Presynaptic Release Probability and Readily Releasable Pool Size Are Regulated by Two Independent Mechanisms during Posttetanic Potentiation at the Calyx of Held Synapse

Abstract

At the immature calyx of Held, the fast decay phase of a Ca 2ϩ transient induced by tetanic stimulation (TS) was followed by a period of elevated [Ca 2ϩ ] i for tens of seconds, referred to as posttetanic residual calcium (Ca res ). We investigated the source of Ca res and its contribution to posttetanic potentiation (PTP). After TS (100 Hz for 4 s), posttetanic Ca res at the calyx of Held was largely abolished by tetraphenylphosphonium (TPP ϩ ) or Ru360, which inhibit mitochondrial Na ϩ -dependent Ca 2ϩ efflux and Ca 2ϩ uniporter, respectively. Whereas the control PTP lasted longer than Ca res , inhibition of Ca res by TPP ϩ resulted in preferential suppression of the early phase of PTP, the decay time course of which well matched with that of Ca res . TS induced significant increases in release probability (P r ) and the size of the readily releasable pool (RRP), which were estimated from plots of cumulative EPSC amplitudes. TPP ϩ or Ru360 suppressed the posttetanic increase in P r , whereas it had little effect on the increase in RRP size. Moreover, the posttetanic increase in P r , but not in RRP size, showed a linear correlation with the amount of Ca res . In contrast, myosin light chain kinase (MLCK) inhibitors and blebbistatin reduced the posttetanic increase in RRP size with no effect on the increase in P r . Application of TPP ϩ in the presence of MLCK inhibitor peptide caused further suppression of PTP. These findings suggest that Ca res released from mitochondria and activation of MLCK are primarily responsible for the increase in P r and that in the RRP size, respectively.

Actin-dependent rapid recruitment of reluctant synaptic vesicles into a fast-releasing vesicle pool

“…The durations of AP e stimuli (step depolarizations from −80 mV to 0 mV) were adjusted in each synapse, such that the first EPSC amplitudes were close to 2 nA (2.35 ± 0.11 nA, 1.5-2.5 nA). This value is comparable to that evoked by a single AP in an unclamped calyx terminal in the presence of 2 mM γ-DGG (14). Examples of EPSC traces evoked by these protocols in the same synapse are superimposed in Fig.…”

“…7B) calyx of Held synapses. Although short-term depression at immature synapses is dominated by desensitization of AMPA receptors (17), cyclothiazide could not be used to prevent desensitization because of its off-target effects on ion channels in the calyx of Held under current clamp (14,18). We examined the effects of latB and CMZ on EPSCs evoked by a 100-Hz train in the presence of 2 mM γ-DGG.…”

Short-Term Presynaptic Plasticity

“…Pharmacological studies implicated protein kinase C (PKC) in PTP at several synapses (Alle et al 2001;Brager et al 2003;Beierlein et al 2007;Korogod et al 2007). However, the involvement of PKC in PTP was uncertain because the selectivity and specificity of PKC activators and inhibitors was called into question (Brose and Rosenmund 2002;Lee et al 2008), and PTP was unaffected by PKC inhibitors at some synapses (Reymann et al 1988a,b;Eliot et al 1994;Lee et al 2008). The use of knockout animals established that calcium-dependent PKC accounts for 80% of PTP at the calyx of Held .…”

“…At the calyx of Held synapse, a 5-min, 20-Hz stimulation produces a 30% increase in the size of the RRP (Habets and Borst 2005). Pool size changes have been studied more extensively for 100-Hz stimulation for 4 sec, which induces approximately twofold enhancement that is only partially explained by an increase in the probability of release (Lee et al 2008;. Tetanic stimulation did not alter the amplitude of the RRP determined with a large presynaptic depolarization, but it did increase the size of a rapidly releasing pool while reducing the size of the slowly releasing pool (Lee et al 2010), and increased the effective pool size determined by stimulation with high-frequency trains (Lee et al 2008;.…”

“…Pool size changes have been studied more extensively for 100-Hz stimulation for 4 sec, which induces approximately twofold enhancement that is only partially explained by an increase in the probability of release (Lee et al 2008;. Tetanic stimulation did not alter the amplitude of the RRP determined with a large presynaptic depolarization, but it did increase the size of a rapidly releasing pool while reducing the size of the slowly releasing pool (Lee et al 2010), and increased the effective pool size determined by stimulation with high-frequency trains (Lee et al 2008;. Myosin light chain kinase (MLCK) (Lee et al 2008(Lee et al , 2010 and calcium-dependent PKC have been implicated in the increases in effective pool size that contribute to PTP.…”