The Pool of Fast Releasing Vesicles Is Augmented by Myosin Light Chain Kinase Inhibition at the Calyx of Held Synapse

Abstract

Synaptic strength is determined by release probability and the size of the readily releasable pool of docked vesicles. Here we describe the effects of blocking myosin light chain kinase (MLCK), a cytoskeletal regulatory protein thought to be involved in myosin-mediated vesicle transport, on synaptic transmission at the mouse calyx of Held synapse. Application of three different MLCK inhibitors increased the amplitude of the early excitatory postsynaptic currents (EPSCs) in a stimulus train, without affecting the late steady-state EPSCs. A presynaptic locus of action for MLCK inhibitors was confirmed by an increase in the frequency of miniature EPSCs that left their average amplitude unchanged. MLCK inhibition did not affect presynaptic Ca(2+) currents or action potential waveform. Moreover, Ca(2+) imaging experiments showed that [Ca(2+)](i) transients elicited by 100-Hz stimulus trains were not altered by MLCK inhibition. Studies using high-frequency stimulus trains indicated that MLCK inhibitors increase vesicle pool size, but do not significantly alter release probability. Accordingly, when AMPA-receptor desensitization was minimized, EPSC paired-pulse ratios were unaltered by MLCK inhibition, suggesting that release probability remains unaltered. MLCK inhibition potentiated EPSCs even when presynaptic Ca(2+) buffering was greatly enhanced by treating slices with EGTA-AM. In addition, MLCK inhibition did not affect the rate of recovery from short-term depression. Finally, developmental studies revealed that EPSC potentiation by MLCK inhibition starts at postnatal day 5 (P5) and remains strong during synaptic maturation up to P18. Overall, our data suggest that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release at a CNS synapse.

Endogenous Rho-Kinase Signaling Maintains Synaptic Strength by Stabilizing the Size of the Readily Releasable Pool of Synaptic Vesicles

“…Coaddition to the bath solution of a specific inhibitor of MLCK fully prevented/reverted all functional and ultrastructural changes induced by ROCK inhibition. These results are consistent with those reported at the calyx of Held synapse, in which MLCK was shown to control the size of the pool of fast releasing vesicles (Srinivasan et al, 2008). The finding that MLCK inhibition per se failed to modify both p-MLC levels and the amplitude of eEPSCs further suggests that basal activity of MLCK in our experimental preparation is low enough so that its effects on the actomyosin apparatus were relegated to the synaptic vesicle pool.…”

“…It is thus possible that ROCK modulates neurotransmitter release and electrical activity of neuronal circuits through its cytoskeletaldependent regulation of presynaptic vesicle pools. This hypothesis gains support from the finding that MLCK controls the size of the pool of fast releasing vesicles at the calyx of Held (Srinivasan et al, 2008).…”

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

“…However, we did not observed any difference in late steady-state neurotransmission during 100 Hz stimulation in the presence of MLCK inhibitor or blebbistatin, suggesting that inhibition of MLCK has little effect on the vesicle recruitment during TS. Consistent with this view, Srinivasan et al (2008) have recently reported that ML-7 affects neither the steady-state EPSCs nor the rate of recovery from synaptic depression. Therefore, we imagine that the mechanisms underlying posttetanic increase in the RRP size might be different from that underlying replenishment of fast SV pool after depletion.…”

“…5Bc). ML-7 increased the basal EPSC amplitude by 28.5 Ϯ 12.1 and 42.3 Ϯ 15.6% in the presence of Kyn or ␥-DGG, respectively, probably because of recruitment of the fast-releasing SV pool (Srinivasan et al, 2008). This can result in occlusion of posttetanic increase in the RRP size.…”

Presynaptic Depression in Phasic Motor Nerve Terminals and Influence of 5-HT on Vesicle Dynamics

“…A possible experiment to determine whether the mechanisms responsible for the rundown are related to the need for energy for vesicular docking and recycling (i.e., they are ATP-dependent processes) could be one in which the rapid release of caged ATP within depressed terminals is induced as was similarly approached with release of Ca2+ from caged compounds [46]. It would be interesting to examine, within this preparation, the possibility of the myosin light chain kinase induced inhibition of vesicular movement as shown in the Calyx of held synapse [47]. Comparative studies with NMJs in Drosophila that show marked synaptic depression might prove to be fruitful [4,48].…”