Functional diversity of GABA activated Cl− currents in Purkinje versus granule neurons in rat cerebellar slices

Neuron,

Vol. 12, 117-126,

January,

1994, CopyrIght

0 1994 by Cell Press

Functional Diversity of GABA-Activated Cl- Currents in Purkinje versus Granule Neurons in Rat Cerebellar Slices Ciulia Puia, Erminio Costa, and Stefano Vicini FIDIA Georgetown Institute for the Neurosciences Georgetown University School of Medicine Washington, D.C. 20007

Summary In rat cerebellar slices, we compared wholecell recordings of spontaneous inhibitory postsynaptic currents (sIPSCS) with Cl- currents resulting from pulses of GABA (1 mM, <2 ms) to outside-out patches from Purkinje and granule neurons. slPSCs in Purkinje cells decayed with a single fast exponential, as previously reported, whereas in granule cells slPSC decay was best described by the sum of a fast and a slow exponential curve, with a variable contribution of the slow component to the peak current. CABA pulses to nucleated patches from granule cells elicited Cl- currents with de cays similar to slPSC decays, whereas in patches from Purkinje neurons GABA pulses produced Cl- currents decaying largely with a fast component, but often followed by a slower exponential. CABA concentration steps produced rapidly desensitizing currents in patches from both cerebellar neurons. In distinct cerebellar neurons, specific functional properties of CABAA receptors may relate to the presence of distinct receptor subtypes. Introduction yhminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian CNS, hyperpolarizes neuronal membranes byopeningacl-channel intrinsic to the GABAA receptor (Sivilotti and Nistri, 1991). This receptor is a hetero-pentameric complex that contains two allosteric modulatory sites for clinically relevant drugs (Costa, 1989; Sieghart, 1992). Molecular cloning studies have established that 16 genes encode the subunits assembled in GABAA receptors (reviewed in Olsen and Tobin, 1990, and Burt and Kamatchi, 1991). The combinatorial manner by which this large subunit diversity is assembled to form GABAA receptors suggests that a high number of structurally different receptor subtypes may exist in vivo. This great heterogeneity might correspond to functionally distinct postsynaptic receptors in inhibitory synapses. A number of studies have investigated the functional properties of GABAA receptors at inhibitory synapses by studying spontaneous and evoked inhibitory postsynaptic currents (IPSCs) in voltage-clamped neurons. Among these studies Cl--mediated IPSCs were described in hippocampal, cortical, and cerebellar neurons in primary culture (Segal and Barker, 1984; Vicini et al., 1986; Cull-Candy and Usowicz, 1987). IPSCswere also studied in hippocampal neurons (Col-

lingridge et al., 1984; Edwards et al., 1990; Ropert et al., 1990; Otis and Mody, 1992; Pearce, 19931, septal neurons (Schneggenburger et al., 1992), and Purkinje neurons (Farrant and Cull-Candy, 1991; Vincent et al., 1992) from rat brain slices. When the results of these studies are compared, the major difference found between GABA-mediated IPSCs in various preparations is the time constant of the decay of the synaptic current. This constant is largely variable, with values ranging from 4 to 30 ms, and in some inhibitory synapses becomes biphasic owing to the presence of a slower time constant. After taking into account the variability of these results related to various experimental conditions, such as temperature, holding potential value, and direction of Cl- flow, one cannot rule out the possibility that at least part of the large range of IPSC kinetic values is related to distinct properties of GABA* receptors, perhaps corresponding to specific functional characteristics associated with selected molecular forms of GABAA receptor subunits. To investigate further the functional heterogeneity of GABAergic synapses, we chose to study GABAergic inhibitory synapses in cerebellar Purkinjeand granule neurons. In the mammalian cerebellum two major types of GABA-mediated synaptic inhibition occur: the first is on Purkinje cells by stellate and basket cells, and the second is operated by the type II Golgi cells on granule cells (Palay and Chan-Palay, 1974). The circuits in which the Golgi, stellate, and basket cells participate have under tight control the basic mossy fiber-granule cell-Purkinje cell pathway and are deeply involved in motor coordination (Eccles et al., 1967; Ito, 1984). In situ hybridization studies (Laurie et al., 1992) and immunocytochemical techniques (Thompson et al., 1992; Fritschy et al., 1992) have shown a different distribution for the mRNAs encoding GABAA subunits and the subunit proteins themselves in Purkinje versus granule cells. In particular, the presence of a6 as well as 8 subunits is detected in cerebellar granule cells but not in Purkinje cells. Furthermore, a recent study with specific antibodies (Baude et al., 1992) indicates that immunoreactivity for both al and a6 is present at synapses innervated by type II Golgi cell terminals in granule neuron dendrites (Baudeet al., 1992), implying that GABA released from Golgi neurons may act on several GABA,+ receptor subtypes. Thus, the structure of the postsynaptic GABAA receptor may become the agent that characterizes the inhibitory message carried by GABA released from type II Golgi neurons. The goal of our work was to characterize the properties of the GABAA receptor channels at the inhibitory synapses located in cerebellar Purkinje and granule neurons by a comparison of the biophysical properties of slPSCs and GABA-activated currents in outsideout patches excised from these neurons. We will describe functional differences of inhibitory synapses

ii8

A

PURKINJE r*-

-

GRANULE

r-

Figure 1. Whole-Cell Recordingsof Recorded from Cerebellar Neurons

slPSCs

(A) slPSCs from a Purkinje neuron (left) and from agranule neuron (right). (B)Theeffect of bath perfusion with bicuculline methiodide (20 PM). (C)The average of 20 synaptic currents superimposed; the curve fitting used single and double exponentials.

-I

250

pA

-1

100 ms

IOOP

100 ms

T 59ms 90 pA

1Oms

I

in granule slices.

versus

Purkinje

neurons

% Slow

in rat cerebellar

Results We have investigated the characteristics of GABAactivated Cl- currents in specific neurons in rat cerebellar slices. Purkinje and granule neurons were visually identified by their location and by easily detectable morphological characteristics, such as the size of the cell body. In many Purkinje cells most parts of the large dendritic tree were clearly seen in slightly different focal planes. Care was taken to record from superficial cells to minimize access resistance, which was typically less than 10 MD. Our results derive from a data set of 49 Purkinje cells and 42 granule cells. The average resting potential measured immediately after establishing the whole-cell recording configuration of Purkinje cells was -61 f 7 mV (mean + SD) and the input resistance was 431 + 54 MQ; granule cells had a resting potential of -67 k 8 mV and an input resistance of 3.9 + 0.9 CD.

Spontaneous IPSCs in Purkinje versus Granule Neurons in Cerebellar Slices We investigated the characteristics of spontaneous IPSCs (slPSCs) with whole-cell recordings from Purkinje and granule neurons voltage clamped at -60 mV. A sample of slPSCs recorded from a Purkinje cell is shown in Figure IA. As previously reported (Farrant and Cull-Candy, 1991; Vincent et al., 1992), in most Purkinje neurons sIPSCS were highly variable in the peak amplitude, being 172 f 123 pA in our data base. Bath perfusion with the GABAA receptor antagonist bicuculline methiodide (10 PM) completely and reversibly abolished sIPSCS (Figure IB). In Purkinje neu-

54

lOIll*

rons under adequate voltage-clamp control, as estimated by the analysis of the capacitative currents to a 10 mV hyperpolarizing voltage step, the decay time oftheaveraged sIPSCswasdescribed byasingleexponential curve (Figure IC) with decay time constant of 7.9 f 1.9 ms (mean f SD, n = 7). Similar results have been previously published (Vincent et al., 1992). In Figure IB, slPSCs from a granule neuron are shown. The peak amplitude of the average of 50 sIPSCS recorded in each granule neuron ranged from 11 to 151 pA, with a mean of 55 f 37 pA in our data base42 granule cells. Bath perfusion with 10 uM bicuculline methiodide completely and reversibly abolished slPSCs in granule neurons (Figure IB). In these neurons, averaged sIPSCS had a rise time of 0.6 f 0.3 ms (range 0.3 to 1.3 ms). In granule neurons, the decay phase of sIPSCS recorded at a holding potential of -60 mV was best described by the sum of two exponentials (Figure IC), with a fast time constant of 7.0 + 1.6 ms and a slow time constant of 59 f 16 ms. In the presence of tetrodotoxin CrrX; 1 PM) sIPSCS in granule neurons were reduced in both amplitude and frequency of occurrence but mantained two kinetic component decay, with a fast time constant of 6.4 f 0.9 ms and a slow time constant of 65 f 19 ms (n = 3), and the relative proportion of the slow component to the average sIPSC was on average of 57% * 19%. Cerebellar granule cells were electronically compact, as suggested by the poor correlation between the distribution of the amplitude versus the various kinetic parameters (Figure 2) investigated in a sample of 100 sIPSCS recorded from a single granule neuron and by total electrotonic length measurements (Silver et al., 1992). Therefore, the slower component of sIPSC decays could not arise solely from unclamped synaptic sites. A slow component was always present, and its average contribution to the peak current was

GABA-Mediated 119

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B

Figure plitude

2. Correlation and Kinetic

between Parameters

slPSC

Am-

Relationship of the amplitude versus the fast decay time constant (A), the slow decay time constant (B), the rise time (C), and the percent slow component (D) derived from the doubleexponential fitting of 100 slPSCs recorded in a cerebellar granule neuron. The linear regression fit is superimposed with the data, and the correlation coefficient (r) is also indicated.

r

=0.02

r023

3 &-FYTTr

Amplitude (pA)

a hallmark of each granule cell investigated, ranging from 15% to 100% of the total peak amplitude, with an average of 60% L- 23%. In Figure 3A we report the distribution of the relative contribution of the slow component to the peak current investigated in a sample of 100 slPSCs recorded from a single granule neuron. Figure 3B shows the distribution of the averaged contribution of the slow component to slPSCs in our global sample of 42 granule neurons. CABA-Activated Currents in Outside-Out Patches from Purkinje and Granule Neurons We studied GABA-activated Cl- currents in outsideout patches excised from Purkinje and granule neurons of rat cerebellar slices. The fast application of brief (<2 ms) pulses of GABA (1 mM concentration in the extracellular solution) combined with TTX (1 PM) elicited Cl-currents in both Purkinjeand granule neurons. However, whereas in Purkinje neurons we obtained peak Cl- currents ranging from 30 to 160 pA (see an example in Figure 4A), in granule neurons the channel density was much lower, yielding currents of only a few picoamperes in the best cases. To overcome this limitation, we used outside-out nucleated patches of granule cells (see Experimental Procedures), in which we could record currents from 50 to 700 pA. In 10 granule cells (Figure 4A), the fast application of GABA (1 mM) elicited Cl- currents with a fast rise time (0.4 f 0.2 ms) and a rapid decay that resembled the slPSCs measured in whole-cell recordings (Figure IA). In fact, from the analysis of the average of 5 repetitive fast GABA applications on each nucleated outside-out patch, the decay of the Cl- currents was best described by the sum of two exponentials (Figure 4B), with a fast time constant of 3.7 + 1.82 ms and a

slow time constant of 102 f 48 ms. The contribution of the slow component to the peak amplitude of the average Cl-currents in nucleated outside-out patches excised from granule neurons was 42% f 8.4%, with a variability of 27%-57%.

A I

% Slow component Figure 3. VariabilityoftheContributionoftheSlowComponent to SIPSC (A) The distribution of the percent slow component derived from the double-exponential fitting of 100 slPSCs in a cerebellar granule neuron. (B) The distribution of the percent slow component derived from the double-exponential fitting of the average slPSCs in 42 cerebellar granule neurons.

A

PURKINJE

GRANULE

-r-1 zs

of Inward Currents of CABA (1 mM) on Excised from a PurNucleated Outsidea Granule Neuron

In (A) 4-5 traces are shown superimposed; (B) shows their average, with the decay fitted by a double exponential. Above the superimposed traces in (A)are shown thecurrents generated by the liquid junction potential due to a 50~1 dilution of the CABA-containing solution measured after blowing out the patch. This gives an indication of the duration of the pulse application. Calibration bars apply to all traces, but not to the 10 pA current pulse.

100 pA

3ms T s 70 ms

Zf

31ms

% Slow

Figure 4. Comparison Elicited by Brief Pulses an Outside-Out Patch kinje Neuron and on a Out Patch Excised from

9 i

% Slow

In outside-out patches from Purkinje neurons (n = 6), however, the decay of currents produced by GABA pulses did not completely match the slPSC decay in these neurons because of the presence of a slowly decayingcomponent.Fromtheanalysisoftheaverage of 5 repetitive GABA pulses on each outside-out patch (Figure 4A), the decay of the Cl- currents was best described by the sum of two exponentials (Figure 4B), with a fast time constant of 5.1 f 2.3 ms and a slow time constant of 95 f 36 ms. However, in Purkinje cells, the contribution of the slow component to the peak amplitude of the average Cl- currents was only 10% + 7.4%, ranging from 0 to 25%. The rise time of these averaged Cl- currents in Purkinje neurons was 0.7 + 0.4 ms. Fast Desensitization of the GABAA Receptor To characterize further the differences in the intrinsic properties of GABA* receptors in outside-out patches excised from cerebellar neurons and to study the response to GABA pulses (<2 ms), we also investigated the Cl- currents elicited by a 1 mM GABA step lasting for a few seconds (Figure 5A). In 14 Purkinje neurons, the step applications of CABA elicited Cl- currents with a fast rise time (0.5 f 0.3 ms) and a biphasic decay (Figure 5). In these cells the fast component of Clcurrent decay had a time constant of 5.6 of:2.7 ms and an initial slow time constant of 158 f 97 ms, followed by a sustained plateau (which we did not investigate). The contribution of the slow component to the peak amplitude of the average Cl- currents was 58% + 14.9%. In 18 nucleated outside-out patches excised from granule cells, step applications of GABA also produced Cl- currents with a fast rise time (0.4 f 0.2 ms) and a biphasic decay (Figure 5B). In these cells the fast component of the Cl- current decay had a time

34

constant of 5 + 2.1 ms and an initial slow time constant of 184 f 173 ms, also followed by a sustained plateau (which we did not investigate). The contribution of the slow component to the peak amplitude of the average Cl- currents was 73% f 14.9%. In 4additional Purkinje and 3 granule cells, the step application of GABA had a slower onset, as measured at the end of the recording (see Experimental Procedures),therebyproducingCI-currentswitharisetime >5 ms. In these patches, the current showed only a slow decaying component, followed by the sustained plateau (data not shown). slPSCs in Nucleated Outside-Out Patches from Granule Neurons In 5 outside-out nucleated patches, we noticed that, after the excision, we could detect an increase of the background noise and record clearly distinct slPSCs by bringing the patch once.again close to the surface oftheslicewherethecell bodyofthegranuleneurons was located (Figure 6). These currents ranged from 10 to 150 pA and were reversibly blocked by bicuculline methiodide (20 PM; data not shown). Perhaps they reflected the spontaneous vesicular release of GABA from the synaptic terminals in the slices being exposed by the cell enucleation procedure. The kinetics of these sIPSCS in the nucleated patches remarkably resembled those of the sIPSCS recorded in the wholecellvoltage-clampconfiguration.Theirdecaywas best described by the sum of two exponentials, with a fast time constant of 7.5 + 0.9 ms and a slow time constant of 69 f 23.1 ms. In each patch, the slow component of sIPSC decay was always present, and its contribution to the peak current was the same as in the sIPSCS recorded in the whole-cell mode, ranging from 15% to 100% of the total peak amplitude, with an average of 60 + 23.2 pA.

CABA-Mediated

Currents

in Cerebellar

Slices

121

A

PURKINJE

Figure 5. Comparison of Inward Currents Elicited by Concentration Steps of CABA (1 mM) on an Outside-Out Patch Excised from a Purkinje Neuron and on a Nucleated Outside-Out Patch Excised from a Granule Neuron

GRANULE

-J

50 ms

B Zf

Ts

6.6 ms 59 ms

% Slow

46

Diazepam Differentially Affects the Time Course of slPSCs in Purkinje versus Granule Neurons In 7 Purkinje and 6 granule neurons, we investigated the action of diazepam (IO PM) on the time course of slPSCs recorded in voltage-clamped neurons. As previously described, in both cell types these currents had very variable peak amplitudes. Therefore, we did not investigate the diazepam effect on slPSC peak amplitude. Upon bath perfusion with a saturating concentration of the benzodiazepine, we noticed a prolongation of the time course of the synaptic currents in both Purkinje and granule neurons that fully recovered after prolonged perfusion with the control solution (data not shown). As shown in Figure 7, a detailed analysis of the decay time of the average of 50 slPSCs in the cerebellar neurons investigated revealed that, whereas in Purkinje cells (n = 7) the prolongation of the average current was achieved by increasing the single decay time constant from 10 f 1 to 18 f 0.6, in granule cells (n = 6) the fast decay time constant increased from 4 f 1.7 to 8 * 2.5, but the slow component remained unchanged after the diazepam application (48 f IO before and 47 f 7 after diazepam). In granule neurons, the relative contribution of the slow component to slPSC peak amplitude was also not affected by diazepam (71 f 15 before and 75 k 15 after, n = 6). Recently, furosemide (500 PM) was shown to cancel selectively the fast component of IPSCs in hippocampal neurons (Pearce, 1993). However, the slPSCs from both cerebellar neurons failed to be affected by furosemide (500 PM, n = 4 Purkinje neurons and n = 5 granule neurons; data not shown). Discussion In the mammalian cerebellum, the synaptic inhibition impinging on cell bodies and dendrites of Purkinje neurons originates from basket and stellate inter-

50

ms

In (A) 3 traces are shown superimposed; (B) shows their average, with the decay fitted by a double exponential. Above the superimposed traces in (A) are shown the currents generated by the liquid junction potential due to a 5O:l dilution of the GABA-containing solution measured after blowing out the patch. This gives an indication of the duration of the pulse application. Calibration bars apply to all traces, but not to the 10 pA current pulse.

‘Cf 4.8ms Ts 107ms % Slow 55

neuron terminals (Palay and Chan-Palay, 1974; Somogyi et al., 1989), whereas the inhibitory Golgi type II interneuron terminals are afferents to the granule neuron dendrites in cerebellar glomeruli (Palay and Char+Palay, 1974; Somogyi et al., 1989). Additional inhibitory synapses to granule cell dendrites have also been shown to arise from axon collaterals of Purkinje neurons (Palay and Chan-Palay, 1974) as well as from GABAergic neurons located in the deep cerebellar nuclei (Hamori and Takacs, 1989). Our results show a functional diversity between inhibitory synapses in

B C

Figure 6. A Comparison of slPSCs Recorded in a VoltageClamped Granule Neuron in Rat Cerebellar Slices Before and After Excison of a Nucleated Outside-Out Patch (A)slPSCsinagranuleneuron.(B)Thecurrenttracea~erexcision of the outside-out nucleated patch from the granule neuron shown in (A). (C) slPSCs produced in the outside-out nucleated patch shown in (B) by repositioning the patch where the cell body was located before excision.

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Figure 7. Whole-Cell Recordings of slPSCs Recorded from Cerebellar Neurons in the Presence and Absence of Diazepam

GRANULE

A

+ Diazepam

(A) slPSCs in a Purkinje neuron (left) and in a granule neuron (right). (B) The effect of bath perfusion with diazepam (IO FM). (C) Superimposed average of 20 synaptic currents in the two experimental conditions. Averaged currents are scaled to the same peak amplitude. The vertical calibration bars in (C) refer to the currents in the presence of diazepam.

10 FM

B 50 pA 500 Ins

C 150 pA

20pA’ I 70 ms

voltage-clamped was documented

Purkinje and granule neurons, which by the slPSC kinetic characteristics.

Kinetically Distinct slPSCs in Cerebellar Neurons slPSCs have been previously described in voltageclamped Purkinje neurons (Farrant and Cull-Candy, 1991; Vincent et al., 1992). We confirm these results by showing that in these neurons slPSCs had a decay time constant of 7-12 ms. To our knowledge, there are not reports of voltage-clamp studies of slPSCs in cerebellar granule neurons. Our data show that the decay of slPSCs in these neurons is best described by two exponentials, with time constants of 7 and 60 ms, with a variable contribution of the slower decay component to the total peak amplitude. These results indicate an intriguing similarity between slPSC decay in granule neurons of the cerebellum and granule neurons of the hippocampus (Edwards et al., 1990). Vincent et al. (1992), discussing the differences between their resultson slPSCs in Purkinje neuronsand those of Edwards et al. (1990) on slPSCs in hippocampal granule neurons, proposed that these kinetic differences might be related to distinct intrinsic properties of the GABAA receptor channels in thetwo preparations. We believe that an analogous interpretation could be extended to the difference we observed between GABAergic currents generated in Purkinje and granule neuron synapses. In fact, the poor correlation of rise and decay times versus amplitude that we observed for slPSCs in cerebellar granule neurons (Figure 2) was also shown for slPSCs recorded in hippocampal granule neurons by Edwards et al. (1990), who considered it to indicate that slPSCs were generated in close proximity to the cell body in a region under adequate voltage clamp. Presumably a similar consideration also applies to cerebellar granule neurons, in which synapses are located on the short dendrites

70 rns

(Palay and Chan-Palay, 1974). However, recent modeling of space-clamp errors associated with whole-cell recording of synaptic currents in neurons revealed the possibility that in some cases the correlation of kinetic parameters may provide misleading conclusions (Spruston et al., 1993). In any case, total electrotonic length measurements and recording of excitatory synaptic currents support the notion that a high quality space clamp can be achieved in whole-cell recordings from cerebellar granule neurons (Silver et al., 1992). The proposal that the presence of fast and slow decay components in slPSCs in granule neurons is related to distinct intrinsic properties of the GABAA receptor channels is further supported by the observation that in a nucleated patch excised from a granule neuron, in which the voltage control is optimal, the slPSCs were similar to those recorded in the wholecell mode. slPSC Decay Times Relate to the Properties of CABA Receptor Channels In GABAergic synaptic transmission, the decay of slPSCs is dependent upon a complex series of events. Neurotransmitter diffusion and reuptake, together with the kinetic properties of the postsynaptic channels, might concur to determine the time course of the synaptic current. To dissect out the various components that determine the slPSC decay, we attempted to mimic synaptic transmission by using GABA pulses to outside-out membrane patches excised from cerebellar neurons. In doing so, we assumed that the transmitter concentration which produces an slPSC is in the millimolar range and that the duration of the vesicular release at the synapse is very short (- 1 ms) and similar to that recently calculated for the vesicular release of glutamate from gluta-

CABA-Mediated 123

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matergic synapses (Clements et al., 1992). Furthermore, we also surmised that the properties of the CABA* receptors in the outside-out patches excised from the cell body were similar to those of the channels operating at synaptic sites. While these assumptions are not yet validated, our results show that in granule neurons there is a good correlation between the kinetic properties of slPSCs and those of the currents evoked by GABA pulses to outside-out nucleated patches. In fact, in these neurons the fast Clcurrents activated by GABA decayed with two exponential components, with kinetics and relative proportion of the slow component similar to those of the slPSCs. In Purkinje neurons, however, while we confirmed the results of Vincent et al. (1992) showing the absence of slPSCs with biphasic decay, we described a biphasic decay of the Cl-currents generated by GABA pulses on outside-out patches. The relative proportion of the slow decay component never exceeded 25% of the total peakamplitude. Distinct properties of synaptic and extrasynaptic GABAA receptors could be responsible for the differences observed between slPSCs and channel currents recorded in patches from Purkinje neurons, although this proposal requires further investigation. In any case, our results indicate that, at least for granule neurons, distinct relaxation properties of GABAA receptor channel currents in outside-out patches approximate the distinctkineticsobservedintheaveragesIPSCandtherefore lend support to the hypothesis that functionally diverse CABAA receptor subtypes underlie the properties of GABA-mediated inhibitory synaptic currents. In addition, we cannot rule out the possibility that synaptic and extrasynaptic GABA,+ receptors are distinct also in granule neurons and that in these neurons the slow slPSC decay component results from synaptically released GABA on extrasynaptic receptors. Fast Desensitization of CABAA Receptors To investigate further the properties of GABA* receptors expressed in cerebellar neurons, we compared theCI-current induced by sustained stepapplications of high GABA concentrations to patches excised from Purkinje and granule neurons. An important result of this experimental protocol was that the Cl- current desensitized during the GABA application (see Figure 5). The GABAA receptor desensitization was similar in both cell types. A slow time course (hundreds of milliseconds) of desensitization of the GABA,+ receptor response has been described previously (Mierlak and Farb, 1988; Oh and Dichter, 1992; Frosch et al., 1992). We report a very rapid (a few milliseconds) component of desensitization for the GABAA receptor response induced by a high concentration of GABA, which is followed by the previously described slower component and a sustained plateau. We, as well as others, failed to observe a fast desensitizing component when the onset of the GABA concentration step was longer lasting (a few milliseconds). A slower rising GABA step might be the reason why a fast GABAA

receptor desensitization was not described previously. In fact, one may suggest that a subgroup of the receptor population enters into a desensitized state during the slow onset of GABA application, preventing the observation of the fast desensitizing response. This finding is reminiscent of the fast desensitization of the response to excitatory amino acids (Tang et al., 1989; Trussell and Fischbach, 1989; Mayer and Vyklicky, 1989). We do not know whether the fast desensitization observed during sustained GABA steps also occurs during the physiological release of GABA at inhibitory synapses. Before this question can be addressed, the concentration dependence limits of fast desensitization as well as the GABA concentration at the synapses has to be thoroughly investigated. Our results, however, indicate that GABA response desensitization is similar in Purkinjeand granulecells. In conclusion, from the experiments in excised membrane patches, it is evident that the onset and the duration of the GABA application are crucial determinants of the kinetics of the Cl- current. As a consequence, at inhibitory synapses of granule but not of Purkinje neurons, the slow component in the decay phase of slPSCs might alternatively relate to distinct relaxation properties of the postsynaptic channels or to a kinetically distinct decrease in neurotransmitter concentration regulated by GABA reuptake (Isaacson et al., 1993). Different Subtypes of GABA* Receptors in Cerebellar Synapses Since in granule neurons we observed both fast and slow GABA-activated Cl- currents, one might surmise that they are the product of a mixed activation of two GABAA receptor populations. This is also supported by the wide distribution of the slow decay contribution to the total synaptic currents recorded from different granule cells. Kinetically distinct GABA responses in Purkinjeand granule neurons might derive from GABAA receptor structural diversity (reviewed in Olsen and Tobin, 1990, and Burt and Kamatchi, 1991), which determines specific biophysical properties in the associated ion channel. Various studies (Bovolin et al., 1992; Wisden et al., 1992; Thompson et al., 1992) have shown that many subunits are common to granule and Purkinje neurons. However, the a6 subunit is uniquely expressed by the granule cells (Wisden et al., 1992). Interestingly, in hippocampal granule neurons, in which slPSCs also have a double exponential decay (Edwards et al., 1990; but see Otis and Mody, 1992), the localization of a4 subunits, which possess a high degree of homology with a6 subunits (Ymer et al., 1989; Liiddens et al., 1990), has been reported (Wisden et al., 1992; Laurie et al., 1992). The 8 subunit mRNA is also selectively present in both cerebellar and hippocampal granule neurons (Laurieet al., 1992). Slow GABA-activated Cl-currents, however, could be independent from the subunit assembly of the receptor and could be instead consequent to reversible posttranslational modifications, such a phosphoryla-

124

tion (Swope et al., 1992). In support of these alternatives, we observed a small proportion of slower relaxing GABA-activated Cl- current in patches from Purkinje neurons, in which neither the a6 nor the 6 subunit has been detected. Further work with recombinant GABAA receptors will be required to determine whether the different functional profiles detected at cerebellar inhibitory synapses are related to the molecular structure of receptors specifically located in certain neuronal populations. Allosteric Regulation of GABAA Receptors in Cerebellar Neurons An important property of the GABAA receptor is that it is allosterically regulated by clinically important compounds, such as benzodiazepines (Costa et al., 1975). In this regard, it has been demonstrated that the molecular composition of the receptor is strongly related to the potency and efficacy of the allosteric modulation by benzodiazepines and betacarboline derivatives (Pritchett et al., 1989a, 1989b; Puia et al., 1991). Therefore, diversity in GABAA receptor responsiveness to benzodiazepines arises from the structural composition of these receptors. Among these distinct benzodiazepine receptor types (reviewed by Doble and Martin, 1992), those lacking they2 subunit as well as those comprising the a6 or 6 subunit have very poor sensitivity to diazepam (Pritchett et al., 1989a; Shivers et al., 1989; Puia et al., 1991; Kleingoor et al., 1991; Ducic et al., 1993; Kleingoor et al., 1993). Our results, provide evidence of a selective prolongation of the fast slPSCs decay by diazepam in Purkinje neurons. The lack of effect of diazepam on slowly decaying slPSCs in granule neurons indicates a specificity in the allosteric modulation of GABAA receptors located in postsynaptic sites in granule neurons, probably related to a specific subunit combination that confers insensitivity to benzodiazepines. Our resultsdemonstrate IPSCs in cerebellargranule neuron with different proportions of fast and slow components. This functional difference might allow inhibitory interneurons in the granule layer to produce specific patterns of inhibition and to select granule cell groups. In s~.~pport of this view, Eccles et al. (1967) demonstrated the existence of nonoverlapping compartments of inhibited granule cells, similar to the glomeruli of the olfactory bulb and the barrels in the neocortex, with structural and functional unity. Experimental

Procedures

Brain Slices Sagittal slices of cerebellum (200-300 pm) were prepared from 14-to la-day-old Sprague-Dawley rats as described by Edwards et al. (1989). Cerebellar neurons were viewed with an upright microscope equipped with differential interferencecontrast Nomarski optics (UEM, Zeiss, Federal Republic of Germany) and an electrically insulated water immersion 40x objective with a long working distance (2 mm). Solutions and Drugs Experiments were performed

at room

temperature

(22”C-24OC)

using an extracellular medium composed of 120 mM NaCI, 3.1 mM KCI, 1.25 mM K2HPO+ 26 mM NaHCOI, 5.0 mM dextrose, 1.0 mM MgCI?, and 2.0 mM CaClz and containing 10 PM 6 cyano7-nitroquinoxaline-2,3dione (CNQX, Tocris, UK) and 20 PM 3-[( f )-2carboxypiperazin4yl]-propyl-I-phosphonic acid (CPP) to block excitatory amino acid-mediated synaptic transmission. The solution was maintained at pH 7.4 by bubbling with 5% COZ, 95% 0,. The slice was completely submerged in a total volume of 500 ~1 and continuously perfused at a rateof 5 ml/min. Bicuculline methiodide, TTX (Sigma, St. Louis, MO), diazepam (in 0.1% dimethylsulfoxide; a gift of the late Dr. Haefely, Hoffman La Roche Laboratories, Basel, Switzerland), and furosemide solution (10 mg/ml; Abbott Laboratories, IL) were diluted in the extracellular medium and were superfused through parallel inputs to the perfusion chamber until effective replacement of the solution was obtained. For fast application of CABA, we used a piezoelectric translator (P-24530Stacked Translator, Physik Instrumente, Federal Republic of Germany) to position double barrel theta tubing in front of the excised patch quickly. One barrel contained extracellular medium with added TTX, and the other contained this solution and 1 mM CABA, similar to the solution described for fast glutamate application by Lester and Jahr (1992) and Colquhoun et al. (1993). After each patch recording, on and off rates as well as pulse duration were measured by “blowing out” the patch and recording currents generated by the liquid junction potential due to a 5O:l dilution of the CABA-containing solution (Lester and Jahr, 1992). On and off rates of the system were typically less than 0.2 ms. Outside-out patches from Purkinje neurons were excised followingtheproceduredescribed byHamilletal.(1981). Nucleated outside-out patches were instead isolated following the detailed protocol described for mouse forebrain neurons in primary culture by Sather et al. (1992). Electrophysiology Voltage-clamp recordings of sIPSCS were performed using the whole-cell recording configuration of the patch-clamp technique (Hamill et al., 1981) with a patch-clamp amplifier (EPC 7, List Electronics, Darmstadt, Federal Republic of Germany) after capacitance and series resistance compensation. Series resistance was checked for constancy throughout the experiments. Electrodes were pulled from borosilicate glass capillaries (Wiretrol II, Drummond, Broomall, PA) and were filled with a solution containing 145 mM CsCI, 1 mM MgCI,, 5.0 mM ECTA, 2.0 mM Na-ATP, and 10 mM HEPES (to pH 7.2 with CsOH). We chose Cs+ as the major cation to improve the quality of the voltage clamp and to prevent GABAs receptor-mediated currents. GABA-activated currents were recorded in outside-out patches excised from neurons in the slicewith a patch pipettecontaining the same solution as for the whole-cell recording experiments. Current traces from whole-cell and outside-out patches were recorded on a VR-10 data storage system (Instrutech Co., Haverhill, MA). Data Analysis Current traces were filtered at 3 kHz (-3 dB, 8 pole, low pass Bessel filter; Frequency Devices) and stored in an LSI II/73 computer (INDEC System, Sunny Vale, CA) after digitization (IO kHz) with a Data Translation analog to digital converter. Decay time constants of sIPSCS and CABA-activated currents were determined from exponential fitting with the 11173 system by using an entirely automated least squares procedure (see Vicini and Schuetze, 1985, for further details). This method uses a Simplex algorithm (Caceci and Cacheris, 1984) to fit the data to either a singleor double-exponential equation of the form I(t) = I, exp(-t/G + IJ exp(-t/r,), where If and I, are the amplitudes of the slPSC fast and slow components, and G and T. are their respective decay time constants. Peak amplitudes were measured at the absolute maximum of the currents, taking into account the noise of the baseline and noise around the peak. Rise times were measured as the time elapsed from 20% to 80% of the peak amplitude of the response

GAEA-Mediated 125

Currents

in Cerebellar

Slices

Acknowledgments We wish to thank Charles T. Livsey for comments on the manuscript. This work was supported in part by NIMH grant #ROl MH49486-OIAI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

June

14, 1993; revised

October

28, 1993.

aminobutyric and triple antibodies.

acid receptors immunofluorescence Proc. Natl. Acad.

Frosch, M. P., Lipton, tion of CABA-activated neurons. J. Neurosci.

identified in neurons by double staining with subunit specific Sci. USA 89, 6726-6730.

S. A.,and Dichter, M.A. currents and channels 72, 3042-3053.

(1992). Desensitizain cultured

cortical

Hamill, 0. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patch-clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflijgers Arch. 397, 85-100. Hamori, J., and Takacs, J. (1989). Two types of GABA-containing axon terminals in cerebellar glomeruli of cat: an immunogold study. Exp. Brain Res. 74, 471-479.

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