Quantitative analysis of synaptic zinc in the brain by covalent chemistry-based semisynthetic biosensors

Abstract Labile zinc ions (Zn²⁺) are stored in glutamatergic vesicles at specific excitatory synapses in the central nervous system and released into synaptic clefts in an activity-dependent manner. Although Zn²⁺ is suggested to modulate various neuroreceptor functions, its precise roles remain unclear due to a lack of tools capable of quantitatively analyzing Zn²⁺ with synapse-level spatial resolution. Here, we developed neuroreceptor-based semisynthetic sensors that record synaptic Zn²⁺ dynamics by covalent chemistry in the living mouse brain. Using a chemical knock-in strategy, we introduced activity-based Zn 2+ -probes with distinct affinities into endogenous α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid or γ-aminobutyric acid type A receptors. These in-brain-constructed Zn²⁺ sensors enabled, to our knowledge, for the first time, quantitative, region-specific mapping of Zn²⁺ released into synaptic clefts. Imaging-based analyses revealed differences in Zn²⁺ concentrations at excitatory and inhibitory synapses across hippocampal regions during kainate-induced seizures, providing new insights into the physiological and pathological functions of synaptic Zn²⁺.