Presynaptic NR2A-containing NMDA receptors implement a high-pass filter synaptic plasticity rule

Abstract

The detailed characterization of synaptic plasticity has led to the replacement of simple Hebbian rules by more complex rules depending on the order of presynaptic and postsynaptic action potentials. Here, we describe a mechanism endowing a plasticity rule with additional computational complexity--a dependence on the pattern of presynaptic action potentials. The classical Hebbian rule is based on detection of conjunctive presynaptic and postsynaptic activity by postsynaptic NMDA receptors, but there is also accumulating evidence for the existence of presynaptic NMDA receptors in several brain structures. Here, we examine the role of presynaptic NMDA receptors in defining the temporal structure of the plasticity rule governing induction of long-term depression (LTD) at the cerebellar parallel fiber-Purkinje cell synapse. We show that multiple presynaptic action potentials at frequencies between 40 Hz and 1 kHz are necessary for LTD induction. We characterize the subtype, kinetics, and role of presynaptic NMDA receptors involved in the induction of LTD, showing how the kinetics of the NR2A subunits expressed by parallel fibers implement a high-pass filter plasticity rule that will selectively attenuate synapses undergoing high-frequency bursts of activity. Depending on the type of NMDA receptor subunit expressed, high-pass filters of different corner frequencies could be implemented at other synapses expressing NMDA autoreceptors.

Fig. 1.

LTD induction requires high-frequency PF activity and NMDAR activation. (A and B) LTD was induced only when PFs fired at least twice. (A) LTD induction protocol consisted of pairing (at 1 Hz for 2 min) a PC depolarization with a double PF stimulation. The second PF stimulus (solid line) was applied at the middle of the PC depolarization whereas the position of the first (dashed line) varied. (Bi) Time course of the normalized EPSC amplitude in experiments where pairing was done with single (black; n = 6) or double PF stimuli at a 5-ms interval (white; n = 9). (Bii) Representative recordings from the experiments in Bi. The PF stimulation during the pairing protocol consisted of single PF stimuli (Upper) or double PF stimuli at a 5-ms interval (Lower). Traces are averages of 10 sweeps, before pairing (solid) and 30 min after pairing (dashed). (C) The magnitude of LTD depends on the interval between the two PF stimuli. Normalized EPSC amplitude after pairing with single PF stimulus (0-ms interval, n = 6), or with double PF stimuli at intervals of: 1 ms (n = 8), 5 ms (n = 9), 15 ms (n = 5), 30 ms (n = 6), or 60 ms (n = 4). Asterisks indicate statistical significance of the depression magnitudes. *, P < 0.01; **, P < 0.005; Sign test. (D) NMDARs but not mGluR1s are required for LTD induction. LTD is prevented by D-APV but not by CPCCOEt. Depression induced by pairing with double PF stimuli at 5-ms and at 1-ms intervals, in control conditions (white; n = 9, n = 8, respectively), in the presence of 200 μM D-APV (black; n = 5, n = 4, respectively) or in the presence of 50 μM CPCCOEt (gray; n = 6). (E) LTD induced upon GCL stimulation is still frequency and NMDAR dependent (Ctrl n = 8, APV n =4, 60 ms n = 4). For D and E, *, P < 0.02; Mann–Whitney U test.

Fig. 2.

NR2A- but not NR2B-containing NMDAR are required for LTD induction. (A) The NR2A antagonist zinc prevents LTD induction. Time course of the EPSC amplitude in control conditions (white; n = 9; same data as in Fig. 1) or in the presence of 300 nM free buffered zinc (black; n = 5). In each case, pairing was done with double PF stimuli at a 5-ms interval. (B) LTD is still induced in the presence of the NR2B antagonist Ro25–6981. Time course of the EPSC amplitude in control conditions (white; n = 9; same data as in Fig. 1) or in the presence of 300 nM Ro25–6981 (gray; n = 6). The application of Ro25–6981 started at least 15 min before induction. In each case, pairing was done with double PF stimuli at a 5-ms interval. (C) Records from representative experiments in control conditions (Top), in 300 nM free zinc (Middle) or 300 nM Ro25–6981 (Bottom). Traces are averages of 10 sweeps, just before pairing (solid) and 30 min after pairing (dashed). (D) Depression induced by pairing with double PF stimuli at a 5-ms interval in control conditions (white; n = 9), in 300 nM free zinc (black; n = 5) or in 300 nM Ro25–6981 (gray; n = 6). *, P < 0.02; Mann–Whitney U test.

Fig. 3.

Immunohistochemistry reveals the presence of NR1, NR2A, and NR2B at presynaptic sites of PF-PC synapses. (A) Fluorescence image of a segment of a dendrite in a neurobiotin-filled PC (green) combined with immunohistochemistry against the NR2A subunit of the NMDAR (red) in a coronal slice. (Scale bar: 1 μm.) (B–E) Electron microscopy of peroxidase-amplified immunostaining of NMDAR subunits. Note the PF varicosities (*) with synaptic vesicles and the postsynaptic densities of PC spines (Sp). (B and C) NR1 staining. (D and E) NR2A staining. (Scale bars, 100 nm.) (F) Quantification of peroxidase labeling of PF boutons: 48.6% of the PF boutons in a given region (177/364 PF-PC synapses) were reactive for NR1, 23.1% (63/273 PF-PC synapses) for NR2A, and 10.0% (26/261 PF-PC synapses) for NR2B antibodies.

Fig. 4.

Deactivation rates of recombinant NMDA receptors at 32 °C. (A) NMDAR currents (average of 5–10 consecutive traces) elicited by 100 ms-long applications of L-glutamate (1 mM) in the presence of 100 μM glycine to small lifted HEK-293 cells transfected with different NMDAR subunit DNAs (NR1–1a plus NR2A, NR2B, or NR2C). (B) NMDAR currents in A normalized to the amplitude at the end of the agonist application. Values of the deactivation time constants (τ_off) are mean ± SEM. n = 10 for NR1+NR2A and NR1+NR2B; n = 8 for NR1+NR2C. (C) The range of PF stimulation intervals resulting in LTD fits with the NR1+NR2A deactivation time course. The interval dependence of LTD induction (Fig. 1C) is plotted with the deactivation time courses of NMDAR currents (Fig. 4B). The start of current decay was aligned with the 0-ms interval for LTD induction and 0 NMDA current was aligned vertically with zero LTD.

Fig. 5.

NMDA autoreceptors as burst detectors. Schematic view of PF membrane potential and associated states of presynaptic NMDARs. (A) Time course of the PF membrane potential (V_{m, PF}) during high or low frequency activity (200 Hz vs. 16.7 Hz). (B) Gating of NMDA autoreceptors activated by glutamate released by the action potentials. Receptor kinetics have been extracted from those of recombinant NR1+NR2A receptors measured in Fig. 4 (τ_on = 1.4 ms; τ_off = 28.6 ms). (C) Voltage-dependent Mg block of NMDARs. (D) Current flow through NMDARs occurs only when receptors are gated and the Mg block is relieved. The effective interval between two successive PF action potentials for NMDAR conduction is defined by the residence time of glutamate on NMDARs. The first action potential provides the glutamate. By the time the receptor opens, the membrane is repolarized and Mg blocks the channel. The second action potential relieves the Mg block and Ca flows through the channel into the PF terminal only if it arrives while glutamate is still bound to the receptor (200 Hz). If the second action potential arrives after glutamate unbinding (16.7 Hz), little current flows.