A new naturally occurring GABAA receptor subunitpartnership with high sensitivity to ethanol

Joseph Glykys1,5, Zechun Peng2,5, Dev Chandra3, Gregg E Homanics3, Carolyn R Houser2,4 & Istvan Mody1

According to the rules of GABAA receptor (GABAAR) subunit assembly, a4 and a6 subunits are considered to be the natural

partners of d subunits. These GABAARs are a preferred target of low, sobriety-impairing concentrations of ethanol. Here we

demonstrate a new naturally occurring GABAAR subunit partnership: d subunits of hippocampal interneurons are coexpressed

and colocalized with a1 subunits, but not with a4, a6 or any other a subunits. Ethanol potentiates the tonic inhibition mediated

by such native a1/d GABAARs in wild-type and in a4 subunit–deficient (Gabra4–/–) mice, but not in d subunit–deficient (Gabrd–/–)

mice. We also ruled out any compensatory upregulation of a6 subunits that might have accounted for the ethanol effect in

Gabra4–/– mice. Thus, a1/d subunit assemblies represent a new neuronal GABAAR subunit partnership present in hippocampal

interneurons, mediate tonic inhibitory currents and are highly sensitive to low concentrations of ethanol.

The amino acid neurotransmitter g-aminobutyric acid (GABA) isresponsible for mediating most of the chemical inhibition in theCNS. GABAARs form a Cl–- and HCO3

–-permeable ion pore that isassembled from five subunits (heteropentameric): a1–6, b1–3, g1–3,d, e, y1–3, p and r1–3 (ref. 1). However, no more than a few dozenreceptor combinations are likely to exist in the mammalian brain. It isthought that this is due to precise rules for subunit assembly (forexample: two as and two bs assemble with either one g or one d) and tospecific subunit partnership2,3.

One such rule of specific partnership is that d subunits co-assemblewith either a4 or a6 subunits4,5. Consistent with this, d and a4 subunitshave very similar distributions in the thalamus, striatum, outer layers ofthe cortex and dentate molecular layer (ML), whereas the d and a6subunits are colocalized in cerebellar granule cells (CGCs)4,6–8. Some-times, a specific partnership is needed to express a certain subunit onthe cell surface. Thus, in mice lacking the a6 subunit, d subunits are alsomissing from the surface of CGCs5.

In the hippocampus, ML interneurons express d subunits9,10 andlarge amounts of a1 subunits10–13. In general, a1-containing receptorsare widely accepted to co-assemble with g2 subunits, but not d subunits,mostly at synapses14, as g2 subunits interact with the anchoring proteingephyrin for synaptic clustering15. As g and d subunits are thought to bemutually exclusive from GABAAR assemblies16,17, and as d subunits arenot anchored at synapses, receptors that contain d subunits are mainlylocated extra- or perisynaptically7,18,19. In light of such contrastingdifferences in the localization of a1 and d subunits—synaptic andextrasynaptic, respectively—the natural assembly of a1/d subunits wasconsidered unlikely.

Synaptic receptors mediate phasic inhibition, whereas extra- andperisynaptic receptors mediate tonic inhibition20–22. The d subunit–containing GABAARs have consistently been shown to mediate tonicinhibition in dentate gyrus granule cells (DGGCs)23–26, CGCs26 andthalamic neurons27,28. The d subunit–containing GABAARs can bemodulated by physiological concentrations of neurosteroids and by lowconcentrations of ethanol in DGGCs as well as in CGCs23,26,29. This isconsistent with the modulation of a4/d- and a6/d-containing GABAARby ethanol in expression systems30,31.

Anatomical, physiological and pharmacological data on thepartnership of d subunits with a4 or a6 subunits has led to a neglectof other potential assembly partners of the d subunits. Yet in expressionsystems, d subunits readily assemble with other a subunits (such as a1)and even show modulation by neurosteroids31–33. To date, however,there is no evidence for functional a1/d subunit assembly occurringunder natural conditions in the brain. As both a1 andd subunits are present in ML interneurons, we wanted to find outwhether functional a1/d subunit–containing GABAARs exist andwhether such receptors also mediate the tonic inhibition recordedin ML interneurons. Furthermore, because sobriety-impairing concen-trations of ethanol enhance tonic inhibitory currents in neuronsexpressing GABAAR d subunits and the potentiation is abolished inthe absence of the d subunit23,30, we also examined the ethanolsensitivity of the tonic inhibition in ML interneurons. Using anatomi-cal, electrophysiological and pharmacological approaches, our findingsdemonstrate a new natural partnership between GABAAR a1 andd subunits and show that this receptor combination is also sensitiveto low ethanol concentrations.

Received 2 October; accepted 15 November; published online 10 December 2006; doi:10.1038/nn1813

1Interdepartmental PhD Program for Neuroscience and Department of Neurology and 2Department of Neurobiology, David Geffen School of Medicine at the Universityof California, Los Angeles, California 90095, USA. 3Departments of Anesthesiology and Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.4Research Service, VA Greater Los Angeles Healthcare System, West Los Angeles, Los Angeles, California 90073, USA. 5These authors contributed equally to this work.Correspondence should be addressed to I.M. (mody@ucla.edu).

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RESULTS

a1, d subunits and tonic inhibition in ML interneurons

In normal C57BL/6 mice, we observed diffuse labeling of both the d anda4 subunits throughout the ML of the dentate gyrus and much of thislabeling is presumed to be localized on the dendrites of DGGCs(Fig. 1a). We also detected some d subunit–labeled interneurons inthe ML and along the base of the granule cell layer (Fig. 1a), althoughthese interneurons were often nearly concealed by diffuse d subunitlabeling. Despite repeated experiments and the use of two different a4subunit–specific antisera, we detected no a4-labeled interneurons.Instead, interneurons in the ML and other regions of the hippocampalformation were strongly labeled for the a1 subunit (Fig. 1a). Thislabeling was distinct and outlined the dendritic processes as well as thesoma of interneurons in the ML and subgranular zone, as previouslydescribed in rats10,11,13.

Qualitative analyses suggested that virtually all ML interneuronswere labeled for both a1 and d subunits. To confirm this, we firstdetermined the proportion of GABA neurons (identified by GAD67, aspecific marker for GABA neurons) in the ML that were labeled for thea1 subunit. Our experiments indicated that 99.7% of the GAD67-labeled ML neurons (n ¼ 300) were labeled for the a1 subunit(Supplementary Fig. 1 online). Using similar double-labeling studies,we demonstrated that 100% of a1-labeled interneurons (n ¼ 316) inthe ML were also labeled for the d subunit (Supplementary Fig. 1online). ML interneurons thus seem to be very homogeneous in theirexpression of a1 and d subunits.

Consistent with a high concentration of a1 subunits that areresponsible for speeding up the decay of inhibitory post-synapticcurrents (IPSCs)34, spontaneous IPSCs (sIPSCs, Vh ¼ –70 mV)recorded in C57BL/6 (wild type) ML interneurons had a significantlyfaster decay time than sIPSCs recorded in DGGCs, which are rich inother a subunits, including a4 (interneurons 5.52 ± 0.29 ms, n ¼ 6

versus DGGCs 6.72 ± 0.23 ms, n ¼ 7;P¼ 0.009 unpaired t-test; Fig. 1b). In additionto rapidly decaying IPSCs, these interneuronshad a tonic inhibitory current of 9.81 ±1.51 pA (n ¼ 17; Fig. 1c) that could berevealed by blocking all GABAAR with a highconcentration of bicuculline methiodide(BMI; 100–200 mM). These findings are con-sistent with the presence of high affinity extra-or perisynaptic receptors that are sensitive tolow-micromolar GABA concentrations.

The abundance of the a1 subunit and thevirtual absence of a4 in the ML interneuronswere consistent with the idea of a potentialpartnership between d and a1 in GABA recep-tors of these neurons. To determine whetherother a subunits showed a similar localizationin the ML interneurons, we conducted doubleimmunofluorescence labeling studies of thea2, a3, a4 or a5 subunits with either d or a1(as a marker for the interneurons). The resultsdemonstrated a complete lack of a2 labeling inML neurons that expressed d subunits. Wedetected extremely low a3 labeling in the MLinterneurons, but this labeling differed fromthe distinct a3 labeling of other groups ofinterneurons, such as some in the hilus. Thusit is possible that the limited labeling is non-specific. Although we saw strong a1 labeling

that outlined the surface of the cell bodies and proximal dendrites ofML interneurons, we saw essentially no a4 subunit labeling withinthese interneurons or along their surface. Likewise, a5 labeling was verylow, but small amounts of cytoplasmic labeling could be detected. Itcannot be determined whether such labeling represents low levels ofspecific labeling or slight nonspecific labeling. In contrast to a1 and d,we observed no surface labeling for any of the other subunits (a2, a3,a4 and a5) (Fig. 2). These findings demonstrate that the a1 subunit isthe most abundant a subunit in ML interneurons and that itslocalization resembles that of the d subunit much more closely thando those of the other a subunits.

Ethanol potentiates tonic inhibition in interneurons

The d subunit–containing GABAARs mediate tonic inhibition inDGGCs7,18,19 and CGCs18,26 and are expressed extra- or perisynapti-cally. We first examined whether d subunits were present at the GABAsynapses of ML interneurons by recording sIPSCs from wild-type miceand comparing them to IPSCs recorded in mice lacking the d subunit(Gabrd–/–). As in other cells where d subunits do not seem to belocalized at synapses18,19, the frequency and kinetics of the sIPSCs werenot different between wild-type and Gabrd–/– ML interneurons(frequency: wild-type 12.6 ± 0.64 Hz versus Gabrd–/– 18.0 ± 6.14 Hz,P ¼ 0.418 unpaired t-test; peak amplitude: wild-type 34.9 ± 4.82 pAversus Gabrd–/– 39.7 ± 7.10 pA, P ¼ 0.585; RT10–90: wild-type0.59 ± 0.03 ms versus Gabrd–/– 0.58 ± 0.04 ms, P ¼ 0.861; tw: wild-type 5.57 ± 0.11 ms versus Gabrd–/– 5.30 ± 0.32 ms, P¼ 0.462 unpairedt-test; n ¼ 6; Fig. 3a and Table 1).

In other cell types where d–containing GABAARs are present andmediate tonic inhibition26, this tonic inhibition is potentiated by lowconcentrations of ethanol23,29. Thus, we next determined the effect ofsobriety-impairing ethanol concentrations (20 and 30 mM; equivalentto blood alcohol concentrations of 0.09% and 0.14%, respectively) on

5 s

20 pA

2 %10 pA

ControlBMI

DGGC

a

b cInterneuron

5 ms

δ α4 α1

M

G

Figure 1 Distribution of a1, a4 and d GABAAR subunits in ML interneurons and the properties of phasic

and tonic inhibitions. (a) Diffuse immunoreactivity for all three subunits is present in the dentate ML (M),

consistent with localization on granule cell dendrites in this region. Scattered interneurons in the ML

(examples at arrows) and along the base of the granule cell layer (G) are labeled for the d and a1 subunits,but no a4 subunit–labeled interneurons are evident. Scale bar, 50 mm. (b) Average sIPSCs normalized by

their peak amplitudes recorded (at Vh ¼ 70 mV) in a wild-type (WT) ML interneuron and a DGGC showing

differences in their decay time constants (5.62 ms for the interneuron and 6.69 ms for the DGGC).

Vertical scale bar, 10 pA for the interneuron and 11 pA for the DGGC. (c) Left, recording from a WT ML

interneuron before and after perfusion of 100 mM BMI to show the tonic inhibitory current; right, Gaussian

fits to the baseline current all-point histograms showing the amplitude of tonic inhibition.

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the tonic inhibition of ML interneurons. Ethanol was perfused after astable baseline control recording (CON) of the tonic current had beenobtained. In wild-type ML interneurons, tonic inhibitory currents werepotentiated by 30 mM ethanol (CON 10.5 ± 2.0 pA versus ethanol20.1 ± 3.5 pA, P ¼ 0.002 paired t-test, n ¼ 10, a potentiation ratio2.05 ± 0.3; Fig. 3b,c) and by 20 mM ethanol (CON 8.8 ± 3.1 pA15.4 ± 4.8 pA, P ¼ 0.011, paired t-test, n ¼ 7, potentiation ratio1.85 ± 0.3; Fig. 3c). Ethanol (30 mM) did not alter the sIPSCfrequencies or kinetics (Fig. 3b and Table 1). To ensure that ethanoldid not enhance the tonic current by reducing GABA uptake, werepeated the 30 mM ethanol experiments in the presence of 10 mMNO-711 (a blocker of GAT-1). The tonic currents under these condi-tions were more than four times larger than those recorded with 5 mMGABA alone, indicating that GABA uptake lowers the ambient GABAconcentration to considerably less than 5 mM (ref. 35). In the presenceof NO-711, ethanol (30 mM) still caused a significant potentiation oftonic inhibitory currents in ML interneurons (CON 44.9 ± 15.9 pAversus ethanol 85.3 ± 23.5 pA, P ¼ 0.016 Wilcoxon matched-pairs signed-ranks test, n ¼ 7; potentiation ratio 2.38 ± 0.7). Aspreviously reported23, 30 mM ethanol significantly potentiated toniccurrents in DGGCs (CON 17 ± 5.6 pA versus ethanol 29.5 ± 8.4 pA,P ¼ 0.008, paired t-test, n ¼ 7, potentiation ratio 2.09 ± 0.4; Fig. 3c),but 20 mM ethanol did not (CON 15.5 ± 3.7 pA versus ethanol16.6 ± 4.1 pA, P ¼ 0.158, paired t-test, n ¼ 5, potentiation ratio1.09 ± 0.04; Fig. 3c).

Mediation of tonic inhibition by d subunit–containing GABAARs is aprerequisite for ethanol potentiation23,29,30 (but see ref. 36) and ethanolpotentiation of tonic inhibition is absent in DGGCs of d subunit–deficient (Gabrd–/–) mice23. Therefore, we wanted to determinewhether the potentiation of tonic inhibitory currents by ethanol seenin ML interneurons also requires the presence of d subunit–containingGABAARs. We saw no significant potentiation of the tonic in-hibitory current by 30 mM ethanol in whole-cell voltage-clamprecordings from ML interneurons of Gabrd–/– mice (CON 12.0 ±3.5 pA versus ethanol 14.0 ± 3.8 pA; P ¼ 0.136 paired t-test; n ¼ 7;potentiation ratio 1.19 ± 0.2; Fig. 3d). As in wild-type neurons, thesIPSCs frequencies and kinetics were unaltered by ethanol (Table 1).These results are consistent with the idea that d subunit–containingreceptors mediate tonic inhibitory currents in ML interneuronsand further support the requirement of d subunits for the potentiationof tonic inhibition by low ethanol concentrations. It should also benoted that the presence of a sizeable tonic current in Gabrd–/– inter-neurons indicates that in the absence of d subunits, other subunitcombinations can mediate tonic inhibition23,35, although they areinsensitive to ethanol.

High ethanol sensitivity persists in Gabra4–/– interneurons

The immunohistochemical analyses indicate that the d and a1 subunitare expressed in ML interneurons, but the lack of immunohistochem-ical labeling of the a4 subunit is by no means proof of its absence in theinterneurons. To obtain additional evidence for the lack of a4 subunitsin ML interneurons, and thus for a lack of d and a4 subunit partnershipin these cells, we used the recently generated Gabra4–/– mice37. Wehypothesized that in the absence of a4 subunits, neurons that containa4/d receptors would show a decreased expression of the d subunit.This would be similar to the findings in d subunit–deficient mice,where the amount of a4 subunit present decreased substantially inprecisely those regions where the d subunit is normally expressed8,38.The normal diffuse pattern of a4 labeling in the ML of wild-type micewas absent in the Gabra4–/– mice (Fig. 4a), confirming that thea4 protein is lost in these mice37. As hypothesized, we observed asubstantial decrease in diffuse d subunit labeling in the ML in the a4-deficient mice (Fig. 4b), which is consistent with a strong partnershipof the a4 and d subunits in DGGCs dendrites19. In contrast, d subunitlabeling of interneurons persisted (Fig. 4c), as would be expected if thea4 subunit were not the primary partner of the d subunit in GABAARsof ML interneurons. In the Gabra4–/– mice, we saw d subunit labelingnot only in cell bodies of the interneurons, but also in their dendriticprocesses (Fig. 4c). This dendritic labeling was more evident ina4 subunit–deficient mice than in wild-type mice because of thedecreased diffuse d subunit labeling in the ML of Gabra4–/– mice.These findings provide strong support for the suggestion that thea4 subunit is not a major partner of the d subunit in ML interneuronsof the dentate gyrus.

δ α1 δ,α1

δ α2 δ,α2

α3α1 α1,α3

α4α1 α1,α4

δ α5 δ,α5

a

b

c

d

e

Figure 2 a1, but not other a subunits, is strongly expressed on the surface of

interneurons in the dentate ML. Interneurons were identified by either

d (rows a,b,e) or a1 (rows c,d) labeling (left column). (a) Distinct labeling for

both a1 and d subunits is present on the surface of an interneuron and

colocalization of the two subunits (yellow) is demonstrated in the right panel.

(b) No a2 labeling is evident in the cytoplasm or on the surface of the

d-labeled interneuron. (c) a3 labeling is virtually absent from the cytoplasm

and surface of the a1-labeled interneuron as well as the surroundingneuropil. (d) a4 labeling is not detected on the surface or within the

cytoplasm of the a1-labeled interneuron. (e) a5 labeling is not evident on

the surface of the interneuron, but slight labeling can be detected in the

cytoplasm of the d-labeled interneuron. Scale bar (all panels), 4 mm.

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We then used immunofluorescence studies with confocal micro-scopy to determine whether the a1 and d subunits were still colocalizedin the interneurons of Gabra4–/– mice (Fig. 5). In these animals,labeling for both the a1 and d subunits was present in ML interneuronsand the subunits were colocalized along their surface (Fig. 5). InGabra4–/– mice, ML interneurons were distinguished by strong labelingfor both a1 and d subunits (Fig. 5a). At high magnification, colocaliza-tion of a1 and d subunits was evident on the surface of the interneurons(Fig. 5b). Although this colocalization does not constitute unequivocalproof that the subunits are part of the same receptor, the labelingstrongly supports such a partnership.

Consistent with the immunohistochemical studies, we observed atonic current, including a significant potentiation by 30 mM ethanol, inML interneurons from Gabra4–/– mice that was similar to that recordedin wild-type mice (CON 8.8 ± 2.2 pA versus ethanol 21.5 ±5.8 pA; P ¼ 0.036 paired t-test; n ¼ 5; potentiation ratio 2.59 ± 0.35;Fig. 6). This is in marked contrast to the lack of potentiation of tonicinhibitory currents by 30 mM ethanol inDGGCs of Gabra4–/– mice (CON 8.9 ± 2.1versus ethanol 8.8 ± 2.3 pA; P ¼ 0.915 pairedt-test; n ¼ 5; potentiation ratio 0.97 ± 0.07).Phasic inhibition, and its lack of modulationby 30 mM ethanol, were similar in wild-typeand Gabra4–/– ML interneurons (Fig. 6b,c;Table 1). Thus, our electrophysiological andanatomical results rule out the idea that a4subunits mediate tonic inhibitory currents inML interneurons. Instead, a1/d-containingGABAARs are most likely to be responsiblefor the tonic inhibitory current recorded inthese cells.

We conducted an overall statistical analysisof the tonic inhibitory currents recorded in theML interneurons of wild-type, Gabra4–/– and

Gabrd–/– mice, and saw no differences among the three genotypes(F2,26 ¼ 0.374, P¼ 0.692; one-way ANOVA). The potentiation ratios oftonic inhibition by ethanol (30 mM), however, were statisticallydifferent between the genotypes (F2,19 ¼ 5.9833, P ¼ 0.010; one-wayANOVA). A Dunnett’s post hoc test, assuming the wild-type ratio as ourcontrol potentiation ratio, identified only Gabrd–/– interneurons assignificantly different (wild-type versus Gabrd–/– P ¼ 0.047; wild-typeversus Gabra4–/– P ¼ 0.317). A similar analysis showed that there wereno significant differences between the phasic currents recorded in theML interneurons of the three genotypes (Table 1).

a6 subunits do not mediate ethanol effects in Gabra4–/– mice

The a6 subunit is a major partner of the d subunit in the cerebellumand is considered homologous to the a4 subunit, which is localizedpredominantly in the forebrain5,6. Therefore, we had to consider thepossibility that the a6 subunit is upregulated in the forebrain tocompensate for the loss of the a4 subunit in Gabra4–/– mice. In these

Figure 3 Ethanol potentiates the tonic inhibition

in wild-type (WT) but not in GABAAR d subunit–

null mutant (Gabrd–/–) ML interneurons. (a) Left,

average sIPSCs (Vh ¼ –70 mV) from a WT and a

Gabrd–/– ML interneuron showing similar kinetics;

right, cumulative probability plots of the values of

inter-event intervals (on a log scale), showing no

difference in the frequency of events between thegenotypes. (b) Voltage-clamp recordings from WT

ML interneurons showing the ethanol-induced

potentiation of the tonic inhibitory current. Left,

baseline current plotted at 500-ms intervals in

control (A), 30 mM ethanol (B) and BMI

conditions (Vh ¼ –70 mV; horizontal bars indicate

perfusion of the drugs); right, Gaussian fits to the

all-point histograms of the baseline current during

each condition (numbers indicate the difference

current between Gaussian means). Below left,

recording low-pass filtered at 1 KHz in control and

ethanol conditions; below right, average sIPSC

from both conditions, showing a lack of ethanol

effect in the same cell shown above. (c) Above,

effects of 20 and 30 mM ethanol on tonic

inhibitory currents recorded in ML interneurons

(n ¼ 7 and 10, respectively). Below, DGGC tonic inhibitory current is potentiated by 30 but not 20 mM ethanol (n ¼ 7 and 5, respectively). (d) Left, same

as the upper panel of b but from a Gabrd–/– ML interneuron showing a lack of ethanol-induced potentiation of the tonic current. Right, lack of 30 mM

ethanol potentiation of ML interneurons tonic inhibitory currents recorded in Gabrd–/– mice (n ¼ 7). The values are plotted as mean ± s.e.m., and the boxrepresents 25th, 50th and 75th percentiles. Asterisk represents statistical significance in paired t-tests (P values are given in the main text); NS, not

statistically significant.

Table 1 Properties of sIPSCs recorded in ML interneurons of different genotypes

Wild-type Gabrd–/– Gabra4–/–

CON EtOH CON EtOH CON EtOH

Frequency (Hz) 12.6 ± 0.64 13.9 ± 1.25 18.0 ± 6.14 18.6 ± 6.06 12.1 ± 3.24 13.9 ± 3.45

Peak amplitude (pA) 34.9 ± 4.82 40.0 ± 6.46 39.7 ± 7.10 41.6 ± 6.51 44.4 ± 3.11 51.3 ± 6.71

RT10–90 (ms) 0.59 ± 0.03 0.59 ± 0.04 0.58 ± 0.04 0.62 ± 0.06 0.61 ± 0.02 0.60 ± 0.02

tw (ms) 5.57 ± 0.11 5.60 ± 0.17 5.30 ± 0.32 5.35 ± 0.40 5.08 ± 0.22 5.31 ± 0.28

n 6 6 5

sIPSCs were recorded in the presence of 3 mM kynurenic acid and 5 mM GABA (Vh ¼ –70 mV). Values are mean ± s.e.m.There were no statistical differences between CON and ethanol (EtOH) conditions in each genotype (P 4 0.05 for eachparameter; paired t-test). Control condition sIPSC frequency and kinetics between the three genotypes were not found tobe statistically different (frequency: F2,14 ¼ 0.642, P ¼ 0.541; peak amplitude: F2,14 ¼ 0.722, P ¼ 0.503; RT10–90%: F2,14 ¼ 0.141, P ¼ 0.87; tw: F2,14 ¼ 1.035, P ¼ 0.381; one-way ANOVA). These results demonstrate that neitherthe a4 nor the d subunit have a major role in shaping inhibitory synaptic events in ML interneurons.

a b

10 pA

50 s

2 %

WTBMIEthanol 30 mM

10 pA16 pA

1

2

2.5 ms5 pA

WTWT

Gabrd –/–

Gabrd –/–

40 pA200 ms

5 ms

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11

2

2

1.0

0.5

0

Cum

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prob

abili

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(ms)

3

Control Ethanol20 mM

Control Ethanol30 mM

Control Ethanol30 mM

0

20

40

60

800

20

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**

*In

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A)

DG

GC

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(pA

)

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NS100 s

10 p

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0

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rd–/

– I Nto

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Gabrd –/–

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mice, a6 was highly expressed in the cerebellar granule cell layer, whereits labeling overlapped with that of the a1 subunit (Fig. 7a), as in wild-type mice. We saw no a6 subunit labeling, however, in the ML of thedentate gyrus in the a4 subunit–deficient mice and no colocalizationwith a1 was evident in the interneurons of this region (Fig. 7a). Thissuggested that the a6 subunit had not replaced the a4 subunit in MLinterneurons in a4 subunit–deficient animals.

We also obtained pharmacological evidence for the lack ofa6 subunits in Gabra4–/– ML interneurons. Furosemide (100 mM) isa known blocker of tonic inhibitory currents that are mediated bya6 subunit–containing GABAARs in CGCs39,40, where a6 subunits arecolocalized with d subunits and the resulting tonic inhibition ispotentiated by ethanol29,30. We thus examined the effects of furosemideon tonic inhibition recorded in Gabra4–/– ML interneurons, reasoningthat if a6 subunits were upregulated in Gabra4–/– ML interneurons,furosemide should reduce tonic inhibition in these cells. The tonicinhibition recorded in Gabra4–/– ML interneurons was not affectedby 100 mM furosemide, however (CON 14.4 ± 3.6 pA versus FURO

15.1 ± 2.4 pA; P ¼ 0.551 paired t-test; n ¼ 4; Fig. 7b). These find-ings are consistent with our anatomical data and strongly argueagainst an upregulation of a6 subunits in the ML interneurons ofGabra4–/– mice.

DISCUSSION

Our main conclusions are (i) GABAAR d subunits are expressed in MLinterneurons without their ‘obligatory’ forebrain partners, the a4subunits; (ii) a1/d subunits colocalize and most likely form functionalreceptors, with a yet-to-be-determined b subunit, that underlie tonicinhibitory currents in ML interneurons; (iii) this natural subunitcombination is highly sensitive to sobriety-impairing concentrationsof ethanol (20–30 mM), perhaps even more than the a4/d combinationfound in DGGCs; (iv) as in other neurons, phasic inhibition is notsensitive to 30 mM ethanol.

The GABAAR kinetics and pharmacology are determined by theirdistinctive subunit composition as well as other intra- and extracellularmodulators41. The d subunit–containing GABAARs have been shownto have orders-of-magnitude higher affinities for GABA than receptorscontaining other subunits and do not show desensitization30, which isconsistent with their extrasynaptic localization in both DGGCs as wellas CGCs and their mediation of tonic inhibition18,19,25. The d subunitsare predominantly found in areas that are rich in a4 or a6 subunits,where they are considered to form specific GABAAR partnerships5,6,8.

Our study strongly suggests that the composition of d subunit–containing GABAARs differs between principal cells and interneurons.The d subunits preferentially assemble with a4 subunits in principalcells of the hippocampal formation, as in other brain regions4,8,38,which is consistent with the localization of these two subunits inDGGCs. In contrast, d and a1 seem to be the preferred partners in ML

α4 α4

δ

δδ

δ

M

M

G

M

G

Gabra4–/–

Gabra4–/–

Gabra4–/–Gabra4–/–

WT

WT

a

b

c

Figure 4 Comparison of a4 and d subunit labeling in the dentate gyrus of

wild-type (WT) and Gabra4–/– mice. (a) Diffuse a4 subunit labeling is present

in the dentate ML (M) of the WT mouse but is absent in the Gabra4–/–

animal, indicating complete loss of the a4 subunit protein in these mice.

(b) Diffuse d subunit labeling in the ML of the WT mouse is decreased in the

Gabra4–/– mouse, as might be expected if the a4 and d subunits form a

strong partnership in granule cell dendrites in this region. However,

d subunit–labeled interneurons continue to be evident in the Gabra4–/– mouse(examples at arrows). (c) In the a4-deficient mouse, d subunit labeling is

present in interneurons in the ML (examples at arrows) and at the base of the

granule cell layer. Labeled dendrites of the interneurons can be visualized in

the ML, providing additional support for specific labeling in these neurons.

Scale bars: a,b, 100 mm; c, left, 50 mm; right, 20 mm.

α1

M

α1,δδ

α1 α1,δδ

a

b

Figure 5 Potential colocalization of the a1 subunit and d subunits in ML

interneurons of Gabra4–/– mice. (a) Despite a decrease in diffuse d subunit

labeling in the dentate ML (M) of a Gabra4–/– mouse, both a1 and d subunitsare strongly labeled in the same interneurons (arrows). (b) At higher magni-

fication, a1 and d subunit immunoreactivity is evident on the surface of an

interneuron, and colocalization is indicated by yellow labeling in the right

panel. Scale bars: a, 20 mm; b, 5 mm.

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interneurons. Previous co-immunoprecipitation studies have suggestedthat not all d subunits are associated with a4 in the hippocampus andhave raised the question of which subunit besides a4 might co-assemble with d subunits4. Because of the relatively low number ofinterneurons in most tissue samples, alternate subunit combinations,including the association of d and a1 shown in our study, might not bedetected in biochemical analyses of tissue homogenates. Clues havecome, however, from earlier immunohistochemical studies in whichdistinct labeling of the a1 subunit has been found on many interneur-ons and their processes in the hippocampal formation10,11,13. Severalstudies have also alluded to the possible lack of a4 subunit labeling ininterneurons in the dentate gyrus9, despite expression of the d subunitin these neurons8–10. Nevertheless, it has been difficult to rule out thepresence of the a4 subunit in interneurons, in part because of a4subunit labeling in the neuropil immediately surrounding theinterneuron cell bodies. In the current study, the Gabra4–/– miceproved to be particularly useful for demonstrating that, despite thecomplete loss of the a4 subunit, d subunit immunoreactivity remainedin the interneurons and was colocalized with a1 labeling along the

surface of these cells. These findings, along with the lack of distinctlabeling for other a subunits, provide strong support for a selectivepartnership of the d and a1 subunits in ML interneurons.

It is not uncommon to use these two subunits in expression systems,where the GABAAR a1 subunit readily combines with d and b subunitsto form functional receptors31–33. Very small amounts (B0.8%) ofa1bxd receptors are thought to be present in the mouse cerebellum, buttheir functional properties remain unclear42. Ours is the first report toshow both a1/d subunit colocalization and functionality in a group ofhippocampal interneurons. We also demonstrate that the a1/d partner-ship is responsible for the GABAAR-mediated tonic inhibitory currentin these interneurons. We did not find any expression of the GABAARa4 subunit in ML interneurons, despite strong expression of the a4subunit in DGGCs. Furthermore, the similar baseline tonic currentsand ethanol potentiation ratios between wild-type and Gabra4–/– micetend to rule out the expression of a1/a4/bx/d receptors in wild-typeinterneurons. If very small amounts of a4 subunits are expressed in theinterneurons, these subunits do not seem to contribute to the tonicinhibitory currents.

Recently, a4b2/3d and a6b3d GABAAR–mediated currents wereshown to be enhanced by low concentrations of ethanol30,31 and,consequently, these GABAARs are theorized to be one of the mainsites of ethanol action in the brain43. In contrast, the a1b2d GABAARdoes not show this very high sensitivity to ethanol in some expressionsystems31, but 30 mM ethanol potentiates by B88% the a1b3dGABAAR–mediated currents when these receptors are expressed inXenopus oocytes29. Here we demonstrate that the a1/d-mediated tonicinhibitory current of ML interneurons is slightly more sensitive tosobriety-impairing concentrations of ethanol than the a4/d subunit–mediated tonic current of DGGCs23, providing evidence for othernaturally occurring GABAAR combinations that could be involved inmediating the effects of ethanol in the brain. The slightly highersensitivity to ethanol of the a1/d-mediated tonic currents of MLinterneurons as compared to the a4/d-mediated tonic currents ofDGGCs may arise from partnerships with different b subunits(b3 instead of b2) that are thought to influence ethanol sensitivity30.

The question arises of whether the a1/d subunit–containingGABAARs of the ML interneurons are the only receptors mediatingthe tonic inhibition. In spite of the low immunohistochemical labelingfor a5 subunits in ML interneurons, some a5bxd receptors may exist.However, it is highly unlikely that such receptors would be sensitive toethanol. The CA1 pyramidal cells highly express a5 subunits and alsomoderately express d subunits, which means that a5bxd receptorscould be present in these cells. Tonic currents of CA1 pyramidal cells,however, are insensitive to the ethanol concentrations used in ourstudy23. For the effects of ethanol on ML interneurons, we couldconsider other receptor combinations (such as a5bxgx) that havealready been shown to underlie tonic inhibition in other hippocampalneurons35, or the numerous receptors with low GABA affinity (forexample, a1b2g2). None of these receptors, however, are sensitive tothe low ethanol concentrations used in our studies23,30. Therefore, theethanol sensitivity of the ML interneuron tonic current at low ambientGABA concentrations should provide clues to the identity of theunderlying receptors. If only a small fraction of the current were tobe mediated by ethanol-sensitive receptors, the amount of potentiationof such a small fractional current would have to be enormous to yield a100% enhancement of the total current. As the amount of potentiationof the tonic current by 30 mM ethanol in ML interneurons (B100%)is in the same range (B88%) as that produced by the same doseof ethanol in a1b3d subunit–containing GABAARs of expressionsystems29, we conclude that the bulk of the tonic current in ML

c

40 pA

200 ms

10 pA

5 ms

0

10

20

30

40

Control Ethanol

Toni

c cu

rren

t (pA

)

d

Ethanol 30 mM

a BMI

25 pA

8 pA

2 %

50 s

10 pA

*

2

1

1

2

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2

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Figure 6 Ethanol-induced potentiation of tonic inhibition in ML interneurons

persists in mice lacking the a4 GABAAR subunit (Gabra4–/–). (a) Voltage-

clamp recordings from Gabra4–/– ML interneurons showing ethanol-induced

potentiation of the tonic inhibitory current. Left, baseline current plotted at

500-ms intervals during control (A), 30 mM ethanol (B) and BMI conditions

(Vh ¼ –70 mV; horizontal bars indicate perfusion of the drugs); right,

Gaussian fits to the all-point histograms of the baseline current recorded

during each condition (numbers indicate the difference current between

Gaussian means). (b) Voltage-clamp recording segments low-pass filtered at

1 KHz in control and ethanol conditions. (c) Averaged sIPSCs recorded under

the two conditions from the same cell show no effect of ethanol. (d) Box-

chart of all tonic currents recorded. Box represents 25th, 50th and 75th

percentiles with the superimposed mean ± s.e.m. Circles connected by lines

represent paired individual values of the tonic inhibitory currents recorded in

a given cell under the two conditions. *P ¼ 0.036, paired t-test, n ¼ 5.

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interneurons must be mediated by d subunit–containing GABAARswith a high ethanol sensitivity. The residual tonic current found in MLinterneurons and DGGCs of Gabrd–/– mice most likely results from thecompensatory upregulation of other extrasynaptic GABAARs contain-ing a5 subunits that are known to mediate tonic currents in otherhippocampal neurons35. Indeed, ML interneurons or DGGCs ofGabrd–/–/Gabra5–/– mice have no residual tonic current (Glykys andMody, unpublished observations).

The lack of potentiation of tonic inhibition by ethanol in both MLinterneurons and DGGCs of Gabrd–/– mice23 is consistent with thenotion that the d subunit is necessary for ethanol sensitivity23,30,31 (butsee ref. 36). The ethanol sensitivity of d subunit–containing GABAARsthat are located in different CNS regions and cell types may translateinto the complex and diverse behavioral effects of ethanol. Mice lackingthe GABAAR d subunits show altered behaviors in response to ethanoleffects, including a reduced preference for voluntary ethanol consump-tion, reduced withdrawal hyperexcitability after chronic ethanol expo-sure and a reduction in the anticonvulsant effects of ethanol. Ethanoltolerance, sedation and anxiolysis are unaffected44, however. Futurestudies will need to focus on the brain-region- and cell-specific basis forthe various behavioral effects of ethanol.

The tonic inhibition ofGabra4–/– DGGCs is also insensitive to 30 mMethanol. This is consistent with the severe reduction of d subunit

labeling in these cells following the loss of a4 subunits and with thereduction in the magnitude of tonic inhibition compared with wildtype37. The lack of ethanol-induced potentiation of tonic currents inGabra4–/– DGGCs indicates that there is little, if any, a1/d partnership inthese cells even after d’s normal partner, a4, has been lost. It is puzzlingwhy, in the absence of a4 subunits, the a1 subunits of DGGCs cannotpromote the membrane expression of d subunits, as the a1/d partner-ship seems to occur readily in ML interneurons. Perhaps, in DGGCs,a1 subunits are confined to a different cellular compartment or arerelative low in abundance and thus are unable to replace the missinga4 subunits to form ethanol-sensitive assemblies with d subunits.

We cannot fully exclude the pairing of d subunits with a subunitsother than a1 in ML interneurons. Our anatomical findings, how-ever, indicate that there are no detectable amounts of a2, a3 or a5subunits in these neurons. To more directly address the association ofa1/d subunits in ML interneurons, we have considered carrying outexperiments in mice deficient in GABAAR a1 subunits. We have notpursued this idea, however, because of the confounding effects of thedrastic compensatory changes in other GABAAR subunits, particularlythe a4 subunits45,46, in Gabra1–/– mice and the considerable changes inother, unrelated genes that have been described in this genotype47,48.

The lack of differences between the synaptic currents of the MLinterneurons of all of the genotypes tested indicates that a1/d-contain-ing GABAARs are exclusively localized extra- or perisynaptically and donot participate in phasic inhibition under normal conditions. More-over, because ML interneurons of Gabra4–/– mice did not show anyalterations in either phasic or tonic inhibitions, these data stronglysupport the anatomical findings that these interneurons lacka4 subunits, both inside and outside the synapses. The lack of ethanoleffects on the phasic inhibition of ML interneurons supports the notionthat the synaptic GABAARs of these cells are made up of a1bxg2combinations, similar to the ethanol-insensitive synaptic GABAARassemblies of CA1 pyramidal neurons and CGCs23,29,49,50.

In summary, we have demonstrated that ML interneurons express anovel and functional GABAAR partnership composed of a1bxd sub-units that mediates their tonic inhibitory currents and is modulated bysobriety-impairing concentrations of ethanol. The net effect of ethanol-sensitive interneurons on the hippocampal network still needs to bedetermined. Our study has focused on ML interneurons that are likelyto influence the strength of the perforant path input to the dentategyrus. Although these neurons are relatively few in number, theiraxonal plexus is extensive51. Thus, control of these interneuronsthrough an ethanol-sensitive tonic inhibition could influenceexcitability in widespread regions of the dentate gyrus. Ethanol, by

40 pA

200 ms

b

1

2

c

a

BMI

12 pA

1 2

50 s

10 pA

1 %

Furosemide 100 µM

11 pA

α1, α6

α1, α6

α6α1

α1 α6

G G G

Gabra4 –/–

Gabra4 –/–

Figure 7 Lack of compensatory upregulation of a6 subunits in ML

interneurons of mice lacking a4 GABAAR subunits. (a) Distinct

immunofluorescence labeling of a1 and a6 subunits is evident in the

cerebellum (upper) of a Gabra4–/– mouse and a strong overlap of labeling

(yellow label) is evident in the granule cell layer (G) of the cerebellar cortex.

In the dentate gyrus (lower), strong a1 labeling is present on the surface of

ML interneurons (arrows), but no a6 labeling is evident, indicating that the

a6 subunit is not being aberrantly expressed in interneurons of the dentategyrus in a4 subunit–deficient mice. Scale bars: upper, 200 mm; lower,

20 mm. (b) Voltage-clamp recordings of tonic inhibitory currents from

Gabra4–/– ML interneurons are insensitive to the a6 subunit–specific

blocker furosemide. Left, baseline current plotted at 500-ms intervals in

control (A),100 mM furosemide (B) and BMI conditions (Vh ¼ –70 mV;

horizontal bars indicate perfusion of the drugs); right, Gaussian fits to the

all-point histograms of the baseline current recorded during each condition

(numbers indicate the difference current between Gaussian means).

(c) Voltage-clamp recordings low-pass filtered at 1 KHz in control and

furosemide conditions.

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enhancing tonic inhibitory currents, could also shunt these inter-neurons, producing an altered temporal pattern of inhibition inDGGCs. The precise function of this newly described ethanol-sensitiveGABAAR partnership in the effects of ethanol, its wider distribution inthe brain, and whether other a subunits may partner with d subunits toform other naturally occurring ethanol-sensitive GABAAR assembliesare all open questions of great interest that will have to be addressed infuture studies.

METHODSImmunohistochemistry and confocal microscopy. Adult male mice (C57BL/6,

Gabra4+/+ and Gabra4–/–; generated as recently described37) were deeply

anesthetized with sodium pentobarbital and perfused transcardially with

4% paraformaldehyde in 0.1 M Na-phosphate buffer (pH 7.3). All animal

use protocols were approved by the University of California, Los Angeles

(UCLA) Chancellor’s Animal Research Committee. Following cryoprotection,

brains were sectioned at 30 mm on a cryostat, and free-floating sections were

processed for single-label immunohistochemistry with previously described

avidin-biotin-peroxidase complex (ABC) methods or double-label immuno-

fluorescence methods, which both included water bath antigen-retrieval treat-

ment9. The following previously characterized primary antisera8,10–12 were

used: guinea pig antisera to a1 (1:50,000), a2 (1:10,000) and a5 (1:3,000)

and a rabbit antiserum to a1 (1:10,000) subunits, all provided by J.-M. Fritschy;

rabbit antisera to a3 (1:1,000), a4 (1:2,000) and d (1:4,000) subunits, all

provided by W. Sieghart; rabbit antisera to a4 (AB5457,1:2,000) and a6

(AB5610, 1:15,000) subunits and a mouse antibody to GAD67 (MAB5406,1:

4,000), all from Chemicon. For single-labeling with ABC methods, the sections

were incubated in primary antisera overnight at room temperature (22–23 1C).

After rinsing, sections were incubated in biotinylated secondary antisera of the

appropriate species, all diluted 1:1,000, for 1 h. Sections were then incubated in

ABC Vectastain Elite solution, 1:200, for 1 h (Vector Laboratories), processed

with diaminobenzidene tetrahydrochloride (DAB) and mounted on slides for

light microscopic analysis.

Double immunofluorescence9 methods were used to identify ML interneur-

ons and determine their subunit expression. To determine the proportion of

GABA neurons in the ML that were labeled for the a1 subunit, we combined

immunolabeling for GAD67 with that for a1. To determine and compare the

localization of a1–6 and d subunit expression in ML interneurons, we

combined immunolabeling for a1 with either a3, a4, a6 or d labeling and

labeling for d with either a2, a4 or a5 labeling. Sections were incubated in a

mixture of primary antisera overnight at room temperature and then for 3–4 d

at 4 1C. Sections were next incubated in a solution containing goat secondary

antisera to IgGs of the appropriate species, conjugated to Alexa Fluor 488 and

Alexa Fluor 555 (both 1:1,000, Molecular Probes) at room temperature for 4 h.

After rinsing, sections were mounted on slides, coverslipped with antifade

medium (Prolong Gold; Molecular Probes) and examined with a Zeiss LSM

510 Meta confocal microscope. Confocal images were analyzed with Zeiss LSM

5 Image Examiner software.

For quantitative analysis of the proportion of GAD67-containing neurons in

the ML that express a1, two double-labeled sections from each of four mice

were analyzed. All GAD67-labeled neurons in the ML on both sides were

identified (n ¼ 300), and each of these neurons was examined for a1 labeling

by multiple-channel confocal scanning with a 40� oil objective and then

analyzed with the Zeiss software mentioned above. Quantitative analysis of

double labeling of a1 and d subunits in ML interneurons was conducted in a

similar way.

Slice preparation. Adult male (1–4 months old) C57BL/6, Gabrd–/– and

Gabra4–/– mice were studied. The Gabrd–/– (ref. 52; Jackson Labs) mice were

bred under the care of the UCLA Division of Laboratory Animal Medicine.

Gabra4–/– mice were generated at the Univ. Pittsburgh as recently described37.

Mice were anesthetized with halothane and decapitated according to a protocol

approved by the UCLA Chancellor’s Animal Research Committee. The brain

was removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) con-

taining 126 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 1.25 mM

NaH2PO4, 26 mM NaHCO3 and 10 mM D-glucose with pH 7.3–7.4 when

bubbled with 95% O2 and 5% CO2. Coronal brain slices, 350 mm thick, were

cut with a Leica VT1000S Vibratome (Leica Microsystems) in aCSF containing

3 mM kynurenic acid (Sigma). Slices were transferred to a modified interface

chamber kept at room temperature and then the temperature was gradually

increased to 30 ± 1 1C. Slices were stored in this modified interface chamber for

at least 1 h before being transferred to the recording chamber.

Whole-cell recordings. Interneurons within the middle one-third to two-thirds

of the hippocampal ML were visually identified (Olympus; BX51WI, IR-DIC

videomicroscopy; 40� water immersion objective) and recorded with an

Axopatch 200A amplifier (Axon Instruments). Slices were continuously per-

fused (B4–5 ml min–1) at 32–34 1C with 95% O2/5% CO2–bubbled aCSF-

containing kynurenic acid (3 mM) and other drugs, as indicated. Microelec-

trodes (3–5 MO when filled) contained the following internal solution:

140 mM CsCl, 1 mM MgCl2, 10 mM HEPES, 0.1 mM EGTA, 4 mM NaCl,

2 mM magnesium ATP, 0.3 mM sodium GTP, pH B7.27, B275 mOsm.

Whole-cell voltage-clamp recordings were performed at –70 mV. Series

resistance and whole-cell capacitance were estimated from fast transients

evoked by a 5-mV voltage command step using lag values of 7 ms and then

compensated to 70–80%. Recordings were discontinued if series resistance

increased by more than 25% through an experiment or the compensated

resistance surpassed 25 MO at any time during the experiment. Drugs were

perfused after a stable control recording period of at least 1 min.

Data acquisition and analysis. All recordings were low-pass filtered at 3 KHz

and digitized on-line at 10 KHz using a PCI-MIO 16E-4 data acquisition board

(National Instruments). (i) Tonic current measurement: a custom-written

macro running under IGOR Pro v5.02 was used to load the digitized recording

and to delete seal tests. An all-points histogram was plotted for every 10,000

points (every 1 s at 10 kHz sampling) and a Gaussian was fitted to the side of

the distribution not skewed by synaptic events. The Gaussian peak represented

the mean holding current over 1 s. For each second, these mean holding current

values were plotted and the resulting graph was de-trended, and the overall

mean holding current over the entire control recording period was normalized

to 0 pA. The measurement of the tonic current was determined as follows.

A 20–30 s period (200,000–300,000 points for the control and drug conditions)

and a 10–15 s period (100,000–150,000 points in the presence of bicuculline

methiodide (BMI)) were used for all-points histogram plots. The histogram

had a skewed distribution toward synaptic events. A Gaussian was fitted only to

the unskewed portion of the distribution. The mean of this Gaussian fit was

used as the value for the tonic current. In a given neuron, we obtained the

magnitude of the tonic current by subtracting the tonic current from that

recorded in the presence of BMI. (ii) Detection and measurement of sIPSCs: all

sIPSCs were detected in 30–60-s recording segments. Event frequency, 10–90%

rise time (RT10–90) and weighted decay time constant (tw) values were

measured. Tau (t) was determined by fitting an exponential function to the

average sIPSCs decay phase. Detection and analysis were performed using

custom-written LabView-based software (EVAN). All data are shown as

mean ± s.e.m. Statistical significance was assessed by paired Student t-test,

unpaired Student t-test assuming unequal variances, Wilcoxon matched-pairs

signed-ranks test, and by one-way ANOVA with a Dunnett’s post hoc when

comparing multiple variables to a control condition. The level of significance

was set at P o 0.05.

Drugs. GABA (Sigma) at a concentration of 5 mM was added to the aCSF

where indicated and slices were preincubated in this solution for B6 min

before the start of the experiment. 1-(2-{[(Diphenylmethylene)imino]oxy}

ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO-711)

and BMI were purchased from Sigma. Stock solutions of furosemide (Sigma)

were prepared in 100% DMSO and added to the aCSF where indicated (final

concentration of DMSO 0.2%).

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSWe thank W. Sieghart (Medical Univ. of Vienna, Austria) and J.-M. Fritschy(Univ. Zurich, Switzerland) for the gifts of the GABAAR antibodies, and R.W.Olsen (UCLA) for helpful discussions. This project was supported by the Gonda

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Fellowship awarded to J. G., US National Institutes of Health (NIH) GrantsNS30549, NS35958 and the Coelho Endowment to I. M., NIH Grant NS051311and US Veterans Administration Medical Research Funds to C.R.H., and NIHGrant AA14003 to G.E.H.

AUTHOR CONTRIBUTIONSJ.G. performed the electrophysiology, Z.P. carried out the immunohistochemistry,and D.C. and G.E.H. produced the Gabra4–/– mice. J.G., Z.P., C.R.H. andI.M. carried out the data analysis, study conceptualization and figurepreparation. J.G., C.R.H. and I.M. wrote and edited the manuscript.G.E.H. provided comments on the manuscript, and G.E.H., C.R.H. andI.M. provided financial support.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureneuroscience

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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