Activity-dependent recruitment of extrasynaptic NMDA receptor activation at an AMPA receptor-only synapse

Abstract

We have identified an excitatory synapse in cerebellar molecular layer interneurons at which the level of presynaptic activity determines the receptor type involved in the postsynaptic response. When small numbers of parallel fibers are activated, EPSCs are mediated solely by AMPA receptors (AMPARs), despite our finding that NMDA receptors (NMDARs) are present in the dendrites of these cells. The EPSC kinetics are fast (tau decay = 0.82 +/- 0.05 msec at room temperature), consistent with the role these interneurons are thought to play in precisely timed inhibitory control of Purkinje cells. NMDARs are activated only when glutamate release is increased either by facilitation with brief high-frequency trains or by recruiting more presynaptic fibers with higher stimulus intensities. Under these conditions, EPSCs consist of a fast-rising AMPAR-mediated current followed by a slow component mediated by both NMDARs and AMPARs. Inhibitors of glutamate transport increase the amplitude and prolong the time course of the compound EPSCs. In contrast, the properties of fast AMPAR EPSCs resulting from the activation of few inputs remain unchanged when glutamate uptake is blocked. Our results suggest that, at these synapses, the postsynaptic density contains AMPARs alone. It is only when transmitter release is high enough for glutamate to diffuse to the extrasynaptic space and to reach concentrations sufficient to activate extrasynaptic receptors that NMDARs are involved in the postsynaptic response. We suggest that such a spatial separation of receptor types may provide a mechanism for rapid changes in EPSC properties, depending on the amount of synaptic activity.

Fig. 1.

Spontaneous EPSCs and those evoked in interneurons by low-intensity stimulation are mediated by AMPA receptors alone.A, Individual sweeps in which EPSCS were successfully evoked by threshold stimulation of the parallel fibers. EPSCs under control conditions (left) and in the presence of 50 μmd-AP-5 (right) are shown at holding potentials of −60 and +50 mV. The stimulus artifact is lost because of the restricted time window shown. B, Overlay of averages of >30 sweeps in both conditions shows that NMDA receptor antagonists have no effect on the EPSC time course. C, Examples of consecutive single spontaneous EPSCs (sEPSCs) occurring in an interneuron recorded at −60 mV in nominally Mg²⁺-free conditions. The normalized average EPSCs are shown on the far right with the fitted biexponential function indicated by the dotted line. Addition of 50 μmd-AP-5 (bottom row) had no effect on the sEPSC, and there was no significant difference in the fitted average.

Fig. 2.

An NMDAR-mediated component is not revealed by increased glycine concentration A, Average responses in an interneuron to low-intensity parallel fiber stimulation at 0.5 Hz in the presence of 10 μm (top panel) and 50 μm (bottom panel) glycine at a holding potential of −60 mV. B, Overlay of normalized average EPSCs under the two different glycine concentrations and after addition of d-AP-5 shows that no NMDAR current was revealed when glycine concentration was increased.

Fig. 3.

Ionophoretic mapping of NMDAR-mediated responses in a molecular layer interneuron. A, Sagittal view of an interneuron filled with Alexa Fluor 594 during whole-cell patch recording. The dendrites and soma are shown in the top panel, and a section of the axonal arbor is shown in thebottom panel. The locations of glutamate application by ionophoresis are indicated by the arrows.B, Averages of 25 currents recorded in response to 25 msec pulses of glutamate (100 mm in electrode) ejected onto the dendrites, soma, and axon of the illustrated cell. Holding potential was −60 mV, and the antagonists TTX (0.5 μm), picrotoxin (100 μm), and CNQX (10 μm) were present in nominally Mg²⁺-free solution.

Fig. 4.

Increased synaptic activity results in a compound EPSC mediated by both AMPA and NMDA receptors. A, Mean EPSCs (averages of 25–50 sweeps) resulting from parallel fiber stimulation using the different stimulus intensities shown (V_hold = +50 mV). Top traces are controls, and bottom traces are in the presence of 10 μm CNQX (note different calibration), showing the appearance of an NMDA component at 30 V stimulus intensity.B, Summary of charge carried by AMPAR and NMDAR channels with respect to stimulus intensity (n = 4). Values for AMPAR-mediated charge transfer were obtained by subtracting values in CNQX from those in control conditions. T, Threshold low-intensity stimulation.

Fig. 5.

Frequency dependence of NMDA receptor-mediated EPSCs. A, Trains of four parallel fiber stimuli evoked CNQX-insensitive currents in interneurons voltage clamped at +50 mV, which increased in amplitude with increasing frequency of stimulation. The current integral (smooth line) is superimposed on the mean synaptic currents (averages of 25–30 sweeps).B, Charge transfer normalized to that obtained at 100 Hz is represented with respect to stimulation frequency for seven cells. Error bars indicate SEM.

Fig. 6.

Effect of glutamate transporter blockade on interneuron EPSCs. A, Average EPSCs evoked at 0.5 Hz at three increasing stimulus intensities. Superimposedthin and thick traces are averaged data in the absence and presence of 50 μmdl-TBOA, respectively. Top traces show the effect ofdl-TBOA under control conditions, and bottom traces show the effect on the NMDAR-mediated component isolated in 20 μm CNQX. B, Responses of the same cell as in A to 50 Hz trains of parallel fiber stimuli.dl-TBOA application prolonged the compound EPSC train at all stimulus intensities and, in this cell, enabled activation of an NMDA component that was previously absent at V1. Stimulus artifacts have been blanked, and stimulus timing is marked by the filled triangles.

Fig. 7.

The effect of inhibition of glutamate transporters is correlated with the amount of glutamate released. A, The relationship between initial NMDAR-mediated charge resulting from a single stimulus at 0.5 Hz for different stimulation intensities. Each data point represents one stimulation intensity for each of the seven cells included. Correlation coefficients (R) and degree of significance (p) were obtained using nonparametric tests (KS). B, Same as A,but for 50 Hz trains. C, The effect of stimulus intensity on the time taken for NMDAR-mediated current resulting from 50 Hz trains to reach peak amplitude.