Project Summary Pharmacological probes are widely used to study the nervous system. Despite often exhibiting exquisite specificity for target receptors, due to diffusion, traditional small molecule drugs act slowly and with spatial imprecision. This impedes neuropharmacological studies in vivo, particularly those involving high-resolution electrophysiological, imaging, and behavioral tracking methods. Such studies greatly benefit from the ability to correlate measurements with well-defined, time-locked stimuli that can be readily varied in intensity and duration. To meet this need, we are developing “caged” drugs that can be applied systemically in an inactive form and subsequently released in the brain with high spatial and temporal precision using short light flashes. To enable compatibility with optical measurements of neural function involving fluorescent probes, we will develop new photochemical protecting groups that are wavelength tuned to be spectrally orthogonal to common green and red fluorophores, in terms of both one-photon and two-photon excitation. We will further optimize these “caging” groups to facilitate the brain penetrance of the resulting caged drugs. We will evaluate the utility of new caging groups by incorporating them into 2nd generation caged opioid drugs that should provide significant experimental advantages over our 1st generation variants, which could only be photoactivated with ultraviolet light. We will rigorously validate new caged opioid drugs using in vitro, ex vivo, and in vivo experimental paradigms, culminating in behavioral assays and fiber photometry recordings of opioid-evoked neurochemical signaling. In addition, we will develop caged ketamine derivatives that can be used to study the neural mechanisms underlying dissociative states, as well as ketamine’s rapid antidepressant actions. Caged ketamine variants will also be evaluated using in vitro, ex vivo, and in vivo experimental paradigms, including measurements of spinogenesis and neural activity in the prefrontal cortex. These efforts will involve the development of an optical configuration for simultaneous two-photon imaging and one photon photolysis through implanted prisms. To maximize end-user uptake, performance criteria for both caged drug families are determined through extensive consultation with the scientific community.