Abstract
Synapsins are synaptic vesicle (SV) proteins organizing a component of the reserve pool of vesicles at most central nervous system synapses. Alternative splicing of the three mammalian genes results in multiple isoforms that may differentially contribute to the organization and maintenance of the SV pools. To address this, we first characterized the expression pattern of synapsin isoforms in the rat calyx of Held. At postnatal day 16, synapsins Ia, Ib, IIb and IIIa were present, while IIa—known to sustain repetitive transmission in glutamatergic terminals—was not detectable. To test if the synapsin I isoforms could mediate IIa-like effect, and if this depends on the presence of the E-domain, we overexpressed either synapsin Ia or synapsin Ib in the rat calyx of Held via recombinant adeno-associated virus-mediated gene transfer. Although the size and overall structure of the perturbed calyces remained unchanged, short-term depression and recovery from depression were accelerated upon overexpression of synapsin I isoforms. Using electron microscopic three-dimensional reconstructions we found a redistribution of SV clusters proximal to the active zones (AZ) alongside with a decrease of both AZ area and SV volume. The number of SVs at individual AZs was strongly reduced. Hence, our data indicate that the amount of synapsin Ia expressed in the calyx regulates the rate and extent of short-term synaptic plasticity by affecting vesicle recruitment to the AZ. Finally, our study reveals a novel contribution of synapsin Ia to define the surface area of AZs.
Highlights
Synapsins are a family of abundant neuron-specific phosphoproteins whose expression varies across synapse types and brain regions with synapsin Ia, Ib, IIa, IIb, and IIIa being most abundant (De Camilli et al, 1983; Sudhof et al, 1989; Matus-Leibovitch et al, 1995, 1997; Hosaka and Sudhof, 1998)
IDENTIFICATION AND DISTRIBUTION OF ENDOGENOUS SYNAPSIN ISOFORMS IN THE CALYX OF HELD To identify the synapsin isoforms contributing to synaptic transmission at the rat calyx of Held at P16, we determined the expression and distribution pattern of endogenous synapsins within the presynaptic terminal, using immunohistochemistry (Figure 1)
Overview images show that multiple synapsin isoforms were present in the medial nucleus of the trapezoid body (MNTB) of P16 rats, where they distributed to the calyx of Held terminals, seen as oval shapes
Summary
Synapsins are a family of abundant neuron-specific phosphoproteins whose expression varies across synapse types and brain regions with synapsin Ia, Ib, IIa, IIb, and IIIa being most abundant (De Camilli et al, 1983; Sudhof et al, 1989; Matus-Leibovitch et al, 1995, 1997; Hosaka and Sudhof, 1998). We characterized the contribution of synapsin I isoforms to SV distribution and synaptic transmission during and following high-frequency activity For this purpose we overexpressed synapsin I isoforms fused to fluorescent reporters, to manipulate SV clustering and/or mobility within the calyx of Held, a giant terminal in the auditory brainstem circuit mediating binaural sound localization (Borst and Soria van Heave, 2012). The IIa isoform—shown to sustain repetitive transmission in glutamatergic terminals (Gitler et al, 2008)—could not be detected in the calyx Overexpression of both synapsin I isoforms at the calyx of Held resulted in a redistribution of SVs within the presynaptic terminal, leading to an increased short-term depression in response to high frequency stimulation trains and faster recovery. We conclude that synapsin I isoforms—and synapsin Ia in particular—affect short-term plasticity by facilitating activitydependent release and acceleration of SV refilling following highfrequency activity (de Lange et al, 2003; Rizzoli and Betz, 2004; Denker and Rizzoli, 2010)
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