Optimizing cAMP and PKA sensors for cell-specific dissection of neuronal circuit in vivo

Neuromodulation imposes powerful control over brain function by dynamically adjusting the gain and plasticity of brain circuits. Defects in neuromodulation are associated with many neurodegenerative and neuropsychiatric diseases. It is increasingly appreciated that an in-depth understanding of neuromodulation would involve visualizing these events during animal behavior. Currently, in vivo interrogation of extracellular neuromodulator releases has become possible thanks to the development of neuromodulator sensors. However, neuromodulators exert their functions by regulating intracellular signaling events, such as those mediated by cAMP and protein kinase A (PKA), in a cell-specific manner: the same neuromodulator may trigger distinct signaling events via different receptors in different cell types. Cell-specific monitoring of neuromodulatory signaling is needed. To date, cAMP and PKA sensors have been developed and started to allow initial in vivo imaging applications. However, the signal remains low, hampering the simultaneous measurements of a large number of cells or the visualization events confined in subcellular compartments. Here, we propose to optimize a FRET-based multi-fluorophore sensor for PKA activity and a single-fluorophore sensor for cAMP that provide complementary information. We will prioritize developing sensors compatible with fluorescence lifetime imaging, which is increasingly recognized to provide unparalleled advantages for imaging signaling events in vivo. We will employ structure-guided molecular dynamic analyses to identify critical positions within each sensor and use a modern high-throughput platform to screen for effective variants. We will benchmark the new variants against previous sensors. Leading candidates will be systematically characterized, benchmarked against current best sensors, and validated in vitro and in vivo. This proposal harnesses the complementary expertise from individual team members, including experimental structural biology, molecular dynamics, sensor development and characterization, and in vivo two-photon lifetime imaging. Successful tools will be used to generate adeno-associated viruses (AAVs) and other cell type-specific expression vehicles for easy use and dissemination to the broader research community. If successful, our proposed study will propel the ability to visualize cAMP/PKA signaling activities in vivo with previously unattained spatial and temporal resolution for mechanistic studies of brain function and dysfunction. This ability to monitor neuromodulatory signaling will complement the measurements of extracellular neuromodulators and neuronal electric activities to enhance our understanding of brain function underlying animal behavior. Project Number: 1UM1MH138931-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Mental Health (NIMH) | Principal Investigator: Haining Zhong (+4 co-PIs) | Institution: OREGON HEALTH & SCIENCE UNIVERSITY, PORTLAND, OR | Award Amount: $2,039,804 | Activity Code: UM1 | Study Section: Special Emphasis Panel[ZMH1 ERB-S (04)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11223110