Separation of Presynaptic and Postsynaptic Contributions to Depression by Covariance Analysis of Successive EPSCs at the Calyx of Held Synapse

Abstract

Synaptic short-term plasticity is considered to result from multiple cellular mechanisms, which may include presynaptic and postsynaptic contributions. We have recently developed a nonstationary EPSC fluctuation analysis (Scheuss and Neher, 2001) to estimate synaptic parameters and their transient changes during short-term synaptic plasticity. Extending the classical variance-mean approach, a short train of stimuli is applied repetitively, and the resulting EPSCs are analyzed for means, variances, and covariances. This provides estimates of the quantal size and quantal content for each EPSC in the train, and furthermore, an estimate of the number of release sites. The latter is less sensitive to heterogeneity in the release probability than that of the variance-mean approach. Here, we applied this analysis to the calyx of Held synapse in brainstem slices of young rats (postnatal day 8-10). We found significant negative covariance in the amplitude of successive EPSCs in a train. The analysis showed that the 10-fold depression in the EPSC amplitude during 100 Hz trains at elevated extracellular Ca 2ϩ concentration resulted from a 2.5-fold reduction in quantal size caused by postsynaptic AMPA receptor desensitization and saturation, and a fourfold reduction in quantal content, which was partially relieved by application of cyclothiazide. The number of release sites estimated by covariance analysis was Ϸ2000 and significantly larger than estimates from variancemean parabolas.Key words: synaptic transmission; short-term plasticity; quantal analysis; covariance analysis; release site; release probability; quantal size Short-term synaptic plasticity is considered as an important element of information processing in neuronal circuits (Abbott et al., 1997;Tsodyks and Markram, 1997). Multiple cellular mechanisms have to be considered to contribute to synaptic short-term plasticity. Because these act most likely simultaneously and in parallel (Zucker, 1989;Fisher et al., 1997), their individual contributions cannot readily be studied in separation. Especially at synapses with large quantal contents, presynaptic and postsynaptic contributions to short-term plasticity are difficult to separate. Ideally, one would like to obtain independent measures of the number of released quanta (quantal content) and of the average quantal size to indicate presynaptic and postsynaptic efficacy of synaptic transmission, respectively. These parameters can, in principle, be provided by fluctuation or quantal analysis of EPSC amplitudes. However, variance-mean analysis according to a binomial model of synaptic transmission (for review, see Clements and Silver, 2000) has so far mainly been applied to steadystate sequences recorded under a variety of conditions (Silver et al., 1998;Reid and Clements, 1999;Oleskevich et al. 2000). Sequences of double pulses (Oleskevich et al., 2000) and repetitive trains of stimuli (Clamann et al., 1989;Meyer et al., 2001) have also been used, but no analysis dedicated to short-term plasticity in such nonstationa...

Roles of the Fast-Releasing and the Slowly Releasing Vesicles in Synaptic Transmission at the Calyx of Held

“…2). Different types of fluctuation analysis confirmed the release rates estimated from the deconvolution method (Neher and Sakaba, 2001a,b;Scheuss et al, 2002). Furthermore, variance analysis of the late phase of EPSCs attained during a 300 Hz train of AP-like stimuli gave estimates of release rates similar to those obtained from the deconvolution method (this study).…”

“…During the train, 3297 Ϯ 634 vesicles were released, on average (n ϭ 6), with 2208 Ϯ 468 vesicles released synchronously according to the above criterion. The amount of synchronous release was comparable to AP-evoked release in previous studies (Scheuss et al, 2002;Taschenberger et al, 2002). The fraction of synchronous release to total release varied among different cell pairs (50 -80% of total) (compare Figs.…”

“…In seven cell pairs, a 300 Hz train was repeated more than five times, and quantal size was estimated to be 18.6 Ϯ 1.8 pA; this is similar to the value reported in the presence of cyclothiazide and kynurenic acid (15 pA) (Neher and Scheuss et al, 2002). The release rate from the variance analysis was estimated to be 6.1 Ϯ 1.1 vesicles/ms, similar to the value estimated from the deconvolution method (6.3 Ϯ 1.1 vesicles/ms).…”

“…The presynaptic patch pipette contained a low Ca 2ϩ buffer (0.05 mM EGTA), and the extracellular solution contained cyclothiazide and kynurenic acid to prevent postsynaptic AMPA receptor desensitization and saturation Joshi and Wang, 2002;Scheuss et al, 2002;Taschenberger et al, 2002). In Figure 1 A, the presynaptic terminal was depolarized to ϩ40 mV for 1 ms to mimic an AP.…”

“…EPSCs were evoked in response to the presynaptic Ca 2ϩ influx and had an amplitude of 2.82 Ϯ 1.06 nA (n ϭ 6). Considering that kynurenic acid blocks EPSC amplitudes by ϳ50% Scheuss et al, 2002), the (corrected) amplitude is comparable to that evoked by a single AP in an unclamped terminal (3-10 nA) (Borst et al, 1995;Schneggenburger et al, 1999;Scheuss et al, 2002;Taschenberger et al, 2002). The deconvolution method was used to estimate quantal release rates .…”