Neighboring cerebellar purkinje cells communicate via retrograde inhibition of common presynaptic interneurons

Neuron,

Vol. 11, 885-893, November,

1993, Copyright

0 1993 by Cell Press

Neighboring Cerebellar Purkinje Cells Communic&e via Retrograde Inhibition of Common Presynaptic Interneurons P. Vincent and A. Marty Laboratoire de Neurobiologie Ecole Normale Supbrieure 46 rue d’Ulm 75005 Paris France

Summary Paired tight-seal whole-cell recordings were obtained from neighboring Purkinje cells in cerebellar slices. Under voltage clamp, spontaneous inhibitory postsynaptic currents resulted from the activity of CABAergic interneurons, stellateand basket cells. Up to 80% of inhibitory postsynaptic currents in paired recordings were in register. This correlation was not affected by antagonists of glutamate receptors, faded with distance, and was abolished by tetrodotoxin. Earlier work showed that voltage-gated Ca*+ entry into a Purkinje cell elicits a transient presynaptic inhibition of inhibitory postsynaptic currents. It is now shown that this inhibition is not restricted to the stimulated cell, but that it is transmitted to its neighbors. The results indicate that Purkinje cells exchange information by an unconventional mechanism involving retrograde control of inhibitory synapses. Introduction Purkinje cells receive inhibitory GABAergic input from two classes of interneurons of the cerebellar molecular layer, basket cells and stellate cells (Andersen et al., 1964; Eccles et al., 1966; Midtgaard, 1992). Each interneuron synapses onto several Purkinje cells, and each Purkinjecell is inhibited by several interneurons (Palay and Chan-Palay, 1974). It seems therefore likely that neighboring Purkinje cells could have a common result share of inhibitory inputs. But no experimental concerning the existence or functional consequences of such concerted synaptic currents is presently available. We have examined this question by performing simultaneous whole-cell recordings in two neighboring Purkinje cells of cerebellar slices. As it turns out, such cells do display synchronous spontaneous inhibitory synaptic currents (IPSCs). This finding is of particular interest in view of previous work from our laboratory showing that, following depolarization of postsynaptic Purkinje cells, the synaptic currents elicited by interneurons undergo complex changes (Llano et al., 1991a; Vincent et al., 1992). During an initial period lasting about 1 min, synaptic currents are depressed. This inhibition quicklygiveswaytoa pronounced and long lasting potentiation (Llano et al., 1991a; Vincent et al., 1992; Kanoet al., 1992). Whereas the latter effect is clearly postsynaptic, the former effect originates in presynaptic neurons as indicated by the following

evidence: the postsynaptic response to exogenous GABA applications is enhanced, not reduced, during the inhibition; the inhibition involves a decrease in the frequency of IPSCs; and at low presynaptic stimulation levels, the number of failures of evoked IPSCs is enhanced during inhibition. The presynaptic inhibition also occurs in the presence of tetrodotoxin (TTX), but is not observed in the absence of external Ca*+ or in the presence of Cd2+ (Llano et al., 1991a). These observations led to the hypothesis that Ca*+ entry into Purkinje cells elicits the release of a retrograde messenger that inhibits transmitter release in presynaptic terminals (Llano et al., 1991a). Since it appears that the electrical activity of common interneurons elicits synchronized synaptic currents in neighboring Purkinje cells, the question arises as to whether Ca*+ entry in one Purkinje cell can influence the pattern of synaptic currents recorded in another Purkinje cell nearby. The results of the present work show that this is indeed the case, suggesting the existence of unconventional inforrnation processing in the cerebellar cortex based on retrograde, rather than forward, synaptic transmission. Results Correlated Spontaneous Synaptic Currents in Two Purkinje Cells The main part of the spontaneous IPSCs recorded in Purkinje cells is due to the activity of inhibitory interneurons and is carried by Cl- (Konnerth et al., 1990; Llano et al., 1991a; Vincent et al., 11992). When recorded with a high Cl- concentration pipette solution, these IPSCs range from about 20 pA (the detection threshold imposed by background noise) to several nanoamperes. Figure 1 illustrates such IPSCs in two simultaneous recordings taken from neighboring cells. There is a striking correspondence between the timesofonsetof IPSCsinthetwocells. Intheexample shown, most of the events appeared to have a partner in the other trace. These events are marked with arrows. Time difference histograms were constructed from such recordings by measuring the times between the onset of each IPSC recorded in the first trace, taken as reference, and the closest event in the other trace (see Experimental Procedures). Figures 2A and 2B show the histograms obtained from al paired recording similar to that of Figure 1 when1 taking one cell or the other as reference. In each cas,e, the histogram has a large narrow peak centered around 0 and a smaller, wider component. To ascertain that the peaks do reflect a correlation between the two recordings, a histogram wasconstructed after introducing an arbitrary time shift of 10 ms in one of the traces. In this case, the peak was reduced in height, and its position was shifted by 10 ms on the time axis (Figure 2C; com-

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examined. The difference in the width of the background components in Figures 2A and 2B reflects the difference between the average frequencies of IPSCs in the two cells examined. On the basis of these results, IPSCs were separated the peak in two classes. A time window encompassing of thetime difference histogram was defined. Usually, this window was set at a total width of 4 ms. Events with time differences within the window were mainly coupled IPSCs, whereas events with time differences outside the window were classified as single IPSCs. However, some uncoupled events took place during the 4 ms window. In view of the results of Figure 2D, suggesting that uncoupled events are equally distributedoverthe *2mswindow,thefollowingprocedure was adopted. A horizontal linewas drawn at the breaking points between the wide component and the central peak. In the peak region, the area comprised between this line and the histogram was taken as the numberof coupled events, C.The integral of the histogram outside the +2 ms window, plus the area under the line in the central region, was taken as the number of uncoupled events, U. The coupling ratio was defined as R = C/(C + U). Note that in two simultaneous recordings the number of single events is not necessarily the same in each trace, so that the two ratios RI and R2 obtained in each cell differ. For example, in the histogram of Figure 2A, R1 was 39%, whereas R2 was 27% in the histogram of Figure 2B.

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Holding potential: -70 mV. Distance between the cells: 33 pm. Arrows mark the onset of spontaneous synaptic currents that are almost simultaneous in the two recordings. CNQX (IO BM) and APV (100 vM) were included in the bath solution.

parewith Figure2A).Thus, thecentral peaksof Figures 2A and 26 clearly reflect correlated events. When a larger time shift (50 ms or more) was introduced between the two traces, only the wide component remained (data not shown). This component thus reflects the random activity of the test trace that is

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Timedifference histograms between IPSCs were constructed as explained under Experimental Procedures. (A and B) Histograms obtained in the control bath solution, containing CNQX and APV. (A) and (B)showthetimedifference histogramsobtained when taking one or the other cell as reference. (C) Time difference histogram obtained from cell 1 with the same protocol as in (A), except that a IO ms time shift was artificially introduced between the two traces. (D) Time difference histogram taken in 0.2 PM TTX (cell 2). In TTX, the frequency of IPSCs was much lower than in the control, so that the slow component of the histogram extended up to 1 s. No fast component near 0 was obtained. The histograms in (A) and (8) were calculated from a period of 124 s of paired recording and contain, respectively, 3602 and 5155 events. The histogram in (D) was calculated from 162 s of paired recording and contains only 350 entries, owing to the decrease in IPSC frequency in TTX. The bin width in (A)-(C) was 0.2 ms.

Retrograde a87

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in the Cerebellum

Effects of Postsynaptic Depolarization

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Coupling ratios (two for each pair) are plotted as a function of the distance between the centers of somata of recorded cells. The results from 1 atypical cell pair, in which only bursts of activity were synchronized, have not been included. The regression line (r = 0.63) indicates a mean coupling ratio of 40% for directly adjacent cells (d = 25 pm) to a value of 0% at a distance of 290 pm. Open circles indicate experiments in control saline, and closed trianglesdescribe resultsobtained in CNQXand APV.

To test whether the cell to cell correlation was dependent on the firing of presynaptic interneurons, coupled cell pairs were exposed to TX (200 nM). This resulted invariably in the abolition of any correlation between paired traces (Figure 2D, n = 4; note time scale difference with other panels in Figure 2), indicating that the firing of presynaptic action potentials is necessary for the coupling of IPSCs. In 23 pairs separated by 25-50 pm (measured between soma centers), only one showed no sign of correlation between IPSCs. In another pair,onlyoccasional bursts were synchronous. In the remaining 21 pairs, the value of R varied between 13% and 78%, with a mean of 41%. Coupling ratios decreased as the distance between recorded Purkinje cells was increased. A regression line through the data (Figure 3) intersected the horizontal axis at a distance of 290 pm. The interneurons of the molecular layer receiveglutamatergic excitatory inputs from granule cells and from climbing fibers (Palay and Chan-Palay, 1974; Ito, 1984). To exclude a possible role of distal excitatory inputs in the synchronization of IPSCs, experiments were performed in the presence of 6-cyano-7-dinitriquinoxaline-2,3-dione (CNQX) (IOpM) and o,L-aminophosphonovalerate (APV) (100 PM), as exemplified in Figure 1 and Figure 2. For comparison, other experiments were performed in control saline. On average, R was found to take similar values with (Figure 3, triangles) and without (Figure 3, circles) the glutamateblocking agents. These results indicate that the correlation between IPSCs originates in the inhibitory interneurons and not in further excitatory afferents.

It was previously reported (Llano et al., 1991a; Vincent et al., 1992) that depolarizing a Purkinje cell resulted in a marked decrease of the frequency ancl mean amplitude of IPSCs. Evidence from this earlier work strongly suggests that this inhibitory effect is presynaptic, as summarized in the Introduction. If two neighboring Purkinje cells received IPSCs from a common interneuron, it seemed possible that presynaptic inhibition of the interneuron resulting from the electrical activity of one postsynaptic cell could induce an alteration of the IPSCs recorded in the neighboring cell. To approach this question, effects of depolarizing voltage trains were analyzed in paired recordings of Purkinje cells separated by 25-150 pm. In each cell, the level of IPSC activity was determined by summing the amplitudes of individual IPSCs over successive periods of time (10 s). The inhibition of the synaptic current activity, called I hereafter, was measured as the percentage of this parameter, which was reduced by the voltage train. In the example shown (Figure 4), 77% of the synaptic activity was inhibited in cell 1 by a train of voltage (I, = 0.77), which was stimulated pulses. Simultaneously, 50% of the synaptic activity in cell 2 was inhibited (12 = 0.50), even though the potential was not changed in that cell. The recovery time of the cross-inhibition in cell 2 was similar to that of the auto-inhibition in cell 1. A similar reduction of the IPSCs was observed in cell 1 following !stimulation of cell 2 (data not shown). In cell 1, the overall inhibition of IPSCs was accounted for by a 60% reduction of their mean amplitude and by a 35% reduction of their mean frequency. In cell 2, the corresponding ratios were 29% and 20%, respectively (see sample records a, b, and c in Figure 4). These figures (corroborated by the results of similar analyses in two additional pairs) suggest that reductions of amplitude and frequency contribute in similar proportions to crossand auto-inhibition. The reduction in mean amplitude does not contradict the notion of a presynaptic site for the inhibition, as this reduction apparently results from the breakdown of spontaneous IPSCs into events of smaller quanta1 content (Llano et al., 1991a). In none of the recordings did we see any indication of an electrical coupling between Purkinje cells, or of inward synaptic currents elicited in cell 2 by stimulating cell 1. However, in about half of the experiments, small outward currents (up to 50 pA) were detected in cell 2following voltage pulses in cell 1. These currents started rising during each pulse, peaked from 0 to 200 ms after the end of the depolarization, and returned to the baseline within 1 s. They were not observed if cell 1 was dialyzed with N-methyl-Dglucamine (NMDG+) instead of Cs+, but they were present if the axon of cell 1 was cut just at the exit point from the soma (a procedure that will be detailed below). The latter result indicates that the outward current is not a synaptic current elicited by a collateral of the axon of cell 1, whereas the former result indi-

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The IPSC amplitudes were summed in 10 s intervals to assess the synaptic activity in two neighboring Purkinje cells (distance: 30 pm). Cell 1 was subjected to eight successive voltage steps to 0 mV of 100 ms duration during the period indicated by the horizontal bar in the upper record. This resulted in a marked reduction of the summed IPSC amplitude, which was fully reversible within 2 min. (Note that IPSC amplitudes were not measured during or shortly after the voltage pulses). The summed IPSC amplitudes were transiently reduced to about 50% of the control in cell 2 as a result of the train in cell 1. In cell 2, there was a slow and steady decline of the synaptic activity. To correct for this effect, the synaptic activity during a period of 3 min before the train was analyzed. The regression line for these data is shown in the cell 2 plot. The inhibition lasts for about 1 min after the end of the train, whether measured in cell 1 or in cell 2. Sample traces obtained in cell 2 during the periods labeled a, b, and c are shown in the lower panel.

cates that the current could be a consequence of the extrusion of Cs+ from cell 1 during the pulses. In a simple model to be presented below, the amount of cross-inhibition, I*, is dependent on the presence of interneurons common to the two recorded cells. It then follows that I2 = II . RZ, in which R2 is the coupling ratio measured in cell 2. Figure 5A shows that the results follow this prediction within experimental error. Figure 56 shows that I2 declines with the distance between Purkinje cells. I2 is better correlated to the II . RZ product (Figure 5A) than to distance (Figure 5B).

Figure 5. Cross-Inhibition Inhibition and Coupling

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(A) Relation between cross-inhibition, auto-inhibition, and coupling ratio. For each pairexamined, theinhibitionof IPSCactivity was measured as shown in Figure 4 as a result of a train of eight depolarizing pulses given in one cell, called cell 1. The autoinhibition, I,, and cross-inhibition, 12,were determined, as well as thecoupling ratio R2measured in cell2 beforethetrain. Finally, I2 was plotted as a function of the product I, Rz. Open circles result from experiments using standard conditions; closed squares correspond to pairs in which the pulsed cell had its axon cut off; closed triangles correspond to pairs in which the pulsed cell contained NMDC+ instead of Cs+. In several experiments, one cell of a pair and its partner were stimulated, giving rise to separate entries in the plot. Up to three trains were applied for one given pair configuration, and a total of 24 experiments were analyzed. The continuous line is a linear regression to all data points (r = 0.64). An equally good representation of the data is given by the line x = y (dotted line). (B) Relation between cross-inhibition and distance between Purkinje cells. Same experiments as in (A). The continuous line is a linear regression to all data points (r = 0.32).

The resuits of Figure 5A suggest that a signal associated with the presynaptic interneuron is conveyed from cell 1 to cell 2 during cross-inhibition. This signal could be an internal messenger generated in the interneurons, where it would diffuse from one presynaptic terminal to the other along the axonal arborization. Alternatively, it could be an electrical message interfering with the propagation of depolarization and/or Ca2+ entry in the axonal arborization. In an attempt to decide between these alternatives, we have

Retrograde

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bition in control saline was 25% f 2% (mean f SEM; data from Figure 5B with separations of 25-40 vm). These results indicate that the cross-inhibition is much weaker in lTX than in the control and therefore support the notion that the signal propagating along the presynaptic interneuron is electrical rather than chemical.

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Figure 6. Lack of Cross-Inhibition

(s)

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Trains of depolarizing pulses were applied in the presence of 200 nM TTX in 5 pairs of neighboring Purkinje cells (separations less than 40 pm). Summed IPSCs were calculated for the pulsed cells (cells 1, upper panel) and for their neighbors (cells 2, lower panel). The data were aligned with respect to the pulse train, normalized, and averaged. The average auto-inhibition was I, = 48%. The mean I2 value was only 6% compared with 25% in the control (Figure 5B, data at 25-40 pm). Regression lines to the three first and three last histogram points are drawn to illustrate the downward trend of the summed IPSCs.

performed auto- and cross-inhibition experiments in TTX. In a first series of experiments, auto-inhibition was compared with and without TFX (200 nM). The summed IPSCs were, on average, reduced by 62% * 9% by depolarizing trains in the control period (22 measurements from nine cells). When perfusing TTX in the bath, the summed IPSCs fell on average to 19% of the control. In lTX, depolarizing trains reduced the synaptic activity by45% f 23% (15 measurements from the same nine cells). These results confirm that TTX does not abolish the auto-inhibition (Llano et al., 1991a) and further show that the extent of autoinhibition is comparable in the control and in TTX. Next, we performed paired experiments in TTX to test whether the cross-inhibition is present when action potentials are blocked. In none of the 5 pairs investigated could any cross-inhibition be detected, even though the auto-inhibition was clear. However, because of the low level of miniature IPSCs in lTX, the results of individual experiments were difficult to quantitate. The results were therefore pooled together (Figure6). The auto-inhibition measured on the pooled results was I, = 48%, and the cross-inhibition was I2 = 6%. For comparison, the mean cross-inhi-

Presynaptic Inhibition Is Not Due to a Polysynaptic Pathway or to Ion Accumulation in Pericellular Spaces Before considering further mechanisms by which postsynaptic stimulation of Purkinje cells is transmitted to presynaptic interneurons, two potential sourcesofartifactwereexamined. First, ion accumulation/depletion following voltage pulses (Ten Bruggencate et al., 1976) could alter the membrane permeability of presynaptic neurons. During pulses performed in our normal recording conditions (isotonic CsCl pipette solution), outward currents of several nanoamPeres were observed. These currents are presumably carried byCs+and/or by residual K+. Outward currents may be abolished by external tetraethylammonium (5 mM) without any obvious effect on the presynaptic inhibition (data not shown). It is also possible to reduce outward currents by using NMDG+ instead of Cs+ as the major internal cation. Under such conditions, Purkinje cell depolarization still produces large inhibition of its own IPSCs, as well as those of neighboring cells (Figure 5A, triangles; Figure 7A). These results show that the inhibition of IPSCs is not due to accumulation of Cs+ in the pericellular space. The possibility that Ca2+ depletion in the intercellular space could be responsible for the inhibition of IPSCs was considered next. A crucial test for this possibility is whether the inhibition can be blocked by a strong intracellular Ca*+ buffer such as BAPTA. In an earlier series of experiments, we were unable to demonstrate a blocking effect of 30 m M BAPTA (Llano et al., 1991a). We have readdressed this issue by using recording pipettes of large diameter and by making sure that the access conductance never fell below 0.1 PS during recording. The internal solution contained a 40 m M BAPTA, 10 m M Ca*+ solution, with a calculated free Ca2+ concentration of 100 nM. Recordings with BAPTA were alternated with control recordings. With the standard internal solution, large inhibitions of spontaneous IPSCs were obtained following a train stimulus of the Purkinje cells for up to 30 min of recording. With the BAPTA solution, however, the inhibition sharply declined with time and became hardly detectable after 10 min of recording. On average, the inhibition measured between 12 and 14 min of recording was 11% f 3% (SEM; n = 4) with the BAPTA solution, compared with 52% + 4% (n = 5) between 12 and 20 min of recording in control cells. The means are significantly different (Student’s t test,, p < O.OOl), suggesting that BAPTA blocks the presynaptic inhibition. These results confirm that the inhibition is Ca*+ dependent, as was previously inferred from experi-

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In both experiments, depolarizing pulses applied in cell 1 (bars) resulted both in autoinhibition and in cross-inhibition of IPSCs recorded in a neighbor cell (cell 2, lower graphs). (A) The stimulated cell was filled with a NMDG’ pipette solution instead of the usual Cs+ solution. (B) The stimulated cell had its axon cut off near its attachment to the soma.

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ments in Ca*+-free saline (Llano et al., 1991a). They also strongly suggest that the site of action of Ca*+ is in the Purkinje cell, rather than in the extracellular space, and thus provide convincing evidence against the Ca2+ depletion hypothesis. A second possible mechanism would be that interneuron firing is regulated by a polysynaptic pathway. Purkinje cell axons have collaterals that branch off the main axon in the granule cell layer and that establish inhibitory synaptic connections with various cell types. In the rat, Purkinje axon collaterals have been found to terminate near Colgi cells, Lugaro cells, neighboring Purkinje cells, and, on rare occasions, near interneurons of the molecular layer (Palay and Chan-Palay, 1974). The fact that Purkinje cell depolarization still results in inhibition of IPSCs in the presence of TFX (Llano et al., 1991a) strongly suggests that the inhibition is not based on a polysynaptic pathway including Purkinje cell axon collaterals. To rule out this possibility, recordings were performed after cutting off the axon just as it exited from the Purkinje cell (Regehr et al., 1992). After this operation, depolarization of the Purkinjecell still inhibited itsown IPSCs and also resulted in an inhibition of IPSCs in neighboring Purkinje cells (Figure 7B). On average, the amount of inhibition obtained in axotomized cells was the same as in intact cells (Figure 5A, closed squares and open circles, respectively). Thus, neither cross-inhibition nor auto-inhibition involves feedback inhibition conveyed by axon collaterals of the stimulated Purkinje cell. Discussion The present work shows that inhibitory synaptic rents in neighboring Purkinje cells are correlated that depolarization-induced inhibition of these

curand cur-

rents may be transferred from one Purkinje cell to the next. We now consider these two findings in turn. Synaptic currents recorded in neighboring cells tend to be synchronous, presumably because they are due to the firing of interneurons innervating both recorded cells. Since the correlation is observed in the presence of CNQX and APV, the synchrony originates at the level of the interneurons and not in neuronal elements upstream (i.e., parallel or climbing fibers). Because the correlation is abolished by TTX, it requires the generation of action potentials. The simplest interpretation of the results is that coupled events resu!t from the firing of one common interneuron. In the rat, axons of basket cells and of deep stellate cells typically have an extension of 200-300 pm along the Purkinje cell layer (Palay and ChanPalay, 1974). This is in good agreement with the present finding that the coupling ratio decreases to 0 for a distance of 290 pm (Figure 3). It cannot be ruled out, however, that some coupled events originate in two distinct presynaptic neurons linked together by electrical synapses. Morphological studies indicate the presence of gap junctions between inhibitory interneurons (Sotelo and Llinss, 1972). Because basket cells have very extensive contacts with Purkinje cells, their contribution to R should be large. In the rat, there are only six to ten basket cells contacting one Purkinje cell (Palay and Chan-Palay, 1974), and this number is presumably further reduced when cutting the slice. Thus, large relative fluctuations in the number of basket cells common to adjacent Purkinje cells have to be expected. These fluctuations could account for the dispersion of data points in Figure 3. The second finding of the present work is that Purkinje cell depolarization can influence the pattern of IPSCs in a neighboring Purkinjecell (cross-inhibition).

Retrograde 891

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Ca2+ entry into cell 1 leads to the production of substance S, which may diffuse to several presynaptic sites of inhibitory interneurons (scheme I). Alternatively, S may diffuse only to presynaptrc sites in contact with cell 1 and use the interneuron as a relay to influence synaptic boutons on cell 2 (scheme II).

In cases in which IPSCs of neighboring Purkinje cells were weakly synchronized (presumably because the cells happened to have a small number of common presynaptic interneurons), this effect was not observed. Thus, cross-inhibition is related to the correlation between IPSCs, rather than to distance. To interpret the results of Figure 5 further, two limiting cases will be considered. Both schemes assume that depolarized Purkinje cells release a substance S that is able to inhibit presynaptic neurons (Figure 8). In scheme I, S diffuses to various synaptic sites of the stimulated cell (cell 1) or of its neighbors (cell 2), where it reduces the efficacy of synaptic transmission. In this scheme, the IPSCs recorded in cell 2 will be decreased according to the distance between the origin of S and the various synaptic sites on cell 2. According to this scheme, I2 should be correlated tightly to the distance between cells 1 and 2. This distance is difficult to assess exactly, however, because S may be released from thedendritesof cell 1, in which casethedistance to be covered by S may be substantially shorter than the distance from soma to soma used in the abscissa of Figure 58. Scheme II, in contrast with scheme I, assumes that S only needs to cross the gap from cell 1 tothepresynapticterminalsin itsimmediatevicinity. The inhibition is then transmitted from the terminals to the entire interneurons. Thus, the synapses of cell 2 innervated by interneurons common with cell 1 are inhibited. In this scheme, the only events in cell 2 that will be inhibited correspond to the coupled events recorded in the control period, and these events are inhibited as strongly as events in cell 1, leading to between the the relation, 12 = II . R2. The agreement results and this prediction must be taken as an indication in favor of scheme II, even though other possibilities such as scheme I are not excluded. The mechanism by which Purkinje cells regulate their own IPSCs is still unclear. Control experiments performed in the present and in previous work exclude the following mechanisms: First, the loss of voltage-clamp control following Ca2+ entry. Earlier evi-

denceagainstthispossibilityincludestheobservation that no change in the input conductance or capacitance of the cell can be detected during a large part of the inhibitory period (Llano et al., 1991a). The present work still strengthens this evidence, because it is inconceivablethat adefectof spaceclamp in cell 1 could alter the IPSCs in cell 2. Second, polysynaptic pathway involving axon collaterals of the Purkinje cell. Our previous evidence against this mechanism was that the inhibition remained in the presence of TTX. We now show in addition that both self- and crossinhibition are still observed in axotomized cells. Third, ion accumulation/depletion in extracellular spaces brought about by postsynaptic voltage pulses. Stimulation of the climbing fiber afferents is known to evoke a transient rise of the external K+ concentration and a transient decrease of the Ca*+ concentration in the molecular layer (Ten Bruggencate et al., 1976; Stockle and Ten Bruggencate, 1980). These concentration changes have, however, atimecourse of recovery that is at least one order of magnitude faster than the decay of the presynaptic inhibition described here, and it is difficult to imagine why the concentration changes near the terminals should be so much slower than the bulk changes recorded with a macroscopic electrode in the molecular layer. The above results with tetraethylammonium ion and NMDC+ provide further evidence against a role of K+ or Cs+ in presynaptic inhibition. Finally, evidence against a mechanism based on depletion of Ca*+ is provided by the present finding that large concentrations of BAPTA block the presynaptic inhibition. Likewise, in hippocampal pyramidal cells, in which a Ca*+-dependent inhibition of GABAergic inputs very similar to that studied here is observed, the inhibition is blocked by strong internal Ca *+ buffers (Pitler and Alger, 1992). These mechanisms being excluded or unlikely, the main possibility that remains is that Purkinje cell depolarization results in the releaseof an inhibitory substance capable of interacting with presynaptic terminals (Llano et al., 1991a). The identity of this substance has remained elusive. Whatever this substance may be, however, the present results indicate that neighboring Purkinje cellscan communicate by influencing the electrical activity of common presynaptic interneurons. Even though the presence of retrograde messengers has been theobject of much interest (Williams et al., 1989; Garthwaite, 1991), experimental evidence supporting such unconventional transmission in the CNS is very scarce. The observed inhibition of presynaptnc release could result from a modification of the exocytosis mechanism (Scanziani et al., 1992; Llano and Gerschenfeld, 1993b) and/or of the membrane conductance of the interneuron. Recent results suggest that in TTX, release is independent of voltage-dependent Ca*+ influx in the presynaptic terminals of hippocampal neurons (Scanziani et al., 1992) and of cerebellar stellate cells (Llano and Gerschenfeld, 1993a). The finding that the auto-inhibition is observed in TTX

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(Figure 6) therefore supports the hypothesis of a direct action on the release process. On the other hand, Figure 6 also shows that TTX strongly inhibits the cross-inhibition. This finding suggests that part of the inhibition is linked to membrane depolarization and involves an alteration of membrane properties. In summary, the results suggest that postsynaptic depolarization has two distinct inhibitory effects on the interneuron. First, it inhibits exocytosis directly. This effect is local and is observed in TTX. The second effect is exerted on the axonal membrane to prevent Ca2+ entry. It is propagated and is not observed in TTX. Both effects may be mediated byacommon effector S. Correlation between synaptic inhibitory inputs and principal cells of layered neuronal tissue may be a widespread phenomenon. Similar to the present results, Miles (1990) found a strong correlation between inhibitory synaptic potentials recorded in neighboring pyramidal cells of hippocampal slices. It is in the same preparation that depolarization of pyramidal cells was recently reported to induce a strong inhibition of GABAergic IPSCs (Pitler and Alger, 1992). The self-inhibition of hippocampal pyramidal, like that of cerebellar Purkinje, neurons appears to rely on Ca2+ entry and is not matched by a reduction of the responses to exogenous GABA applications. in view of the striking similarities between the two sets of results, it is tempting to speculate that the crossinhibition described in the presentworkwill also turn out to be present in the hippocampal preparation. Experimental

Procedures

Sagittal slices (160-180 vrn thick) were taken from the vermis of cerebella from rats aged 13-20 days. The slices were visualized through a 40x objective in an upright microscope for patchclamprecording(Hamilletal.,1981;Edwardsetal.,1989).Superficial Purkinje cells were selected. To form a seal, a large pressure (about 0.1 atm) was applied in the recording pipette as it was moved toward the neuronal membrane. The pipette was advanced until the stream coming out of its tip created an invagination of the cell membrane. The pressure was then released. Clamping the pipette current to about -200 pA and applying occasional suction episodes (about -0.01 atm) to the pipette led to the formation of a tight seal within a few minutes. Transition to the whole-cell mode was obtained with a sharp pulse of suction in the pipette. The standard pipette solution contained 70 m M CsCI, 70 m M Cs-MOPS, 1 m M EGTA, 0.6 m M MgC12, 4 m M Mg-ATP, and 0.4 m M CTP (pH 7.3). In some experiments, the CsCl concentration was 140 mM, and no MOPS was used. The pipette resistance was near 2 MTJ as measured in the bath solution, and 3-10 MB during whole-cell recording. During recording, 60%-90% of the series resistance was compensated for. This resulted in effective voltage control over the entire cell with a time constant of 2-5 ms (Llano et al., 1991b). The standard extracellular saline contained 125 m M NaCI, 2.5 m M KCI, 2 m M CaC&, 1 m M MgCI,, 1.25 m M NaH,PO+ 26 m M NaHC03, and 25 m M glucose. This solution was continuously bubbled with a gas containing 95% 02, 5% CO2 before entering the experimental chamber. Its pH was 7.4. The chamber was perfused continuously during recording. All experiments were performed at room temperature (20°C-25’C). Liquid junction potentials between pipette and bath solutions were less than 5 mV and were left uncorrected. All recordings were taken at a holding potential of -70 mV. With a Cl- Nernst potential of -16 mV (half MOPS solution) or

of +I mV (isotonic Cl- solution), large inward Cl- currents were elicited by the activity of presynaptic CABAergic interneurons. When using the isotonic Cl- pipette solution, the spontaneous IPSCs were so large that they occasionally elicited dendritic Ca*’ spikes. To prevent spiking, a low concentration of bicuculline (1 PM) was used in the bath in some of these experiments, ieading to a reduction of the IPSC amplitude by a factor close to 2. Purkinje ceils were stimulated with depolarizing voltage pulses to 0 mV. Eight pulses of 100 ms duration were given with 2 s intervals. The trains were repeated at intervals of 3-4 min. The firsttrain elicitedacomplexinhibition-potentiation sequenceof IPSCs (Vincent et al., 1992). One example is shown in Figure 7A. Subsequent trains only led to the IPSC inhibition studied in the present paper (see Vincent et al., 1992). Current traces were continuously recorded on videotape for off-line analysis. Synchronous reacquisition of two current traces was done using Axolab-1 interface and Axess 1.01 program (Axon Instuments, Inc). The sampling rate was 0.2 ms for each channel. Currents were filtered with two independent 8 pole Bessel filters at a cutoff frequency of 1 kHz. In the analysis of single recordings, traces were usually reacquired using the Fetchex program (pCLAMP package, Axon Instruments) at a sampling rate of 0.5 ms and a cutoff frequency of 0.5 kHz. Spontaneous IPSCs were detected and analyzed by a program written by one of us (P. V.). This program is available on request. It detects spontaneous events by moving a window on the current trace. A fixed number of points are averaged both at the beginning and at the end of the window, leaving a gap of a few points in between. The difference between the two averages, which is a form of derivative of the current trace, is compared with a threshold. The program moves the window to the right one point at a time, until it finds a value of the derivative that exceeds the threshold. At this point, an event is stored with the time of onset and amplitude, calculated as follows. The left average is kept as the baseline value for the event amplitude. The time at the first point of the right average is kept as the time of onset. The right average is moved to the right until it reaches a maximum value, corresponding to the peak current. From this point, the moving window starts again screening the file. The left average was usually calculated on 8-15 points. A long baseline is less noisy, but is more perturbed by preceeding events if the IPSCs frequency is high. The right average was usually calculated on 3-6 points. Short averages give a more precise definition of the peak for sharp events. However, a sufficient number of points should be averaged, or else noise during the onset could be detected incorrectly as the peak of the event. The gap between the two averages helps to detect events with slow onset and was usually set to O-3 points. Parameters were set checking several parts of the recording under visual control. Toanalyze the summed IPSCs, the amplitudes of spontaneous events detected in time periods of 10 s in duration were added together. The resulting histograms showed a downward trend (e.g., Figure 4; Figure 6). In the analysis of the data, this trend was corrected for by calculating a regression to the data outside stimulation periods. In the analysis of two simultaneous recordings, time difference histograms were calculated as follows. The events in one of the two traces, called reference trace, were scanned. For each event in the reference trace, the event closest in time in the other trace (the test trace) was determined, and the time difference, whether positive or negative, between reference and test events was entered. The program then proceeded by moving to the next event in the reference trace. To break off Purkinje cell axons, a pipette having an opening diameter of about 20 pm was filled with external saline and approached to the cell. Nearby granule cells were removed by applying sequences of pressure and suction in the pipette (Edwards et al., 19891, and the axon was finally cut off (Regehr et al., 1992). Only cells in which theaxon could clearly bevisualized before cutting and/or in which the axon stump could clearly be seen emerging from the cell after cutting were used in these experiments. In some cases (including the example illustrated in Figure 6B), visualization of axon stumps was improved by

Retrograde 893

Inhibition

in the Cerebellum

dialyzing the cells with Lucifer yellow the preparation with epifluorescence

(1 mglml) and examining illumination.

Acknowledgments This work was supported by the CNRS (URA 295) and by the EEC (grant SCI 652). We thank Drs. B. Barbour, I. Llano, and C. Lena for useful comments on the manuscript. 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.

S. M. (1992). Presynaptic inhibition of miniature excitatory synaptic currents by baclofen and adenosine in the hippocampus. Neuron 9, 919-927. Sotelo, C., and Llinas, R. (1972). Specialised membrane junctions between neurones in the vertebrate cerebellar cortex. J. Cell Biol. 53, 271-289. Stockle, H., and Ten Bruggencate, G. (1980). Fluctuations of extracellular potassium and calcium in the cerebellar cortex related to climbing fiber activity. Neuroscience 5, 893-901. Ten Bruggencate, G., Nicholson, Climbing fiber evoked potassium Pfliigers Arch. 367, 107-109.

C., and Stockle, H. (1976). release in cat cerebellum.

References

Vincent, P., Armstrong, C. M., and Marty, A. (1992). Inhibitory synaptic currents in rat cerebellar Purkinje cells: modulation by postsynaptic depolarization. J. Physiol. 456, 453-471.

Andersen, P., Eccles, J. C., and Voorhoeve, P. E. (1964). Postsynaptic inhibition of cerebellar Purkinje cells. J. Neurophysiol. 27, 1138-1153.

Williams, J. H., Errington, M. L., Lynch, M. A., and Bliss, T. V. P. (1989). Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus. Nature 347, 739-742.

Received

April

12, 1993; revised

August

27, 1993.

Eccles, J. C., Llinas, R., and Sasaki, K. (1966). lntracellularly recorded responses of thecerebellar Purkinjecells. Exp. Brain Res. 7, 161-183. Edwards, F. A., Konnerth, A., Sakmann, B., and Takahashi, T. (1989). A thin slice preparation for patch-clamp recordings from neurones of the mammalian central nervous system. Pfltigers Arch. 474, 600-612. Garthwaite, J. (1991). Glutamate, nitric oxide and cell-cell signalling in the nervous system. Trends Neurosci. 74, 60-67. Hamill, 0. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981). Improved patch-clamptechniquesfor high-resolution current recording from cells and cell-free membrane patches. Pfliigers Arch. 391, 85-100. Ito, M. (1984). The Cerebellum Raven Press).

and Neural

Control

(New York:

Kano, M., Rexhausen, U., Dreessen, J., and Konnerth, A. (1992). Synaptic excitation produces a long-lasting rebound potentiation of inhibitory synaptic signals in cerebellar Purkinje cells. Nature 356, 601-604. Konnerth, A., Llano, currents in cerebellar 87, 2662-2665.

I., and Armstrong, C. M. (1990). Synaptic Purkinje cells. Proc. Natl. Acad. Sci. USA

Llano, I., and Cerschenfeld, H. M. (1993a). Inhibitory synaptic currents in stellate cells of rat cerebellar slices. J. Physiol. 468, 177-200. Llano, I., and Gerschenfeld, H. M. (1993b). B-adrenergicenhancement of inhibitory synaptic activity in rat cerebellar stellate and Purkinje cells. J. Physiol. 468, 201-224. Llano, I., Leresche, N., and Marty, A. (1991a). Calcium entry increases the sensitivity of cerebellar Purkinje cells to applied CABAanddecreasesinhibitorysynapticcurrents. Neuron6,565574. Llano, I., Marty, A., Armstrong, C. M., and Konnerth, A. (1991b). Synaptic- and agonist-induced excitatory currents of Purkinje cells in rat cerebellar slices. J. Physiol. 434, 183-213. Midtgaard, J. (1992). Stellate cell inhibition of Purkinje the turtle cerebellum in vitro. J. Physiol. 457, 355-367.

cells in

Miles, R. (1990). Variation in strength of inhibitory synapses in the CA3 region of guinea-pig hippocampus in vitro. J. Physiol. 437, 659-676. Palay, S. L., and Chart-Palay, V. (1974). Cerebellar ogy and Organization (Berlin: Springer).

Cortex,

Cytol-

Pitler, T. A., and Alger, B. E. (1992). Postsynaptic spike firing reduces synaptic CABAh responses in hippocampal pyramidal cells. J. Neurosci. 72, 4122-4132. Regehr, W. C., Konnerth, A., and Armstrong, C. M. (1992). Sodium action potentials in the dendrites of cerebellar Purkinje cells. Proc. Natl. Acad. Sci. USA 89, 5492-5496. Scanziani,

M., Capogna,

M., Gahwiler,

B. H., and Thompson,