NMDA receptor activation by spontaneous glutamatergic neurotransmission

Abstract

Under physiological conditions N-methyl-D-aspartate (NMDA) receptor activation requires coincidence of presynaptic glutamate release and postsynaptic depolarization due to the voltage-dependent block of these receptors by extracellular Mg(2+). Therefore spontaneous neurotransmission in the absence of action potential firing is not expected to lead to significant NMDA receptor activation. Here we tested this assumption in layer IV neurons in neocortex at their resting membrane potential (approximately -67 mV). In long-duration stable recordings, we averaged a large number of miniature excitatory postsynaptic currents (mEPSCs, >100) before or after application of dl-2 amino 5-phosphonovaleric acid, a specific blocker of NMDA receptors. The difference between the two mEPSC waveforms showed that the NMDA current component comprises approximately 20% of the charge transfer during an average mEPSC detected at rest. Importantly, the contribution of the NMDA component was markedly enhanced at membrane potentials expected for the depolarized up states (approximately -50 mV) that cortical neurons show during slow oscillations in vivo. In addition, partial block of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor component of the mEPSCs did not cause a significant reduction in the NMDA component, indicating that potential AMPA receptor-driven local depolarizations did not drive NMDA receptor activity at rest. Collectively these results indicate that NMDA receptors significantly contribute to signaling at rest in the absence of dendritic depolarizations or concomitant AMPA receptor activity.

FIG. 1.

Long-term stability of miniature excitatory postsynaptic current (mEPSC) properties at rest. A: mEPSPs recorded under current-clamp conditions before (control, top trace) or after addition of dl-2 amino 5-phosphonovaleric acid (AP-5, bottom trace). Right: the average (±SE) resting membrane potential before and after addition of AP-5 (P > 0.5). B: control mEPSCs recorded at −82 mV in 2 mM Mg²⁺ under voltage-clamp conditions (top trace). Bottom left traces: events extracted from the section indicated by the dotted lines on an expanded time scale. Bottom right: alignment of 100 mEPSCs (black traces) plus average (gray trace) chosen from the same experiment shown above. C: average mEPSCs corresponding to recordings in the control condition (without AP-5). Left and middle: the averages of events respectively chosen during 8–13 and 23–28 min after reaching whole cell configuration. Both traces were overlapped on the right. To test the stability of the signal, the middle trace was subtracted from left trace, and the resultant trace shown (“difference”). Inset: the comparison of charge transfer at times 8–13 min or 23–28 min (n = 7; P > 0.5).

FIG. 2.

The N-methyl-d-aspartate (NMDA) receptor-mediated component of mEPSCs under physiological conditions. A: panel depicts average mEPSCs recorded before and after 50 μM AP-5 application as well as the difference between the 2 averages from representative experiments at the indicated holding potentials and extracellular Mg²⁺ concentrations. B: averages of the AP-5-sensitive component at the indicated experimental conditions (numbers of experiments indicated in parenthesis). C: average (left) or relative (right) charge transfer by the NMDA receptor component during mEPSC under different conditions. Statistical significance was assessed with 1-way ANOVA (P < 0.001). The Dunnett's post hoc test was used to compare the no-AP-5 condition, as the control (no-NMDA receptor component was revealed) to all other conditions. Experiments at −67 mV through −42 mV were significantly different from the no-AP-5 control.

FIG. 3.

Changes in the properties of individual mEPSCs after AP-5. A and C: the amplitudes and rise times of mEPSCs were not different before and after application of (n = 10). This suggests that a fast non-NMDA component dominates these 2 parameters at −67 mV (1.25 mM Mg²⁺). B and D: absolute charge transfer during a mEPSC and mEPSC decay times were significantly reduced in the presence of AP-5 (both, ∼20% difference). E: cumulative distributions of charge transfer by mEPSCs before and after application of AP-5 at −67-mV holding potential. This graph shows that the AP-5 induced reduction in total charge transfer by individual mEPSCs was homogenously distributed across all mEPSC sizes. F and G: cumulative distributions of rise times and decay times of mEPSCs before and after application of AP-5 at −67-mV holding potential. These graphs show that the AP-5 application had a minimal effect on the distribution of rise times, whereas it significantly decreased the decay times of individual mEPSCs. In both cases, however, mEPSCs were affected in a homogenous manner.

FIG. 4.

Impact AMPA current inhibition on NMDA receptor-mediated component of mEPSCs. A and E: application of 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX, 1.8 μM) and 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX, 350 nM) caused partial-block of the non-NMDA receptor component of mEPSCs in the presence of AP-5. In these conditions, mEPSCs were reduced by 40 to 60%. B and F: the NMDA receptor mediated component of mEPSCs analyzed in the presence of low concentrations of CNQX or NBQX. Traces depict normalized mEPSCs before (control) or after addition of 50 μM AP-5 (+AP-5). The difference (light gray trace) represents the AP-5-sensitive component. C and G: average charge transfer by NMDA receptors (NMDAR-Q) is unaffected by application of CNQX or NBQX (n = 4; in all cases). D and H: the presence of CNQX or NBQX reduced the amplitudes of the non-NMDA receptor mediated component of mEPSCs and thus increased the relative contribution of NMDA receptors to total charge transfer.