Localized covalent targeting of NMDA receptors reprograms behavior

Spatiotemporal control of neuronal activity has largely relied on genetically defined cell populations, limiting the ability to interrogate native biomolecules within intact circuits. Pharmacological approaches with high spatial precision are therefore needed to preserve physiological network architecture. Here, we demonstrate confined neuromodulation through two-photon molecular tattooing (sequential localized photocrosslinking). We introduce AL-701, an aryl azide derivatized photoreactive NMDA receptor antagonist that covalently binds receptors upon two-photon laser irradiation. In zebrafish larvae, molecular tattooing enabled spatially restricted inhibition of NMDA receptors in a hindbrain subregion regulating the acoustic startle reflex. This localized inhibition led to persistent suppression of NMDA receptor-dependent habituation, demonstrating that regionally restricted modulation of receptor function can alter behavior. Our results show the proof-of-concept for localized photocrosslinking within cell clusters in a vertebrate nervous system and thereby establish molecular tattooing as a robust chemical biology tool for circuit modulation and optically controlled reprogramming of behavior in vivo.