrodes (final resistances, 6–8 M) were pulled from borosilicate glass (Sutter Instrument Company, Novato, CA) on a horizontal Flaming-Brown microelectrode puller (model P-97; Sutter Instrument Company), using a two-stage pull protocol. Electrode tips were filled with a filtered internal recording solution consisting of 153.3 mM CsCl, 1.0 mM MgCl₂, 10.0 mM HEPES, and 5.0 mM EGTA (pH adjusted to 7.2 with CsOH; osmolarity, 285–295 mOsM). The electrode shank contained 3 mM ATP Mg²⁺ salt, 0.1 mM GTP Na⁺ salt. Neurons were voltage-clamped to –65 mV with an Axopatch 1D or Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). Whole cell capacitance and series resistance were compensated by 70 to 75% at 10-µs lag. Recording was performed when series resistance after compensation was 20 M or less. Access resistance was monitored with a 10-ms, –5-mV test pulse once every 2 min, and if the series resistance increased by 25% at any time during the experiment, the recording was terminated. Currents were filtered at 5 kHz and then digitized using a Digidata 1322 digitizer and acquired using Axoscope 8.0 software (Molecular Devices) on an IBM PC-compatible computer hard drive.

For recording of GABA_A receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) and tonic GABAergic current, 50 µM DL-2-amino-5-phosphonopentanoic acid (DL-AP5) and 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione (Tocris-Cookson Inc., Ellisville, MO) were included in the ACSF to block N-methyl-D-aspartate and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptor-mediated currents, respectively. All other reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Acquisition and Analysis. The digitized current traces were analyzed with Mini Analysis software (Synaptosoft, Leonia, NJ). The software was used to detect and analyze sIPSCs. To detect sIPSCs, a detection threshold five times the root-mean-square (RMS) amplitude was used. RMS noise (I_rms) was defined as a square root of the average of the squares of the deviation from the I_avg (amplitude) over the chosen time interval using the equation

where I_avg is an average amplitude, I_n is amplitude in an individual point, and n is the number of measurements in an epoch.

To study the tonic inhibition, transient events were manually removed from the current trace, so that it consisted only of holding current, the current required to voltage-clamp the cell. Two features of the holding current were studied: the mean current and the noise. Mean current was measured in 100-ms epochs with 1-s interval between epochs in 30 epochs. The measurements were taken 30 s before and 5 min after application of a drug. The segments of a current trace that contained synaptic event were omitted. Mean current (I_avg) was defined as an arithmetic mean of peak-to-peak amplitudes of individual points during that epoch. To assess the effect of a drug on I_avg in an individual neuron, the distribution of I_avg before application of the drug during the baseline period was compared with that after drug application by means of a Kolmogorov-Smirnov (KS) test. To compare the data obtained from a group of neurons, mean of I_avg from all neurons before and after drug application was compared.

A second measure of holding current, I_rms, was studied. The time interval (epoch) for each measurement was 50 ms and contained 250 amplitude measurements (5-kHz digitization rate). The time interval between two epochs was 500 ms. Sixty epochs were analyzed for each experimental condition (60 control and 60 after a drug application in each cell). To assess the effect of a drug on I_rms in an individual neuron, the distribution of I_rms in epochs before the application of a drug (during the baseline period) was compared with that after drug application by means of KS test. To compare data obtained from a group of neurons, I_rms values in individual epochs before and after drug application were averaged. Mean I_rms, mean I_avg, and mean sIPSC frequency and amplitude values were compared using t tests unless specified otherwise.

	Results

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Tonic Inhibition Revealed by Competitive Antagonists. Several recent studies have used competitive antagonists to measure tonic inhibition. To measure tonic inhibition, sIPSCs interspersed with the mean current were recorded from DGCs in the hippocampal slices by blocking excitatory neurotransmission with 50 µM DL-AP5 and 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione. The neurons were voltage-clamped to –65 mV, under nearly symmetrical Cl^– conditions, at a room temperature (20–22°C; see Materials and Methods for details). After the input resistance became stable, sIPSCs and mean current were recorded for 5 min, and then 100 µM bicuculline was bath-applied. A typical recording with 100 µM bicuculline application is shown in Fig. 1A. In response to bicuculline, a slow outward current occurred, accompanied by loss of sIPSCs and reduction in baseline noise.

View larger version (48K): [in this window] [in a new window]   Fig. 1. Bicuculline (100 µM) inhibits sIPSCs mean baseline current (Iavg). A, current trace recorded from a dentate granule cell. Bicuculline (100 mM) (solid line) eliminated synaptic currents and decreased Iavg. Decrease of Iavg was accompanied by decrease of baseline noise (Irms). Averaged 5-ms epochs of Iavg are scattered wider before application of bicuculline. B, competitive GABAA receptor antagonist SR 95531 (100 µM; solid line) inhibited synaptic currents with no effect on Iavg in a DGC. Note that unlike bicuculline, SR 95531 did not affect Iavg. C, SR 95531 inhibited Irms in DGCs. The traces display baseline noise during a 50-ms epoch before application of SR 95531 (top trace) and in the presence of 100 µM SR 95531 (bottom trace). Irms (shown by arrows between two solid lines) was defined as a square root of the average of squares of the deviation of individual points from the average value over 50-ms period. Note larger Irms in the bottom trace compared with the top trace in B.

Application of another competitive GABA_A receptor antagonist, SR 95531 (100 µM), to 5 DGCs produced similar results on synaptic currents in each cell (Fig. 1B). Unlike bicuculline, SR 95531 did not have an effect on I_avg in three DGCs (82.8 ± 6.9 versus 80.1 ± 3.6 pA; p = 0.57; paired t test). The I_rms was significantly decreased (Fig. 1C). Data were pooled from all five cells, and during baseline period the mean I_rms was 6.671 ± 0.11 pA and during SR 95531 application it was 5.496 ± 0.07 pA (KS test; D = 0.24; p < 0.001).

High-Affinity GABA_A Receptors. Studies with bicuculline and SR 95531 confirmed that tonic inhibition was present in DGCs; however, these drugs blocked synaptic inhibition along with tonic inhibition. It has been suggested that the GABA_A receptors specifically mediating tonic inhibition possess the high affinity for GABA that makes them less sensitive to competitive GABA_A receptor antagonists than those receptors mediating synaptic inhibition. However, this high affinity should render these receptors mediating tonic inhibition more sensitive to agonists than synaptic receptors. We determined whether a low concentration of GABA (1 µM), similar to that found in the extracellular space, could selectively activate GABA_A receptors mediating tonic inhibition without altering synaptic currents.

After making a baseline recording for 5 min, a low concentration of GABA (1 µM) was bath-applied, and 5 min was allowed to elapse to allow the drug to equilibrate within the slice. The holding current and sIPSCs in the subsequent 5 min were compared with those during the 5-min baseline period before GABA application. A typical experiment is shown in Fig. 1. Note that there was no apparent change in I_avg on visual examination of the trace (Fig. 2A). A detailed quantitative examination of the I_avg confirmed the visual impression. The distribution of I_avg in 30 epochs before GABA application was compared with that in 30 epochs after application KS test (Fig. 2B), and the two distributions were not different (71.3 ± 11.2 versus 69.3 ± 6.7 pA; D = 0.04; p = 0.94). The experiment was repeated in four DGCs, and 1 µM GABA did not change I_avg. in any of the cells.

View larger version (44K): [in this window] [in a new window]   Fig. 2. GABA (1 µM) did not change Iavg but increased Irms. A, current trace recorded from a dentate granule cell voltage-clamped to –65 mV in the presence of 50 µM DL-AP5 and 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione, before (baseline recording) and during application of 1 µM GABA (solid line); the current trace did not seem to change. B, 1 µM GABA did not change Iavg. Measurements of Iavg in 30 epochs (1 epoch is 100 ms) at 1-s intervals during the baseline period (solid line) and in the presence of 1 µM GABA (dotted line). Note that the Iavg did not change after application of 1 µM GABA. C, current traces from the same DGCs display increased Irms after application of 1 µM GABA. The top trace shows a 10-s recording before application of GABA, and the bottom trace shows a 10-s recording in the presence of 1 µM GABA. D, current traces display baseline noise during a 50-ms epoch before application of GABA (top trace) and in the presence of 1 µM GABA (bottom trace). Irms (shown by arrows between two solid lines) was defined as a square root of the average of squares of the deviation of individual points from the average value over a 50-ms period. Note larger Irms in the bottom trace compared with the top trace in B. E, KS test was used to compare Irms before and after application of GABA in a neuron. Irms in 60 epochs (1 epoch is 50 ms) at a 500-ms interval in the presence of 1 µM GABA (dotted line) was more than that during baseline period (solid line). F, distribution of RMS noise in cell during the baseline period (black columns) and in the presence of 1 µM GABA (white columns) were compared using a frequency distribution histogram with a bin size of 0.2 pA to demonstrate the rightward shift of Irms in the presence of 1 µM GABA. G, membrane slope conductance before and after application of 1 µM GABA was compared. DGCs were voltage clamped to –65, –35, and –15 mV, and 60 epochs of Irms were obtained at each holding potential during the baseline period and in the presence of 1 µM GABA, and Irms values were plotted against holding potential. During the baseline period, the slope of the Irms (current)-voltage relationship was 18.1 pS, and application of 1 µM GABA increased it to 26.2 pS.

Visual inspection of the trace also suggested that the baseline trace was thicker after application of 1 µM GABA, and when expanded further it seemed to possess greater noise (Fig. 2C). The I_rms during baseline period was 5.30 pA (Fig. 2D, top trace), and 5 min after application of 1 µM GABA it was 6.0 pA. The distribution of I_rms measurements from each of 60 epochs before and after GABA application was compared using the KS test, indicating that they were significantly different (D = 0.68; p = 0.001; Fig. 2E). This finding was confirmed in four DGCs, and in each one the KS test revealed that I_rms was significantly increased. To further understand the impact of 1 µM GABA on I_rms, an I_rms amplitude frequency distribution histogram was constructed (Fig. 2F), which demonstrated that 1 µM GABA increased the frequency of large I_rms epochs. In four DGCs tested, the mean I_rms increased from 6.29 ± 0.02 to 6.66 ± 0.03 pA (p < 0.0001) in response to 1 µM GABA.

Using a different approach, we studied the slope of the current-voltage relationship (slope conductance) to confirm that 1 µM GABA indeed increased GABA_A receptor conductance. Recordings were obtained in DGCs at baseline and after application of 1 µM GABA voltage-clamped to –65, –35, and –15 mV. I_rms in 60 epochs was obtained for each holding potential, and these values were plotted against the membrane holding potential (Fig. 2G). During the baseline period, the I_rms decreased proportionally to the driving force (r² = 1) and ordinate intercept was 2.9 pA. The regression line crossed the abscissa at close to 0 mV, the chloride reversal potential, suggesting that passage of chloride ions through the membrane contributed to I_rms. The slope of the RMS noise (current)-voltage relationship in the control condition was 18 pS. GABA (1 µM) increased I_rms at each holding potential, and the ordinate intercept was also close to 0 (2.9 mV). The slope of the RMS noise (current)-voltage relationship in the presence of 1 µM GABA in this neuron was 26.2 pS; therefore, 1 µM GABA increased the slope conductance. These findings were confirmed in four DGCs, and in each cell 1 µM GABA increased the slope conductance of I_rms.

We tested whether increased I_rms by 1 µM GABA was accompanied by inhibition of sIPSCs, because low concentrations of GABA are known to desensitize synaptic GABA_A receptors and diminish synaptic currents in DGCs (Overstreet and Westbrook, 2001). The mean amplitude of sIPCSs remained unchanged after application of 1 µM GABA, 31.2 ± 5.3 pA in baseline and 33.7 ± 8.6 pA (approx. 500 events; paired t test; p = 0.82; n = 4). The mean decay time constant was also unchanged (9.9 ± 0.9 versus 10.12 ± 1.1 ms; p = 0.88). The frequency of sIPSCs was 1.38 ± 0.7 Hz at baseline and 1.49 ± 0.7 Hz after application of 1 µM GABA (p = 0.92). Therefore, 1 µM GABA selectively enhanced baseline noise but did not have an effect on synaptic currents.

GABA Concentration-I_rms Relationship. Theoretical studies on receptor-gated channel noise and subsequent recordings predict that the relationship between agonist concentration and I_rms is parabolic, with minimum noise occurring at low and high concentrations of the agonist and maximum noise occurring at intermediate concentrations (Traynelis and Jaramillo, 1998). As noted above, 1 µM GABA increased mean I_rms by 0.37 pA. In three DGCs, 10 µM GABA increased mean I_rms by 1.69 pA at the maximum change of I_avg. However, when the concentration of GABA was increased further to 30 µM, the increase in mean I_rms was smaller, 0.41 pA. These results indicated that the GABA concentration versus I_rms noise change relationship was parabolic, thus further confirming that GABA_A receptor conductance contributes to I_rms.

Penicillin Modulates Tonic and Synaptic Inhibition. Bicuculline and SR 95531 blocked synaptic inhibition along with tonic inhibition. When the GABA_A receptor is going through open and desensitized states, the agonist remains bound to it (agonist trapping) and competitive antagonists cannot bind to the receptor or close it (Bianchi and Macdonald, 2001). Therefore, bicuculline cannot selectively inhibit tonic currents, which are mediated by bound open receptors. We reasoned that a drug that preferentially binds to GABA_A receptors in the open state is likely to discriminate between receptors mediating tonic and synaptic inhibition. Penicillin G is a noncompetitive antagonist of the GABA_A receptor (Macdonald and Barker, 1977) that inhibits GABA_A receptors by blocking the chloride channel in all open states (Twyman and Macdonald, 1992). Penicillin at a concentration as low as 250 µM reduces total average current in a single channel by 38% (Twyman et al., 1992).

The effect 300 µM penicillin on tonic and synaptic currents was studied in seven DGCs. A typical experiment is shown on Fig. 3, where penicillin did not change the I_avg but it did diminish I_rms (Fig. 3, A and B). The decrease was significant for each cell, as confirmed by KS test with p < 0.001. The mean I_rms decreased from 4.364 ± 0.30 to 3.609 ± 0.21 pA (p = 0.002; n = 7). At the same time, penicillin effects on synaptic currents were subtle. It did not change peak amplitude and frequency of sIPSCs but significantly decreased decay time constant (Fig. 3C). The mean frequency of sIPSCs during the baseline period in seven neurons was 1.190 ± 0.19 Hz, and in the presence of 300 µM penicillin it was 1.25 ± 0.25 Hz (p = 0.84). The mean amplitude of sIPSCs was unchanged, 45.1 ± 13 pA during the baseline period and 43.7 ± 14 pA (p = 0.95) in the presence of penicillin. However, penicillin is an open channel blocker, and it is believed to inhibit channels after activation. We tested whether the decay of sIPSCs was accelerated by penicillin. The average decay time constant of sIPSCs decreased from 9.310 ± 0.9 to 5.240 ± 0.9 ms (p = 0.002; n = 4). The decrease of decay resulted in decreased charge transfer. It decreased from 512.1 ± 56.9 to 256.0 ± 33.4 pA/ms (p = 0.006; n = 4).

View larger version (40K): [in this window] [in a new window]   Fig. 3. The open channel blocker penicillin inhibits Irms, synaptic currents but does not affect Iavg. A, current trace recorded from a DGC. Penicillin (300 µM; solid line) did not change Iavg. B, expanded current traces before application of penicillin (top trace) and 5 min after application of penicillin (bottom trace). Irms (shown by arrows between solid and dashed lines) was smaller in the bottom trace compared with the top trace. C, penicillin (300 µM) did not change peak amplitude of sIPSCs but sharply decreased decay time constant. Current traces recorded from a DGC before application of penicillin (left) and 5 min after application of penicillin (right). Note faster decay in the presence of penicillin.

After 5 min of baseline recording, 10 µM GABA was applied for 10 min. This resulted in an inward shift in I_avg, which reached a maximum (trough) and then decayed to persistent current that did not return to baseline I_avg for the duration of application of GABA (Fig. 4, A and B). In five DGCs tested, the peak I_avg evoked by 10 µM GABA was 134.7 ± 0.81 pA. The I_avg decayed to a persistent current (90.25 ± 0.57 pA), which was measured 5 min after application of 10 µM GABA was started, and the residual nondesensitizing component I_avg was 44.5 ± 1 pA (n = 5). Therefore, the extent of desensitization of I_avg was 33%. The channel noise can decrease because of opening or closing of channels; therefore, I_rms was not used to study desensitization of receptors.

View larger version (23K): [in this window] [in a new window]   Fig. 4. GABA (10 µM) augmented baseline RMS noise and mean current. A, current trace recorded from DGC before (baseline period) and during application of 10 µM GABA (solid line). Note that 10 µM GABA resulted in an inward current that reached a maximum and then decayed to persistent current, which did not return to the baseline. B, expanded fragments from current trace in A; bottom trace recorded in the presence of 10 µM GABA demonstrated larger noise than the top trace recorded during baseline period. C, plot of KS test of Iavg in 30 epochs before and in 30 epochs after application of 10 µM GABA were compared with KS test. Note the significant increase of Iavg after application of 10 µM GABA (dotted line). D, normalized sIPSCs (average of approx. 500 sIPSCs) from the current trace recording shown in A. Thin line represents sIPSCs recorded in the baseline, and the thick line represents sIPSCs after application of 10 µM GABA. Note the reduction of peak amplitude after application of 10 µM GABA.

In each of eight DGCs studied, 10 µM GABA inhibited sIPSC amplitude and frequency (Fig. 4, C and D). The mean amplitude of sIPSC decreased from 64.3 ± 6.6 to 47.1 ± 6.1 pA (p < 0.05) (Fig. 4E), and the frequency decreased from 1.26 ± 0.3 to 0.36 ± 0.04 Hz (p = 0.01). The total charge transfer mediated by sIPSCs before the application of GABA was 120.24 pA/s, and in the presence of GABA it was 42.39 pA/s. Therefore, prolonged application of 10 µM GABA desensitized 65% of synaptic currents, substantially more than desensitization of mean current, which was 33%, suggesting synaptic currents were more susceptible to desensitization than mean current.

We confirmed these findings using a higher concentration of GABA. In five DGCs, higher concentration of GABA (100 µM) produced a 1642 ± 164 pA inward shift of I_avg at the maximum (trough), which decayed to a persistent 676.5 ± 148.5 pA current. The effect of 100 µM GABA on sIPSCs was more dramatic, because they disappeared in all five cells. The mean frequency of sIPSCs before GABA application was 0.92 ± 0.12 Hz, and no transient event could be detected after the mean current reached the peak.

Activation of presynaptic GABA_B receptors by GABA could have reduced applied to DGCs in the presence of CGP 55845 (10 µM) to block GABA_B receptors. There was no change in frequency of sIPSCs when recordings without CGP 55845 were compared with recordings in the presence of CGP 55845 (0.92 ± 0.17 Hz, n = 8 and 1.22 ± 0.05 Hz, n = 3; p = 0.36). The sIPSCs were completely inhibited by 100 µM GABA in the presence of 10 µM CGP 55845. Therefore, desensitization of postsynaptic GABA_A receptors alone without activation of GABA_B receptors could explain the loss of synaptic currents.

Saturating Concentration of GABA to Measure Total Residual Conductance. Studies with 10 and 100 µM GABA suggested that GABA could be used to differentially desensitize two kinds of receptors that mediate tonic and phasic inhibition in DGCs. GABA activated both kinds of receptors, but a fraction of receptors desensitized rapidly, whereas others were persistently active in the presence of GABA. We determined the residual GABA_A receptor conductance in the presence of a saturating concentration of GABA. GABA (1 mM) produced a large, whole cell inward I_avg with maximum amplitude of 1628 ± 194 pA (n = 8; see an example in Fig. 5A). Continuous application of 1 mM GABA (10 min) produced desensitization of GABA_A receptors. However, residual current remained regardless of duration of GABA application. This residual current had mean amplitude of 534 ± 126 pA. This residual current was probably mediated by a combination of slowly and rapidly desensitizing receptors.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Saturating concentration of GABA revealed a slowly desensitizing conductance. A, current trace from a DGC showing baseline recording followed by application of 1 mM GABA (solid line), which evoked a large whole cell current that decayed to a persistent, slowly desensitizing current. Note that sIPSCs gradually disappeared in during application of 1 mM GABA and were eliminated in the persistent slowly desensitizing component, suggesting that synaptic GABAA receptors were desensitized by 1 mM GABA. The difference between the baseline and the persistent, slowly desensitizing component (dashed lines) was measured. B, i, current trace from a DGC voltage-clamped to 4 mV (near reversal potential for Cl–) with application of conductance pulses (5 mV; 2 ms; 1 Hz) before (baseline period) and during application of 1 mM GABA (solid line). ii, expanded potions of a current trace from i demonstrated a large increase in conductance in response to GABA by reduction of conductance (right trace), but the conductance remained increased compared with the baseline period. C, histograms demonstrating membrane resistance (in megaohms) at baseline (black) at maximally increased conductance evoked by 1 mM GABA (white) and in persistently increased conductance (dashed).

Pharmacological Properties GABA_A Receptors Mediating Tonic Currents. A metabolite of progesterone, 3a-OH-5a-pregnan-20-one or allopregnanolone, is a potent allosteric modulator of GABA_A receptors. Allopregnanolone, in concentrations of the physiological range (10–30 nM), enhances whole cell GABA_A receptor currents elicited from DGCs (Mtchedlishvili et al., 2001). Nanomolar concentrations of neurosteroid THDOC enhance GABA_A receptor currents elicited from recombinant receptors containing the subunit (Wohlfarth et al., 2002); thus, we tested whether low concentrations of allopregnanolone could modulate tonic inhibition in DGCs.

A physiological concentration of allopregnanolone (10 nM) was applied to a DGC after a 5-min recording of sIPSCs, which did not alter I_avg but increased mean I_rms (Fig. 6, A and B). The effect of allopregnanolone was studied in five DGCs, and it increased I_rms in each of the five DGCs tested (KS test). When the data from individual cells were pooled, 10 nM allopregnanolone increased I_rms from 9.165 ± 0.5 to 9.612 ± 0.7 pA (p < 0.001; n = 5). When data from five cells were combined, allopregnanolone (10 nm) increased mean I_avg from 46.8 ± 3.1 to 48.3 ± 0.6 pA (p = 0.4). A higher concentration of allopregnanolone, 30 nM, also increased mean I_rms from 7.887 ± 0.47 to 8.121 ± 0.4335 pA (t test; p = 0.003; n = 5) without any effect on mean I_avg (69.1 ± 8 and 71.6 ± 3.2 pA; t test; p = 0.38).

View larger version (52K): [in this window] [in a new window]   Fig. 6. Effect of allopregnanolone on GABAA receptor-mediated tonic current. A, typical recording of current trace before (baseline) and during bath application of 10 nM allopregnanolone (solid line) to a DGC demonstrating no change in Iavg. B, expanded fragments from current trace in A; bottom trace recorded in the presence of 10 nM allopregnanolone demonstrated larger noise than the top trace recorded during baseline period. C, frequency distribution histogram of Irms in 60 epochs during the application of 10 nM allopregnanolone (white columns; bin size, 0.2 pA) demonstrated rightward shift compared with epochs in the baseline period (black columns). D, typical recording of current traces from a DGC before and after application of 300 nM allopregnanolone. The presence of tonic GABAA receptor-mediated current was confirmed by application of 100 µM bicuculline. Note the downward deflection of the holding current.

Furosemide and Loreclezole. Furosemide is a noncompetitive antagonist, which inhibits GABA_A receptor currents elicited from 4 subunit-containing recombinant receptors (Wafford et al., 1996) and does not discriminate between and subunit-containing receptors (Korpi and Luddens, 1997). In six DGCs, furosemide (300 µM) decreased I_rms from 7.611 ± 0.54 pA during baseline to 6.498 ± 0.35 pA (p < 0.05; Fig. 7A), but it did not change I_avg, which was 143.7 ± 23 pA during baseline and 145.6 ± 35 pA (p = 0.38) during drug application. The presence of tonic GABA_A receptor-mediated current was confirmed by bath application of bicuculline (100 µM) in the presence of 300 µM furosemide. Bicuculline abolished all synaptic currents and decreased mean I_avg by 47 ± 8.31 pA (n = 3).

View larger version (42K): [in this window] [in a new window]   Fig. 7. Furosemide (300 µM) decreased GABAA receptor-mediated Irms in DGCs. A, typical recording of a current traces from a DGC before (baseline) and during application of 300 µM furosemide indicated by a solid line. Note that 300 µM furosemide did not change Iavg, and bicuculline (100 µM) applied at the end of the experiment demonstrated the presence of tonic inhibition. B and C, expanded fragments from the trace shown in A show diminished Irms during the furosemide application (bottom traces) than in the baseline period (top trace). Note decreased Irms in the bottom trace compared with the top trace in C (distance between solid and dashed lines). D, frequency distribution histogram of Irms during the furosemide application (white columns; bin size, 0.2 pA) was shifted to the left compared with that in the baseline period (black columns). Loreclezole (30 µM) decreased GABAA receptor-mediated Irms in DGCs.

Loreclezole Decreased Tonic Current. Anticonvulsant loreclezole enhances ₂ or ₃, but it inhibits ₁ subunit-containing GABA_A receptors (Fisher et al., 2000). Loreclezole (30 µM; Fig. 8) decreased I_rms from 7.573 ± 0.75 to 7.355 ± 0.63 pA (p < 0.05) in four DGCs. Loreclezole at 30 µM had the tendency to reduce mean I_avg from 174.0 ± 1.1 pA during baseline to 168 ± 0.57 pA during drug application (p > 0.05; n = 4).

View larger version (31K): [in this window] [in a new window]   Fig. 8. A, typical recording of current trace from a DGC before (baseline) and during application of 30 µM loreclezole (solid line). Note that 30 µM loreclezole did not change Iavg. B and C, expanded fragments from the trace in A show diminished Irms during the loreclezole application (bottom trace) compared with that in the baseline period (top trace). Note decreased Irms in bottom trace compared with top trace in C (distance between solid and dashed lines). D, frequency distribution histograms of Irms during loreclezole application (white columns; bin size, 0.2 pA) was shifted to the left compared with that in the baseline period (black columns).

View larger version (38K): [in this window] [in a new window]   Fig. 9. Diazepam (100 nM) did not affect GABAA receptor-mediated tonic current in DGCs. A, current trace from a DGC before (baseline) and during application of 100 nM diazepam indicated by solid line. B and C, expanded fragments from the trace in A show unchanged Irms during diazepam application (bottom trace) compared with the baseline period (top trace). D, frequency distribution histograms of Irms during diazepam application (white columns; bin size, 0.2 pA) was unchanged compared with that in the baseline period (black columns).

View larger version (37K): [in this window] [in a new window]   Fig. 10. Zolpidem (100 nM) did not affect GABAA receptor-mediated tonic current in DGCs. A, current trace from a DGC before (baseline) and during application of 100 nM zolpidem indicated by a solid line. Bicuculline (100 µM) was applied in the presence of zolpidem (solid line) to confirm presence of tonic inhibition. B and C, expanded fragments from the trace in A show unchanged Irms during zolpidem application (bottom trace) compared with the baseline period (top trace). D, frequency distribution histograms of Irms during zolpidem application (white columns; bin size, 0.2 pA) was unchanged compared with that in the baseline period (black columns).

	Discussion

Top Abstract Materials and Methods Results Discussion References

Tonic GABA_A Receptor Conductance Contributes to Membrane Noise. Membrane current fluctuations have long been used to study the properties of ion channels and receptors, but there are few studies demonstrating GABA_A receptor contribution to membrane noise. Several experiments in the present study suggested that persistently open GABA_A receptor channels contribute to the membrane noise (I_rms) in DGCs in hippocampal slices. The current fluctuations recorded from DGCs diminished in proportion to the chloride driving force, suggesting that passage of chloride ions through the membrane contributed to I_rms. Penicillin, which blocks open GABA_A receptors, reduced I_rms. GABA enhanced I_rms, suggesting that activation GABA_A receptors could contribute to I_rms. Pharmacological studies suggested that only a subset of GABA_A receptors contribute to baseline membrane noise. I_rms was enhanced by neurosteroid allopregnanolone, whereas benzodiazepine site agonists diazepam and zolpidem, which enhance synaptic GABA_A receptor currents in DGCs, did not enhance membrane noise.

We found that the membrane noise was more sensitive to GABA, penicillin, and allosteric modulators of GABA_A receptors than mean current, I_avg. Previous studies using competitive antagonists such as bicuculline found that these drugs reduce I_avg as well as membrane noise, I_rms (Stell and Mody, 2002). However, competitive antagonists block synaptic currents before inhibiting tonic currents. Penicillin altered I_rms without any change in I_avg. Penicillin had a minimal effect on synaptic currents; it did not decrease peak amplitude or frequency of IPSCs, but it hastened IPSCs decay, which resulted in a 2-fold decrease in total charge transfer. In contrast, bicuculline completely eliminated synaptic currents, and this could have contributed to changes in I_avg. Likewise, furosemide, another noncompetitive GABA_A receptor antagonist, reduced I_rms but not I_avg.

High-Affinity GABA_A Receptors. Experiments with low concentrations of GABA (1 µM and 300 nM) revealed a high-affinity GABA_A receptor on DGCs. Low concentrations of GABA increased membrane noise, but this could have resulted from closure of channels because of desensitization. Increased I_rms is not simply because of GABA_A receptor channel openings because the relationship between the noise variance (RMS noise) and mean current amplitude is parabolic, described by the binomial theorem [² = i² Np(1–p), where is ² is I_rms, N is number of channels, i is single channel currents, and p is probability of single channel opening. That is, the current fluctuations at lowest and highest concentrations of a ligand are minimal because most channels are in a closed or open state, but the fluctuations are maximal at intermediate concentrations of a ligand (for review, see Traynelis and Jaramillo, 1998). Several lines of evidence suggested that the augmentation of I_rms by 1 µM GABA was because of an opening of receptor channels and not because of closure of open channels. Comparison of membrane slope conductance in the presence and absence of GABA demonstrated increased conductance, suggesting that GABA opened more channels than it had shut. Furthermore, 1 µM GABA did not alter the frequency or amplitude of sIPSCs, suggesting that this concentration did not desensitize synaptic GABA_A receptors. Finally, increasing the concentration of GABA to 10 µM resulted in a greater increased membrane noise than that observed with 1 µM GABA.

A number of studies report the extracellular GABA concentration to be in the range from tens of nanomoles to a few micromoles. Extracellular GABA concentration was reported to be 30 nM in neocortex, measured by high-performance liquid chromatography (Zhang et al., 2005). Based on microdialysis studies, it has been suggested that the extracellular concentration of GABA in the hippocampus varies in the 2.1 to 3.8 µM range (Shin et al., 2002). It seems that extracellular GABA concentration in vivo varies in the 0.3 to 3 µM range (Timmerman and Westerink, 1997). Studies in the past used blockade of GABA uptake to modulate tonic current in DGCs (Nusser and Mody, 2002), but the exact change in extracellular concentration of GABA in these studies is unknown. The current study demonstrated that a low concentration of exogenously applied GABA could activate chloride conductance.

This study also suggested that DGCs express GABA_A receptors with a high affinity to GABA. A likely explanation for high GABA affinity of receptors mediating tonic inhibition is that they contain the ₄ subunit. Recombinant GABA_A receptors containing the ₄ have a higher affinity for GABA than those containing the ₁ subunit (Wafford et al., 1996). The mRNA coding for the ₄ and polypeptide has been localized to DGCs in the past. Inhibition of tonic GABA_A receptor-mediated currents by furosemide suggests that ₄ subunit-containing GABA_A receptors participate in mediating tonic currents. Furosemide is known to inhibit ₄ subunit-containing receptors and was demonstrated to inhibit GABA_A receptor currents in dentate granule cells, which express the ₄ subunit (Wafford et al., 1996; Kapur and Macdonald, 1999). In addition to furosemide sensitivity, recombinant GABA_A receptors containing the ₄ subunit are insensitive to diazepam and zolpidem, which was also the case in our experiments with tonic currents recorded from DGCs.

Slowly Desensitizing Receptors on DGCs. A large residual GABA_A receptor conductance was found in the equilibrium state after prolonged application of 100 µM and 1 mM GABA. This residual current was probably mediated by both synaptic and extrasynaptic receptors. Increasing the extracellular concentration of GABA inhibits synaptic GABA_A receptors (Overstreet and Westbrook, 2001) by slow desensitization (Overstreet et al., 2000). Furthermore, the disappearance of synaptic currents was temporally correlated with reduction of the whole cell conductance, suggesting these receptors were being desensitized. Finally, previous studies have suggested that ₁ and ₂ subunit-containing receptors are expressed in GABAergic synapses on DGCs. Recombinant receptors containing these subunits desensitize rapidly and to a substantial extent (90%) (Bianchi et al., 2002). In contrast, the subunit-containing receptors desensitize very slowly (Saxena and Macdonald, 1996; Haas and Macdonald, 1999) and to a lesser extent (35%) (Bianchi and Macdonald, 2002). However, studies with recombinant receptors have shown desensitization of 2 subunit-containing receptors is never complete. Because subunit-containing receptors produce larger currents compared with subunit-containing receptors, even after 90% decrement of the current, the residual current corresponding to slow and ultra-slow desensitization rates of subunit-containing receptors can be of similar amplitude as residual current mediated by subunit-containing receptors, which loose only 35% of current in response to 28-s application of 1 mM GABA (Bianchi and Macdonald, 2002). Therefore, it is likely that in our preparation, residual current after prolonged application of 1 mM GABA is generated by mixed population of and subunit-containing receptors. However, the current study does not permit to estimate the extent of participation of each subunit type-containing receptor because the ratio of expression of each subunit-containing receptor is unknown. Separate study will be needed to answer this question.

Pharmacological Properties of GABA_A Receptors Mediating Tonic Currents. Pharmacological properties of persistently open GABA_A receptors in DGCs were similar to those of recombinant receptors containing the subunits. Recombinant receptors containing the subunit are highly sensitive to neurosteroid enhancement. Enhancement of RMS noise by 10 nM allopregnanolone suggested that persistently open GABA_A receptors on DGCs are sensitive to a physiological concentration of allopregnanolone. This high sensitivity to allopregnanolone is likely to be caused by the presence of subunit-containing GABA_A receptors (Wohlfarth et al., 2002). Lack of diazepam and zolpidem sensitivity of receptors also suggested absence of ₂ subunit and presence of subunit in these receptors.

We did not observe changes in mean current when low concentrations (10 and 30 nM) of allopregnanolone were applied; however, the RMS noise was increased. The mean current was increased by a 300 nM concentration of allopregnanolone (see Results). In contrast, low concentrations of the neurosteroid THDOC increased mean current in DGCs (Stell et al., 2003), which may suggest differential sensitivity of GABA_A receptors to THDOC and allopregnanolone. We have previously reported that 10 nM allopregnanolone strongly enhanced whole cell currents in DGCs (Mtchedlishvili et al., 2001), but it was not clear whether the enhancement was because of effect of allopregnanolone on synaptic currents, tonic currents, or both. The current study demonstrated that the enhancement of whole cell currents at least in part was because of enhancement of tonic currents.

The loreclezole inhibition of tonic currents suggested the expression of ₁ subunit in receptors mediating tonic currents. Loreclezole is an anticonvulsant that enhances peak GABA_A receptor currents elicited by subsaturating concentrations of GABA by acting at an allosteric regulatory site on ₂ and ₃ subunits (Wafford et al., 1994; Fisher and Macdonald, 1997). Peak currents elicited from receptors containing the ₁ subunit were not enhanced by loreclezole because the receptors lack the positive modulatory site present on the ₂ and ₃ subunits. In addition to potentiation of peak currents, loreclezole inhibited steady-state GABA_A receptor currents by acting at a site distinct from the positive modulatory site on ₂ and ₃ subunits and increased the apparent desensitization. This inhibitory action of loreclezole occurred regardless of the subunit. Thus, GABA_A receptors containing the ₁ subunit may be inhibited by loreclezole or stay unaffected by it. Spontaneously opening GABA_A receptors are present in hippocampal neurons (Macdonald et al., 1989; Birnir et al., 2000). It is possible that spontaneously opening GABA_A receptors participate in the tonic current described in this article.

Conclusions. Ligand-gated ion channels can mediate at least three kinds of tonic currents: currents mediated by spontaneously open channels in the absence of the ligand, slowly desensitizing currents mediated by ligand-bound channels, and residual equilibrium currents recorded after desensitization of channels. These three forms of "tonic" currents may be mediated by overlapping pools of receptors as demonstrated in the current study. The current study demonstrated that on DGCs, some GABA_A receptors are persistently open. These channels have high affinity for GABA; therefore, they may be ligand-bound open channels. Properties of persistently open GABA_A receptors on DGCs were similar to recombinant receptors containing ₄, ₁, and subunits. A persistent current could be recorded after desensitization of GABA_A receptors on DGCs.

	Footnotes

 
This study was supported Grants R01-NS040337 and R01-NS044370 (to J.K.).

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.016683.

ABBREVIATIONS: DGC, dentate granule cell; THDOC, tertrahydrodeoxycorticosterone; ACSF, artificial cerebrospinal fluid; sIPSC, spontaneous inhibitory postsynaptic current; DL-AP5, DL-2-amino-5-phosphonopentanoic acid; RMS, root mean square; KS, Kolmogorov-Smirnov; SR 95531, 2-(3-carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide; CGP 55845, (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid.

Address correspondence to: Dr. Zakaria Mtchedlishvili, Department of Neurology, Box 800394, University of Virginia-Health Sciences Center, Charlottesville, VA 22908. E-mail: zm5e{at}virginia.edu

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