Final Report Summary - VESDYN (Synaptic vesicle acidification and refilling dynamics) Synapses in the central nervous system are highly specialized structures dedicated to fast chemical transfer of information from one neuron to another. Synaptic transmission is initiated when an action potential triggers synaptic vesicle fusion and thus neurotransmitter release from a presynaptic nerve terminal. Neurotransmitters are packaged into synaptic vesicles by specific transporters embedded in the vesicular membrane. The loading of neurotransmitters is potentially a rate-limiting step in neurotransmission when rapidly recycling synaptic vesicles are involved. The mechanisms of vesicular refilling have been widely studied using biochemistry applied to isolated synaptic vesicles. Nonetheless, the biophysical properties of vesicle recycling in physiological conditions are poorly understood. Thus, the aim of the VesDyn project was to approach this question in an experimental situation which approximates synaptic vesicle recycling in vivo. In intracellular compartments and notably synaptic vesicles, a proton electrochemical gradient is produced by the vacuolar-type H+-ATPase (v-ATPase) in order to power transport processes across the membrane. The proton electrochemical gradient consists of an electrical and a chemical component, namely the membrane potential and the pH gradient, the ratio of which influences differentially the vesicular uptake for different neurotransmitters. For vesicular refilling, however, not only protons are essential but also chloride ions regulate transmitter transport through modulation of the proton electrochemical gradient and of vesicular transporter activity. Yet, we still don’t know how the vesicular neurotransmitter concentration is regulated in order to modulate synaptic transmission. In our project, we investigated the mechanisms of synaptic vesicle reacidification and refilling in live cultured hippocampal neurons using pH-sensitive fluorescent reporters coupled to synaptic vesicle proteins. The main excitatory neurotransmitter is glutamate. First, pharmacological inhibition of vesicular glutamate transporters (VGLUTs) unmasked an unexpected channel-like stoichiometrically uncoupled chloride conductance. During vesicular recycling, chloride present in the extracellular medium is engulfed inside synaptic vesicles. The chloride efflux turns out to be mostly responsible for generation of the membrane potential and in the first place enables glutamate loading up to an isosmotic concentration of about 1800 molecules per synaptic vesicle. Experiments on neurons lacking VGLUT1 indicated that the chloride conductance is bestowed by VGLUT itself. Second, chloride substitution experiments revealed that glutamate loading is coupled to a proton antiport with a stoichiometry close or equal to one, shunting away the massive charge build-up by the huge proton influx (200 s-1) through a single V-ATPase. Third, the acidification kinetics measured under physiological conditions directly reflect the glutamate loading kinetics, which implies an initial glutamate-proton exchange rate of about 300 molecules per second, i.e. 30 per second per VGLUT assuming ten transporters per vesicle. Finally, while the transport of glutamate relies primarily on the membrane potential, the transport of the inhibitory transmitter GABA relies more equally on both components of the proton electrochemical gradient. Accordingly, we found that chloride engulfed from the extracellular space doesn’t modulate significantly the recycling dynamics of GABAergic synaptic vesicles.Overall, our study highlights a tight coupling between chloride engulfed in recycling synaptic vesicles and proton gradient generation that together regulate replenishment of vesicles with glutamate. In addition, our results obtained under physiological conditions enable us to infer some basic properties of VGLUT which functions as a glutamate-proton exchanger with a chloride channel activity. The importance of vesicular loading is demonstrated by the lethal consequences of inactivating vesicular transporters. The significance of our study is reinforced by the interaction of psychostimulants and certain environmental toxic substances with vesicular uptake. Therefore, elucidating properties of vesicular refilling in conditions that approximate those found in vivo will help shedding light onto the influence of neurotransmitter loading on synaptic transmission and on the effect of psychostimulants or toxic substances on function of the nervous system. In conclusion, our approach considers basic questions related to synaptic vesicle cycle. It may well be the first tangible contribution to understand how quantal size is controlled under physiological conditions and how it contributes to synaptic plasticity. Therefore, it is our hope that the successfully completed project attracts considerable interest among other specialists of this and related fields and that the new knowledge stimulates further research towards understanding the impact of synaptic vesicle refilling modulation on the function of integrated systems.