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

Abstract

Glutamatergic synaptic terminals harbor reluctant synaptic vesicles (SVs) that contribute little to synchronous release during action potentials but are release competent when stimulated by sucrose or by direct intracellular application of calcium. It has been noted that the proximity of a release-competent SV to the calcium source is one of the primary factors that differentiate reluctant SVs from fastreleasing ones at the calyx of Held synapse. It has not been known whether reluctant SVs can be converted into fast-releasing ones. Here we show that reluctant SVs are recruited rapidly in an actindependent manner to become fast-releasing SVs once the pool of fast-releasing SVs is depleted by a short depolarization. Recovery of the pool of fast-releasing SVs was accompanied by a parallel reduction in the number of reluctant SVs. Quantitative analysis of the time course of depletion of fast-releasing SVs during high-frequency stimulation revealed that in the early phase of stimulation reluctant SVs are converted rapidly into fast-releasing ones, thereby counteracting short-term depression. During the late phase, however, after reluctant vesicles have been used up, another process of calmodulindependent recruitment of fast-releasing SVs is activated. These results document that reluctant SVs have a role in short-term plasticity and support the hypothesis of positional priming, which posits that reluctant vesicles are converted into fast-releasing ones via relocation closer to Ca 2+ -channels.readily releasable pool | synaptic vesicle dynamics S ynaptic strength is determined by quantal size, the number of release-competent synaptic vesicles (SVs), and their release probability (Pr). The estimate for the number of release-competent SVs, which also is referred to as the "readily releasable pool (RRP) size," depends heavily on the method used for its determination. The RRP size estimated by a cumulative plot of the excitatory postsynaptic currents (EPSC) amplitudes evoked by high-frequency afferent fiber stimulation (RRP cum ) is smaller than that estimated by the application of a hypertonic sucrose solution or presynaptic strong depolarization (1-3). This discrepancy reflects the presence of reluctant SVs, which are scarcely released by an action potential (AP) at glutamatergic synaptic terminals. Consistently, deconvolution analysis of EPSCs evoked by a long depolarizing pulse at the calyx of Held revealed that release-competent SVs can be separated into fast-and slowreleasing SV pools (FRP and SRP, respectively) (4). The FRP vesicles are responsible for phasic release during a high-frequency train of action potentials (APs), whereas the SRP vesicles contribute primarily to asynchronous release, when the intracellular concentration of calcium ions ([Ca 2+ ]) is increased globally during the late period of the train (2). Thus, SRP vesicles can be regarded largely as reluctant SVs at the calyx of Held.Despite the evidence for the presence of reluctant SVs at glutamatergic synapses, their role in short-term plasticity i...

Superpriming of synaptic vesicles after their recruitment to the readily releasable pool

“…1C). In agreement with previous reports (6,16), latrunculin B (15 μM; n = 7) inhibited CDR and SDR, and CaMip (20 μM; n = 7) abolished CDR ( Fig. 1D).…”

“…1A; see also Table S1 ]. We showed previously that the preDP3 completely depletes the FRP while releasing very few SRP SVs (6). The preDP10 depletes the SRP and the FRP, and the preDP30 induces Ca 2+ -dependent pool recovery, as shown previously.…”

“…The present study indicates that such SVs are fully matured only when they are positioned close to the Ca 2+ source. We demonstrate that the time course for such full maturation or superpriming of newcomer SVs is slower (τ = 3.6 s) than that of cytoskeleton-dependent conversion of reluctant SVs into FRP SVs (τ = 60 ms) (6). Therefore, we propose a two-step model for refilling of the FRP: rapid "positional priming," which brings vesicles closer to Ca 2+ sources, followed by slower superpriming, which enhances the Ca 2+ sensitivity of vesicles.…”

“…CaM inhibitors specifically affect CDR (6,16) and have little effect on SDR and the recovery of τ fast (Fig. 2B).…”

“…We found recently that SVs in the SRP rapidly convert into the FRP after specific FRP depletion by a short depolarizing pulse (6). Such rapid refilling of the FRP with SRP vesicles, which is referred to as SRP-dependent recovery (SDR), was suppressed by actin depolymerization or inhibition of myosin, implying that SDR involves a transport process, steering docked and partially primed vesicle toward Ca 2+ channels.…”