The small GTPase protein Ras is important for many neuronal processes essential to the regulation of synaptic connections such as strengthening of synaptic transmission, formation of new synapses and regulation of cell excitability. Ras is also important for protein synthesis and gene transcription required for long-term maintenance of synaptic plasticity. Consistent with essential roles of Ras signaling in synaptic plasticity, failures in Ras signaling are associated with diseases causing cognitive impairments and learning deficits such as autism, X-linked mental retardation and neurofibromatosis 1. Although the importance of Ras signaling in synaptic plasticity is well recognized, it is not clear how Ras decodes and relays calcium dynamics to regulate its diverse downstream effects. In neurons, Ras signaling is involved in signaling events spanning different compartments, including spines, dendrites and the nucleus. Thus, the spatiotemporal dynamics of Ras signaling are likely to be important in determining its downstream effects. To study Ras signaling mechanism in neurons, we have recently developed a fluorescence technique that allows us to image Ras activity with single synapse resolution in living neurons deep in brain tissue, using this technique, the objective of this proposal is to understand the mechanisms of spatiotemporal regulation of Ras signaling. Our hypothesis is that the spatiotemporal pattern of Ras signaling is shaped by 1) Ras activation controlled by calcium-dependent signaling networks involving multiple kinases and feedback loops, and 2) spatial spreading of Ras activation due to the diffusion and trafficking of Ras and Ras regulators. To test this hypothesis, we will image Ras activity in spines and dendrites in response to activation of glutamate receptors on a single spine using 2-photon glutamate uncaging. Our preliminary data suggested that Ras activation occurs at the stimulated spine, subsequently spreading into its parent dendrite and nearby spines. The specific aims of this proposal are to 1) identify upstream signaling that activates Ras in individual spines, 2) determine the mechanisms and roles of the spatial regulation of Ras in dendrites, and 3) elucidate mechanisms underlying differential activation of the Ras GTPase family. This work will advance our understanding of how Ras couples calcium with synaptic plasticity, and ultimately with learning and memory. Moreover, our study will provide insights into the molecular mechanisms underlying Ras-related mental disorders.