Following exocytosis of neurotransmitter, vesicular retrieval is essential to support continued transmission and maintainence of nerve terminal integrity. The goal of this proposal is to clarify features of synaptic transmission and vesicular recycling at single synapses between hippocampal neurons in culture. We will consider the classical picture of exo/endocytosis, wherein vesicles fuse completely into the plasmalemma, instantaneously release their contents, are slowly retrieved by clathrin-coated pits and slow recycle through endosomes. In addition, we will also look beyond these events, to examine critically the set of possible phenomena often called """"""""kiss and run."""""""" These include fusion pores that slowly dilate or quickly close, exocytotic events that preserve vesicular integrity, retrieval of vesicles within 1 s or less, and rapid reuse of the recaptured vesicles for multiple rounds of transmitter release. Photoconversion of styryl dyes (FM series) will cause selective staining of individual vesicles that have undergone exocytosis and retrieval, and will establish a morphological basis for various vesicle pools at the electron-microscopic level. Vesicles that have undergone fast vesicular retrieval will be specifically photoconverted to help clarify the cell biology of this incompletely understood process. Kinetic properties of fusion pore closure over periods ranging down from seconds to milliseconds will be studied with FM dyes of varying departitioning rates and by application of a novel method wherein fluorescence of an indicator (VAMPGFP) attached to the vesicle lumenal face is extinguished by entry of a small molecule quencher. Experiments will test the idea that rapid retrieval of vesicles in the readily releasable pool is followed by reutilization of the same vesicles for repeated neurotransmission. The role of fusion pore dynamics in regulation of quantal synaptic transmission will be tested to see if restricted openings (and possibly, rapid closure) contribute to modulation of exocytotic release and variations in cleft transmitter concentration that have been observed during paired pulse modulation and long-term potentiation. The proposed morphological and kinetic studies will provide new perspectives on fusion pore properties, vesicular recapture and reuse. Modulation of these processes may be critical for certain forms of synaptic plasticity, important for CNS function in both health and disease.
