Three‐Photon Infrared Stimulation of Endogenous Neuroreceptors in Vivo

To interrogate neural circuits and crack their codes, in vivo brain activity imaging must be combined with spatiotemporally precise stimulation in three dimensions using genetic or pharmacological specificity. This challenge requires deep penetration and focusing as provided by infrared light and multiphoton excitation, and has promoted two‐photon photopharmacology and optogenetics. However, three‐photon brain stimulation in vivo remains to be demonstrated. We report the regulation of neuronal activity in zebrafish larvae by three‐photon excitation of a photoswitchable muscarinic agonist at 50 pM, a billion‐fold lower concentration than used for uncaging, and with mid‐infrared light of 1560 nm, the longest reported photoswitch wavelength. Robust, physiologically relevant photoresponses allow modulating brain activity in wild‐type animals with spatiotemporal and pharmacological precision. Computational calculations predict that azobenzene‐based ligands have high three‐photon absorption cross‐section and can be used directly with pulsed infrared light. The expansion of three‐photon pharmacology will deeply impact basic neurobiology and neuromodulation phototherapies.