PROJECT SUMMARY The reliability and precision of synaptic transmission are required in circuits of the auditory brainstem in order to encode timing with submillisecond accuracy. Auditory information is encoded by action potentials phase-locked to sound frequency at high rates. Accordingly, synaptic vesicles need to be recycled and refilled rapidly. Accumulating studies have uncovered the processes of vesicle fusion and recycling; however, the control of the contents of synaptic vesicles has received considerably less attention. Reasons for this gap in our understanding include the small size of synaptic vesicles and of conventional synapses, the complex ionic basis for loading of neurotransmitter into vesicles, and the difficulty in manipulating and assessing vesicle loading in physiological conditions. We have recently found that a Na+/H+ exchanger expressed on synaptic vesicles promotes vesicle filling with glutamate. Using the calyx of Held, a giant glutamatergic synapse in the auditory brainstem that permits direct pre- and postsynaptic recordings and manipulation of the presynaptic cytosol, we showed that glutamate loading is facilitated by intracellular Na+ over the physiological concentration range. Na+ influx through presynaptic plasma membrane HCN channels affects presynaptic Na+ concentration, regulates glutamate uptake, and thus controls miniature excitatory postsynaptic currents. Here we propose that during high-frequency signaling, when large amounts of glutamate are released, Na+ accumulates in terminals and facilitates glutamate uptake into synaptic vesicle, accelerating vesicle replenishment and sustaining reliable synaptic transmission. We further hypothesize that the control of vesicle loading, release and recycling can be affected by hearing loss. This work will establish a new fundamental role of Na+ to link activity and synaptic function under physiological and pathological conditions.