Project Summary: Project 4 - Novel Genetically Encoded Indicators for Interrogating Neuron-Astrocyte Communication Across Timescales Astrocytes, the most abundant cell type in the brain, have long thought to be primarily passive support cells. Considerable evidence from the labs has shown that astrocyte-synapse displays a dynamic and bi-directional relationship, with local synaptic transmission and neuromodulation being capable of shaping astrocytic activity and PAP structural plasticity, and astrocyte shaping synapse formation and modulating plasticity and signaling via secreted factors and adhesion molecules. These critical advances in understanding astrocyte biology in vivo are primarily due to recent applications of modern techniques initially designed for studying neurons to direct manipulation and interrogation of astrocytes. Though the concept of astrocytes as integral and modulatory components of neural circuit is emerging, a mechanistic understanding of causative and correlative roles of astrocytes in operating neural circuit and contribution to the complex behaviors is still lacking, which necessities and drives the development of improved tools. Thus, a large-scale protein engineering effort to develop an improved tool to address unsolved questions to achieve a mechanistic understanding of causal and correlative roles of astrocyte in neuronal circuit function and contributions to behavior is being proposed. Provided items include: 1. a set of optimized red-shifted glutamate, GABA, DA, and NE sensors, 2. a set of green and red-shifted synaptic glutamate/GABA sensors to probe neuron-astrocyte connectivity and extracellular NT transients at tripartite synapses, and 3. interrogate cross-talk between PKA and calcium in astrocytes and optimized green and red-shifted kinases sensors for in vivo applications. These new sensors will be applied to study 1) how experience-dependent changes that drive complex patterns of neurotransmitter or neuromodulatory signaling lead to the changes in astrocytic activity and 2) how astrocytes modulate synaptic activity via structural plasticity across various temporal scales. The contribution is significant because these improved tools will permit new hypotheses being tested in astrocyte biology. This toolset will provide needed tools to facilitate experiments proposed here and provide a rich resource to the field to bring full swing the investigation of astrocyte-neuron interaction underlying complex behavioral and cognitive processes that are inaccessible via currently existing approaches.