Fluorescent Indicators for Imaging Synaptic Zinc in Cortical Sound Processing

摘要 虽然突触锌的重要性，作为一种新出现的神经调节剂贯穿大脑，已被广泛 值得注意的是，突触锌释放对自然产生的刺激的反应在很大程度上仍然存在 难以捉摸。源于荧光蛋白的基因编码的锌指示剂(GEZI)是一种流行的检测工具 胞浆和胞内细胞器中的锌成像。然而，锌分泌物的荧光成像 由于当前GEZI的局限性(例如，不足)，活体动物的大脑尚未实现 胞外膜定位、不匹配亲和力和/或动态范围和光稳定性不足)。 这个跨学科、多PI、为期4年的R01项目，由具有遗传编码专业知识的艾慧旺博士领导 指示剂和荧光成像以及Thanos Tzounopoulos博士在研究锌在 听觉处理，目标是(1)开发新一代GEZI，以解决成像分泌的障碍 以及(2)将新的GEZI与我们创新的ZnT3 CKO小鼠整合，这是第一次 时间，允许外源基因在表达ZnT3的神经元中依赖于Cre和Dre依赖的表达 区域和细胞类型特异的条件基因敲除，以确定锌2的细胞和电路特异性 塑造皮层声音处理的动力学。 该项目将导致一种新的能力，即在清醒状态下对大脑中突触释放的锌进行成像 动物。我们优化GEZI外质定位的创新策略可能会推广到增强 其他基因编码的指标。此外，由于突触锌在整个脑区都是一个强有力的调节剂。 我们对初级听觉皮质(A1)在声音处理过程中的锌动力学的研究结果将有所改善 对突触锌在感觉皮质以外的皮质信息处理中作用的理解。我们 期待我们的研究能促进一系列与锌相关的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.