Neurotransmitter Release Site Replenishment and Presynaptic Plasticity

Abstract

An action potential (AP) triggers neurotransmitter release from synaptic vesicles (SVs) docking to a specialized release site of presynaptic plasma membrane, the active zone (AZ). The AP simultaneously controls the release site replenishment with SV for sustainable synaptic transmission in response to incoming neuronal signals. Although many studies have suggested that the replenishment time is relatively slow, recent studies exploring high speed resolution have revealed SV dynamics with milliseconds timescale after an AP. Accurate regulation is conferred by proteins sensing Ca²⁺ entering through voltage-gated Ca²⁺ channels opened by an AP. This review summarizes how millisecond Ca²⁺ dynamics activate multiple protein cascades for control of the release site replenishment with release-ready SVs that underlie presynaptic short-term plasticity.

Keywords: Ca2+ channels; Ca2+ sensors; action potential; active zone; dynamin; myosin; presynaptic plasticity; presynaptic proteins; synaptic vesicle.

Figure 1

Scheme of synaptic vesicle dynamics in the active zone demonstrated by the ‘zap-and-freeze’ method. (At rest) Synaptic vesicles (SVs) beneath the active zone (AZ) are transit between docked and undocked states. Docked SVs are ready to fuse in response to action potential (AP). (0–5 ms) Synchronous SV fusion, often of multiple SVs, starts within hundreds of microseconds of an AP. Fused SVs collapse into the plasma membrane by 11 ms. (11 ms) From 5 to 11 ms, SV fusion, toward the center of the AZ, is asynchronized. SVs start to be recruited. (14 ms) SVs are docked to fully replace the used SV for fusion. These docked SVs reduces within 100 ms. Reproduced from Kusick et al., 2020 [10].

Figure 2

Active zone proteins. The active zone is a highly organized structure that docks synaptic vesicles (SVs) close to fusion machinery proteins (SNAREs) and Ca²⁺ channels. This establishes the tight spatial organization required for fast exocytosis upon Ca²⁺ entry, and it provides molecular machinery to set and regulate synaptic strength. Reproduced from Wang S et al., 2016 [26].

Figure 3

Active zone protein CAST plays a role in synaptic vesicle loading. The presynaptic neurons were transfected with CAST^S45 DNA, 2 days before EPSP recording. (A) Phosphonegative-CAST^S45A relieved the second EPSP reduction, while phosphomimetic-CAST^S45D potentiated the reduction. Paired EPSP ratio was plotted against paired-AP interval. (Bar: SEM. * p < 0.05; unpaired student’s t-test. *green, CAST^S45A vs. CAST^WT; * navy, CAST^S45D vs. CAST^WT) (B) EPSP recovery from depletion of SVs. A 5-min train of APs at 5 Hz was applied, as indicated, for SV depletion. EPSP amplitude recovered with two different rates: fast (pink arrow) and slow phases (blue arrow) (left). The fast recovery rate is estimated by an exponential fit to the increase in EPSP amplitude from 0 to 8 s after the depletion (right). (* p < 0.05, ** p < 0.01, *** p < 0.001.; unpaired student’s t-test. *dark gray, CAST^S45D or CAST^WT vs. un-transfection; *green, CAST^S45D vs. CAST^S45A; *gray, CAST^S45D vs. CAST^WT) (C) Cascaded reactions of CAST phosphorylation. Adapted from Mochida et al., 2016 [56].

Figure 4

Myosin IIB and VI support release-ready SV restoration. Presynaptic neurons were transfected with control, myosin IIA-, IIB-, or VI-siRNA 2–3 days before the EPSP recordings. (A) Myosin VI, but not IIB, knockdown reduced release-ready SVs after AP-evoked transmitter release. The averaged paired-EPSP ratio was plotted against the paired-AP interval. (Bar: SEM * p < 0.05; Bonferroni post hoc test after one-way ANOVA. VI- knockdown vs. control IIA- or IIB- knockdown) (B) Myosin IIB and VI knockdown impaired transmitter release during APs firing at 10 Hz, resulted in a sudden reduction in third and second EPSP amplitude, respectively. (C) Myosin IIB and VI knockdown delayed recovery from the RRP depletion. After a 1 min control recording at 1 Hz, a 4-min AP train at 5 Hz was applied to deplete SVs. EPSP amplitudes were normalized to the mean EPSP amplitudes before the 4-min train. Recovery rate of release-ready SVs is estimated by double exponential growth fit to the increase in averaged EPSP amplitude after the depletion. +Dyn1: double knockdown of dynamin-1 and myosin; +Dyn3: double knockdown of dynamin-3 and myosin.

Figure 5

Neural activity selects Myosin IIB and VI in distinct dynamin isoform-mediated SV resupply pathways. Schematic drawing of SV resupply to the release site by myosin VI and IIB through distinct SV membrane recycling pathways mediated by dynamin isoforms. *purple, SV reloading activated by slow Ca²⁺ rise; *orange, SV reloading activated by rapid Ca²⁺ rise. ?, Clathrin-coated vesicle endocytosis is under debated [3,13]. SV clusters are classified into two pools: release-readily vesicles on the active zone membrane [57] and replacement site vesicles [8]. Adapted from Lu et al., 2009 [60]; Tanifuji et al., 2013 [85]; Mori et al., 2014 [86]; Hayashida et al., 2015 [64].

Figure 6

AP modulates synaptic transmission via Ca_V2.1 channel regulation. Ca_V2.1 channel, wild type (WT), alanine mutation at the IQ-like motif (IM), or deletion of the CaM-binding domain (ΔCBD) was expressed in presynaptic neurons and EPSPs were recorded in the presence of a blocker for native Ca_V2.2 channels. (A) Paired-AP alters the second EPSP amplitude. Timing of the second AP-induced depression and facilitation of synaptic transmission. Depression was prevented by ΔCBD, while facilitation was prevented by IM-AA mutation of the pore-forming α1 subunit. (B) Model for Ca²⁺/CaM-dependent inactivation and facilitation of the Ca_V2.1 channel and neurotransmitter release. (C) Biphasic synaptic transmission during 1-s APs at 30 Hz changed to synaptic depression by IM-AA or synaptic facilitation by ΔCBD mutation. Adapted from Mochida et al., 2008 [121].

Figure 7

Milliseconds Ca²⁺ dynamics activates multiple proteins for controlling release-ready SVs. Myosin IIB and VI activated by Ca²⁺/CaM dependent on APs firing pattern translocate SVs to restore release-ready state SVs. In contrast, APs-induced CAST phosphorylation brakes release-ready state SV restoration. Elevation of Ca²⁺ with a single AP activates Ca²⁺/CaM/myosin VI and SAD-B at the AZ and controls the speed of SV reloading into the RRP (upper scheme), resulting in modulation of transmitter release efficacy for an incoming AP (bottom left traces). Repetitive APs control the efficacy by myosin IIB activation (upper scheme) and possibly by other proteins in the AZ for sustainable synaptic transmission (bottom right traces). All these key protein function and property of Ca²⁺ channels in the AZ regulated by AP via Ca²⁺ sensors action determine release efficacy and underlie presynaptic short-term plasticity. (RP, replacement state SV pool [8]; RRP, release-ready state SV pool [57]; KD, knockdown).