Project Summary Plasticity is a fundamental aspect of neuronal circuits across all species. It is at the base of learning and memory, sensory adaption, and many disease-related processes such as addiction, chronic pain or regeneration. On the molecular level biochemical mechanisms have been well described, but little is known on how these are coordinated in space and time within neuronal circuits of living brains. To elucidate the circuit operation of plasticity in vivo we here propose to develop and validate highly sensitive, red FRET/FLIM sensors for simultaneous use with green calcium sensors, allowing imaging of neuronal calcium activity and biochemical signaling dynamics in individual synapses and neuronal populations. We will focus on plasticity-linked biochemical events involved in synaptic plasticity and spine morphogenesis. FRET/FLIM sensors will allow for the accurate measurement of small changes, even in the presence of brain movement during behavior. In aim 1, we will develop highly sensitive red-emitting FRET/FLIM sensors using mammalian cell based protein libraries and structurally-guided large scale screening. Aim 2 will involve validation of sensors ex vivo and in vivo. Finally, sensors will be validated for use in studies on synaptic and neuronal plasticity during spatial navigation tasks in awake mice. We plan for iterative cycles of improvements in which input from biophysical analysis of sensors, validation in slices and in vivo and feed-back from early roll-out users with different animal models will be used to create successive sensor generations with ever increasing performance. Combined in vivo 2P FRET/FLIM of plasticity and 2P fluorescence imaging of calcium activity will provide a powerful new approach to study synaptic and neuronal plasticity in living organisms.