Imaging and recording of synaptic zinc in the brain by covalent chemistry-based semisynthetic biosensors

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. However, the precise roles of synaptic Zn²⁺ remain unclear due to a lack of tools capable of analyzing Zn²⁺ with synapse-level spatial resolution in vivo. Here, we developed neuroreceptor-based semisynthetic biosensors that function as integrating dosimeters, recording synaptic Zn²⁺ by covalent chemistry in the living mouse brain. Using a chemical knock-in strategy, we introduced activity-based Zn²⁺ 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 region-specific mapping of Zn²⁺ in synaptic clefts in the living mouse brain. Imaging-based analyses revealed differences in apparent Zn²⁺ levels at excitatory and inhibitory synapses across hippocampal regions during kainate-induced seizures, providing insights into the physiological and pathological functions of synaptic Zn²⁺.