In the central nervous system, most excitatory synapses terminate on dendritic spines, tiny (~0.1 femtoliter) protrusions emanating from the dendritic surface. Calcium influx into spines activates signaling networks composed of tens of species of molecules to induce synaptic plasticity and other adaptive reactions. Although many of the molecules required for synaptic plasticity have been identified, the signaling mechanisms by which calcium dynamics in spines are decoded and translated into specific cellular responses are unknown. To further our understanding of signal transduction in spines, we have developed a technique to measure protein-protein interactions in individual spines using fluorescence resonance energy transfer (FRET). By combining fluorescence lifetime imaging with 2-photon laser scanning microscopy, we can achieve robust FRET detection with single- synapse resolution deep in brain slices. Using this technique, we have imaged the activation of the GTPase protein Ras in response to calcium influx into spines. Ras is an essential component of the signaling pathways that lead to rapid synaptic potentiation , the, formation of new synapses, and the regulation of dendritic excitability. When 2- photon glutamate uncaging is used to stimulate a single spine, Ras activation occurs initially at the stimulated spine, and subsequently spreads more than 10um into the parent dendrite and nearby spines. The same stimulation induces both structural and functional plasticity in the stimulated spine, as indicated by a long-term (~60 min) spine enlargement associated with a potentiation of postsynaptic glutamate receptor current. To understand how Ras activation spreads and the role it may play in synaptic plasticity, we are now developing techniques to image molecules upstream and downstream of Ras.