Discovery of functional cell types in the inferior colliculus with combined molecular-genetic and electrophysiological approaches

结合分子遗传学和电生理学方法发现下丘功能细胞类型

• 批准号: 9300564
• 负责人: Nace L Golding
• 金额: $ 18.97万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9300564
• 关键词: Address; Affect; Anatomy; Area; Auditory; Brain; Calcium-Binding Proteins; Cell Nucleus; Cells; Cellular Morphology; Cereals; Characteristics; Classification; Communication impairment; Complement; Cues; Data; Electrophysiology (science); Elements; Exhibits; Fluorescence; Foundations; Future; Genetic; Genetic Techniques; Gerbils; Goals; Inferior Colliculus; Interneurons; Label; Light; Lighting; Location; Maps; Mediating; Midbrain structure; Molecular Genetics; Morphology; Nature; Neurons; Pathway interactions; Pattern; Phenotype; Physiological; Physiology; Population; Property; Proteins; Recombinant adeno-associated virus (rAAV); Role; Slice; Stream; Structure; Synapses; Testing; Tinnitus; Training Activity; Viral; Virus; Whole-Cell Recordings; auditory pathway; auditory thalamus; calbindin; calbindin-D28K; cell type; excitatory neuron; experimental study; genetic approach; hearing impairment; in vivo; inhibitory neuron; intersectionality; neural circuit; neurochemistry; novel; optogenetics; patch clamp; promoter; sound; tool

Project Summary The central nucleus of the inferior colliculus (ICC) is a midbrain nucleus that serves as a pivotal point of convergence for a large number of ascending and descending auditory pathways. Understanding how computations carried out in the ICC relate to the underlying circuitry has been unusually difficult, in large part due to the lack of clear definitions of cell types. Indeed, there appears to be no correlations between intrinsic firing properties and cell morphology, even across excitatory and inhibitory cells. We propose that functionally distinct classes of neurons in the ICC can be identified and genetically accessed using viruses with promoters to different neurochemical markers. To address this hypothesis, we will combine electrophysiological and anatomical approaches with interdependent recombinant adeno-associated viruses (rAAV) targeting excitatory and inhibitory classes of calbindin D-28k ICC neurons (CB-excitatory and CB- inhibitory). CB is a calcium binding protein expressed in subsets of neurons of the ICC and in many other brain areas. Our preliminary results show that viruses carrying a CB promoter element target ICC neurons that exhibit a single firing phenotype, and that these neurons may comprise both excitatory and inhibitory subclasses. In Aim 1, we will combine in vivo labeling of ICC neurons with rAAV viruses, fluorescence guided patch recordings in IC slices, and anatomical analyses to fully characterize the two pools of CB neurons, including their physiology, morphology and axonal projections. In parallel, labeled neurons will be characterized according to their complement of expressed neurochemical markers. In Aim 2, we will use our molecular-genetic tools to drive channelrhodopsin-2 selectively in CB-excitatory or CB-inhibitory neurons. We will use structured illumination to map CB neurons' local inputs and targets in the ICC as well as to facilitate paired whole-cell patch recordings from connected neuron pairs, enabling characterization of synaptic properties between physiologically identified network partners. These experiments will serve as a blueprint for identifying and characterizing other functionally distinct circuits in the ICC. Our goal is to establish a foundation for future studies involving cell-specific optogenetic manipulations in vivo, where we can assess the roles of CB neurons and other defined ICC cell types in auditory computations.

项目摘要 下丘中央核（ICC）是中脑核团，作为中枢神经系统的中枢点， 大量上行和下行听觉通路的会聚。了解如何 在ICC中进行的与底层电路相关的计算在很大程度上是异常困难的 由于缺乏对细胞类型的明确定义。事实上，似乎没有内在的相关性， 放电特性和细胞形态，甚至跨越兴奋性和抑制性细胞。 我们提出，ICC中功能不同的神经元类别可以被识别和遗传访问 使用带有不同神经化学标记启动子的病毒。为了解决这个假设，我们将联合收割机 利用相互依赖的重组腺相关病毒的电生理学和解剖学方法 在另一个实施方案中，本发明提供了靶向钙结合蛋白D-28 k ICC神经元的兴奋性和抑制性类别（CB-兴奋性和CB-兴奋性）的重组腺相关病毒（rAAV）。 抑制的）。CB是一种钙结合蛋白，在ICC的神经元亚群和许多其他脑组织中表达， 地区我们的初步结果表明，携带CB启动子元件的病毒靶向ICC神经元， 表现出单一放电表型，并且这些神经元可以包括兴奋性和抑制性神经元。 子类。在目标1中，我们将联合收割机结合ICC神经元的体内标记与rAAV病毒，荧光引导的免疫荧光技术， IC切片中的斑片记录，以及解剖分析以充分表征CB神经元的两个池， 包括它们的生理学、形态学和轴突投射。同时，标记的神经元将被 根据其表达的神经化学标记物的互补来表征。在目标2中，我们将使用 分子遗传工具，以驱动channelrhodopsin-2选择性地在CB兴奋性或CB抑制性神经元。我们 将使用结构化照明来映射CB神经元在ICC中的局部输入和目标， 来自连接的神经元对的配对全细胞斑片记录，使得能够表征突触 生理上识别的网络伙伴之间的属性。 这些实验将作为识别和表征其他功能不同的电路的蓝图 在ICC。我们的目标是为未来的细胞特异性光遗传学研究奠定基础。 在体内的操作，在那里我们可以评估CB神经元和其他定义的ICC细胞类型的作用， 听觉计算