Phenylazopyridines are photoisomerizable compounds with high potential to control biological functions with light. We have obtained a series of phenylazopyridines with light dependent activity as negative allosteric modulators (NAM) of metabotropic glutamate receptor subtype 5 (mGlu₅). Here we describe the factors needed to achieve an operational molecular photoisomerization and its effective translation into in vitro and in vivo receptor photoswitching, which includes zebrafish larva motility and the regulation of the antinociceptive effects in mice. The combination of light and some specific phenylazopyridine ligands displays atypical pharmacological profiles, including light-dependent receptor overactivation, which can be observed both in vitro and in vivo. Remarkably, the localized administration of light and a photoswitchable compound in the peripheral tissues of rodents or in the brain amygdalae results in an illumination-dependent analgesic effect. The results reveal a robust translation of the phenylazopyridine photoisomerization to a precise photoregulation of biological activity.

Significance: Two-photon fluorescent probes to image intracellular calcium and other biochemical processes have revolutionized cell biology. In neurobiology, 2-photon calcium indicators produce signals that indirectly reflect action potential firing in neurons deep in the brain, and have become essential to record and correlate large-scale neural activity at high spatiotemporal resolution. Here we show that 2-photon excitable, diffusible photoswitches allow the activation and deactivation of neurotransmitter receptors with higher subtype-specificity and spatial resolution compared with previous molecular tools, and combined with calcium indicators enables to both control and readout activity using 2-photon excitation. Specifically, we used a potent and selective antagonist to switch on/off at the micrometer scale the activity of mGlu ₅ receptors expressed endogenously in neurons and astrocytes of intact brain tissue.