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

High Resolution Image Download MS PowerPoint Slide Despite its crucial role in neurotransmission and neural processing, direct in vivo measurement of Zn 2+ transients has been hindered by the limited responsiveness and stability of available fluorescent indicators. Here, we report hpGRISZ ( h igh- p erformance gr een i ndicator for s ynaptic Z n 2+ ), an ultraresponsive, thermostable, turn-on green fluorescent biosensor engineered for high brightness, large dynamic range, and affinity tuned 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 enables robust wide-field, two-photon, and fiber photometry recordings in awake mice. Using hpGRISZ, we visualized synaptic Zn 2+ dynamics across multiple brain regions and neuronal circuits, revealing direct evidence of Zn 2+ 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 Zn 2+ and its roles in neural circuit function in vivo.