Open Access

Barbara Jánosi

• Synaptic transmission is a fundamental process that involves the transfer of information from a presynaptic neuron to a target cell through the release of neurotransmitters. The SV cycle is a complex series of events that enables the recycling of SVs, allowing for the sustained release of neurotransmitters. This process is mediated by a variety of proteins and enzymes, and its regulation is critical for maintaining proper synaptic function. Despite extensive research efforts, many aspects of the SV cycle and the underlying synaptic proteins remain poorly understood, highlighting the need for continued investigation into this important process. During this work, multiple aspects of synaptic transmission were studied by performing behavioural, pharmacological, optogenetic, electrophysiological and ultrastructural assays on Caenorhabditis elegans. First, the role of two proteins (ERP-1 and RIMB-1) were analysed in the synaptic vesicle cycle. Second, a new optogenetic tool, the pOpsicle assay was described, which enables the direct visualization of synaptic vesicle (SV) release. Activity-dependent bulk endocytosis (ADBE) enables the endocytosis of SV membrane and proteins in a fast manner during intense stimulation, resulting in bulk endosomes (also so-called large vesicles, LVs). Recycling proteins can be characterized by its site of action, whether they act at the plasma membrane (participating at the LV formation), or at the LV membrane (participating at the SV formation). ERP-1 (the C. elegans ortholog of Endophilin B) was recently identified as a possible SV recycling factor, its contribution to synaptic transmission has not been analysed before. During this project the function and possible cooperation of three proteins, ERP-1, UNC-57 (the C. elegans ortholog of Endophilin A) and CHC-1 (the C. elegans ortholog clathrin heavy chain) were studied, with a special emphasis of the site of action. It has been confirmed that these proteins participate together in synaptic vesicle recycling. Endophilins (ERP-1 and UNC-57) act both at the PM and the LV level, but while UNC-57 has been identified as the main player, ERP-1 rather has a minor role and acts as a back-up protein. CHC-1 functions the LV level in the first place, but it can compensate for the loss of UNC-57 and acts as a back-up protein at the PM. RIM-binding protein is an evolutionarily conserved active zone protein, which interacts directly with RIM and N, P/Q, as well as L-type Ca2+ channels. RIM-BP and RIM have redundant functions in different model organisms including C. elegans, however, while the loss of UNC-10 (the C. elegans ortholog of RIM) led to drastic behavioural defects, the loss of RIMB-1 (the C. elegans ortholog of RIM-BP) led only to mild phenotypes. During this work the synaptic function of RIMB-1 and its interaction with UNC-10 and UNC-2 (C. elegans ortholog of the CaV2 1 subunit) were extensively investigated. It has been shown that RIMB-1 contributes to the precise localization of VGCCs in cooperation with UNC-10. Furthermore, it has been demonstrated, that RIMB-1 plays different roles in cholinergic and GABAergic neurons, thus it contributes to maintain a proper excitation/inhibition balance. There are numerous available assays, which enable the indirect analysis of synaptic transmission, however, a tool, that enables the direct visualization of SV release, is highly desired. pOpsicle is a method which combines the optogenetic stimulation of cholinergic neurons with real-time visualization of SV release. A pH-sensitive fluorescence protein, pHuji, was inserted into the second intravesicular loop of the synaptic vesicle membrane protein, synaptogyrin (SNG-1). The fluorescence of pHuji is quenched inside the vesicles, but once they are released, the pH increases and pHuji can be detected. pOpsicle enables not only the direct visualization of SV exo-, and endocytosis events, but also the identification of putative SV recycling proteins.