PROJECT SUMMARY Neuromodulation, such as that mediated by the neuromodulators norepinephrine, acetylcholine, and dopamine, imposes powerful control over brain function. It regulates the excitability, synaptic plasticity, and other aspects of neuronal function. Defects in neuromodulation are associated with many neuropsychiatric diseases. Neuromodulators exert their functions by regulating intracellular signaling events via their corresponding G protein-coupled receptors (GPCRs). Although in vivo interrogation of extracellular neuromodulators has started to become possible, the effects of neuromodulators on subcellular signaling and neuronal function are cell type-specific. Monitoring the cell type-specific outcomes of neuromodulatory subcellular signaling events remains difficult. There is also a lack of practical tools for antagonizing neuromodulatory signaling events with high temporal resolution in vivo, which is required for establishing the causal relationship between the signaling events and neuronal functions or animal behavior. To overcome these problems, we propose to develop novel genetically encoded sensors for examining the activities, in vivo with single-neuron resolution, of an understudied neuromodulatory signaling pathway: the protein kinase C (PKC) pathway. Although prototypic genetically encoded PKC sensors based on Förster resonance energy transfer (FRET) have been used for experiments in vitro, their application in vivo has been difficult due to lower signal-to-noise ratios under the more challenging in vivo imaging conditions. Building on our previous successful experience in developing sensors for the cAMP and protein kinase A (PKA) pathway for in vivo imaging, we will employ a multi-pronged approach to characterize and improve PKC sensors for in vivo imaging. In addition, we will develop novel genetically encoded actuators for both the PKA and PKC pathways that are effective only when they are stimulated by blue light. We will validate the utility of these tools for monitoring or manipulating neuromodulatory activities in awake mice during behavior. The successful tools will be packaged into viral vectors for their easy introduction in vivo, and will be disseminated to the research community. If successful, our efforts will provide the research community with a previously unattainable ability to conduct large-scale monitoring and manipulation of neuromodulatory signaling activities in the brain at the cellular and circuit levels. This ability to quantify and manipulate neuromodulatory signaling will complement the measurements of extracellular neuromodulators and neuronal electric activities to enhance our understanding of brain function underlying animal behavior.

PROJECT NARRATIVE Animal behaviors are regulated by neuromodulation, and defects in neuromodulation are associated with many neuropsychiatric diseases. We aim to develop novel sensors and tools for detecting and manipulating, respectively, the subcellular signaling activities induced by neuromodulators in their native habitat – the intact brains of living animals. These tools will enable future dissection of the neuromodulatory mechanisms underlying brain function and dysfunction.