STRUCTURAL AND MOLECULAR DYNAMICS OF SYNAPTIC VESICLE EXO- AND ENDOCYTOSIS AT HIPPOCAMPAL CA3 TO CA1 SYNAPSES

When neurons communicate through synaptic connections in the brain, synaptic vesicle (SV) exocytosis occurs within milliseconds of a stimulus, presenting a major challenge for studying activity-dependent molecular and structural changes during SV release. In our “Flash and Freeze-fracture” technique, we combine optogenetic stimulation of neurons in acutely cut brain slices with timed high-pressure freezing, followed by freeze-fracture replica immunolabeling and transmission electron microscopy. In this ongoing project, we first identified structural correlates of SV fusion sites (fusion pores) and endocytic events at CA3-CA1 synapses in acute brain slices of adult Grik4-Cre/Ai32 mice. We present preliminary results on the formation of SV fusion pores and endocytic pits at distinct time points after optogenetic stimulation. Specifically, we found an increased frequency of SV fusion pores 5 – 8 ms after optogenetic stimulation compared to earlier and later time points. In contrast, presynaptic endocytic pits were measured 0.1 – 10s post optogenetic stimulation. For molecular analysis, we performed immunolabeling of Flash and Freeze-fracture replicas to detect the location of presynaptic calcium channels (Cav2.1), SV-associated proteins (vGluT1), active zone proteins (RIM, CAST and Neurexin) and endocytosis markers (clathrin, endophilins, dynamins, syndapin1). In addition, we present preliminary results on the positions of endo- and exocytosis pores relative to the active zone proteins. Our preliminary results provide insights into molecular and structural dynamics of SV exo- and endocytosis during evoked neurotransmission.