An Ultraresponsive Green Biosensor for Robust in Vivo Imaging of Synaptic Zinc Dynamics

Despite its crucial role in neurotransmission and neural processing, direct in vivo measurement of Zn²⁺ transients has been hindered by the limited responsiveness and stability of available fluorescent indicators. Here, we report hpGRISZ (<u>h</u>igh-<u>p</u>erformance <u>gr</u>een <u>i</u>ndicator for <u>s</u>ynaptic <u>Z</u>n²⁺), an ultraresponsive, thermostable, turn-on green fluorescent biosensor engineered for high brightness, large dynamic range, and affinity tuned 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 enables robust wide-field, two-photon, and fiber photometry recordings in awake mice. Using hpGRISZ, we visualized synaptic Zn²⁺ dynamics across multiple brain regions and neuronal circuits, revealing direct evidence of Zn²⁺ signaling during auditory processing in the cortex and during aversive responses in the amygdala. hpGRISZ thus establishes a versatile platform for dissecting the spatiotemporal dynamics of synaptic Zn²⁺ and its roles in neural circuit function in vivo.