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Journal of Physiology (1991), 434, pp. 183-213 With 13 figures Printed in Great Britain
SYNAPTIC- AND AGONIST-INDUCED EXCITATORY CURRENTS OF PURKINJE CELLS IN RAT CEREBELLAR SLICES
BY ISABEL LLANO*, ALAIN MARTY*, CLAY M. ARMSTRONGt AND ARTHUR KONNERTHt From *the Laboratoire de Neurobiologie, Ecole Normale Supe'rieure, Paris, France and the Wax-Planck Institut fur Biophysikalische Chemie, Gottingen, Germany (Received 17 January 1990) SUMMARY
1. Postsynaptic currents originating from activation of the two major excitatory inputs to Purkinje cells were studied in thin slices of rat cerebellum, using the tightseal whole-cell recording technique. Two types of excitatory postsynaptic currents were analysed: those evoked by stimulation of the granule cell-parallel fibre system (PF-EPSC) and those elicited by stimulation of the climbing fibres (CF-EPSC). 2. Both types of postsynaptic currents had a linear current-voltage relation, reversing at membrane potentials close to 0 mV. Their time course of activation was independent of the membrane potential. 3. For both types of postsynaptic currents, the time course of decay was well described by a single exponential function, with a time constant which increased as the membrane potential was made more positive. 4. Postsynaptic currents arising from stimulation of the climbing fibre generally had a slightly faster time course of onset and decay than those associated with stimulation of the granule cell-parallel fibre system. The average values of the 10-90% rise time were 1-8+004 ms (meanss .D. n = 7) for PF-EPSCs and 0 8 + 0 3 ms (n = 9) for CF-EPSCs. Time constants of decay, at a holding potential of -60 mV, had values of 8-3+ 1-6 ms (n = 7) and 6 4+ 1 1 ms (n = 9) for PF-EPSCs and CF-EPSCs respectively. 5. CF-EPSCs and PF-EPSCs had the characteristics described above in slices derived from animals aged 9-22 days old and 9-15 days old, respectively. The PFEPSCs in animals older than 15 days had very slow time courses and positive apparent reversal potentials, suggesting that they originated from distal locations, not under accurate voltage control. 6. In order to assess the quality of the voltage clamp, responses to hyperpolarizing pulses from -70 mV were analysed. The capacitive currents could be fitted by the sum of two exponentials, and were interpreted with an equivalent electrical circuit comprising two main compartments (soma and proximal dendrites on one hand, distal dendrites on the other). Analysis of synaptic currents in terms of this model suggested that the recorded time course of decay was approximately correct. t Permanent address: Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA. MIS 8201
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7. CF-EPSCs as well as PF-EPSCs were insensitive to the NMDA receptor antagonist 3-3(2-carboxypiperazine-4-yl)propyl- 1 -phosphonate (CPP), but were blocked in a dose-dependent reversible manner by the non-NMDA antagonist 6cyano-7-nitroquinoxaline-2,3-dione (CNQX). 8. The responses of Purkinje cells to exogenous application of various glutamate agonists were tested in the presence of tetrodotoxin (TTX) and bicuculline to minimize polysynaptic contributions (8- to 11-day-old rats). The Purkinje cells had a high sensitivity to the glutamate agonists quisqualate and kainate, but were insensitive to N-methyl-D-aspartate (NMDA). 9. In the absence of TTX and bicuculline, NMDA induced large responses with a clear polysynaptic nature. These responses were identified as arising mainly from the activation of GABAergic interneurones either directly or following excitation of granule cells. A large fraction (80-90 %) of the polysynaptic response to NMDA was blocked by bicuculline, while only 10-20 % of such response was blocked by CNQX. These results suggest that cerebellar inhibitory interneurones respond directly to NMDA. INTRODUCTION
The aim of the present study was to establish the basic properties of the postsynaptic currents which can be evoked in Purkinje cells upon stimulation of its two main excitatory afferents, under conditions that allow the membrane potential and the intracellular composition of the Purkinje cell to be controlled. To fulfill this purpose, we have used a recently reported method (Edwards, Konnerth, Sakmann & Takahashi, 1989) which renders possible the application of patch-clamp recording techniques (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) to neuronal cells in acutely prepared slices of brain tissue. The analysis of postsynaptic currents is complicated in adult Purkinje cells by the extensive dendritic arborization, which is likely to escape from a somatic voltage-clamp system. This is especially important in view of the fact that in the adult state a large part of the excitatory input (specifically that arising from parallel fibres) arrives to dendritic locations which are far away from the cell soma. To minimize this problem, we have carried out most of our studies in cerebella derived from young animals, where synapses from various afferents are more likely to be localized at regions of the dendritic arborization which are electrically close to the cell body. Using this experimental approach, excitatory synaptic currents can be recorded from Purkinje cells upon extracellular stimulation of climbing fibres and of the granule cell-parallel fibre system. The synaptic currents evoked by each of these afferent pathways can be easily identified on the basis of their dependence on stimulus intensity and their response to paired stimuli (Konnerth, Llano & Armstrong, 1990b). In the present report we describe these excitatory postsynaptic currents as recorded with a tight-seal whole-cell voltage clamp and discuss the reliability of their voltage dependence and kinetic properties. Based on pharmacological evidence, these currents are identified as arising from the activation of non-NMDA glutamate receptors. These results are complemented by an analysis of the sensitivity of Purkinje cells to various glutamate agonists.
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METHODS
Slices were prepared from rat cerebellum following the general procedures described by Edwards et al. (1989). Briefly, rats (8-22 days old) were killed by decapitation after cervical dislocation, the cerebellar vermis was quickly dissected out and immersed for a couple of minutes in ice-cold bicarbonate-buffered saline (BBS; see composition below). The tissue was subsequently glued to the stage of a vibroslicer and sliced at a thickness of 130-140 ,um. Sagittal as well as transverse slices (i.e. cut along the parallel fibre axis) were used in the present studies. The slices were incubated in oxygenated BBS saline at 36 °C for at least 45 min prior to the beginning of experimental recordings. Patch-clamp recordings from Purkinje cells were performed on slices visualized at 40 x magnification with Nomarski optics using an upright microscope (either a Zeiss 'Standard 14' or a Zeiss 'Axioscope'). Under these conditions, recognition of the various cell layers within the cerebellar cortex as well as identification of individual cells was easily achieved, as shown in Fig. 1. Two pipettes are seen in the photograph, a small patch pipette in contact with a Purkinje cell, and a larger (10 ,um) pipette in the granule cell layer. To the right in the photograph, in the same plane of focus, is another Purkinje cell with a prominent dendritic shaft extending into the molecular layer. The surface of the Purkinje cell to be studied was cleaned with glass pipettes of 8-10 ,am diameter prior to seal formation, following the general technique described by Edwards et al. (1989). During recordings, the slices were maintained at room temperature (20-22 °C) and were continuously perfused at a rate of 2 ml/min with the standard bicarbonate-buffered saline (BBS) which contained (in mM): 125 NaCl, 2 5 KCl, 2 CaCl2, 1 MgCl2, 1-25 NaH2PO4, 26 NaHCO3 and 25 glucose; a mixture of 95 % 02 and 5% C02 was bubbled through the solution to keep its pH at 7-4. Unless otherwise noted, bicuculline (10-16 /tM) was added to the external solution, in order to eliminate GABAergic postsynaptic currents. In the series of experiments designed to test the sensitivity of Purkinje cells to exogenous agonists, the slices were bathed in Mg2+-free BBS, which had the same composition as the BBS described above, except that Mg2+ was omitted from the solution. The tight-seal whole-cell recording technique (Hamill et al. 1981) was used for all experiments presented in this paper. Seal resistances were always larger than 10 GfQ. Recording pipettes were pulled from borosilicate glass, coated with Sylgard resin (Dow Corning Chemical Co.) and firepolished to a final tip diameter of approximately 2-5 ,um. They had a resistance of 2-3 MQ when filled with the internal saline, which had the following composition (in mM): 140 CsCl, 2 MgCl2, 1 CaC12 10 Cs-EGTA, 10 Cs-Hepes and 4 Na-ATP (pH of 7-3). In some experiments, performed to assess the passive properties of Purkinje cells, an internal K+ solution was used. This solution contained (in mM): 140 KCl, 2 MgCl2, 1 CaCl2, 10 K-EGTA, 10 K-HEPES and 4 Na-ATP (pH of 7 3). Membrane currents were recorded with a patch-clamp amplifier (EPC-7, List Electronic, Germany) set at a gain of 0 5 or 1 mV/pA. The slow transient cancellation and G-series adjustment of the amplifier were used to compensate the initial portion of the capacitance transient elicited by 10 mV hyperpolarizing pulses, and to estimate the value of the series resistance, as explained in the results section. Typical values for this parameter were 5-10 MiQ. The series resistance compensation control of the amplifier was set at 60-75 %. For the study of passive properties of Purkinje cells, current transients elicited by hyperpolarizing steps were filtered with an 8-pole Bessel filter (Frequency Devices, USA) set at a corner frequency of 5 kHz and digitized at a sampling rate 50 ,us per point. Purkinje cell postsynaptic currents were evoked by electrical stimulation, using a bipolar electrode consisting of a 7-10 ,um tip diameter glass pipette (see Fig. 1) and a remote 100 4am thick platinum wire. Square pulses of 100-300 Its duration and amplitudes ranging from 1-5 to 12 V were applied, while the glass pipette was moved within the visual field until the synaptic current was evoked with minimum stimulus intensity. The EPSCs were filtered at 3-5 kHz and digitized at a sample rate of 50 jus per point. In most cases, analysis of the synaptic signals was performed after subtraction from the raw data of the stimulation artifact, using a series of records taken at the reversal potential of the current. Rise times were measured as the time required for the current to change from 10 to 90% of its peak amplitude. Fits of model exponentials to the experimental data were performed using an analysis program based on an error-minimization algorithm. For fits of the decay of EPSCs, the first 1-3-1-5 ms
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following the peak of the current were excluded from the fit. In some cases, a double exponential fit to both rising and decaying phases of EPSCs was performed. Application of various drugs and transmitter agonists was )erformed by changing the bath to a solution containing the relevant compound. Given the perfusion rate (2 ml/min) and the volume (700 jdl) of the chamber, exchange of the external solution could be achieved within less than 1 min.
XV.
Fig. 1. View of the experimental preparation and recording configuration. Photograph of a transverse cerebellar slice from a 14-day-old rat, taken at 400 x magnification. The upper part of the figure corresponds to the molecular layer, and the lowest portion to the granule cell layer. A patch pipette is in contact with a Purkinje cell and a stimulating pipette (10,am diameter) is positioned in the granule cell layer. Calibration bar (upper left) equals 20 ,um. The glutamate agonists N-methyl-D-aspartic acid (NMDA), quisqualic acid (Quis) and kainic acid (Kai) were purchased from Cambridge Research Biochemicals. The NMDA-receptor antagonist 3-3(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP), the non-NMDA antagonist 6-eyano7-nitroquinoxaline-2,3-dione (CNQX) and the GABAA receptor antagonist bieuculline were purchased from Tocris Neuramin. Tetrodotoxin (TTX) was purchased from Sigma Chemical Co. RESULTS
Passive properties of the Purkinje cell membrane The interpretation of recorded synaptic currents necessitates a basic knowledge of the passive properties of Purkinje cells. In this section, we describe the capacitive and resistive currents of these neurones, and present a qualitative interpretation of
GLUTAMATE SYNAPSES IN PURKINJE CELLS18 187 the results in terms of an equivalent circuit modelling which is made explicit in the
Appendix. Figure 2A presents the current response of a Purkinje cell to a voltage step from - 70 to - 80 mV. The decay of the current was biphasic, and could be described by A
500 pA
5 ms B
=
4.6 ms
C
,r=
2.6 ms
D
1 Ms
Fig. 2. Capacitive currents in Purkinje cells. Current responses to 10 mV hyperpolarizing voltage pulses from a holding potential of - 70 mV are shown at various stages of the series resistance compensation. Each trace is the average of seven sweeps. A, uncompensated. B, after cancellation of the fast capacitive current. C, after capacitive current cancellation and series resistance compensation (70 %). D, expanded traces for the early part of the capacitive currents in A and B. A double exponential fit of the uncompensated current is shown in A (where the fit cannot be distinguished from the experimental trace) and in D, left trace. Amplitude coefficients and time constants for the two exponentials fitted were: - 437 pA, 0-26 ms and - 690 pA, 4-51 ins. Single exponential fits to the current decay are superimposed on the current records in B, C and in D, right trace. When going from B to C, the time constant of decay, T2' decreased from 4-6 to 2-6 ins. The small sustained inward current visible in A represents a leakage conductance of 314 MCI. This measurement was taken after 1 min of whole-cell recording. Later on this resistance increased to over 1 Gfl (traces in B and C were taken 2 and 3 min after that in A respectively) presumably as a result of Cs+ ions diffusing into the cell. Transverse slice from a 12-day-old rat.
I. LLANO AND OTHERS 188 the sum of two exponential components with time constants of 0'3 and 45 ms. The fit is shown as a smooth continuous line on top of the experimental trace of Fig. 2A. Its initial portion is displayed at an expanded time scale in the left trace of Fig. 2D. In general, the decay was well fitted by the sum of two exponentials. The ratio between the two time constants was quite large, of the order of 20 (average values TABLE 1. Passive properties of Purkinje neurones n C1(pF) C2(pF) R1(MfQ) R2(MQ) R3(MQ) 2(ms) T'2(ms) Tl(ms) 146+91 494+ 100 9 7 +0-9 4 0+ 1-0 441 + 177 0 40+0-20 6-83+ 1-75 3-31 +0-86 5
Sagittal slices Transverse 87 + 54 373 + 113 7-4 + 1-2 6-6 + 3-9 283 + 90 0-20 + 0-03 4-64 + 1-80 2-12 + 1 00 8-9 slices Means + S.D. for each series of n cells are given. rl is the time constant of the fast component of the capacitive transient. T2 is the time constant of the slow component of the capacitive transient, and T'2 is the time constant of this component after series resistance compensation. T'2 values for sagittal slices have been calculated as explained in the text and have not been directly measured during the experiment. CsCl internal solution.
for the two time constants are given in Table 1). It is particularly remarkable that the main part of the decay (the second component) can be described by a single exponential. On average, the fast component contributed only 2 % of the total capacitive charge. No deviation from the model exponential could be detected in the late portion of the major component. As discussed later, this indicates that the entire dendritic tree is controlled with similar efficacy by the somatic voltage clamp. A second finding which comes out of these experiments is that Purkinje cells have a larger input resistance than hitherto reported with standard microelectrode impalements. In sagittal slices, the average input resistance was 167 + 50 MQ (mean + S.D.; n = 5) with internal KCl solutions and 441 + 177 MQ (n = 5) with CsCl solutions. These measurements were collected during the first 3 min of whole-cell recording. With CsCl pipettes there was a tendency of the input resistance to increase, as exemplified in Fig. 2. The equivalent circuit used to account for the passive current results, as discussed below, is depicted in Fig. 13 (Appendix). Essentially, the model distinguishes two regions in the Purkinje cell. Region 1, which has a surface area corresponding to a capacitance C1, represents the soma and the main proximal dendrites. It is separated from the head stage amplifier by the pipette access resistance R1. Region 2, which is associated with capacitance C2, represents the main part of the dendritic tree. It is linked to region 1 by resistor R2, which represents a lumped contribution of the many individual resistances arising between the main dendrites and each membrane region of the distal dendrites (see Discussion). Finally, R3 represents the lumped resistance of the dendritic tree membrane. As will be described in the Appendix, numerical values of the various cell parameters R1,R2,R3,C1 and C2 can be obtained from the parameters of a double exponential fit to the decay of the current. These parameters are listed in Table 1 for both transverse and sagittal slices. After a sudden displacement of Vo, V2 rises after a short lag following an exponential time course (Fig. 13, Appendix). The time constant of this exponential, T2, essentially sets the speed of the voltage-clamp system. If no series resistance
GLUTAMATE SYNAPSES IN PURKINJE CELLS
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compensation is performed, 72 = C2 (R1 +JR2) in the case that R3 is infinite. With optimum series resistance compensation, r2 could be decreased to C2R2, roughly an improvement by a factor of two. The procedure of C, cancellation is illustrated in Fig. 2B and D. Note that for the proper adjustment of CQ cancellation (performed
B
A
/(nA) 2
V (mV)
*
-80 -60 -40 -201
1 A
20 40 60
*-2
Ms~~~~~~~~
10
L-4 Fig. 3. Current-voltage relation of the PF-EPSC. A, currents recorded at membrane potentials of -70, -50, -30, -10, + 10 and + 30 mV. Each trace is the average of three records. B, relation between the Purkinje cell membrane potential and the peak amplitude of the evoked synaptic current. Transverse slice from a 15-day-old rat.
using the G-series and C-slow compensation controls of the amplifier) there is no discontinuity in the current trace at the onset of the voltage pulse (see arrow in Fig. 2D). The decay phase of the current can be described by a single exponential component (time constant of 4-6 ms in the example shown), corresponding to the time constant of the slow component of the raw capacitive transient. After 70% compensation of R1, T2 was found to decrease from 4-6 to 2-6 ms (Fig. 2C). This is in excellent agreement with the value of 2-5 ms predicted using eqn (10) below and numerical parameters obtained from the double exponential fit to the uncompensated transient. Values of T2 in transverse slices after capacitance cancellation and 65-75 % series resistance compensation (Tr, Table 1) averaged 2-12 ms (n = 9). The corresponding values averaged 3-31 ms in sagittal slices (n = 5). The 'r values for sagittal slices are derived from the uncompensated capacitive current transients rather than from direct measurement because the latter determination was usually not performed during the experiments. Postsynaptic currents arising from stimulation of the granule cell-parallel fibre system As reported elsewhere (Konnerth et al. 1990b), in voltage clamped Purkinje cells, postsynaptic currents whose amplitude is graded with the stimulus intensity can be reliably evoked by external stimulation within the granule cell layer or in the molecular layer. The graded nature of the postsynaptic currents is in accord with previous reports of the excitatory postsynaptic potentials which are obtained from Purkinje cells in vivo (reviewed by Eccles, Ito & Szentagothai, 1967 and by Ito, 1984)
I LLANO AND OTHERS
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as well as in cerebellar slices upon stimulation of parallel fibres (e.g. Crepel, Dhanjal & Garthwaite, 1981; Kimura, Okamoto & Sakai 1985a). We will thus refer to this graded signal as evoked synaptic current resulting from activation of the granule cell-parallel fibre system (PF-EPSC).
B 20.
A
+40 mvl
\
r(ms)
}~~~~~~~~r
109m
10-
7.5ms i o ~ ~ ~~~~T= -40 mVl
ln
\ 10 ms
2 -100
-60
-20
20
60
V (mV)
Fig. 4. Time course of decay of the PF-EPSC. A, evoked synaptic currents obtained from the same cell as illustrated in Fig. 3, at membrane potentials of + 40 mV (upper trace) and -40 mV (lower trace). Each trace is the average of three records. The smooth line superimposed on each of the experimental traces represents the fit of the decay phase of the current to a single exponential function. The two parameters derived from the fit, initial amplitude and time constant, were 1-66 nA and 10 9 ms at + 40 mV and - 1'87 nA and 7 5 ms at -40 mV. B, semilogarithmic plot of the time constant of decay as a function of membrane potential. r values were derived from the fit of the decay to a single exponential function, as illustrated in A. Transverse slice from a 15-day-old rat.
Figure 3A presents the PF-EPSC recorded from a Purkinje cell at different membrane holding potentials (from -70 to 30 mV). The absolute peak current amplitude increases linearly as the membrane potential is shifted away from the level at which the current reverses (O mV; see Fig. 3B). At all membrane potentials, the current activates with a lag of 1'8 ms from the start of the stimulating pulse, increases to a peak value 3-2-3-5 ms after the onset of the response, and decays thereafter to baseline levels. Although the membrane potential does not affect the onset of the synaptic current, the decay of the current is slower at more depolarized potentials. The effect of membrane potential on the time course of PF-EPSC decay is analysed in Fig. 4. The traces show EPSCs at -40 mV (Fig. 4A, lower trace) and + 40 mV (Fig. 4A, upper trace). In both cases, the decay phase of the current is well fitted by a single exponential, plotted as a smooth continuous line on the experimental traces. The time constant of decay (rd) obtained from the single exponential fit was 1-4 times slower at + 40 mV (Td of 10-9 ms) than at -40 mV (Td of 7-5 ms). As illustrated by the semilogarithmic plot presented in Fig. 4B, Td varied monotonically with
GLUTAMATE SYNAPSES IN PURKINJE CELLS 191 membrane potential. A linear regression fit of the data points (continuous line) yields an e-fold change in Td per 271 mV. PF-EPSCs having similar properties to those described above were recorded in slices derived from young animals (11-16 days old). In seven cells analysed, the latency between the stimulus and the onset of the response was 2-3 + 0-5 ms (mean+ S.D.), and the 10-90 % rise time was 1P8 + 0 4 ms. The time constant of decay at -60 mV, derived from single exponential fits, was 8-3 + 1-6 ms. In all of the cells studied, the reversal potential of the response was close to 0 mV. In older animals (over 20 days old), a similar type of stimulation elicited currents which exhibited a slower time course, taking 8-10 ms to reach their peak, and which reversed at values of membrane potentials above + 40 mV. This suggested that they were arising from a region of the cell escaping voltage control. Postsynaptic currents arising from stimulation of the climbing fibre Activation of the climbing fibre system via electrical stimulation of the white matter in cerebellar slices is known to elicit a powerful excitation of Purkinje cells. The postsynaptic potentials which have been recorded intracellularly upon such stimulation are of an all-or-none nature (e.g. Llinas & Sugimori, 1980; Crepel et al. 1981; Kimura et al. 1985a) and can thus be easily distinguished from the graded response which accompany activation of the parallel fibre pathway. Under our experimental conditions, all-or-none synaptic currents which we have interpreted as the stimulation of a climbing fibre can be easily evoked in Purkinje cells (Konnerth et al. 1990 b). The synaptic currents evoked by climbing fibre stimulation (CF-EPSC) are generally quite large (greater than 2 nA at -60 mV) and are capable of activating voltage-dependent conductances in the Purkinje cell. An example of this type of behaviour is illustrated in Fig. 5A, which presents a series of responses evoked by' stimulation 90 ,tm away from the Purkinje cell soma, when its membrane potential is held at -60 mV. While the response on the two smaller traces has a simple time course, the two larger ones show clear indications of the activation of regenerative membrane conductances at loci remote from the soma, which are not under voltage control. These responses with a regenerative component were usually found at the beginning of whole-cell recording and they tended to disappear later on. The active component of the response could be eliminated by holding the membrane potential at depolarized levels (above -50 mV), as illustrated in Fig. 5B. The records correspond to consecutive traces taken from the same cell as shown in Fig. 5A, but at a holding potential of -30 mV. Under these conditions, the response shows no evidence of active membrane conductances. The average of ten such CF-EPSCs is shown in the lower panel of Fig. 5B. The current rises smoothly, reaching its peak value 1-9 ms after its onset. As was the case for the PF-EPSC, the time course of decay for the CF-EPSC can be well described by a single exponential component, the time constant of decay for this particular cell being 6-5 ms (see figure legend). CF-EPSC could be easily distinguished from the antidromic spikes evoked by direct stimulation of the Purkinje cell axon. The antidromic spikes activate in an allor-none fashion but, in contrast to the synaptically evoked current, they have a much shorter latency (of the order of 200 ,ts from the onset of the stimulating pulse;
I. LLANO AND OTHERS
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see Fig. 5C). In some cases, stimulation elicited antidromic spikes which were followed by signals closely resembling the CF-EPSC with active components described above. In the example presented in Fig. 5D, one stimulus excited only the Purkinje cell axon, resulting in an antidromic spike. Another stimulus in the same
A
-60 mV
B
-30 mV
I 2 nA
I1 nA 10 ms
C
-60 mV
D
I 2 nA
-60 mV
J 2 nA
Fig. 5. A, synaptic currents and active responses elicited by climbing fibre stimulation. Each trace is a single sweep obtained at a holding potential of -60 mV, upon extracellular stimulation of the climbing fibre. The single stimulus is sufficient in two of the traces to elicit 'active responses', as evidenced by the notch in the rising phase of the current. B, upper panel, consecutive traces of the synaptic current evoked in the same cell and with identical stimulating conditions as for A, but at a holding potential of -30 mV. Lower panel, average of ten records obtained during the stimulation series illustrated in the upper panel. The decay phase of the current has been fitted by a single exponential function (smooth line) with a time constant of 6-53 ms and an initial amplitude of - 1-79 nA. C, antidromic spike evoked from direct axonal stimulation in the granule cell layer (two superimposed traces with identical stimulation intensity). These all-or-none signals activate within 200 ,us after the onset of the stimulating pulse. D, in a different location, stimulation elicits an antidromic spike which is sometimes followed by a CF response with active components (two superimposed traces). Data in A and B are from the same cell (transverse slice from a 16-day-old rat). Data in C and D are from another cell (transverse slice; 15-day-old rat).
location excited the Purkinje cell axon and the climbing fibre. In the latter case, the climbing fibre evoked a regenerative response in the axon or, more likely, in the dendrite, away from the point of voltage control.
GLUrTAMATE SYNAPSES IN PUTRKINJE CELLS
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The EPSCs associated with CF stimulation could be reliably elicited in either sagittal or transverse slices obtained from rats aged 9-22 days. In contrast to the graded PF-EPSC (see above), the CF-EPSC had a reversal potential close to 0 mV (ranging from -7 to + 5 mV), throughout the ages studied. The traces presented in I(nA)
B
A
01~~~~~~~~~~~~~~~
r-\ X~~~~~
2
~
~
-80 -60 -40 -20
~~~~~~~~~~~~~~~~~~ I 20
*
40 60 V (MV)
10 ms
-4
Fig. 6. Current-voltage relation of the CF-EPSC. A, currents recorded at membrane potentials of -60, -40, -20, 0, + 20 and + 40 mV. Each trace is the average of four records. B, relation between the membrane potential and the peak amplitude of the evoked synaptic current. Sagittal slice from a 22-day-old rat.
Fig. 6A show the CF-EPSC recorded from a Purkinje cell, in a slice derived from a 22-day-old animal, as the holding potential is increased from -60 to + 40 mV in increments of 20 mV. The relation between peak current amplitude and membrane potential is linear as illustrated in Fig. 6B, the reversal potential for this response being + 7 mV. The decay phase of the CF-EPSC was well approximated by a single exponential function, at all membrane potentials tested. Similar to the observations for PFEPSCs, the time constant of decay (Td) for CF-EPSCs generally increased as the membrane potential was held more positive. This dependence of Td on holding potential is presented in Fig. 7, which illustrates the fit by a single exponential of the decay phase of the CF-EPSC, recorded at +40 (Td = 12-5 ms; Fig. 7A, upper trace) and -40 mV (rd = 9 0 ms; Fig. 7A, lower trace). The increase in Td for this cell, as the membrane potential is varied between -70 and 50 mV, is shown by the semilogarithmic plot of Fig. 7B. An e-fold change in the decay time constant per 289 mV is predicted by a linear regression fit of the experimental data. CF-EPSCs had on average a faster time course than PF-EPSCs recorded from a comparable age group (11-16 days old). In nine CF-EPSCs analysed, the 10-90% rise time was 0-8 + 0-3 ms (mean + S.D.) and the time constant of decay at -60 mV was 6A4+1I1 ms.
PHY 434
1. LL,AN4O ANVD OTHERS
194
Effect of current amplitude and series resistance compensation on the time course of synaptic currents Series resistanee (Rj) compensation had different consequences on climbing fibre and on parallel fibre synaptic currents. In the first case, the most conspicuous effect A
B
vr(m s) +40 mV
125ms
10-
T=~~~-c9-0 ms
\ / -40 mV
/
~~~1nA
2-
-80
-40
0
40
80
V(mV) Fig. 7. Time course of decay of the CF-EPSC. A, synaptic currents recorded at holding potentials of +40 mV (upper trace) and -40 mV (lower trace). Each trace is the average of five records. The smooth line superimposed on each of the experimental traces represents the fit of the decay phase of the current to a single exponential functio"i. The two parameters obtained from the fit, initial amplitude and time constant, were 1-85 nA and 12-5 ms at +40 mV and -2-03 nA and 90 ins at -40 mV. B, semilogarithmic plot of the time constant of decay as a function of membrane potential. rTvalues were obtained from the fit of the decay to a single exponential function, as illustrated in A. Sagittal slice from a 13-day-old rat.
was to increase the size of the synaptic current, without significant alterations on the rising phase. This effect is illustrated in Fig. 8A, which shows CF-EPSCs in the absence of series resistance compensation (trace a) and after compensation of 60% Rs (trace b). In the compensated condition, the peak current amplitude was increased by a factor of 1 4. Note that in the absence of Rs compensation, the current decay deviates from a single exponential function. This is expected since the series resistance error is proportionally larger near the peak of the current and transforms an exponential decay to a non-exponential curve. In order to test the predictions of the model developed in the Appendix, successive corrections according to eqns (12) and (13) were applied to experimental trace a to mimic a 60% Rs compensation. The resulting curve (not shown) had a peak amplitude of 6-2 nA and a Td of 6 0 ms, in excellent agreement with the experimental parameters obtained with 60 % Rs compensation (trace b; 6-2 nA and 6-5 ms). With PF-EPSCs, the effect of Rs compensation was variable. The effect was inconspicuous for responses having a fast rise time but rather marked for the slower responses. One example of the latter case is shown in Fig. 8B. The entire time course
GLUTAMATE SYNAPSES IL PLURKIVJE CELLS
195
of the PF-EPSC could be described by the sum of two exponentials with opposite polarities. In the uncompensated condition (trace a), the corresponding time constants had values of 641 and 6-4 ms. After Rs compensation (70%; trace b), the time constant of the rising phase decreased to 3-3 ms, but there was no significant A
B
b
Fig. 8. Effect of R, compensation on the amplitude and time course of EPSCs. A, CFEPSCs are presented in the absence of Rs compensation (a) and after compensation of R, by 60 % (b). Holding potential, -40 mV. In the uncompensated condition R" was 3-8 MQ. Each trace is the average of four sweeps; 10-90 % rise times were 0-6 and 0 5 ms for a and b respectively. The smooth lines on top of the experimental traces correspond to the fit of the decay phase to a single exponential function, with time constant of 6-o ms. Note that the uncompensated trace deviates significantly from the fit. Transverse slice from a 11-day-old rat. B, PF-EPSCs are presented in the absence of Rs compensation (a) and after 70% compensation (b). Holding potential, -70 mV. In the uncompensated condition Rs was 9 MQ. Each trace is the average of twenty sweeps. The smooth lines superimposed on each experimental trace correspond to the fit of the data to the sum of two exponentials of opposite polarities. The time constants derived from the fits were 6-1 and 6-4 ins for a and 3-3 and 6-2 ms for b. The stimulus artifact has not been subtracted from the raw data and gives rise to a deflection at the onset of the current. Transverse slice from a 15-day-old rat.
change of the current amplitude nor of its decay time constant. Such behaviour, as predicted by eqns (10) and (11) below, is the signature of a synaptic current arising exclusively in the C2 compartment (see Appendix). Thus, the time constants of the rising phase of the PF-EPSC are in reasonable agreement with the values of r2 and T2 obtained from the same cell (4 0 and 3 4 ms respectively). It must be considered that the percentage of feasible Rs compensation is limited (60-75%). The residual Rs is expected to deform the time course and amplitude of the recorded currents, such deformation being a function of the current amplitude (see eqn (12) below). The effects of current amplitude on the decay time course were investigated both for PF-EPSCs and for CF-EPSCs. In the first case, there was no significant correlation between current amplitude and the time constant of decay of the graded response elicited on a given cell, probably due to the relatively small size of the synaptic currents (typically ranging from less than 100 pA to 2 nA at -70 mV). Larger effects were expected when using climbing fibre stimulation, which in some cases elicited currents as large as 8 nA at -70 mV. However, a direct assessment of the relation between current amplitude and time course was usually not possible for a given cell in this case since the response was all-or-none. But on 7-2
,/c
Journal of Physiology (1991), 434, pp. 183-213 With 13 figures Printed in Great Britain
SYNAPTIC- AND AGONIST-INDUCED EXCITATORY CURRENTS OF PURKINJE CELLS IN RAT CEREBELLAR SLICES
BY ISABEL LLANO*, ALAIN MARTY*, CLAY M. ARMSTRONGt AND ARTHUR KONNERTHt From *the Laboratoire de Neurobiologie, Ecole Normale Supe'rieure, Paris, France and the Wax-Planck Institut fur Biophysikalische Chemie, Gottingen, Germany (Received 17 January 1990) SUMMARY
1. Postsynaptic currents originating from activation of the two major excitatory inputs to Purkinje cells were studied in thin slices of rat cerebellum, using the tightseal whole-cell recording technique. Two types of excitatory postsynaptic currents were analysed: those evoked by stimulation of the granule cell-parallel fibre system (PF-EPSC) and those elicited by stimulation of the climbing fibres (CF-EPSC). 2. Both types of postsynaptic currents had a linear current-voltage relation, reversing at membrane potentials close to 0 mV. Their time course of activation was independent of the membrane potential. 3. For both types of postsynaptic currents, the time course of decay was well described by a single exponential function, with a time constant which increased as the membrane potential was made more positive. 4. Postsynaptic currents arising from stimulation of the climbing fibre generally had a slightly faster time course of onset and decay than those associated with stimulation of the granule cell-parallel fibre system. The average values of the 10-90% rise time were 1-8+004 ms (meanss .D. n = 7) for PF-EPSCs and 0 8 + 0 3 ms (n = 9) for CF-EPSCs. Time constants of decay, at a holding potential of -60 mV, had values of 8-3+ 1-6 ms (n = 7) and 6 4+ 1 1 ms (n = 9) for PF-EPSCs and CF-EPSCs respectively. 5. CF-EPSCs and PF-EPSCs had the characteristics described above in slices derived from animals aged 9-22 days old and 9-15 days old, respectively. The PFEPSCs in animals older than 15 days had very slow time courses and positive apparent reversal potentials, suggesting that they originated from distal locations, not under accurate voltage control. 6. In order to assess the quality of the voltage clamp, responses to hyperpolarizing pulses from -70 mV were analysed. The capacitive currents could be fitted by the sum of two exponentials, and were interpreted with an equivalent electrical circuit comprising two main compartments (soma and proximal dendrites on one hand, distal dendrites on the other). Analysis of synaptic currents in terms of this model suggested that the recorded time course of decay was approximately correct. t Permanent address: Department of Physiology, University of Pennsylvania, Philadelphia, PA, USA. MIS 8201
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7. CF-EPSCs as well as PF-EPSCs were insensitive to the NMDA receptor antagonist 3-3(2-carboxypiperazine-4-yl)propyl- 1 -phosphonate (CPP), but were blocked in a dose-dependent reversible manner by the non-NMDA antagonist 6cyano-7-nitroquinoxaline-2,3-dione (CNQX). 8. The responses of Purkinje cells to exogenous application of various glutamate agonists were tested in the presence of tetrodotoxin (TTX) and bicuculline to minimize polysynaptic contributions (8- to 11-day-old rats). The Purkinje cells had a high sensitivity to the glutamate agonists quisqualate and kainate, but were insensitive to N-methyl-D-aspartate (NMDA). 9. In the absence of TTX and bicuculline, NMDA induced large responses with a clear polysynaptic nature. These responses were identified as arising mainly from the activation of GABAergic interneurones either directly or following excitation of granule cells. A large fraction (80-90 %) of the polysynaptic response to NMDA was blocked by bicuculline, while only 10-20 % of such response was blocked by CNQX. These results suggest that cerebellar inhibitory interneurones respond directly to NMDA. INTRODUCTION
The aim of the present study was to establish the basic properties of the postsynaptic currents which can be evoked in Purkinje cells upon stimulation of its two main excitatory afferents, under conditions that allow the membrane potential and the intracellular composition of the Purkinje cell to be controlled. To fulfill this purpose, we have used a recently reported method (Edwards, Konnerth, Sakmann & Takahashi, 1989) which renders possible the application of patch-clamp recording techniques (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) to neuronal cells in acutely prepared slices of brain tissue. The analysis of postsynaptic currents is complicated in adult Purkinje cells by the extensive dendritic arborization, which is likely to escape from a somatic voltage-clamp system. This is especially important in view of the fact that in the adult state a large part of the excitatory input (specifically that arising from parallel fibres) arrives to dendritic locations which are far away from the cell soma. To minimize this problem, we have carried out most of our studies in cerebella derived from young animals, where synapses from various afferents are more likely to be localized at regions of the dendritic arborization which are electrically close to the cell body. Using this experimental approach, excitatory synaptic currents can be recorded from Purkinje cells upon extracellular stimulation of climbing fibres and of the granule cell-parallel fibre system. The synaptic currents evoked by each of these afferent pathways can be easily identified on the basis of their dependence on stimulus intensity and their response to paired stimuli (Konnerth, Llano & Armstrong, 1990b). In the present report we describe these excitatory postsynaptic currents as recorded with a tight-seal whole-cell voltage clamp and discuss the reliability of their voltage dependence and kinetic properties. Based on pharmacological evidence, these currents are identified as arising from the activation of non-NMDA glutamate receptors. These results are complemented by an analysis of the sensitivity of Purkinje cells to various glutamate agonists.
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METHODS
Slices were prepared from rat cerebellum following the general procedures described by Edwards et al. (1989). Briefly, rats (8-22 days old) were killed by decapitation after cervical dislocation, the cerebellar vermis was quickly dissected out and immersed for a couple of minutes in ice-cold bicarbonate-buffered saline (BBS; see composition below). The tissue was subsequently glued to the stage of a vibroslicer and sliced at a thickness of 130-140 ,um. Sagittal as well as transverse slices (i.e. cut along the parallel fibre axis) were used in the present studies. The slices were incubated in oxygenated BBS saline at 36 °C for at least 45 min prior to the beginning of experimental recordings. Patch-clamp recordings from Purkinje cells were performed on slices visualized at 40 x magnification with Nomarski optics using an upright microscope (either a Zeiss 'Standard 14' or a Zeiss 'Axioscope'). Under these conditions, recognition of the various cell layers within the cerebellar cortex as well as identification of individual cells was easily achieved, as shown in Fig. 1. Two pipettes are seen in the photograph, a small patch pipette in contact with a Purkinje cell, and a larger (10 ,um) pipette in the granule cell layer. To the right in the photograph, in the same plane of focus, is another Purkinje cell with a prominent dendritic shaft extending into the molecular layer. The surface of the Purkinje cell to be studied was cleaned with glass pipettes of 8-10 ,am diameter prior to seal formation, following the general technique described by Edwards et al. (1989). During recordings, the slices were maintained at room temperature (20-22 °C) and were continuously perfused at a rate of 2 ml/min with the standard bicarbonate-buffered saline (BBS) which contained (in mM): 125 NaCl, 2 5 KCl, 2 CaCl2, 1 MgCl2, 1-25 NaH2PO4, 26 NaHCO3 and 25 glucose; a mixture of 95 % 02 and 5% C02 was bubbled through the solution to keep its pH at 7-4. Unless otherwise noted, bicuculline (10-16 /tM) was added to the external solution, in order to eliminate GABAergic postsynaptic currents. In the series of experiments designed to test the sensitivity of Purkinje cells to exogenous agonists, the slices were bathed in Mg2+-free BBS, which had the same composition as the BBS described above, except that Mg2+ was omitted from the solution. The tight-seal whole-cell recording technique (Hamill et al. 1981) was used for all experiments presented in this paper. Seal resistances were always larger than 10 GfQ. Recording pipettes were pulled from borosilicate glass, coated with Sylgard resin (Dow Corning Chemical Co.) and firepolished to a final tip diameter of approximately 2-5 ,um. They had a resistance of 2-3 MQ when filled with the internal saline, which had the following composition (in mM): 140 CsCl, 2 MgCl2, 1 CaC12 10 Cs-EGTA, 10 Cs-Hepes and 4 Na-ATP (pH of 7-3). In some experiments, performed to assess the passive properties of Purkinje cells, an internal K+ solution was used. This solution contained (in mM): 140 KCl, 2 MgCl2, 1 CaCl2, 10 K-EGTA, 10 K-HEPES and 4 Na-ATP (pH of 7 3). Membrane currents were recorded with a patch-clamp amplifier (EPC-7, List Electronic, Germany) set at a gain of 0 5 or 1 mV/pA. The slow transient cancellation and G-series adjustment of the amplifier were used to compensate the initial portion of the capacitance transient elicited by 10 mV hyperpolarizing pulses, and to estimate the value of the series resistance, as explained in the results section. Typical values for this parameter were 5-10 MiQ. The series resistance compensation control of the amplifier was set at 60-75 %. For the study of passive properties of Purkinje cells, current transients elicited by hyperpolarizing steps were filtered with an 8-pole Bessel filter (Frequency Devices, USA) set at a corner frequency of 5 kHz and digitized at a sampling rate 50 ,us per point. Purkinje cell postsynaptic currents were evoked by electrical stimulation, using a bipolar electrode consisting of a 7-10 ,um tip diameter glass pipette (see Fig. 1) and a remote 100 4am thick platinum wire. Square pulses of 100-300 Its duration and amplitudes ranging from 1-5 to 12 V were applied, while the glass pipette was moved within the visual field until the synaptic current was evoked with minimum stimulus intensity. The EPSCs were filtered at 3-5 kHz and digitized at a sample rate of 50 jus per point. In most cases, analysis of the synaptic signals was performed after subtraction from the raw data of the stimulation artifact, using a series of records taken at the reversal potential of the current. Rise times were measured as the time required for the current to change from 10 to 90% of its peak amplitude. Fits of model exponentials to the experimental data were performed using an analysis program based on an error-minimization algorithm. For fits of the decay of EPSCs, the first 1-3-1-5 ms
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following the peak of the current were excluded from the fit. In some cases, a double exponential fit to both rising and decaying phases of EPSCs was performed. Application of various drugs and transmitter agonists was )erformed by changing the bath to a solution containing the relevant compound. Given the perfusion rate (2 ml/min) and the volume (700 jdl) of the chamber, exchange of the external solution could be achieved within less than 1 min.
XV.
Fig. 1. View of the experimental preparation and recording configuration. Photograph of a transverse cerebellar slice from a 14-day-old rat, taken at 400 x magnification. The upper part of the figure corresponds to the molecular layer, and the lowest portion to the granule cell layer. A patch pipette is in contact with a Purkinje cell and a stimulating pipette (10,am diameter) is positioned in the granule cell layer. Calibration bar (upper left) equals 20 ,um. The glutamate agonists N-methyl-D-aspartic acid (NMDA), quisqualic acid (Quis) and kainic acid (Kai) were purchased from Cambridge Research Biochemicals. The NMDA-receptor antagonist 3-3(2-carboxypiperazine-4-yl)propyl-1-phosphonate (CPP), the non-NMDA antagonist 6-eyano7-nitroquinoxaline-2,3-dione (CNQX) and the GABAA receptor antagonist bieuculline were purchased from Tocris Neuramin. Tetrodotoxin (TTX) was purchased from Sigma Chemical Co. RESULTS
Passive properties of the Purkinje cell membrane The interpretation of recorded synaptic currents necessitates a basic knowledge of the passive properties of Purkinje cells. In this section, we describe the capacitive and resistive currents of these neurones, and present a qualitative interpretation of
GLUTAMATE SYNAPSES IN PURKINJE CELLS18 187 the results in terms of an equivalent circuit modelling which is made explicit in the
Appendix. Figure 2A presents the current response of a Purkinje cell to a voltage step from - 70 to - 80 mV. The decay of the current was biphasic, and could be described by A
500 pA
5 ms B
=
4.6 ms
C
,r=
2.6 ms
D
1 Ms
Fig. 2. Capacitive currents in Purkinje cells. Current responses to 10 mV hyperpolarizing voltage pulses from a holding potential of - 70 mV are shown at various stages of the series resistance compensation. Each trace is the average of seven sweeps. A, uncompensated. B, after cancellation of the fast capacitive current. C, after capacitive current cancellation and series resistance compensation (70 %). D, expanded traces for the early part of the capacitive currents in A and B. A double exponential fit of the uncompensated current is shown in A (where the fit cannot be distinguished from the experimental trace) and in D, left trace. Amplitude coefficients and time constants for the two exponentials fitted were: - 437 pA, 0-26 ms and - 690 pA, 4-51 ins. Single exponential fits to the current decay are superimposed on the current records in B, C and in D, right trace. When going from B to C, the time constant of decay, T2' decreased from 4-6 to 2-6 ins. The small sustained inward current visible in A represents a leakage conductance of 314 MCI. This measurement was taken after 1 min of whole-cell recording. Later on this resistance increased to over 1 Gfl (traces in B and C were taken 2 and 3 min after that in A respectively) presumably as a result of Cs+ ions diffusing into the cell. Transverse slice from a 12-day-old rat.
I. LLANO AND OTHERS 188 the sum of two exponential components with time constants of 0'3 and 45 ms. The fit is shown as a smooth continuous line on top of the experimental trace of Fig. 2A. Its initial portion is displayed at an expanded time scale in the left trace of Fig. 2D. In general, the decay was well fitted by the sum of two exponentials. The ratio between the two time constants was quite large, of the order of 20 (average values TABLE 1. Passive properties of Purkinje neurones n C1(pF) C2(pF) R1(MfQ) R2(MQ) R3(MQ) 2(ms) T'2(ms) Tl(ms) 146+91 494+ 100 9 7 +0-9 4 0+ 1-0 441 + 177 0 40+0-20 6-83+ 1-75 3-31 +0-86 5
Sagittal slices Transverse 87 + 54 373 + 113 7-4 + 1-2 6-6 + 3-9 283 + 90 0-20 + 0-03 4-64 + 1-80 2-12 + 1 00 8-9 slices Means + S.D. for each series of n cells are given. rl is the time constant of the fast component of the capacitive transient. T2 is the time constant of the slow component of the capacitive transient, and T'2 is the time constant of this component after series resistance compensation. T'2 values for sagittal slices have been calculated as explained in the text and have not been directly measured during the experiment. CsCl internal solution.
for the two time constants are given in Table 1). It is particularly remarkable that the main part of the decay (the second component) can be described by a single exponential. On average, the fast component contributed only 2 % of the total capacitive charge. No deviation from the model exponential could be detected in the late portion of the major component. As discussed later, this indicates that the entire dendritic tree is controlled with similar efficacy by the somatic voltage clamp. A second finding which comes out of these experiments is that Purkinje cells have a larger input resistance than hitherto reported with standard microelectrode impalements. In sagittal slices, the average input resistance was 167 + 50 MQ (mean + S.D.; n = 5) with internal KCl solutions and 441 + 177 MQ (n = 5) with CsCl solutions. These measurements were collected during the first 3 min of whole-cell recording. With CsCl pipettes there was a tendency of the input resistance to increase, as exemplified in Fig. 2. The equivalent circuit used to account for the passive current results, as discussed below, is depicted in Fig. 13 (Appendix). Essentially, the model distinguishes two regions in the Purkinje cell. Region 1, which has a surface area corresponding to a capacitance C1, represents the soma and the main proximal dendrites. It is separated from the head stage amplifier by the pipette access resistance R1. Region 2, which is associated with capacitance C2, represents the main part of the dendritic tree. It is linked to region 1 by resistor R2, which represents a lumped contribution of the many individual resistances arising between the main dendrites and each membrane region of the distal dendrites (see Discussion). Finally, R3 represents the lumped resistance of the dendritic tree membrane. As will be described in the Appendix, numerical values of the various cell parameters R1,R2,R3,C1 and C2 can be obtained from the parameters of a double exponential fit to the decay of the current. These parameters are listed in Table 1 for both transverse and sagittal slices. After a sudden displacement of Vo, V2 rises after a short lag following an exponential time course (Fig. 13, Appendix). The time constant of this exponential, T2, essentially sets the speed of the voltage-clamp system. If no series resistance
GLUTAMATE SYNAPSES IN PURKINJE CELLS
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compensation is performed, 72 = C2 (R1 +JR2) in the case that R3 is infinite. With optimum series resistance compensation, r2 could be decreased to C2R2, roughly an improvement by a factor of two. The procedure of C, cancellation is illustrated in Fig. 2B and D. Note that for the proper adjustment of CQ cancellation (performed
B
A
/(nA) 2
V (mV)
*
-80 -60 -40 -201
1 A
20 40 60
*-2
Ms~~~~~~~~
10
L-4 Fig. 3. Current-voltage relation of the PF-EPSC. A, currents recorded at membrane potentials of -70, -50, -30, -10, + 10 and + 30 mV. Each trace is the average of three records. B, relation between the Purkinje cell membrane potential and the peak amplitude of the evoked synaptic current. Transverse slice from a 15-day-old rat.
using the G-series and C-slow compensation controls of the amplifier) there is no discontinuity in the current trace at the onset of the voltage pulse (see arrow in Fig. 2D). The decay phase of the current can be described by a single exponential component (time constant of 4-6 ms in the example shown), corresponding to the time constant of the slow component of the raw capacitive transient. After 70% compensation of R1, T2 was found to decrease from 4-6 to 2-6 ms (Fig. 2C). This is in excellent agreement with the value of 2-5 ms predicted using eqn (10) below and numerical parameters obtained from the double exponential fit to the uncompensated transient. Values of T2 in transverse slices after capacitance cancellation and 65-75 % series resistance compensation (Tr, Table 1) averaged 2-12 ms (n = 9). The corresponding values averaged 3-31 ms in sagittal slices (n = 5). The 'r values for sagittal slices are derived from the uncompensated capacitive current transients rather than from direct measurement because the latter determination was usually not performed during the experiments. Postsynaptic currents arising from stimulation of the granule cell-parallel fibre system As reported elsewhere (Konnerth et al. 1990b), in voltage clamped Purkinje cells, postsynaptic currents whose amplitude is graded with the stimulus intensity can be reliably evoked by external stimulation within the granule cell layer or in the molecular layer. The graded nature of the postsynaptic currents is in accord with previous reports of the excitatory postsynaptic potentials which are obtained from Purkinje cells in vivo (reviewed by Eccles, Ito & Szentagothai, 1967 and by Ito, 1984)
I LLANO AND OTHERS
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as well as in cerebellar slices upon stimulation of parallel fibres (e.g. Crepel, Dhanjal & Garthwaite, 1981; Kimura, Okamoto & Sakai 1985a). We will thus refer to this graded signal as evoked synaptic current resulting from activation of the granule cell-parallel fibre system (PF-EPSC).
B 20.
A
+40 mvl
\
r(ms)
}~~~~~~~~r
109m
10-
7.5ms i o ~ ~ ~~~~T= -40 mVl
ln
\ 10 ms
2 -100
-60
-20
20
60
V (mV)
Fig. 4. Time course of decay of the PF-EPSC. A, evoked synaptic currents obtained from the same cell as illustrated in Fig. 3, at membrane potentials of + 40 mV (upper trace) and -40 mV (lower trace). Each trace is the average of three records. The smooth line superimposed on each of the experimental traces represents the fit of the decay phase of the current to a single exponential function. The two parameters derived from the fit, initial amplitude and time constant, were 1-66 nA and 10 9 ms at + 40 mV and - 1'87 nA and 7 5 ms at -40 mV. B, semilogarithmic plot of the time constant of decay as a function of membrane potential. r values were derived from the fit of the decay to a single exponential function, as illustrated in A. Transverse slice from a 15-day-old rat.
Figure 3A presents the PF-EPSC recorded from a Purkinje cell at different membrane holding potentials (from -70 to 30 mV). The absolute peak current amplitude increases linearly as the membrane potential is shifted away from the level at which the current reverses (O mV; see Fig. 3B). At all membrane potentials, the current activates with a lag of 1'8 ms from the start of the stimulating pulse, increases to a peak value 3-2-3-5 ms after the onset of the response, and decays thereafter to baseline levels. Although the membrane potential does not affect the onset of the synaptic current, the decay of the current is slower at more depolarized potentials. The effect of membrane potential on the time course of PF-EPSC decay is analysed in Fig. 4. The traces show EPSCs at -40 mV (Fig. 4A, lower trace) and + 40 mV (Fig. 4A, upper trace). In both cases, the decay phase of the current is well fitted by a single exponential, plotted as a smooth continuous line on the experimental traces. The time constant of decay (rd) obtained from the single exponential fit was 1-4 times slower at + 40 mV (Td of 10-9 ms) than at -40 mV (Td of 7-5 ms). As illustrated by the semilogarithmic plot presented in Fig. 4B, Td varied monotonically with
GLUTAMATE SYNAPSES IN PURKINJE CELLS 191 membrane potential. A linear regression fit of the data points (continuous line) yields an e-fold change in Td per 271 mV. PF-EPSCs having similar properties to those described above were recorded in slices derived from young animals (11-16 days old). In seven cells analysed, the latency between the stimulus and the onset of the response was 2-3 + 0-5 ms (mean+ S.D.), and the 10-90 % rise time was 1P8 + 0 4 ms. The time constant of decay at -60 mV, derived from single exponential fits, was 8-3 + 1-6 ms. In all of the cells studied, the reversal potential of the response was close to 0 mV. In older animals (over 20 days old), a similar type of stimulation elicited currents which exhibited a slower time course, taking 8-10 ms to reach their peak, and which reversed at values of membrane potentials above + 40 mV. This suggested that they were arising from a region of the cell escaping voltage control. Postsynaptic currents arising from stimulation of the climbing fibre Activation of the climbing fibre system via electrical stimulation of the white matter in cerebellar slices is known to elicit a powerful excitation of Purkinje cells. The postsynaptic potentials which have been recorded intracellularly upon such stimulation are of an all-or-none nature (e.g. Llinas & Sugimori, 1980; Crepel et al. 1981; Kimura et al. 1985a) and can thus be easily distinguished from the graded response which accompany activation of the parallel fibre pathway. Under our experimental conditions, all-or-none synaptic currents which we have interpreted as the stimulation of a climbing fibre can be easily evoked in Purkinje cells (Konnerth et al. 1990 b). The synaptic currents evoked by climbing fibre stimulation (CF-EPSC) are generally quite large (greater than 2 nA at -60 mV) and are capable of activating voltage-dependent conductances in the Purkinje cell. An example of this type of behaviour is illustrated in Fig. 5A, which presents a series of responses evoked by' stimulation 90 ,tm away from the Purkinje cell soma, when its membrane potential is held at -60 mV. While the response on the two smaller traces has a simple time course, the two larger ones show clear indications of the activation of regenerative membrane conductances at loci remote from the soma, which are not under voltage control. These responses with a regenerative component were usually found at the beginning of whole-cell recording and they tended to disappear later on. The active component of the response could be eliminated by holding the membrane potential at depolarized levels (above -50 mV), as illustrated in Fig. 5B. The records correspond to consecutive traces taken from the same cell as shown in Fig. 5A, but at a holding potential of -30 mV. Under these conditions, the response shows no evidence of active membrane conductances. The average of ten such CF-EPSCs is shown in the lower panel of Fig. 5B. The current rises smoothly, reaching its peak value 1-9 ms after its onset. As was the case for the PF-EPSC, the time course of decay for the CF-EPSC can be well described by a single exponential component, the time constant of decay for this particular cell being 6-5 ms (see figure legend). CF-EPSC could be easily distinguished from the antidromic spikes evoked by direct stimulation of the Purkinje cell axon. The antidromic spikes activate in an allor-none fashion but, in contrast to the synaptically evoked current, they have a much shorter latency (of the order of 200 ,ts from the onset of the stimulating pulse;
I. LLANO AND OTHERS
192
see Fig. 5C). In some cases, stimulation elicited antidromic spikes which were followed by signals closely resembling the CF-EPSC with active components described above. In the example presented in Fig. 5D, one stimulus excited only the Purkinje cell axon, resulting in an antidromic spike. Another stimulus in the same
A
-60 mV
B
-30 mV
I 2 nA
I1 nA 10 ms
C
-60 mV
D
I 2 nA
-60 mV
J 2 nA
Fig. 5. A, synaptic currents and active responses elicited by climbing fibre stimulation. Each trace is a single sweep obtained at a holding potential of -60 mV, upon extracellular stimulation of the climbing fibre. The single stimulus is sufficient in two of the traces to elicit 'active responses', as evidenced by the notch in the rising phase of the current. B, upper panel, consecutive traces of the synaptic current evoked in the same cell and with identical stimulating conditions as for A, but at a holding potential of -30 mV. Lower panel, average of ten records obtained during the stimulation series illustrated in the upper panel. The decay phase of the current has been fitted by a single exponential function (smooth line) with a time constant of 6-53 ms and an initial amplitude of - 1-79 nA. C, antidromic spike evoked from direct axonal stimulation in the granule cell layer (two superimposed traces with identical stimulation intensity). These all-or-none signals activate within 200 ,us after the onset of the stimulating pulse. D, in a different location, stimulation elicits an antidromic spike which is sometimes followed by a CF response with active components (two superimposed traces). Data in A and B are from the same cell (transverse slice from a 16-day-old rat). Data in C and D are from another cell (transverse slice; 15-day-old rat).
location excited the Purkinje cell axon and the climbing fibre. In the latter case, the climbing fibre evoked a regenerative response in the axon or, more likely, in the dendrite, away from the point of voltage control.
GLUrTAMATE SYNAPSES IN PUTRKINJE CELLS
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The EPSCs associated with CF stimulation could be reliably elicited in either sagittal or transverse slices obtained from rats aged 9-22 days. In contrast to the graded PF-EPSC (see above), the CF-EPSC had a reversal potential close to 0 mV (ranging from -7 to + 5 mV), throughout the ages studied. The traces presented in I(nA)
B
A
01~~~~~~~~~~~~~~~
r-\ X~~~~~
2
~
~
-80 -60 -40 -20
~~~~~~~~~~~~~~~~~~ I 20
*
40 60 V (MV)
10 ms
-4
Fig. 6. Current-voltage relation of the CF-EPSC. A, currents recorded at membrane potentials of -60, -40, -20, 0, + 20 and + 40 mV. Each trace is the average of four records. B, relation between the membrane potential and the peak amplitude of the evoked synaptic current. Sagittal slice from a 22-day-old rat.
Fig. 6A show the CF-EPSC recorded from a Purkinje cell, in a slice derived from a 22-day-old animal, as the holding potential is increased from -60 to + 40 mV in increments of 20 mV. The relation between peak current amplitude and membrane potential is linear as illustrated in Fig. 6B, the reversal potential for this response being + 7 mV. The decay phase of the CF-EPSC was well approximated by a single exponential function, at all membrane potentials tested. Similar to the observations for PFEPSCs, the time constant of decay (Td) for CF-EPSCs generally increased as the membrane potential was held more positive. This dependence of Td on holding potential is presented in Fig. 7, which illustrates the fit by a single exponential of the decay phase of the CF-EPSC, recorded at +40 (Td = 12-5 ms; Fig. 7A, upper trace) and -40 mV (rd = 9 0 ms; Fig. 7A, lower trace). The increase in Td for this cell, as the membrane potential is varied between -70 and 50 mV, is shown by the semilogarithmic plot of Fig. 7B. An e-fold change in the decay time constant per 289 mV is predicted by a linear regression fit of the experimental data. CF-EPSCs had on average a faster time course than PF-EPSCs recorded from a comparable age group (11-16 days old). In nine CF-EPSCs analysed, the 10-90% rise time was 0-8 + 0-3 ms (mean + S.D.) and the time constant of decay at -60 mV was 6A4+1I1 ms.
PHY 434
1. LL,AN4O ANVD OTHERS
194
Effect of current amplitude and series resistance compensation on the time course of synaptic currents Series resistanee (Rj) compensation had different consequences on climbing fibre and on parallel fibre synaptic currents. In the first case, the most conspicuous effect A
B
vr(m s) +40 mV
125ms
10-
T=~~~-c9-0 ms
\ / -40 mV
/
~~~1nA
2-
-80
-40
0
40
80
V(mV) Fig. 7. Time course of decay of the CF-EPSC. A, synaptic currents recorded at holding potentials of +40 mV (upper trace) and -40 mV (lower trace). Each trace is the average of five records. The smooth line superimposed on each of the experimental traces represents the fit of the decay phase of the current to a single exponential functio"i. The two parameters obtained from the fit, initial amplitude and time constant, were 1-85 nA and 12-5 ms at +40 mV and -2-03 nA and 90 ins at -40 mV. B, semilogarithmic plot of the time constant of decay as a function of membrane potential. rTvalues were obtained from the fit of the decay to a single exponential function, as illustrated in A. Sagittal slice from a 13-day-old rat.
was to increase the size of the synaptic current, without significant alterations on the rising phase. This effect is illustrated in Fig. 8A, which shows CF-EPSCs in the absence of series resistance compensation (trace a) and after compensation of 60% Rs (trace b). In the compensated condition, the peak current amplitude was increased by a factor of 1 4. Note that in the absence of Rs compensation, the current decay deviates from a single exponential function. This is expected since the series resistance error is proportionally larger near the peak of the current and transforms an exponential decay to a non-exponential curve. In order to test the predictions of the model developed in the Appendix, successive corrections according to eqns (12) and (13) were applied to experimental trace a to mimic a 60% Rs compensation. The resulting curve (not shown) had a peak amplitude of 6-2 nA and a Td of 6 0 ms, in excellent agreement with the experimental parameters obtained with 60 % Rs compensation (trace b; 6-2 nA and 6-5 ms). With PF-EPSCs, the effect of Rs compensation was variable. The effect was inconspicuous for responses having a fast rise time but rather marked for the slower responses. One example of the latter case is shown in Fig. 8B. The entire time course
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of the PF-EPSC could be described by the sum of two exponentials with opposite polarities. In the uncompensated condition (trace a), the corresponding time constants had values of 641 and 6-4 ms. After Rs compensation (70%; trace b), the time constant of the rising phase decreased to 3-3 ms, but there was no significant A
B
b
Fig. 8. Effect of R, compensation on the amplitude and time course of EPSCs. A, CFEPSCs are presented in the absence of Rs compensation (a) and after compensation of R, by 60 % (b). Holding potential, -40 mV. In the uncompensated condition R" was 3-8 MQ. Each trace is the average of four sweeps; 10-90 % rise times were 0-6 and 0 5 ms for a and b respectively. The smooth lines on top of the experimental traces correspond to the fit of the decay phase to a single exponential function, with time constant of 6-o ms. Note that the uncompensated trace deviates significantly from the fit. Transverse slice from a 11-day-old rat. B, PF-EPSCs are presented in the absence of Rs compensation (a) and after 70% compensation (b). Holding potential, -70 mV. In the uncompensated condition Rs was 9 MQ. Each trace is the average of twenty sweeps. The smooth lines superimposed on each experimental trace correspond to the fit of the data to the sum of two exponentials of opposite polarities. The time constants derived from the fits were 6-1 and 6-4 ins for a and 3-3 and 6-2 ms for b. The stimulus artifact has not been subtracted from the raw data and gives rise to a deflection at the onset of the current. Transverse slice from a 15-day-old rat.
change of the current amplitude nor of its decay time constant. Such behaviour, as predicted by eqns (10) and (11) below, is the signature of a synaptic current arising exclusively in the C2 compartment (see Appendix). Thus, the time constants of the rising phase of the PF-EPSC are in reasonable agreement with the values of r2 and T2 obtained from the same cell (4 0 and 3 4 ms respectively). It must be considered that the percentage of feasible Rs compensation is limited (60-75%). The residual Rs is expected to deform the time course and amplitude of the recorded currents, such deformation being a function of the current amplitude (see eqn (12) below). The effects of current amplitude on the decay time course were investigated both for PF-EPSCs and for CF-EPSCs. In the first case, there was no significant correlation between current amplitude and the time constant of decay of the graded response elicited on a given cell, probably due to the relatively small size of the synaptic currents (typically ranging from less than 100 pA to 2 nA at -70 mV). Larger effects were expected when using climbing fibre stimulation, which in some cases elicited currents as large as 8 nA at -70 mV. However, a direct assessment of the relation between current amplitude and time course was usually not possible for a given cell in this case since the response was all-or-none. But on 7-2
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