Abstract
Despite the crucial role of synaptic Zn(2+) in neurotransmission and neural processing, direct in vivo measurement of Zn(2+) transients has remained challenging due to the limited responsiveness of existing fluorescent indicators. Here we report hpGRISZ (high-performance green indicator for synaptic Zn(2+)), an ultraresponsive turn-on green fluorescent biosensor engineered through iterative linker optimization, mutagenesis, and directed evolution. hpGRISZ exhibits exceptional brightness, thermostability, and a large fluorescence response (F/F(0) ≈ 25) with micromolar affinity suitable for detecting extracellular synaptic Zn(2+) release. We comprehensively characterized hpGRISZ in vitro, in mammalian cells, and in vivo. A membrane-anchored version of hpGRISZ traffics robustly to the cell surface under physiological conditions, where it retains strong responsiveness and supports wide-field, two-photon, and fiber photometry recordings in awake mice. These assays revealed synaptic Zn(2+) dynamics across distinct brain regions and neuronal circuits. Together, our findings establish hpGRISZ as a powerful tool for dissecting zinc signaling in neural circuits and as a prototype for next-generation genetically encoded biosensors for in vivo imaging.