Genetic tools for visualization of co-transmission in identified neurons

用于可视化已识别神经元中的共传递的遗传工具

• 批准号: 9090625
• 负责人: Leslie C Griffith
• 金额: $ 24.38万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9090625
• 关键词: Accounting; Animal Model; Antibodies; Automobile Driving; Behavior; Behavioral; Behavioral Genetics; Brain; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Color; Communication; Complex; Courtship; Development; Drosophila genus; Ectopic Expression; Failure; Feeding behaviors; Generations; Genes; Genetic; Glutamates; Imagery; Individual; Invertebrates; Label; Learning; Libraries; Logic; Maps; Mediating; Memory; Methods; Modeling; Modification; Nature; Neurons; Neurotransmitters; Output; Pattern; Peptides; Phenotype; Population; Potassium Channel; Process; Protein Fragment; Proteins; Reporter; Reproduction; Resolution; Role; Sea; Serotonin; Signal Transduction; Site; Sleep; Spatial Distribution; Synapses; Synaptic Vesicles; System; Techniques; Technology; Tetanus Toxin; Traction; Vesicle; addiction; base; gamma-Aminobutyric Acid; homologous recombination; in vitro Assay; information model; memory consolidation; mutant; neurotransmitter release; postsynaptic; promoter; protein reconstitution; public health relevance; reconstitution; relating to nervous system; segregation; tool; transmission process; visual process; visual processing

 DESCRIPTION (provided by applicant): The ability to understand how brains generate behavior both in normal and pathological situations relies on our understanding of the neural computations carried out by behavioral circuits. The nature of chemical communication between neurons is determined by the specific neurotransmitters released onto postsynaptic targets and it was thought for many years that neurons released a single type of transmitter onto all their targets. In recent years, however, it has become clear that key neurons in vertebrate and invertebrate circuits involved in addiction, memory formation, feeding behavior and reproduction contain neurons that violate this rule, releasing multiple neurotransmitters. In this proposal we develop genetic tools to allow mapping of the distribution of specific neurotransmitter release sites in single neurons in multiple colors by modifying the endogenous genetic loci of vesicular transporters for neurotransmitter with fluorescent proteins. This allows both accurate and complete accounting of transmitter release sites since the marker's generation and turnover rely on processes that control the presence of the endogenous protein. Single cell resolution is obtained via an intersectional version of this strategy in which split fluorescent proteins become reconstituted only in specific cells. There is no current technique in any system which can provide this level of resolution. This technology will provide the ability to determine, in complex neurons releasing several chemical substances, the spatial distribution of each of the chemicals and its relationship to downstream targets of that neuron. It will also allow the mapping of temporal changes, either developmental or plasticity-induced, in neurotransmitter release. The technique is developed initially for use in Drosophila, a model organism which has been immensely important for our understanding of both the genetic and circuit basis of behavior, but as a general strategy can also be adapted for use in mammalian brain.

 描述（由应用程序提供）：了解大脑在正常情况和病理情况下如何产生行为的能力取决于我们对行为回路进行的神经调节的理解。神经递质之间化学通信的性质取决于释放到突触后靶标的特定神经递质，并且人们认为神经元将单一类型的发射机释放到其所有靶标上。然而，近年来，很明显，脊椎动物和无脊椎动物电路中的关键神经元还涉及记忆形成，喂养行为和含有违反该规则的神经元的复制，从而释放了多个神经递质。在此提案中，我们开发了遗传工具，以通过修饰具有荧光蛋白的神经递质的内源性遗传转运蛋白的内源性遗传局部遗传遗传局部，以多种颜色的单个神经元中特定神经递质释放位点的分布进行映射。这允许对发射机释放位点进行准确和完整的核算，因为标记的产生和周转率取决于控制内源性蛋白质存在的过程。单细胞分辨率是通过该策略的相交版本获得的，在该策略中，分裂荧光蛋白仅在特定细胞中重构。在任何系统中都没有目前的技术可以提供这种分辨率。技术将提供确定复杂的能力 神经元释放几种化学物质，每种化学物质的空间分布及其与该神经下游靶标的关系。它还将允许在神经递质释放中绘制临时变化，无论是发育性或可塑性诱导的。该技术最初是用于用于果蝇（一种模型生物体）的开发，对于我们对行为的遗传和电路基础非常重要，但是作为一般策略也可以适应用于哺乳动物大脑。