Project Summary/Abstract Transmission of information through neural circuits required for cognition, learning, and motor function occurs via Ca[2+]-dependent release of neurotransmitters and their reception by postsynaptic neurons at specialized junctions termed synapses. Neurons commonly use a broad bandwidth of action potential frequencies to encode information, which together with a limited number of neurotransmitter release sites requires that synaptic release be highly tunable. Munc13-1 is a presynaptic protein essential for excitatory neurotransmitter release and a central determinant of release rates. Specifically, Munc13-1 transitions vesicles into a readily releasable state, and thus regulates enhancement or depression of synapses. Importantly, recent evidence suggests that the activation state of Munc13-1 is itself precisely regulated, perhaps by RIM proteins, which facilitate transition of Munc13-1 from an auto-inhibited homodimer to a vesicular priming active monomer. The objective of this proposal is to elucidate the molecular dynamics by which Munc13-1 is transitioned into its priming-active state in addition to determining the functional significance of Munc13-1[unreadable]s interaction with RIM for activation and priming. To address this we capitalize of a novel technique, TIRF/FRET that can monitor the temporal and spatial characteristics of Munc13-1 transitions from autoinhibitory homodimers to physiologically active monomers. Furthermore, using a combination of genetic manipulations, fluorescence imaging, and electrophysiological measurements we will delineate: 1) the temporal and spatial properties ofMunc13- 1 activation and 2) the molecular mechanism by which RIM enhances Munc13-1-mediated neurotransmitter release. From these studies we expect to develop an understanding of Munc13-1 activation and regulation for vesicle priming, as well as reveal the features of the Munc13-1/RIM interaction important for coupling Ca[2+] signaling and priming to enhance neurotransmitter release. Uncovering the dynamics of how Munc13-1 may impact presynaptic plasticity will provide insights into mechanisms by which synapses are modified to sustain complex information processing. The information sought is fundamentally important to human health, as Munc13-1 is central to neurotransmission &synaptic plasticity. In fact, many neurological diseases and age-related deficits in cognitive ability are tightly linked to deficits in synaptic transmission. Moreover, Munc13 mediated vesicle priming has been shown to regulate the amyloid precursor protein which gives rise to senile plaques composed of Beta-amyloid peptides characteristic of neural tissue in Alzheimer[unreadable]s disease patients. Thus, the importance of understanding Munc13 dynamics goes beyond basic science discovery and will serve future studies focused on identifying causes and drug treatments for dementia.