hpGRISZ: a high-performance fluorescent biosensor for in vivo imaging of synaptic zinc dynamics

Zhang, H.; Kumar, M.; Correia, R.; Yang, S.; Huang, W.; Soares-Cunha, C.; Cody, P. A.; Zhang, Y.; Tian, X.; Zhao, S.; Drobizhev, M.; Rodrigues, A. J.; Tzounopoulos, T.; Ai, H.

doi:10.1101/2025.10.06.680726

bioRxiv · 10.1101/2025.10.06.680726

hpGRISZ: a high-performance fluorescent biosensor for in vivo imaging of synaptic zinc dynamics

Zhang, H.·Kumar, M.·Correia, R.·Yang, S.·Huang, W.·Soares-Cunha, C.·Cody, P. A.·Zhang, Y.·Tian, X.·Zhao, S.·Drobizhev, M.·Rodrigues, A. J.·Tzounopoulos, T.·Ai, H.

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Abstract

Despite the crucial role of synaptic Zn2+ in neurotransmission and neural processing, direct in vivo measurement of Zn2+ transients has remained challenging due to the limited responsiveness of existing fluorescent indicators. Here we report hpGRISZ (high-performance green indicator for synaptic Zn2+), 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/F0 {approx} 25) with micromolar affinity suitable for detecting extracellular synaptic Zn2+ 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 Zn2+ 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.

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Zhang, H., Kumar, M., Correia, R., Yang, S., Huang, W., Soares-Cunha, C., Cody, P. A., Zhang, Y., Tian, X., Zhao, S., Drobizhev, M., Rodrigues, A. J., Tzounopoulos, T., Ai, H.. 2025-10-07. hpGRISZ: a high-performance fluorescent biosensor for in vivo imaging of synaptic zinc dynamics. https://doi.org/10.1101/2025.10.06.680726

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