Fluorescent Indicators for Imaging Synaptic Zinc in Cortical Sound Processing

用于皮质声音处理中突触锌成像的荧光指示剂

• 批准号: 10599603
• 负责人: Huiwang Ai
• 金额: $ 68.42万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10599603
• 关键词: Address; Affinity; Alzheimer' s Disease; Animals; Auditory area; Brain; Cells; Chelating Agents; Color; Cytosol; Development; Directed Molecular Evolution; Engineering; Epilepsy; Frequencies; Generations; Genes; Hearing problem; Image; Imaging Device; Imaging technology; In Vitro; Ischemia; Knockout Mice; Link; Location; Membrane; Mental Depression; Mus; Neurobiology; Neuromodulator; Neurons; Neurotransmitters; Oranges; Organelles; Outcome; Property; Proteins; Research Personnel; Role; SLC30A3 gene; Sensory; Shapes; Signal Transduction; Specificity; Stimulus; Stroke; Synapses; Testing; Time; Transgenic Mice; Variant; Vesicle; Viral Vector; Zinc; auditory processing; awake; base; cell type; extracellular; fluorescence imaging; imaging capabilities; improved; in vivo; information processing; innovation; knockout gene; nervous system disorder; neurotransmission; new technology; novel; presynaptic; protein transport; public health relevance; response; sensory cortex; sensory stimulus; sound; sound frequency; spatiotemporal; synergism; trafficking; two-photon

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.

抽象的 尽管突触 Zn2+ 作为整个大脑中新兴的神经调节剂的重要性已被广泛认识到。 众所周知，响应自然发生的刺激的突触 Zn2+ 释放动态在很大程度上仍然存在 难以捉摸。源自荧光蛋白的基因编码 Zn2+ 指示剂 (GEZI) 是流行的工具 对细胞质和细胞内细胞器中的 Zn2+ 进行成像。然而，Zn2+分泌的荧光成像 由于当前 GEZIs 的局限性（例如，不足），活体动物大脑尚未实现 细胞外膜定位、亲和力不匹配和/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，由具有基因编码专业知识的艾辉旺博士领导 指标和荧光成像以及 Thanos Tzounopoulos 博士在研究 Zn2+ 在 听觉处理，旨在 (1) 开发新一代 GEZI 以解决分泌成像的障碍 体内大脑中的 Zn2+，以及 (2) 将新的 GEZIs 与我们创新的 ZnT3 cKO 小鼠相结合，这是第一个 时间，允许表达 ZnT3 的神经元中外源基因的 Cre 依赖性表达和 Dre 依赖性 区域和细胞类型特异性条件 ZnT3 基因敲除，以确定 Zn2+ 的细胞和电路特异性 塑造皮质声音处理的动力学。 该项目将带来一种新的能力，可以对清醒时大脑中突触释放的 Zn2+ 进行成像 动物。我们优化 GEZIs 外质位置的创新策略可以推广到增强 其他基因编码指标。此外，由于突触 Zn2+ 是整个过程中的有效调节剂 皮层，我们对声音处理过程中初级听觉皮层 (A1) 中 Zn2+ 动态的研究结果将会改善 了解突触 Zn2+ 在感觉皮层以外的皮层信息处理中的作用。我们 期望我们的研究能够促进 Zn2+ 相关神经生物学和神经病学的广泛研究 疾病。