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

Abstract

Recruitment of release-competent vesicles during sustained synaptic activity is one of the major factors governing short-term plasticity. During bursts of synaptic activity, vesicles are recruited to a fast-releasing pool from a reluctant vesicle pool through an actin-dependent mechanism. We now show that newly recruited vesicles in the fast-releasing pool do not respond at full speed to a strong Ca 2+ stimulus, but require approximately 4 s to mature to a "superprimed" state. Superpriming was found to be altered by agents that modulate the function of unc13 homolog proteins (Munc13s), but not by calmodulin inhibitors or actin-disrupting agents. These findings indicate that recruitment and superpriming of vesicles are regulated by separate mechanisms, which require integrity of the cytoskeleton and activation of Munc13s, respectively. We propose that refilling of the fast-releasing vesicle pool proceeds in two steps, rapid actin-dependent "positional priming," which brings vesicles closer to Ca 2+ sources, followed by slower superpriming, which enhances the Ca 2+ sensitivity of primed vesicles.T he release rate of a synaptic vesicle (SV) is governed by two factors, the intrinsic Ca 2+ sensitivity of the vesicle fusion machinery and the distance of the SV to Ca 2+ channels. As Munc13s and Munc18s confer fusion competence on a docked SV, the regulation of release rate by Munc13s and Munc18s is called "molecular priming" (1). It is distinguished from "positional priming," a process that is thought to regulate the proximity of an SV to the calcium source (2, 3). However, it is not known how these two priming mechanisms are manifested in the kinetics of quantal release. Deconvolution analyses of excitatory postsynaptic currents (EPSCs) evoked by long presynaptic depolarizations at the calyx of Held (a giant nerve terminal in the auditory pathway) showed that releasable SVs can be separated into fastreleasing pools (FRPs) and slowly releasing pools (SRPs) (4). The differences in SV priming that underlie the differences in release kinetics between SVs in the FRP and the SRP are currently unclear (3, 5). Wadel et al. (3) found that SVs in the SRP can be released by homogenous Ca 2+ elevation only 1.5 to 2 times slower than SVs in the FRP, even though they are released 10 times slower by depolarization-induced Ca 2+ influx. This was interpreted as evidence that the differences in their release kinetics arise from differences primarily in positional priming. In contrast, Wölfel et al. (5) showed that release with two kinetic components is even observed if the intracellular Ca 2+ concentration is homogenously elevated throughout the calyx terminal, indicating that SVs in the FRP and the SRP differ with regard to their molecular priming.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 ...

Tissue-specific dynamin-1 deletion at the calyx of Held decreases short-term depression through a mechanism distinct from vesicle resupply

“…Interestingly, dynamin KO leads to alteration of actin polymerization and reorganization in other types of cells (34,66). A role for actin in vesicle recruitment has also been reported (73,83,84). Similarly, Lat-B caused an apparent enhancement in short-term depression in our study.…”

Superpriming of synaptic vesicles as a common basis for intersynapse variability and modulation of synaptic strength

“…Slow superpriming induced by activation of the PLC-DAG pathway has been observed before in the calyx of Held under voltage clamp, using step depolarizations (33). In hippocampal synapses, superpriming was shown to depend on the presence of at least one isoform of rab3 (32).…”

“…At the calyx of Held, potentiation of neurotransmitter release by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a more physiological substitute for PdBu, has been interpreted as a slow maturation process of SVs, following their recruitment to the readily releasable pool (33). This process was found to display distinct pharmacology, involving PLC and DAG, and to take several seconds for completion.…”

“…This, together with the considerations above regarding the molecules involved, suggests an attractive scenario for the case that a sequential reaction scheme applies (51): Release-competent SRP vesicles with a fully functional release apparatus undergo a process of positional priming, which moves them closer to Ca 2+ channels by interaction with the active zone through Rim, Rab3, and Rim binding protein (58,59), turning them into FRP vesicles. This process depends on an intact cytoskeleton (33) ] i and by interactions of Munc13 with the abovementioned active zone proteins, possibly including GIT (49). In the scenario of a parallel model, these interactions would provide a dynamically changing number of sites for superpriming.…”

Numbers of presynaptic Ca 2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses

“…S2). Monte Carlo simulations of these results indicated that, at 4 wk, the release probability of docked SVs is reduced for the first and second stimulation, possibly reflecting a decrease of the percentage of high release probability "superprimed" SVs (39,40). In addition, simulations indicated a reduction of the initial occupancy of the replacement site at 4 wk (Fig.…”