Function of Synapsin Proteins in Synaptic Transmission at a Giant CNS Synapse

Abstract

At chemical synapses synaptic vesicles are functionally segregated into distinct pools depending on their mobility and release probability in response to action potentials. At most synapses synapsin proteins cluster and immobilize reserve pool vesicles within the cytoskeletal network, distally from the active zones. Activity-dependent phosphorylation of synapsins releases synaptic vesicles from the complex meshwork of the cytoskeletal components and renders them freely mobile and able to undergo vesicle cycling. There are three synapsin genes in mammals and their alternative splicing results in more than ten isoforms whose functional differences in maintaining synaptic transmission are not fully elucidated. In this study we examined the involvement of synapsins in synaptic transmission using two independent approaches. First, we established the structure-function relationship at synapses, overexpressing synapsin I isoforms (synapsin Ia or synapsin Ib). Second, synaptic transmission and structural integrity of synapses in mice, lacking all three synapsin genes (triple knock-out (TKO)) were examined. The calyx of Held, a giant glutamatergic synapse located in the auditory brain stem, was utilized as a model system. Synapsin I isoforms overexpression was accomplished through transduction of globular bushy cells (GBCs), located in the ventral cochlear nucleus, with recombinant adeno- associated viral particles. The GBCs are projection neurons, which give rise to the calyx of Held in the medial nucleus of the trapezoid body. 10 days after the transduction, overexpression of synapsin I isoforms resulted in a redistribution of SV within the calyx of Held, without changing the size and the overall structure of the perturbed synapses. Ultrastructural analysis, using serial sectioning scanning electron microscopy (S3EM) revealed that synapsin Ia overexpression resulted in decreased numbers of SVs at the active zone without altering the total vesicle number. Therefore, we could conclude that synapsin Ia overexpression was followed by vesicle redistribution within the presynaptic terminal. On the functional level, the overexpression of both synapsin I isoforms had no effect on the properties of spontaneous and evoked EPSCs. However, repeated stimulation at frequencies exceeding 10 Hz led to accelerated short-term depression (STD). Overexpression of either isofroms led also to accelerated recovery from depression after strong stimulation. Brain lysates from synapsin TKO mice revealed a strong reduction in the level of several synaptic vesicle proteins, while proteins of the active zone cytomatrix or of the postsynaptic density remained unaffected. Accordingly, TKO calyces had lower amounts of vGluT1 immunoreactivity while the level of the active zone marker bassoon was unchanged Summary i as shown via 3D reconstructions of TKO calyces. The S3EM analysis confirmed these results revealing a 50 % reduction in the number of synaptic vesicles in TKO calyces. The structural alterations resulting in the absence of synapsins led to accelerated and more pronounced STD at stimulation at frequencies above 100 Hz. Synapsin deletion, contrary to synapsin overexpression, slowed down the recovery of depression. This might prove that synapsin- dependent SVs contribute to the faster replenishment of the readily releasable pool, which maintains synaptic transmission under basic conditions. Despite the structural defects and the alterations in the short-term depression, transmission failures were not observed during the high-frequency trains. These results reveal that in wild-type synapses the synapsin-dependent vesicles account only for a small fraction of the SVs that enter the RRP. In conclusion, synapsins maintain of a specific vesicle population at CNS synapses. However, these vesicles are dispensable from normal basal synaptic transmission and are recruited only when the synapse is exposed to long-lasting high-frequency activity. The synapsin-dependent vesicles are fed into the readily releasable pool to counteract the effects of presynaptic depression and aid the faster recovery of the synapse. Although synapsins may be required for normal synaptic vesicle biogenesis, trafficking and immobilization, they are not essential for sustained synaptic transmission at the calyx of Held.