Exploring Synaptic Vesicle Exocytosis

The strength of a synapse is an important variable that will affect the function of neural circuits. This thesis develops optical techniques to study determinants of presynaptic efficiency. Our methods are largely based on a chimera of a pH-sensitive variant of the green fluorescent protein and the vesicular glutamate transporter. This reporter is incorporated into synaptic vesicles and increases its fluorescence when those vesicles fuse with the presynaptic membrane. Using this reporter, we measure the size of the primed pool of vesicles (n), a privileged subset of synaptic vesicles which are docked at the active zone and can immediately fuse with the membrane, thereby releasing neurotransmitter in response to an action potential. We also estimate the probability that a vesicle within that pool will fuse upon arrival of a stimulus to the presynaptic terminal (Pv). Our studies show that at each bouton in a cultured rat hippocampal neuron there are four primed vesicles per synapses, each of which has a 10% chance of fusing with the membrane in response to a stimulus. These values only represent averages; both Pv and n vary widely between neurons and between synapses made by those neurons. Tomosyn, a molecule intimately involved with the fusion machinery of synaptic vesicles, exercises a negative control on exocytosis. Reducing the levels of this protein, without eliminating it completely from a synapse, leads to increases in Pv, but does not affect n. In addition to measuring synaptic properties of individual neurons, our methods can be used to study Pv and n at the level of single synapses. There is considerable variability among synapses in these properties, even between synapses formed by the same axon. This variability is not correlated with the fraction of P/Q or N-type Ca2+ channel subtypes present at each presynaptic terminal. Furthermore, it cannot be explained by the variance in distances between these channels and primed vesicles. Synapses differ not only in their basic properties, but also in the degree to which their strength can be modulated. In particular, activation of the cAMP pathway leads synapses with lower Pv to potentiate more than synapses with high Pv. In conclusion, the methods developed herein represent a powerful approach to study the molecular determinants of synaptic vesicle exocytosis at the level of individual synapses