LOCALIZATION OF ENDOCYTOSIS- AND EXOCYTOSIS-RELATED MOLECULES IN MEDIAL HABENULA TERMINALS USING FLASH AND FREEZE-FRACTURE REPLICA LABELLING

Neurons require tight regulation in presynaptic terminals for precise release of neurotransmitters. Presynaptic regulatory molecules play a critical role in ensuring proper exocytosis and endocytosis of synaptic vesicles (SV) at these terminals. Exocytosis-related proteins have been extensively studied, however, endocytosis-related proteins are understudied. Historically, endocytosis of SVs have been classified into clathrin-mediated endocytosis (CME), which involves clathrin, a cargo protein coating SVs for retrieval, and clathrin independent endocytosis. However, recent studies question the role of CME in SV recycling at the terminal and suggest different endocytosis-related molecules for the sustained SV release. Therefore, we want to find out how different presynaptic regulatory molecules are localized at the terminal during the time course of exocytosis and endocytosis. For this aim, we use Flash and freeze-fracture replica labelling in medial habenula (MHb) terminals projecting to the interpeduncular nucleus (IPN). We optogenetically stimulate MHb terminals in acute slices, high-pressure freeze, and fracture the frozen slices to produce replicas for immunolabeling to observe the dynamics of these proteins at 5 ms to 10 sec after the light stimulation with nano-scale precision. The MHb-IPN pathway in the mid-brain region offers a myriad of enigmatic presynaptic characteristics including presynaptic localization of R-type calcium channels and GABA_B receptors (GBR), mediating a switch from tonic release mode to phasic release mode under physiological stimulation frequency. Hence, understanding the presynaptic regulation of endocytosis and exocytosis in this region seems riveting. The freeze-fracture replica labelling helps us identify various molecules involved in exocytosis and endocytosis in the MHb terminals.