A genetically encoded far-red fluorescent indicator for imaging synaptically released Zn²

Synaptic zinc ion (Zn²⁺) has emerged as a key neuromodulator in the brain. However, the lack of research tools for directly tracking synaptic Zn²⁺ in the brain of awake animals hinders our rigorous understanding of the physiological and pathological roles of synaptic Zn²⁺. In this study, we developed a genetically encoded far-red fluorescent indicator for monitoring synaptic Zn²⁺ dynamics in the nervous system. Our engineered far-red fluorescent indicator for synaptic Zn²⁺ (FRISZ) displayed a substantial Zn²⁺-specific turn-on response and low-micromolar affinity. We genetically anchored FRISZ to the mammalian extracellular membrane via a transmembrane (TM) ⍺ helix and characterized the resultant FRISZ-TM construct at the mammalian cell surface. We used FRISZ-TM to image synaptic Zn²⁺ in the auditory cortex in acute brain slices and awake mice in response to electric and sound stimuli, respectively. Thus, this study establishes a technology for studying the roles of synaptic Zn²⁺ in the nervous system.