Project Summary Cell signaling pathways in the brain are an essential part of a complex system regulating the activity and coordination of neuronal circuits. During learning and memory synaptic plasticity processes regulate the strength of synaptic connections and modify neuronal circuits. Intracellular signaling pathways play critical roles in regulating synaptic strength and are an important part of the molecular mechanisms underlying learning and memory. Kinase signaling pathways play a central role integrating neuromodulatory and excitatory and inhibitory synaptic inputs to control how neurons and neuronal circuits adapt during behavior. However, direct interrogation of signaling pathways in live awake animals has been challenging due to lack of appropriate tools. Monitoring of multiple signaling pathways in such a setting has not been achieved. The goal of this research proposal is to develop novel tools to simultaneously monitor the activity of several signaling pathways and to use in vivo imaging techniques to visualize cell-specific and circuit-specific activity of these dynamic signaling pathways in live animals during physiologically relevant sensory experience and learning. In initial studies the use of genetically encoded biosensors will be used to monitor signaling pathways in vivo using two-photon microscopy to image an existing biosensor in the somatosensory cortex of mice. New single- color fluorescent protein based biosensors with a greater dynamic range than existing FRET-based sensors will be developed for imaging signaling pathways (PKA, CaMKII, ERK, and mTOR complex 1) involved in synaptic plasticity. The ultimate goal of this research proposal is to introduce these new biosensors into the mouse brain and to monitor both rapid dynamics of signaling pathways on the order of seconds to minutes and long-term stability of signaling pathways on the order of weeks to months using two-photon microscopy in awake behaving animals. This proposed project would be the first investigation of cell-specific and circuit- specific neuronal signaling beyond calcium and voltage changes in live animals. These studies will allow us to uncover mechanisms underlying the regulation of signaling pathways and will shed light on the complexity of signaling pathways during synaptic plasticity in vivo.