Project Summary Cell signaling pathways in the brain are an essential part of a complex system regulating the activity and coordination of neuronal networks. During learning and memory these neuronal networks can be modified through neuronal and synaptic plasticity processes in which information is stored in the synaptic network. Intracellular signaling pathways play critical roles in regulating neuronal excitability and synaptic strength, thereby comprising an important part of the cellular and molecular mechanisms underlying learning and memory. Disruptions in the proper regulation of synaptic plasticity are involved in a number of neurological and psychiatric disorders including autism, schizophrenia, and intellectual disability. Revealing the dynamic activities and interactions of different signaling pathways is therefore crucial for understanding the mechanisms controlling neuronal networks both in health and disease. However, direct interrogation of signaling pathway activity in live animals has been challenging due to a 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 rapid, sensitive in vivo imaging techniques to visualize dynamic activity of these signaling pathways in live animals during physiologically relevant sensory experience and learning. Most existing biosensors for signaling activities are based on fluorescence resonance energy transfer (FRET) and the use of two different fluorescent proteins, which limits their use for monitoring of multiple signaling pathways in parallel. In this proposal, new single-color fluorescent protein-based kinase biosensors with high sensitivity and optimized two-photon excitation properties will be developed for imaging signaling pathways involved in synaptic plasticity, especially PKA, CaMKII, ERK, and PKC. The ultimate goal of this research proposal is to establish the use of these new biosensors in the mouse brain and to monitor both rapid dynamics of signaling pathways on the order of seconds to minutes and the long- term stability of signaling pathways on the order of weeks to months using two-photon microscopy in awake behaving animals. This proposed project will be the first investigation of multiple neuronal activities beyond calcium and voltage changes in live animals. These studies will allow us to examine the regulation of kinase pathways in vivo and will help elucidate the complexity of signaling pathways during synaptic plasticity in the brain.