The crayfish claw opener neuromuscular junction (NMJ) is a biological model for studying presynaptic neuromodulation by serotonin and synaptic vesicle recycling. Serotonin acts on crayfish axon terminals to increase the release of the neurotransmitter glutamate, but a complete understanding of its mechanisms of action are unknown. In order to sustain enhanced neurotransmission over long periods of time, it was hypothesized that serotonin recruits (activates) a population of previously non-recycling vesicles to become releasable and contribute to neurotransmission. To determine if serotonin activates a distinct population of synaptic vesicles, FM1-43 fluorescence unloading experiments were performed on crayfish excitatory opener axon terminals. These experiments could not resolve a serotonin-activated population of synaptic vesicles, but instead revealed that synaptic vesicles change behaviour in axon terminals independent of serotonin, with vesicles becoming less likely to exocytose and unload FM1-43 dye over time. The change in behaviour was hypothesized to be due to conversion of vesicles from a recycling (releasable) status to a reserve (reluctant to release) status. Synaptic vesicle pool localization was then tested using photoconversion of FM1-43 and transmission electron microscopy techniques. The spatial location of FM1-43-labeled vesicles fixed 2 minutes following 20 Hz stimulation did not reveal retention of vesicles specifically near release sites and the distribution of FM1-43-labeled vesicles was not significantly different between early (2 min) and late (180 min) time points. Terminals fixed 30 seconds following stimulation, however, contained numerous endosome-like structures - the most frequently observed structures resembled large vesicles, which were the equivalent of 2-5 regular vesicle sizes. These results suggest that crayfish axon terminals recycle vast amounts of membrane in response to sustained 20-Hz stimulation and endocytosis appears to occur via multiple routes with the most common being through large vesicle intermediates. / Graduate

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