Estimation of quantal size and number of functional active zones at the calyx of Held synapse by nonstationary EPSC variance analysis

At the large excitatory calyx of Held synapse, the quantal size during an e voked EPSC and the number of active zones contributing to transmission are not known. We developed a non-stationary variant of EPSC fluctuation analys is to determine these quantal parameters. AMPA receptor-mediated EPSCs were recorded in slices of young (postnatal 8-10 d) rats after afferent fiber s timulation, delivered in trains to induce synaptic depression. The means an d the variances of EPSC amplitudes were calculated across trains for each s timulus number. During 10 Hz trains at 2 mM Ca2+ concentration ([Ca2+]), we found linear EPSC variance-mean relationships, with a slope that was in go od agreement with the quantal size obtained from amplitude distributions of spontaneous miniature EPSCs. At high release probability with 10 or 15 mM [Ca2+], competitive antagonists were used to partially block EPSCs. Under t hese conditions, the EPSC variance-mean plots could be fitted with parabola s, giving estimates of quantal size and of the binomial parameter N. With t he rapidly dissociating antagonist kynurenic acid, quantal sizes were large r than with a slowly dissociating antagonist, suggesting that the effective glutamate concentration was increased at high release probability. Conside ring the possibility of multivesicular release and moderate saturation of p ostsynaptic AMPA receptors, we conclude that the binomial parameter N (637 +/- 117; mean +/- SEM) represents an upper limit estimate of the number of functional active zones. We estimate that during normal synaptic transmissi on, the probability of vesicle fusion at single active zones is in the rang e of 0.25-0.4.