Structural & functional basis of presynaptic plasticity

Long-term changes in synaptic efficacy are generally thought to underlie learning and memory. Although a consensus is that many forms of long-term synaptic changes involve postsynaptic receptors and structures, very little is known about the modifications that occur at the presynapse hours or days after past activity. We aim to characterize functional and structural changes within a single presynaptic active zone (AZ) following a well-documented form of long-term potentiation in the cerebellum. Increasing evidence indicates that each AZ contains several docking sites where synaptic vesicles bind before being released. We will address the relationship between voltage-gated calcium channels (VGCCs) and docking sites after forskolin treatment and train-induced LTP using optogenetics. We will use synapses formed by parallel fibers (PF) onto Purkinje cells (PCs) and onto molecular layer interneurons (MLIs), where cAMP-dependent LTP has been reported. Experiments are organized into 5 work packages, which will be carried out in parallel by the two partners, Ryuichi Shigemoto (IST Austria) and Alain Marty (Université de Paris). Ryuichi Shigemoto has developed quantitative SDS-digested freeze-fracture replica labelling, which has high efficiency (up to 100%) and nanoscale resolution. We use it for examining the number of VGCCs and their distance from fused vesicles in the AZ membrane, by combining with optogenetic stimulation of PF in vivo and in vitro, using newly developed Flash & Freeze-fracture method. Conventional 3D reconstruction is also used for quantifying docked vesicles. Alain Marty has developed electrophysiological techniques to determine the number of docking sites in a single AZ and to characterize their function in short-term synaptic plasticity. We will also examine changes in presynaptic calcium signaling by two-photon microscopy during early and late phases of LTP. The methods described above are highly original and complementary to each other. Combining these techniques in a previous collaboration, we proposed that each cluster of VGCCs at AZs defines a docking site. In this project, we will extend our study to elucidate mechanisms of LTP by using newly generated transgenic animals for optogenetic stimulation of PF. We will also examine the role of two splice variants of presynaptic VGCCs, EFa and EFb, during LTP using newly generated Cre-dependent exon-switching mice. This project will provide for the first time an integrated structural and functional view of long-term synaptic plasticity at individual AZs of central mammalian synapses. It will also provide insight into cellular mechanisms underlying presynaptic LTP.