Cellular/Molecular

Intracellular Chloride Ions Regulate the Time Course ofGABA-Mediated Inhibitory Synaptic Transmission

Catriona M. Houston, Damian P. Bright, Lucia G. Sivilotti, Marco Beato,* and Trevor G. Smart*Department of Neuroscience, Physiology & Pharmacology, University College London, London WC1E 6BT, United Kingdom

The time-dependent integration of excitatory and inhibitory synaptic currents is an important process for shaping the input– outputprofiles of individual excitable cells, and therefore the activity of neuronal networks. Here, we show that the decay time course ofGABAergic inhibitory synaptic currents is considerably faster when recorded with physiological internal Cl � concentrations than withsymmetrical Cl � solutions. This effect of intracellular Cl � is due to a direct modulation of the GABAA receptor that is independent of thenet direction of current flow through the ion channel. As a consequence, the time window during which GABAergic inhibition cancounteract coincident excitatory inputs is much shorter, under physiological conditions, than that previously measured using highinternal Cl �. This is expected to have implications for neuronal network excitability and neurodevelopment, and for our understandingof pathological conditions, such as epilepsy and chronic pain, where intracellular Cl � concentrations can be altered.

IntroductionThe time course of synaptic GABAA receptor-mediated IPSC willprofoundly influence the excitability of individual neurons andnetworks, by setting the duration of a time window during whichan inhibitory input can most effectively counteract a coincidentexcitatory input.

The IPSC time course is determined by both presynaptic andpostsynaptic factors at inhibitory synapses. These include theconcentration–time profile of GABA in the synaptic cleft, whichis itself affected by the density of GABA transporters and thedegree of synchrony for presynaptic GABA release (Overstreetand Westbrook, 2003; Hefft and Jonas, 2005; Keros and Hablitz,2005), as well as several postsynaptic factors, such as the following:the presence of perisynaptic GABAA receptors, which prolong theIPSC decay phase (Wei et al., 2003), the subunit composition ofsynaptic receptors (Barberis et al., 2007), and the phosphoryla-tion state of GABAA receptor subunits (Jones and Westbrook,1997; Houston et al., 2008). Another factor, which has receivedless consideration and may also influence IPSC decays, is thenature and concentration of permeant ions through the ligand-gated ion channel. These are known to affect the kinetics ofvoltage-gated ion channels (Stanfield et al., 1981; Swenson andArmstrong, 1981), but there are very few reports of such an effecton synaptic ligand-gated channels (Marchais and Marty, 1979;Onodera and Takeuchi, 1979; Schneggenburger and Ascher,

1997). Indeed, for GABA ion channels, such a modulation by Cl�

would not have been readily observed before, as most patch-clamp studies of synaptic inhibition routinely use matching highinternal and external Cl� concentrations to set the Cl� reversalpotential near to zero. This is convenient because it increases theamplitude of the IPSC at the negative holding potentials that aresuitable for whole-cell recording.

Here, we report that intracellular Cl�, by acting directly onthe GABAA receptor, critically affects the GABAergic IPSC decayphase at interneuron–Purkinje cell inhibitory synapses in the cer-ebellum. At low Cl� concentrations, typically approaching thosefound in neurons (�5–10 mM), the IPSC decay phase is rapid,and the duration of the IPSC shorter. We found that by incre-mentally raising the internal Cl� concentration, the IPSC decaytime was proportionately increased. Thus, variations of the intra-cellular Cl� concentration, following physiological or patho-physiological processes, will have a crucial impact on the IPSCprofile with important implications for the dynamics of inhibi-tory transmission throughout the CNS.

Materials and MethodsPreparation of slices. Thin sagittal slices (250 �m) were cut from thecerebellum of postnatal day 12 Sprague Dawley rats using a LeicaVT1200s vibroslicer, in accordance with the UK Animals (ScientificProcedures) Act of 1986. Slices were cut in an artificial CSF (aCSF)solution containing (in mM) 125 NaCl (or 85 NaCl, 75 sucrose), 2.5KCl, 1.25 NaH2PO4, 26 NaHCO3, 1 CaCl2, 5 MgCl2, and 11 glucose,gassed with 95% O2/5% CO2 at 4°C. The slices were then incubated at35°C for 45 min before being allowed to cool to room temperature(23°C) before use. Over this time the solution was slowly changed tostandard aCSF and slices were perfused in the recording bath at roomtemperature containing (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4,26 NaHCO3, 2 CaCl2, 2 MgCl2, and 11 glucose, gassed with 95%O2/5% CO2 (Duguid and Smart, 2004; Bright et al., 2007). For thebicarbonate-free experiments, the ACSF contained (in mM) 135 NaCl,2.5 KCl, 1.25 NaH2PO4, 20 HEPES, 2 CaCl2, 2 MgCl2, and 11 glucose(Kaila et al., 1997), gassed with 100% O2. To block NMDA and AMPA

Received April 7, 2009; revised June 2, 2009; accepted June 5, 2009.This work was supported by the Medical Research Council (UK) to T.G.S. and L.G.S., and by a Royal Society

University Research Fellowship and Wellcome Trust support to M.B. We thank Angus Silver and Jason Rothman forthe loan of the dynamic clamp amplifier. After completion of our study, we learned that unpublished observationssimilar to those described in the present paper were independently made by Joel Charvas, Christophe Pouzat, andAlain Marty.

*M.B. and T.G.S. contributed equally to this work.Correspondence should be addressed to Prof. Trevor G. Smart, Department of Neuroscience, Physiology & Phar-

macology, University College London, London WC1E 6BT, UK. E-mail: t.smart@ucl.ac.uk.DOI:10.1523/JNEUROSCI.1670-09.2009

Copyright © 2009 Society for Neuroscience 0270-6474/09/2910416-08$15.00/0

10416 • The Journal of Neuroscience, August 19, 2009 • 29(33):10416 –10423

receptor activation, 20 �M AP-5 and 10 �M CNQX were added to thestandard aCSF.

Expression of recombinant receptors in HEK293 cells and fast concentrationjumps. HEK293 cells were transfected 6 h after plating using the calciumphosphate precipitation method with 4 �g of DNA of ��� GABAA re-ceptor subunits and EGFP in equimolar ratios per 35 mm dish. Cells werebathed in a solution composed of (in mM) 102.7 NaCl, 20 Na gluconate,2 KCl, 2 CaCl2, 1.2 MgCl2, 10 HEPES, 14 glucose, 15 sucrose, and 20TEA-Cl, pH adjusted to 7.4 with NaOH (osmolarity �320 mOsm). Re-cordings were obtained in the outside-out patch configuration usingeither a high-Cl � pipette solution (107.1 KCl, 1 CaCl2, 1 MgCl2, 10HEPES, 11 EGTA, 20 TEA-Cl, and 2 MgATP) or a low-Cl � solution(121.1 K gluconate, 1 CaCl2, 1 MgCl2, 10 HEPES, 11 EGTA, 6 TEA-Cl,and 2 MgATP). Concentration jumps were performed using a theta tube(outer diameter 2 mm, inner diameter 1.7, septum 0.117, 14-072-01,Hilgenberg) whose tip was cut with a diamond pen to a final diameter of�150 �m. The theta tubes were filled with control solution, and 3 mM

GABA was added to one of the two barrels. Fast drug applications wereperformed by delivering a short (1–2 ms) voltage pulse to a piezo-stepper(Burleigh Instruments). The exchange time was measured by applying a20% diluted solution to the open tip of the recording pipette after rup-ture of the patch and measuring the resulting junction potentials. Exper-iments were included in the analysis if the exchange time was faster than150 �s. A minimum of 10 individual responses at five different voltages(from �100 to �60 mV in 40 mV steps) were averaged and fitted with amixture of two or three exponentials. Weighted time constants are re-ported throughout the text.

Electrophysiology. Whole-cell patch-clamp recordings were made fromsingle cells identified by differential interference contrast microscopy(Nikon Eclipse E 600FN) using a MultiClamp 700A amplifier in thevoltage-clamp configuration (Molecular Devices). Patch pipettes(1.5–2 M�) were filled with solutions containing the following (inmM): 140 CsCl (150 mM Cl �), 140 Cs-gluconate (10 mM Cl �), or 20CsCl and 120 Cs-gluconate (30 mM Cl �); and 4 NaCl, 0.5 CaCl2, 5EGTA, 10 HEPES, 2 Mg2ATP, and 5 QX-314-Cl, pH adjusted to 7.3with CsOH. The osmolarity was adjusted to 310 mOsm with sucrose.To make a 5 mM Cl � internal solution, 4 NaCl was replaced with 4Na-gluconate and 4 QX-314-Cl.

Currents were filtered at 3 kHz (Bessel filter), digitized at 20 kHz andanalyzed using Clampex 8.2 (Molecular Devices). Series resistance aftercompensation was typically between 2 and 3 M� (�1 M� for experi-ments at 37°C) and was constantly monitored throughout recording.Cells were not used for analysis if their series resistance changed by �10 –15%. The capacitance of a typical Purkinje cell at this age was 355 � 37 pA

(n � 7), and cells that deviated from this by�20% were not used for analysis. Cells werecontinuously superfused with CNQX (10 �M)and AP-5 (20 �M) to block glutamate-mediated fast synaptic transmission.

The stimulating electrode was a patch elec-trode filled with aCSF positioned within thelower third of the molecular layer, �150 �mlateral to the Purkinje cell body (Mittmann andHausser, 2007) to preferentially stimulate bas-ket cells that form a GABAergic synapse closeto the soma at the axon initial segment (Angoet al., 2004). Stimulation was applied througha constant current stimulator (DS3, Digi-timer) and gradually increased until anevoked IPSC (eIPSC) could be detected inthe recorded Purkinje cell. This stimulationwas increased until the amplitude of theeIPSC increased substantially presumablydue to recruitment of more basket cells. Thestimulation intensity was then set at �1.5times the threshold, and only cells wherethere was a clear separation between thestimulation transient and the rising phase ofthe eIPSC were used.

Data analysis. The decay times of IPSCs weredetermined in Clampfit 8.2 by fitting a single exponential to the (90 –10%) decay phase. At each holding potential, individual IPSCs were ex-amined and those with any deflection in the rising or decaying phaseswere deleted, as were any failures, before fitting the mean IPSC. AveragedIPSCs included �30 –50 individual sweeps. In cumulative distributionplots the individual decay times of each IPSC were plotted and normal-ized between cells. Spontaneous (s)IPSCs were analyzed using Mini-Analysis (version 6.0.1, Synaptosoft) and were also fitted with a singleexponential (Houston et al., 2008). IPSC events were selected for analysisif there were no deflections in their rising or decaying phases and theydecayed back to the baseline holding current. Events of low amplitude(�10 pA) and slow rise times (10 –90% � 3 ms) were discarded from thisanalysis as they were thought to represent events occurring at more dis-tant synapses and therefore more likely to be affected by dendritic cabling(Llano et al., 2000).

Dynamic clamp experiments. Current-clamp recordings from Purkinjeneurons used a standard intracellular solution composed of (in mM) 130KCl, 10 HEPES, 1 CaCl2, 4 NaCl, 5 EGTA, and 4 MgATP, pH 7.3 withKOH. A GABAergic conductance was simulated with a function of thefollowing form:

y � A � �1 � e�t�t0

�rise� � � e�t�t0

�decay� ,

where t0 represents the start of the waveform, �rise was 1.8 ms (deter-mined from our eIPSC recordings), and �decay was either 8.5 ms (simu-lating low Cl � conditions) or 12.5 ms (replicating our decays in highCl � at �60 mV, data not shown). This waveform was injected through adynamic clamp amplifier (SM-1, Cambridge Conductance) setting thereversal potential to �85 mV (Chavas and Marty, 2003) and a conduc-tance of 15 nS. Trains of spikes were evoked by constant current injectionand superimposed with either a fast or a slow conductance. Excitatory(AMPA-like) conductances had the same functional profile as those usedfor simulating inhibition, but with a rise time of 0.3 ms and a decay timeof 3 ms. The value of the conductance was adjusted to be 10 –20%above the threshold for firing (typically 12–18 nS). The inhibitory con-ductance (either fast or slow) was injected with a variable latency withina 120 ms window and repeated at 4 ms intervals.

Statistics. For the statistical comparison of two means, an unpaired,two-tailed Student’s t test was used, or the nonparametric alternative,Mann–Whitney test, if there was a significant difference in the group SDas determined by an F test. With all statistical tests, two means were

Figure 1. Reducing intracellular Cl � concentration decreases the decay time constant of evoked IPSCs. A, Consecutively evoked(gray) and averaged (black) eIPSCs recorded at �100 mV from Purkinje cells in an acute cerebellar slice using: 5, 10, 30, or 150 mM

internal Cl � solutions. B, Cumulative distributions for eIPSC decay times measured at �100 mV in Purkinje cells recorded with 5,10, 30, or 150 mM internal Cl �. C, Bar chart of the decay-time constants for eIPSCs at �100 (n � 13) and 20 mV (n � 6) holdingpotentials with high (black, 150 mM) or low (gray, 10 mM) internal Cl � concentrations (means � SEM, *p � 0.05). D, Consecutive(gray) and averaged (black) eIPSCs, recorded with either a 10 or 150 mM internal Cl � solutions at 20 mV.

Houston et al. • GABA-Mediated Synaptic Inhibition and Internal Cl� J. Neurosci., August 19, 2009 • 29(33):10416 –10423 • 10417

considered significantly different if p � 0.05.Statistical tests were performed using Graph-Pad Instat version 3.01.

ResultsEvoked, spontaneous, and miniatureIPSC decay times are affected byinternal Cl � concentrationTo determine whether the intracellularCl� concentration affects the kinetics ofIPSCs, we recorded evoked and spontane-ous IPSCs in cerebellar Purkinje cells,which receive inhibitory afferents frombasket and stellate cells in the molecularlayer. We used several different internalCl� concentrations, from the high sym-metrical Cl� frequently used in most elec-trophysiological experiments, to the lowintracellular Cl� normally found in ma-ture Purkinje neurons (Chavas andMarty, 2003); IPSCs were evoked by ap-plying focal stimulation from a patch pi-pette filled with aCSF positioned in thelower third of the molecular layer. At aholding potential of �100 mV, the eIPSCdecay was fit with a single exponential(Fig. 1A) (Puia et al., 1994), with a timeconstant, �, that visibly increased with theinternal Cl� concentration (Fig. 1A,C).This consistent change in the decay timeswith the internal Cl� concentrations wasclearly evident from the lateral shifts in thecumulative distribution for IPSC decaytime constants (Fig. 1B). By reducing in-ternal Cl� from 150 to 10 mM, the median� was reduced from 10.2 to 6.5 ms at �100mV (150 mM: mean � � 10 � 0.5 ms; 10mM: � � 6.6 � 0.2 ms, p � 0.0001, Mann–Whitney test, n � 13), and from 20.5 to 14ms at 20 mV (150 mM: mean � � 20.7 �1.0 ms; 10 mM: � � 14.5 � 1.2 ms, p �0.004, Mann–Whitney test, n � 6) (Fig.1C). At �100 mV, increasing internal Cl�

over a physiologically relevant range from5 to 30 mM, caused � to increase by �59%.

By comparing the eIPSC decay timeconstants at different holding potentials,it was apparent that the IPSC decays were also voltage depen-dent (Fig. 1C,D) [cf. Collingridge et al. (1984) and Otis andMody (1992)]. The ratio of � at �20 mV and �100 mV (�20/�100)was 2.10 � 0.19 with symmetrical Cl� (mean � SEM; n � 6) and2.35 � 0.04 (n � 5) with low internal Cl �, suggesting that thevoltage dependence of the IPSC decay was unaltered by theCl � concentration. In marked contrast, at �100 mV, � wasconsistently reduced by lowering the internal Cl� concentration[�100 mV: 9.7 ms (150 mM Cl�), 8.9 ms (30 mM), 6.3 ms (10 mM

Cl�), 5.6 ms (5 mM)], a feature also noted at 20 mV [20.4 ms (150mM), 13.9 ms (10 mM); n � 5– 6]. Given that internal Cl� af-fected the speed of the eIPSC decay at �100 and 20 mV to asimilar extent, it is clear that this regulation must be independentof the net direction of current flow (Fig. 1D).

We also examined whether the internal Cl� concentrationsimilarly affected spontaneous IPSCs (sIPSCs). In accord with the

eIPSC data, the decay profiles of sIPSCs were described by singleexponentials and displayed a similar voltage sensitivity from�100 mV to �20 mV, with � increasing with membrane depolar-ization (Fig. 2A–D). Recording from Purkinje cells with 150 mM

internal Cl� concentrations the �20/�100 ratio was �2.33 � 0.14(n � 6) (Fig. 2E), and remained constant at 10 mM Cl� concen-tration (2.64 � 0.13, n � 6) (Fig. 2E). As observed from experi-ments with eIPSCs, a low internal Cl� concentration (10 mM),reduced the mean decay time constant for sIPSCs atall holding potentials examined [�100 mV: 9.2 � 0.6 ms (150mM Cl�), 6.4 � 0.5 ms (10 mM Cl�); 20 mV: 21.0 � 1.1 ms (150mM Cl�), 16.6 � 0.6 ms (10 mM Cl�), p � 0.009 at �100 and20 mV, Mann–Whitney test, n � 6] (Fig. 2E). The displacementsin the cumulative distributions for sIPSC decay times also re-vealed a consistent effect of the low Cl� concentration in reduc-ing the median decay time constant [�100 mV, 9.1 ms (150 mM

Figure 2. Intracellular Cl � regulates the decay phase of spontaneous IPSCs. A, sIPSCs (gray) and averaged sIPSC (black)recorded from Purkinje cells with 150 mM Cl � internal solution at �100 mV. The averaged and peak-scaled sIPSCs recorded at�100 and 20 mV (sIPSCs are inverted) are overlaid (inset). B, The same protocol as in A for individual and averaged sIPSCs recordedat 20 mV. C, sIPSCs recorded using 10 mM Cl � in the patch pipette (gray lines), at �100 mV. Averaged and peak-scaled sIPSCs at�100 and 20 mV are overlaid (inset). D, sIPSCs clamped at 20 mV (10 mM Cl �). The scale bar in B applies to all. E, Bar chart of meansIPSC decay-time constants at different holding potentials with high (black) or low (gray) internal Cl �. F, Cumulative probabilityplots for sIPSC decay times at �100 mV and �20 mV with high (black) and low (gray) Cl �.

10418 • J. Neurosci., August 19, 2009 • 29(33):10416 –10423 Houston et al. • GABA-Mediated Synaptic Inhibition and Internal Cl�

Cl�), 6.2 ms (10 mM Cl�); 20 mV: 21.9 ms (150 mM Cl�), 15.9ms (10 mM Cl�); n � 6] (Fig. 2F).

In accord with our results with eIPSCs and sIPSCs, internalCl � also regulated the decay times of miniature IPSCs(mIPSCs). By reducing internal Cl�, the mIPSC decay time con-stant measured at �100 mV was reduced from � (high Cl�) �9.6 � 0.8 ms to � (low Cl�) � 6.8 � 0.4 ms (n � 6; p � 0.018).Similarly, at a holding potential of �20 mV, � (high Cl�) wasreduced from 23.1 � 0.6 ms and � (low Cl�) to 16.5 � 0.7 ms(n � 6; p � 0.0003; unpaired t test).

The increased decay rate for IPSCs recorded with low internalCl� is expected to increase further when the recording tempera-ture is raised from 23°C to 37°C. When eIPSCs were recorded atphysiological temperatures from Purkinje neurons held at �80mV, reducing the internal Cl� from 150 to 10 mM also reducedtheir median decay time constant from 2.7 ms to 1.9 ms ( p �0.01; Mann–Whitney; n � 6 – 8) (Fig. 3A,B). The cumulativedistribution for the decay times (Fig. 3C) indicated that the me-dian � for 10 mM Cl� at 37°C approached the limit we can effec-tively resolve with the level of low-pass filtering imposed by thecombination of whole-cell capacitance and series resistance. It isfor this reason that most of the subsequent experiments wereconducted at room temperature.

IPSC decay times are unaffected by HCO3� permeation

through GABA channelsAlthough Cl� is the major permeating anion through GABAchannels, it is known that HCO3

� ions also have an appreciablepermeability through synaptic GABA channels in mammalianneurons (Bormann et al., 1987; Kaila et al., 1993), with a PHCO3

/PCl ratio of �0.2 (Kaila, 1994). Whether the internal HCO3

�

concentration was also important for theregulation of the IPSC decay time was ex-amined by comparing eIPSCs recordedfrom Purkinje neurons bathed in aCSF orin an O2-gassed/HEPES-based externalsolution to create a nominally HCO3

�-free solution (Fig. 4A–D). In the HCO3

�-free, 10 mM Cl� solution, the amplitudesof the eIPSCs were reduced to 65.8 �7.5% of their value compared in normalaCSF ( p � 0.05, unpaired two-tailed ttest, n � 6 – 8).

However, by using an 150 mM internalCl� concentration, eIPSC amplitudes re-mained unaffected by switching to anHCO3

�-free aCSF (95.2 � 9.8%; p � 0.5,n � 6 – 8). This reflected the relative con-tributions of HCO3

� and Cl � ions to theIPSC and the lower permeability ofHCO3

� through the GABA channel. Thedecay time constant was again reduced, asexpected, by low internal Cl� concentra-tions in normal aCSF (Fig. 4E) (150 mM

Cl�, median � 8.9 ms; 10 mM Cl� me-dian � 6.3 ms). The extent of the reduc-tion in eIPSC decay times by low internalCl� remained unaffected by the HCO3

�-free solution (150 mM Cl�, median � 9.5ms; 10 mM Cl� median � 6.3 ms) (Fig.4E), indicating that as a permeant ion,only Cl� are necessary and sufficient toregulate the decay phase of the eIPSC.

Low Cl � regulates IPSC decay times by directly affectingGABAA receptor functionTo determine whether the Cl� modulation is intrinsic to thesynaptic GABAA receptor, rather than mediated by receptor-associated molecules located at inhibitory synapses (Luscher andKeller, 2004), we expressed �1�2�2L subunits in human embry-onic kidney (HEK) cells. This combination of subunits waschosen to replicate the likely composition of synaptic GABAA

receptors in Purkinje cells (Laurie et al., 1992; Fritschy andMohler, 1995). Outside-out patches were briefly exposed to sat-urating concentrations of GABA (3 mM, 1 ms) to emulate theGABA concentration transient in the synaptic cleft (Jones andWestbrook, 1995). With either 150 mM (Fig. 5A,B) or 10 mM

(Fig. 5C,D) internal Cl� concentrations, the GABA-activatedcurrent (IGABA) decays were best fit by two or three exponentialcomponents. Over a patch potential range from �100 mV to �60mV, the weighted �decay for IGABA increased monotonically in asimilar manner to the decay time constants for evoked and spon-taneous IPSCs. The weighted �decay for inward IGABA at �100 mVwas significantly reduced by 10 mM intracellular Cl� from 54.5 �5.8 (n � 10) to 21.1 � 2.9 ms (n � 10, p � 0.0002, two-tailed ttest) (Fig. 5E). A similar reduction in �decay was observed (from75.4 � 13.6 to 40.9 � 8.3 ms, p � 0.04) when the same patcheswere held at 60 mV causing the direction of the GABA-activatedcurrents to become net outward. The consistent effects of internalCl� concentration on the current decays mediated by both syn-aptic and recombinant GABAA receptors, with their similar sub-unit composition, strongly indicates that Cl� is modulating thereceptor directly, rather than acting via receptor-associated mol-ecules. However, while elements of the postsynaptic density will

Figure 3. Effect of low internal Cl � on IPSC decays at 37°C. Consecutively evoked (gray) and averaged eIPSCs (black) recordedat 37°C from cerebellar Purkinje cells held at �80 mV with a 150 mM (A) or 10 mM (B) internal Cl � solution. C, Cumulativeprobability relationships for eIPSC decay time constants at �80 mV and 37°C with 150 mM (black) and 10 mM (gray) internal Cl �

(n � 6 – 8).

Houston et al. • GABA-Mediated Synaptic Inhibition and Internal Cl� J. Neurosci., August 19, 2009 • 29(33):10416 –10423 • 10419

not be present in HEK cells, it is conceiv-able that some remnants of receptor-associated molecules, such as proteinkinases, may be present in outside-outpatches and these might be affected byCl� concentration thereby affecting mac-roscopic GABA current decays. To ad-dress this, we included the broad spec-trum serine/threonine kinase inhibitor,staurosporine (500 nM), in the patch pi-pette and repeated the rapid GABA appli-cations in the presence of low and highCl� concentrations. The weighted �decay

for inward IGABA at �100 mV was againsignificantly reduced by 10 mM internalCl� from 45.1 � 2.7 (n � 6) to 20.1 � 3.3ms (n � 5, p � 0.0004, unpaired t test),yielding a �(high Cl)/�(low Cl) ratio of2.3, which is very similar to that measuredin the absence of staurosporine (2.5).Thus internal Cl� is most likely modulat-ing the receptor by a direct action ratherthan through receptor-associated signal-ing molecules.

Implications of fast-decaying IPSCs forsynaptic inhibitionGiven that the physiological Cl� concen-tration in an adult neuron is lower thanwe routinely use in our symmetrical Cl�

solutions, it is likely that the real profile ofan IPSC in an intact cell is much shorterthan we previously thought. Therefore,does the speed of the IPSC decay, and thecorresponding effect this has on the timewindow for inhibition, reduce the effec-tiveness of synaptic inhibition in control-ling neuronal excitability? This questionwas addressed by recording from Pur-kinje cells under current-clamp condi-tions and then injecting inhibitory conductances using a dynamicclamp amplifier. We evoked a continuous spike train in the Pur-kinje cell with a constant current pulse (�200 pA) before intro-ducing an artificial inhibitory conductance to inhibit the train.To reproduce the IPSCs expected in a Purkinje cell with eitherhigh (150 mM) or low (10 mM) internal Cl� concentrations, theconductance decay time constants were set to 12.5 and 8.5 ms,respectively (Fig. 6A). Action potentials ceased immediately afterthe onset of the artificial inhibitory conductance, and the latencyto the first spike was significantly increased when the injectedconductance had a longer decay time (average first latencychanged from 41 � 2 to 54 � 4 ms, n � 9) (Fig. 6A,B).

As an alternative approach to assess the impact of the ki-netics of inhibition on cell excitability, we injected a briefexcitatory conductance into the soma of the Purkinje cell undercurrent clamp to emulate the activation of synaptic AMPA-typeglutamate receptors at climbing fiber excitatory synapses and ini-tiate action potential firing (Traynelis et al., 1993). An inhibitoryconductance with either slow or fast kinetics (based on the in-ternal Cl � concentration) was also introduced and its latencyvaried so that the inhibitory conductance was active over a timewindow that encompassed the excitation. A slow inhibitory con-ductance, which preceded an excitatory conductance, inhibited

spike firing within a time window of �37 ms (Fig. 6C,D). For thefaster inhibitory conductance, typical of neurons exposed to lowinternal Cl�, this window is significantly reduced by over half to15 ms.

DiscussionInternal Cl � regulates IPSC decay by a direct effect on theGABAA receptorWe have demonstrated here, for the first time, that the durationof GABAergic synaptic inhibition is strongly influenced by theintracellular concentration of Cl�, the GABAA receptor’s majorpermeating anion. Indeed, by studying a range of internal Cl�

concentrations, we observed that the IPSC decay time constantwas clearly affected by changes to the Cl� concentration over arange that is considered to be physiologically relevant (e.g., be-tween 5 and 30 mM).

It is important to note that GABAA receptor channels are alsopermeable to HCO3

� ions (Kaila et al., 1993), and it is conceivablethat these might have emulated or supplemented the effect ofinternal Cl� on the IPSC decay times, by competing with Cl�

ions for the same binding site(s) on the receptor. Under ourrecording conditions, using aCSF with a partial pressure of CO2

of �37.5 mmHg, the intracellular HCO3� concentration lies be-

tween 8 and 13 mM, at an intracellular pH of 7.2–7.4. This implies

Figure 4. Bicarbonate ions do not affect the IPSC decay time constant. A, In the presence of bicarbonate ions, contained innormal aCSF, consecutively evoked (gray) and averaged eIPSCs (black) were recorded from cerebellar Purkinje cells with 150 mM

internal Cl � solution at �100 mV. B, Evoked IPSCs recorded from the same cell after exchanging aCSF with HCO3�-free solution

(100% O2/HEPES buffered). C, Consecutive (gray) and averaged (black) eIPSCs with 10 mM internal Cl � solution at �100 mV. D,Evoked IPSCs recorded from the same cell after exchange with HCO3

�-free aCSF. E, Cumulative distribution for eIPSC decay timeconstants at �100 mV with 150 mM (black) and 10 mM (gray) internal Cl � recorded in normal aCSF (dashed lines) and HCO3

�-freeaCSF (solid lines).

10420 • J. Neurosci., August 19, 2009 • 29(33):10416 –10423 Houston et al. • GABA-Mediated Synaptic Inhibition and Internal Cl�

the equilibrium potential for HCO3� is between �18 and �31

mV. Therefore, the maximum contribution of HCO3� to the

eIPSC will occur when we are recording with low internal Cl� at�100 mV. However, even under these conditions, the removal ofHCO3

� ions did not affect the decay time constant, indicating thatCl�, in the context of permeant ions, is the major determinant ofthe IPSCs decay kinetics.

A link between the effectiveness of inhibition and internalCl � has been noted previously from early intracellularcurrent-clamp recordings of CA1 pyramidal neurons using 3 M

KCl and 2 M KCH3SO4-filled electrodes (Alger and Nicoll, 1979).A prolonged time course of the depolarizing IPSP was observed inneurons where Cl� leakage had occurred from the KCl pipette.Although the duration of synaptic potentials is also dependent onthe membrane time constant, most likely this prolongation re-flected the phenomenon of internal Cl� affecting IPSC decaysthat we report in the present study.

There are several mechanisms by which Cl� could regulate thedecay phase kinetics of the IPSC. One possibility is that high

internal Cl� prolongs the IPSC decay byslowing the uptake of GABA by the prin-cipal neuronal GABA transporter sub-type, GAT-1 (Guastella et al., 1990),which is thought to have a stoichiometryof 2 Na�:1 GABA:1 Cl� (Cammack etal., 1994). This is unlikely to account forour findings because GABA current kinet-ics also became slower with high internalCl� in excised outside-out patches whereGABA is applied by a rapid applicationsystem and GABA transport plays no rolein the current decay process. This resultalso indicated that the regulation by inter-nal Cl� must be an intrinsic property ofthe GABAA receptor and does not dependon intracellular signaling mechanismsand on receptor-associated molecules,many of which, such as gephyrin, are notexpressed in HEK cells (Thomas andSmart, 2005).

The results obtained with the recombi-nant GABAA receptors suggest that one ormore sites must exist on the receptors thatare capable of binding Cl�. There appearto be no clear structural consensus se-quences for Cl� binding sites (Plested andMayer, 2007), but as internal Cl � regu-lates the IPSC decay, such sites are mostlikely to be found within the intracellu-lar domains between M1 and M2, or be-tween M3 and M4, or conceivablywithin the ion channel and its associatedinternal vestibule. Some support for thelatter structure as a potential site(s) comesfrom prior studies of acetylcholine recep-tors in Aplysia neurons, where the con-centration of the permeating ion wasdirectly related to the mean channel opentime, and occupancy of such a site(s) inthe ion channel was postulated to prolongthe channel open duration (Ascher et al.,1978; Marchais and Marty, 1979).

Physiological impact of regulating the speed ofsynaptic inhibitionWe predict that the regulation of GABAA receptor function byinternal Cl� is likely to have significant consequences for neuro-nal excitability, because the duration over which an inhibitoryinput can effectively counteract coincident neuronal excitation isappreciably shorter with low physiological levels of Cl� com-pared with that observed with symmetrical high Cl�. This isfundamentally important, as intracellular Cl� concentrations aresubject to alteration by the density and activity of Cl� transport-ers (DeFazio et al., 2000), which will influence IPSC time profilesand thereby signal integration.

Our finding that the kinetics of GABAA receptor currents aremodulated by internal Cl� ions has many implications for ourunderstanding of synaptic inhibition. It is clear that we have beenunderestimating the speed of inhibitory transmission. This hasoccurred because of the routine use of symmetrical Cl� record-ing solutions to depolarize ECl close to 0 mV, to ensure that theIPSCs are of large amplitudes for ease of measurement when

Figure 5. Recombinant �1�2�2L GABAA receptor kinetics are modulated by intracellular Cl �. Shown are currents activatedby 3 mM GABA (1 ms pulse) recorded from outside-out patches of HEK cells expressing GABAA receptors. A, GABA currents recordedat �100 to 60 mV (40 mV steps, average of 10 sweeps per voltage) with 150 mM Cl � in the pipette solution decay more slowly atpositive potentials. Inset, An I–V plot for GABA-activated currents (Erev is 7 mV). B, Peak-scaled, superimposed currents at �100and 60 mV. Note the currents recorded at �100 mV have been flipped to match those at 60 mV. C, The same experiment repeatedwith 10 mM intracellular Cl �. Currents reverse at �56 mV (inset). The relative voltage dependence of the weighted decay timeconstant is maintained (compare peak-scaled currents in D), but at all membrane voltages, the decay time is 2–3 times faster in lowCl � (average of 12 sweeps). E, Summary of weighted decay times in 150 mM (black) and 10 mM (gray) Cl � at all the voltagestested. The calibrations in A and B apply to the bars in C and D, respectively.

Houston et al. • GABA-Mediated Synaptic Inhibition and Internal Cl� J. Neurosci., August 19, 2009 • 29(33):10416 –10423 • 10421

holding cells near to their resting membrane potentials. By usingphysiological concentrations of internal Cl�, the shorter timewindow now available for synaptic inhibition will reduce its effecton cell excitability with consequences for synaptic plasticity andpresumably the modeling of neuronal network behavior. Indeed,at physiological body temperatures, the real durations of IPSCsare so short that they now approach 2–3 ms, and therefore be-come difficult to resolve accurately with the level of filtering im-posed by the recording apparatus.

Furthermore, the kinetics of IGABA for recombinant GABAA

�1�2�2L receptors expressed in HEK cells exhibited a similarsensitivity to the internal Cl� concentration. This combinationof GABAA receptor subunits is considered to account for atleast 30% of inhibitory synaptic GABAA receptors in the CNS(Whiting et al., 1995), indicating that their regulation by internalCl�, may well be a universal feature of many inhibitory synapsesacross the CNS. Indeed, a similar phenomenon is also apparentfor the glycine receptor (Pitt et al., 2008), indicating that regula-tion by internal Cl� may be an intrinsic property of all ligand-gated anion channels in the Cys-loop superfamily.

The regulation of the speed of IPSC decays may also berelevant to neurons during postnatal development. The higherinternal Cl� concentrations that characterize this stage of neuro-development (Ben-Ari et al., 2007) would allow GABAA receptoractivation to initiate more prolonged depolarization, eventually

exceeding the threshold for voltage-gated calcium channel acti-vation (Cherubini et al., 1991). Even for more mature neuronswith lower basal Cl� concentrations, the prolonged activation ofGABAA receptors in structurally small dendrites can eventually leadto Cl� loading and membrane depolarization, which is exacerbatedby HCO3

� efflux (Staley and Proctor, 1999). Under these conditions,the time course of the synaptic GABA currents would again be pro-longed with consequences for neuronal excitability.

It has also been reported that ECl can vary between the den-drites and the soma, affecting synaptic inhibition in the sameneuron (Duebel et al., 2006; cf. Glickfeld et al., 2009). We wouldpredict that the time course of, and therefore the time windowfor, synaptic inhibition will be shorter, where the Cl� concentra-tion is lowest and thus IPSCs with dendritic and somatic originscould have different time courses in the same cell. This newlydiscovered role for Cl� will have important implications not onlyfor how synaptic inhibition regulates the activity of neuronalnetworks, but also for understanding how alterations in internalCl� affect early neuronal development (Ben-Ari, 2002) and theprogression of pathophysiological conditions, such as epilepsy(Huberfeld et al., 2007) and chronic pain (Coull et al., 2003).

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Figure 6. Decay times of inhibitory synaptic conductances profoundly affect Purkinje neuron excitability. A, Constant current pulse (1 s, 200 pA) evoked spike firing in a Purkinje neuron with slow(12.5 ms decay time, black) and fast (8.5 ms decay time, blue) inhibitory conductances (Erev ��82 mV) (Chavas and Marty, 2003) injected through a conductance clamp amplifier during the train.The fast conductance is reflected by the shorter inhibition time window [see expanded timescale, right panel; 99 (fast conductance) and 102 (slow) overlaid sweeps at �65 mV]. B, The cumulativedistribution for first spike latencies after conductance injection is significantly displaced to the right for longer-lasting conductances (n �9, time course of the conductance injection is shown below).C, Injecting an excitatory conductance (Erev � 0 mV, �rise � 0.3 ms, �decay � 3 ms, conductance fixed at 20% above threshold, typically 12–18 nS) to induce a single spike with a concurrent slidinginhibitory conductance (fixed at 15 nS) with either slow (left, black) or fast (right, blue) kinetics. Sweeps are separated by 4 ms, and those shown in the top panel correspond to conductances depictedas thick lines in the lower panel. The cell is prevented from firing by the inhibition time window, which is reduced from �50 to �20 ms for the fast conductance injection (measured over 50 – 60trains of injections, alternating between fast and slow conductance injection). D, Summary of the duration of the inhibition windows for fast (blue) and slow (black) conductance injections.

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Houston et al. • GABA-Mediated Synaptic Inhibition and Internal Cl� J. Neurosci., August 19, 2009 • 29(33):10416 –10423 • 10423