Fluorescent Indicators for Imaging Synaptic Zinc in Cortical Sound Processing

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Abstract Although the importance of synaptic Zn2+, as an emerging neuromodulator throughout the brain, has been widely appreciated, the dynamics of synaptic Zn2+ release in response to naturally occurring stimuli remains largely elusive. Genetically encoded Zn2+ indicators (GEZIs) derived from fluorescent proteins are popular tools for imaging Zn2+ in the cytosol and intracellular organelles. However, fluorescence imaging of Zn2+ secretion in the brain in live animals has not yet been achieved due to the limitations of current GEZIs (e.g., insufficient extracellular membrane localization, mismatching affinity, and/or inadequate dynamic range and photostability). This interdisciplinary multi-PI 4-year R01 project, led by Dr. Huiwang Ai with expertise in genetically encoded indicators and fluorescence imaging and Dr. Thanos Tzounopoulos with expertise in studying the role of Zn2+ in auditory processing, aims to (1) develop a new generation of GEZIs to address the hurdles for imaging secreted Zn2+ in the brain in vivo, and (2) integrate the new GEZIs with our innovative ZnT3 cKO mice, which, for the first time, allow for Cre-dependent expression of exogenous genes in ZnT3-expressing neurons and Dre-dependent region- and cell type-specific conditional ZnT3 gene knockout, to identify the cell- and circuit-specificity of Zn2+ dynamics that shape cortical sound processing. The project will lead to a novel capability of imaging synaptically released Zn2+ in the brain in awake behaving animals. Our innovative strategy to optimize the exoplasmic location of GEZIs may be generalized to enhance other genetically encoded indicators. Furthermore, because synaptic Zn2+ is a potent modulator throughout the cortex, our findings on Zn2+ dynamics in the primary auditory cortex (A1) during sound processing will improve the understanding of the roles of synaptic Zn2+ in cortical information processing beyond sensory cortices. We expect our studies to catalyze an extensive array of studies on Zn2+-related neurobiology and neurological diseases.