Fluorescent Indicators for Imaging Synaptic Zinc in Cortical Sound Processing

摘要 虽然突触Zn 2+作为一种新兴的神经调质在整个大脑中的重要性已被广泛研究， 尽管我们认识到，突触Zn 2+释放响应于自然发生的刺激的动力学在很大程度上仍然存在， 难以捉摸来源于荧光蛋白的遗传编码的Zn 2+指示剂（GEZI）是用于检测Zn 2+的流行工具。 对胞质溶胶和细胞内细胞器中的Zn 2+进行成像。然而，荧光成像的Zn 2+分泌在 由于目前GEZI的限制（例如，不足 细胞外膜定位、错配亲和力和/或不足的动态范围和光稳定性）。 这个跨学科的多PI 4年R 01项目，由Huiwang Ai博士领导，具有遗传编码方面的专业知识。 指标和荧光成像和博士Thanos Tzounopoulos与专业知识，在研究锌的作用+， 听觉处理，旨在（1）开发新一代GEZI，以解决成像分泌的障碍 Zn 2+在体内大脑中的作用，以及（2）将新的GEZI与我们创新的ZnT 3 cKO小鼠整合，这是第一次。 时间，允许外源基因在ZnT 3表达神经元中的Cre依赖性表达和Dre依赖性表达。 区域和细胞类型特异性条件性ZnT 3基因敲除，以鉴定Zn 2+的细胞和回路特异性 影响皮层声音处理的动力学。 该项目将导致一种新的成像能力，即在清醒行为中大脑中突触释放的Zn 2 + 动物我们的创新策略，以优化外质位置的GEZI可以推广到提高 其他基因编码的指标。此外，由于突触Zn 2+是贯穿突触的有效调节剂， 皮层，我们对初级听觉皮层（A1）在声音处理过程中锌离子动力学的研究结果将改善 了解突触锌离子在感觉皮层以外的皮层信息处理中的作用。我们 我希望我们的研究能促进一系列广泛的锌相关神经生物学和神经学研究 疾病

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.