Presynaptic effects of NMDA in cerebellar Purkinje cells and interneurons

Abstract

NMDA receptors (NMDARs) are generally believed to mediate exclusively postsynaptic effects at brain synapses. Here we searched for presynaptic effects of NMDA at inhibitory synapses in rat cerebellar slices. In Purkinje cells, application of NMDA enhanced the frequency of miniature IPSCs (mIPSCs) but not that of miniature EPSCs (mEPSCs). This increase in frequency was dependent on the external Mg2+ concentration. In basket and stellate cells, NMDA induced an even larger mIPSC frequency increase than in Purkinje cells, whereas mEPSCs were again not affected. Moreover, NMDA induced an inward current in both types of interneuron, which translated into a small depolarization (approximately 10 mV for 30 microM NMDA) under current-clamp conditions. In paired recordings of connected basket cell-Purkinje cell synapses, depolarizations of 10-30 mV applied to the basket cell soma enhanced the frequency of postsynaptic mIPSCs, suggesting that somatic depolarization was partially transmitted to the terminals in the presence of tetrodotoxin. However, this effect was small and unlikely to account fully for the effects of NMDA on mIPSCs. Consistent with a small number of dendritic NMDARs, evoked EPSCs in interneurons had a remarkably small NMDA component. Evoked IPSCs at interneuron-interneuron synapses were inhibited by NMDA, and the rate of failures was increased, indicating again a presynaptic site of action. We conclude that activation of NMDARs in interneurons exerts complex presynaptic effects, and that the corresponding receptors are most likely located in the axonal domain of the cell.

Fig. 1.

NMDAR activation increases the mIPSC frequency in Purkinje cells. A, Top two traces show mIPSCs recorded from a representative Purkinje cell under control conditions (left) and in the presence of 15 μm NMDA (right). Bottom traces, After washing NMDA, the bath was perfused with a solution containing 50 μmd-APV. Further addition of 15 μm NMDA did not alter the pattern of mIPSCs. B, Pooled results from seven experiments. In the absence of 50 μmd-APV, 15 μmNMDA leads to a 141 ± 45% increase in the frequency of mIPSCs. In the presence of 50 μmd-APV, the increase in the frequency is almost completely blocked (15 ± 4%). All experiments were performed in the presence of 0.2 μm TTX and 10 μm NBQX.

Fig. 2.

d-APV reduces the frequency of mIPSCs in Purkinje cells. A, Raw traces, showing mIPSCs under control conditions (left, 0.2 μm TTX + 10 μm NBQX) and after addition of 50 μmd-APV (right). B, After addition of d-APV, the cumulated amplitude histogram is not modified (left), whereas the cumulated interval histograms indicate a change to a lower event frequency (right). Same cell as in A. Total number of events (5 min recording periods in each condition): 831 in control saline, and 615 in the presence of d-APV. Mean amplitudes: 166 pA in control, and 170 pA in d-APV.

Fig. 3.

NMDAR activation preferentially increases the frequency of small events. A, Average amplitude distribution of mIPSCs of seven cells under control conditions (0.2 μm TTX and 10 μm NBQX). Histograms were taken from 3-min-long recordings and were normalized to the total number of events. Bin width is 10 pA. B, Normalized average amplitude distribution of mIPSCs of the same cells in the presence of 15 μm NMDA. C, Normalized average amplitude distribution of NMDA-induced events, obtained by subtracting the distributions in NMDA from the control ones. D, Comparison of integrals of the plots inA–C (cumulative amplitude histograms). NMDA-induced events are on average smaller than control events.

Fig. 4.

Mg²⁺ dependence of NMDA effect. Plot of NMDA-induced increase in mIPSC frequency in Purkinje cells in different external Mg²⁺ concentrations. Open circles, 0 mm Mg²⁺; gray circles, 1 mm Mg²⁺ (normal BBS);black squares, 8 mm Mg²⁺. For 0 Mg²⁺, only two NMDA concentrations were tested. Experiments were performed in the presence of 0.2–0.5 μm TTX and 10 μm NBQX (n = 3–7).

Fig. 5.

NMDA enhances synaptic activity in stellate and basket cells. A, Synaptic currents recorded from a representative stellate cell under control conditions (0.2 μm TTX, left trace) and in additional presence of 30 μm NMDA (right trace).B, Same as A, except that the recorded cell was a basket cell. Dotted lines in right traces in A and B indicate control holding current to show the NMDA-induced inward current in interneurons (26 and 24 pA, respectively). C, Amplitude distribution of mIPSCs of same stellate cell as in Aunder control conditions (0.2 μm TTX,left) and in additional presence of 30 μmNMDA (right). Three minute recordings were used for each histogram. Ratio of mean amplitudes in NMDA–control, 1:3.

Fig. 6.

In TTX, somatic depolarization of an interneuron increases the mIPSC frequency in a postsynaptic Purkinje cell. All data obtained from paired recordings with presynaptic basket cells (BC) and postsynaptic Purkinje cells (PC). A–C are from the same pair. A1, Average of spikes recorded in the basket cell in cell-attached mode. A2, Superimposed IPSCs recorded in the Purkinje cell. B, Results obtained after superfusion with 0.2 μm TTX, shortly after breaking into the basket cell. Top trace shows the presynaptic depolarization protocol (repetitive cycles from −60 to −30 mV in 10 mV steps, holding for 1 sec at each potential). Gray andblack traces show corresponding current trace recorded in the basket and Purkinje cell, respectively. C, Average mIPSC frequencies in the Purkinje cell (n = 100 1 sec segments) for each 1 sec segment, plotted against the somatic potential of the basket cell. D, Pooled data from seven pairs. Data in D were normalized to the number of events at a presynaptic holding potential of −60 mV.

Fig. 7.

The NMDA component of interneuron EPSCs is weak. EPSCs were evoked in an interneuron by extracellular stimulation in the granule cell layer. A, At −60 mV, EPSCs had rapid decay kinetics, fluctuated markedly among trials, and displayed a fair amount of jitter in their onsets. At +60 mV, only hints of a late component could be obtained in single traces. This component was insensitive to NBQX (10 μm) but was blocked by further addition of d-APV (20 μm). All solutions used contained 15 μm bicuculline to block IPSCs. All four panels show five superimposed traces in each condition.B, Average traces at −60 mV in control conditions (bicuculline alone; thick black line), at +60 mV in control conditions (middle gray line), at +60 mV in the presence of NBQX (thin black line), and at +60 mV after further addition of APV (light gray line). Theinset shows the traces at +60 mV with enlarged vertical scale and slow time base. At +60 mV, the peak currents of the AMPA and NMDA components are 59 and 2.4 pA, respectively, with a ratio of 25-fold. Stimulation artifacts have been clipped off.

Fig. 8.

NMDA reduces evoked IPSCs in interneurons. A, Superimposed representative traces (three in each top panel) in control conditions (10 μm NBQX; left), and after addition of 15 μm NMDA (right). Eachtrace represents the response to a stimulation pair (interval, 20 msec; stimulation frequency, 0.33 Hz). Note that addition of NMDA leads to an increase in the frequency of spontaneous IPSCs. Average responses in control conditions and in the presence of NMDA are shown in the bottom panels. Stimulation artifacts have been clipped off. B, Average failure rate results (responses to the first pulse of pairs) for seven experiments such as in A, including four cells in the presence of 0.5 μm CGP 62349 and three cells without the blocker.C, Summary data for paired-pulse ratios in control conditions and in NMDA.