VESDYN · Synaptic vesicle acidification and refilling dynamics

In newly endocytosed synaptic vesicles, a proton electrochemical gradient is produced by the vacuolar-type H+-ATPase in order to power transport processes across the membrane. The mechanisms responsible for vesicle refilling have been largely investigated on isolated synaptic vesicles. Biochemical investigation indicates that the preference of vesicle filling for each component of the electrochemical gradient, namely the membrane potential and the proton gradient, depends on the specific transport system for classical neurotransmitters. But, in the process of vesicular refilling, not only the pH gradient is of uttermost importance, also a chloride gradient plays a key role. For instance, chloride allows isolated synaptic vesicles to acidify by collapsing the membrane potential. In addition, it influences vesicular glutamate transport velocity and final content. The work on isolated vesicles has led to a solid concept of how vesicles are filled with neurotransmitters. Yet, the dynamics of vesicle acidification and refilling in neurotransmitter in intact preparations (i.e. neurons) is still largely unknown. Because vesicular transmitter content is now recognized to influence synaptic transmission, vesicular acidification and refilling dynamics need to be elucidated in physiological conditions. Using imaging techniques and new fluorescent probes, changes in proton and chloride gradient will be monitored simultaneously to study vesicular refilling dynamics in cultured hippocampal neurons, during spontaneous or frequency-dependent activity. I will test to which extent proton and chloride ions affect vesicular refilling dynamics and whether glutamatergic and GABAergic vesicles shared some common features or if vesicles possess some unique properties by virtue of the presence of selective transporters in their membranes. This project should provide new insights into the mechanisms of vesicular electrochemical gradient generation and neurotransmitter refilling in neurons.