Optical interrogation of neuropeptide dynamics

Neuropeptides are signaling molecules in the brain that regulate a wide range of biological and behavioral processes through interactions with G-protein coupled receptors (GPCRs). Although such neuropeptide-GPCR interactions are highly implicated in neuropsychiatric disorders, their mechanisms of action remain poorly understood due to limited technology for high-resolution study in vivo. To address this, we present a high-throughput optical screening platform that allows for rapid design and optimization of genetically encoded neuropeptide biosensors. Traditional biosensor optimization is labor-intensive, involving placing a single variant in a well and phenotyping each well individually. In contrast, we leverage STARmap, an intact-tissue spatial transcriptomics method, to screen large libraries of biosensor variants through simultaneous phenotyping and genotyping. This enables the design of biosensors for multiple neuropeptides including beta-endorphins, vasopressin, and histamine, assessing their fluorescence responses in vivo to reveal dose-dependent signal upregulation. Cell clustering by genotype indicates diverse responses, allowing us to select the most robust variants for further characterization and optimization. Our platform is fully functional and scalable high-throughput sensor development. The successful design and optimization of GPCR-based biosensors demonstrates robust fluorescence responses to neuropeptides that will be applied to in vivo whole-brain imaging. This work not only provides an adaptable platform for high-throughput biosensor engineering, but also establishes a foundation to map neuropeptide dynamics across brain structures and behaviors, allowing insights into the molecular basis of neuropsychiatric disorders.