Synaptic vesicle recycling is essential to sustain neurotransmission during activity in central synapses. However, it is yet uncertain if the only function of synaptic vesicle trafficking is to maintain fusion competence of synaptic vesicles and structural homeostasis of synapses in the long term. Or can it, in addition, modulate frequency dependence of synaptic responses during short-term synaptic plasticity? Clathrin-mediated endocytosis comprises a ubiquitous means for vesicle recycling in most cell types. This pathway typically possesses well-defined morphological markers (coated pits, endosomal intermediates etc.) and adequate molecular tools are available to probe its properties in synapses. A rapid vesicle-recycling pathway, in contrast, may not employ the same molecular players and structural intermediates and is therefore harder to examine morphologically and molecularly. Therefore, most evidence in support of a fast retrieval and recycling mechanism for synaptic vesicles relies on electrophysiological and optical techniques with rapid time resolution. Our results from experiments using a combination of fluorescence imaging of synaptic vesicle trafficking and electrophysiological detection of short-term synaptic plasticity collectively suggest that a rapid form of synaptic vesicle recycling strongly contributes to neurotransmission in a frequency-dependent manner in hippocampal synapses. These findings can be outlined as follows: 1. 10-50% of styryl dye (FM1-43 or FM2-10) taken up during a bout of intense stimulation (e.g. 10 Hz for 10 s or 30 Hz for 3 s) can be rapidly re-available for exocytosis within 10 seconds after dye uptake. In addition, up to 50% of FM dye taken up during an exhaustive stimulation (30 Hz or 90 mM K+) can be re-released within 15 seconds. 2. Impairment of neurotransmitter refilling into endocytosed synaptic vesicles by inhibiting their re-acidification enables an electrophysiological means to test the role of vesicle reuse in synaptic depression. After inhibition of neurotransmitter refilling, synapses onto hippocampal CA1 pyramidal cells show faster depression with increasing stimulation frequencies. At 20 Hz, compromising neurotransmitter refilling increases depression within 300 ms reaching completion within 2 seconds. In contrast, at 1 Hz, inhibition of neurotransmitter refilling has a delayed effect on depression, which emerges gradually and becomes significant within 100 seconds. These observations suggest that rapid re-availability of endocytosed vesicles for fusion can slow the kinetics of short-term synaptic depression. 3. To image the effect of inhibiting vesicle re-acidification at the level of individual hippocampal synapses, we infected hippocampal cultures with lentivirus expressing synaptic vesicle protein synaptophysin tagged with superecliptic-pHluorin. Superecliptic-pHluorin is a GFP-based pH sensor that is normally quenched at pH 5.5 within the vesicle lumen but fluoresces once vesicles fuse with the plasma membrane and the fluorophore is exposed to extracellular pH (7.4). In response to stimulation at 20 Hz for 2 seconds, synaptophysin-pHluorin fluorescence shows a swift increase and a gradual slow return to baseline in agreement with earlier observations. After incubation with an inhibitor of vesicle re-acidification, however, in response to the same stimulation protocol, synaptophysin-pHluorin fluorescence shows a larger increase detectable within the first 500 ms consistent with rapid internalization of synaptophysin-pHluorin and re-acidification of synaptic vesicle interior in the absence of the inhibitor. In addition, the same experiments outlined above also support the presence of a slow synaptic vesicle trafficking pathway that operates over minutes (e.g. slow decay of synaptophysin-pHluorin fluorescence after stimulation). Therefore, our current efforts are focused on examining the signaling mechanisms that potentially target vesicles to one of the two available pathways for synaptic vesicle recycling. Our initial results point to a strong influence of chronic activity in addition to acute frequency of presynaptic stimulation in determining the pathway through which synaptic vesicles can be recycled. Synaptic activity can influence signal transduction in nerve terminals in several ways, and therefore we are currently examining the roles of candidate mechanisms that may impact synaptic vesicle trafficking and alter resulting dynamics of neurotransmitter release.