An Off–On Fluorescent Probe Reveals Spatiotemporal Signaling of Opioid Receptors In Vivo for Pain Control

Interrogation of the function of neuronal receptors and how they are involved in disease intervention requires spatiotemporally precise imaging in live animal brains. Most activatable fluorescent probes can realize imaging of enzyme biomarkers but face challenges in generating an amplified fluorescence signal on GPCRs. Here, we present the visualization of μ opioid receptor (μOR) activity in zebrafish larvae using P5N3, an antagonist-conjugated pyridinium dye that enables a 25-fold fluorescence increase upon binding in the orthosteric pocket of μOR. This turn-on fluorescence is attributed to the synergistic effects of restricted movement of the pyridinium moiety and its hydrogen bond interactions with amino acid residues in the receptor binding domain, as elucidated by DFT calculations. We observed behaviorally correlated μOR activity in whole-brain recordings of wild-type zebrafish during acetic-acid-induced nociception and identified sinomenine-mediated attenuation with both spatiotemporal and pharmacological precision, highlighting the involvement of the optic tectum region. We propose that leveraging spatiotemporal mapping of μOR binding patterns using the turn-on molecular probe in freely behaving larval zebrafish holds significant promise as an in vivo tool for advancing translational pain research and accelerating the discovery of analgesic drugs.