Calcium is the critical trigger that fuses synaptic vesicles. So, the placement of calcium channels at the synapse can determine the site and probability of vesicle fusions. Importantly, cytosolic calcium is toxic to cells. The entry of calcum into and out of the cell must be carefully regulated to maintain homeostasis. Defects in synaptic calcium regulation underlie some neurodegenerative diseases, such as amyotrophic lateral sclerosis and Alzheimer's Disease. Calcium indicators have been used to study the flux of calcium in cell bodies, but synapses are beyond the resolving power of most standardized techniques. Classically, synaptic calcium was thought to enter the through a single type of channel that localized to the center of the synapse. Cells could increase the total amount of calcium by inserting more channels, or increasing the stimulus. However, our preliminary results indicate that several calcium channels coordinate to control distinct regions of the synapse in a multi-type calcium channel model. I propose to study the location of each type of calcium channel in the synapse, and determine its region of assigned synaptic vesicles relative to the dense projection at the center of the synapse. Different channel types may open only in response to specific types of stimulus, suggesting of an additional level of synaptic plasticity. While some channels may localize to the dense projection, it is predicted that other types will localize to peripheral regions of the synaptic active zone. I will test this model in the following three aims. Aim 1) Where are calcium channels in the synapse? I will use nanoscopic fluorescence correlated electron microscopy to identify the precise location of each calcium channel type involved in neurotransmission at the synapse. Aim 2) Which vesicles are assigned to a channel? I will use flash-and-freeze electron microscopy to stimulate synaptic vesicle fusions in calcium channel mutants only milliseconds before freezing. Aim 3) Which proteins localize specific calcium channels to a region? I will use a candidate gene approach and a forward genetic screen to identify specialized proteins involved in synaptic calcium channel placement.