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Synapses contain a limited number of synaptic vesicles (SVs) that are released in response to action potentials (APs). Therefore, sustaining synaptic transmission over a wide range of AP firing rates and timescales depends on SV release and replenishment. Although actin dynamics impact synaptic transmission, how presynaptic regulators of actin signaling cascades control SV release and replenishment remains unresolved. Rac1, a Rho GTPase, regulates actin signaling cascades that control synaptogenesis, neuronal development, and postsynaptic function. However, the presynaptic role of Rac1 in regulating synaptic transmission is unclear. To unravel Rac1's roles in controlling transmitter release, we performed selective presynaptic ablation of Rac1 at the mature mouse calyx of Held synapse. Loss of Rac1 increased synaptic strength, accelerated EPSC recovery after conditioning stimulus trains, and augmented spontaneous SV release with no change in presynaptic morphology or AZ ultrastructure. Analyses with constrained short-term plasticity models revealed faster SV priming kinetics and, depending on model assumptions, elevated SV release probability or higher abundance of tightly docked fusion-competent SVs in Rac1-deficient synapses. We conclude that presynaptic Rac1 is a key regulator of synaptic transmission and plasticity mainly by regulating the dynamics of SV priming and potentially SV release probability., Competing Interests: CK, MA, TR, CT, PV, DG, MR, HT, NK, SY No competing interests declared, (© 2022, Keine et al.)

At chemical synapses, synaptic vesicles release their acidic contents into the cleft, leading to the expectation that the cleft should acidify. However, fluorescent pH probes targeted to the cleft of conventional glutamatergic synapses in both fruit flies and mice reveal cleft alkalinization rather than acidification. Here, using a reaction-diffusion scheme, we modeled pH dynamics at the Drosophila neuromuscular junction as glutamate, ATP, and protons (H + ) were released into the cleft. The model incorporates bicarbonate and phosphate buffering systems as well as plasma membrane calcium-ATPase activity and predicts substantial cleft acidification but only for fractions of a millisecond after neurotransmitter release. Thereafter, the cleft rapidly alkalinizes and remains alkaline for over 100 ms because the plasma membrane calcium-ATPase removes H + from the cleft in exchange for calcium ions from adjacent pre- and postsynaptic compartments, thus recapitulating the empirical data. The extent of synaptic vesicle loading and time course of exocytosis have little influence on the magnitude of acidification. Phosphate but not bicarbonate buffering is effective at suppressing the magnitude and time course of the acid spike, whereas both buffering systems are effective at suppressing cleft alkalinization. The small volume of the cleft levies a powerful influence on the magnitude of alkalinization and its time course. Structural features that open the cleft to adjacent spaces appear to be essential for alleviating the extent of pH transients accompanying neurotransmission., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Stromalin, a cohesin complex protein, was recently identified as a novel memory suppressor gene, but its mechanism remained unknown. Here, we show that Stromalin functions as a negative regulator of synaptic vesicle (SV) pool size in Drosophila neurons. Stromalin knockdown in dopamine neurons during a critical developmental period enhances learning and increases SV pool size without altering the number of dopamine neurons, their axons, or synapses. The developmental effect of Stromalin knockdown persists into adulthood, leading to strengthened synaptic connections and enhanced olfactory memory acquisition in adult flies. Correcting the SV content in dopamine neuron axon terminals by impairing anterograde SV trafficking motor protein Unc104/KIF1A rescues the enhanced-learning phenotype in Stromalin knockdown flies. Our results identify a new mechanism for memory suppression and reveal that the size of the SV pool is controlled genetically and independent from other aspects of neuron structure and function through Stromalin., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by Ca V 2.1 calcium channels (Ca V 2.1) and is highly dependent on the physical distance between Ca V 2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of Ca V 2.1 through direct interactions with the Ca V 2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant Ca V 2.1 α 1 subunits on a Ca V 2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of Ca V 2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for Ca V 2.1 abundance and coupling. Therefore, our work advances our molecular understanding of Ca V 2.1 regulation of neurotransmitter release in mammalian CNS synapses.

Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca(2+) channels (VGCCs) to vesicles. But because the topography of VGCCs and their proximity to vesicles is unknown, a quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local [Ca(2+)] imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca(2+) buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca(2+) sensors for vesicular release are located at the perimeter of VGCC clusters (<30 nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This "perimeter release model" provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Key points: CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held.CAST/ELKS regulate all three CaV2 subtype channel levels in the presynaptic terminal and not just CaV2.1.The half‐life of ELKS is on the timescale of days and not weeks.Synaptic transmission was not impacted by the loss of CAST/ELKS.CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. Many presynaptic active zone (AZ) proteins have multiple regulatory roles that vary during distinct stages of neuronal circuit development. The CAST/ELKS protein family are evolutionarily conserved presynaptic AZ molecules that regulate presynaptic calcium channels, synaptic transmission and plasticity in the mammalian CNS. However, how these proteins regulate synapse development and presynaptic function in a developing neuronal circuit in its native environment is unclear. To unravel the roles of CAST/ELKS in glutamatergic synapse development and in presynaptic function, we used CAST knockout (KO) and ELKS conditional KO (CKO) mice to examine how their loss during the early stages of circuit maturation impacted the calyx of Held presynaptic terminal development and function. Morphological analysis from confocal z‐stacks revealed that combined deletion of CAST/ELKS resulted in a reduction in the surface area and volume of the calyx. Analysis of AZ ultrastructure showed that AZ size was increased in the absence of CAST/ELKS. Patch clamp recordings demonstrated a reduction of all presynaptic CaV2 channel subtype currents that correlated with a loss in presynaptic CaV2 channel numbers. However, these changes did not impair synaptic transmission and plasticity and synaptic vesicle release kinetics. We conclude that CAST/ELKS proteins are positive regulators of presynaptic growth and are suppressors of AZ expansion and CaV2 subtype currents and levels during calyx of Held development. We propose that CAST/ELKS are involved in pathways regulating presynaptic morphological properties and CaV2 channel subtypes and suggest there is developmental compensation to preserve synaptic transmission during early stages of neuronal circuit maturation. Key points: CAST/ELKS are positive regulators of presynaptic growth and are suppressors of active zone expansion at the developing mouse calyx of Held.CAST/ELKS regulate all three CaV2 subtype channel levels in the presynaptic terminal and not just CaV2.1.The half‐life of ELKS is on the timescale of days and not weeks.Synaptic transmission was not impacted by the loss of CAST/ELKS.CAST/ELKS are involved in pathways regulating morphological properties of presynaptic terminals during an early stage of circuit maturation. [ABSTRACT FROM AUTHOR]

Key points Spatiotemporal glutamate concentration profile in the cleft is a determinant of the EPSC amplitude and time course., Two key parameters required to describe this profile are the number of glutamate in a vesicle ( NGlu) and its diffusion coefficient ( DGlu), both of which are unestablished., Using the rat calyx of Held synapse as a model, the distribution of AMPA receptors was mapped with SDS-digested freeze fracture replica labelling and their performance as glutamate sensors was evaluated with outside-out patch recordings., Based on these data, synaptic responses were simulated using various combinations of NGlu and DGlu, and an optimal range of the NGlu- DGlu combinations that could reproduce the recordings was determined., Using the estimated profile, we show that release from a single vesicle does not saturate the receptors, glutamate spillover does not affect the synaptic conductance amplitude, and synaptic response increases non-linearly with the number of multivesicular releases., Abstract Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle ( NGlu) and its diffusion coefficient ( DGlu) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of NGlu and DGlu with an optimum of 7000 and 0.3 μm2 ms−1 reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals. [ABSTRACT FROM AUTHOR]

In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by CaV2.1 calcium channels (CaV2.1) and is highly dependent on the physical distance between CaV2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of CaV2.1 through direct interactions with the CaV2.1 a1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant CaV2.1 a1 subunits on a CaV2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of CaV2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for CaV2.1 abundance and coupling. Therefore, our work advances our molecular understanding of CaV2.1 regulation of neurotransmitter release in mammalian CNS synapses. [ABSTRACT FROM AUTHOR]