A neurotransmitter-specific intersectional genetic method for neuronal silencing and defining neuronal identity

用于神经元沉默和定义神经元身份的神经递质特异性交叉遗传方法

• 批准号: 8941965
• 负责人: Steve STOWERS
• 金额: $ 21.25万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8941965
• 关键词: Activities of Daily Living; Animal Model; Antibodies; Behavioral; Biological Assay; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA; Development; Diagnosis; Drosophila genus; Engineered Gene; Engineering; Excision; Exons; Genes; Genetic; Genetic Models; Glutamates; Infertility; Investigation; Knowledge; Lead; Mediating; Mental disorders; Methods; Modeling; Mood Disorders; Mus; Neurologic; Neurons; Neurotransmitters; Pattern; Phenotype; Property; Reporter; Site; Temperature; Transgenes; Transgenic Organisms; Zebrafish; base; cholinergic; fly; gene function; genome editing; information processing; interest; loss of function mutation; meetings; mutant; neural circuit; neuron component; null mutation; optogenetics; public health relevance; recombinase; relating to nervous system; targeted sequencing; tool; transcription factor

 DESCRIPTION (provided by applicant): How a neural circuit encodes, transmits, and processes information to make behavioral decisions is far from completely understood. Knowledge of the neurotransmitter usage, and thus the potential functional capacity, of each neuron in a neural circuit will significantly facilitate neural circuit analysis, as will enhancingthe spatial precision with which neuronal subsets can be functionally manipulated. This proposal aims to develop a set of neurotransmitter-specific fly strains for each Drosophila neurotransmitter that utilizes an intersectional genetic method to enable: 1) the complete definition of the neurotransmitter usage of all Drosophila neurons; and 2) neurotransmitter-specific silencing and transgene targeting to intersecting neuronal subsets within a GAL4 expression pattern. The method involves inserting B3 recombinase target sites (B3RTs) into neurotransmitter- specific genes such that the function of the gene is maintained and essential coding sequences are flanked. Subsequent expression of the B3 recombinase using a UAS-B3 recombinase transgene controlled by a GAL4 driver results in excision of the DNA between the B3RTs within all neurons in which the GAL4 driver expresses. The excision creates a complete-loss-of function mutation in the neurotransmitter-specific gene. In addition, the coding sequence for the LexA transcription factor is inserted such that after the B3 recombinase-mediated excision, a LexA driver is created that recapitulates the endogenous expression pattern of the neurotransmitter-specific gene. Although the B3 recombinase-mediated excision will occur in all neurons in which a given GAL4 driver expresses, the resulting LexA driver will express only in the subset of neurons in which a specific neurotransmitter is utilized. These neurons are easily visualized using a fluorescent reporter, but any transgene can be expressed using the LexA driver including transgenes for manipulating neuronal activity such as channelrhodopsin (for optogenetic excitation) or temperature-sensitive shibire (for neuronal silencing). In a genetic background otherwise mutant for a given neurotransmitter-specific gene, neurons within a GAL4 expression pattern that utilize that neurotransmitter will be silenced upon B3 recombinase-mediated excision. Definition of the neurotransmitter identity of each neuron within a neural circuit of interest, and an enhanced ability to manipulate the activity of small subsets of neurons based on the intersection of neurotransmitter usage and GAL4 expression pattern, will make possible finer scale neural circuit analysis than is currently possible with existing methods and thereby the potential for a more in- depth understanding of the functional properties of neural circuits.

 描述（由申请人提供）：神经回路如何编码，传输和处理信息以做出行为决策还远未完全理解。神经回路中每个神经元的神经递质使用情况以及潜在功能能力的知识将显著促进神经回路分析，这将增强神经元子集可以在功能上操纵的空间精度。该提案旨在为每种果蝇神经递质开发一组神经递质特异性果蝇品系，其利用交叉遗传方法来实现：1）所有果蝇神经元的神经递质使用的完整定义;和2）神经递质特异性沉默和转基因靶向GAL 4表达模式内的交叉神经元子集。 该方法涉及将B3重组酶靶位点（B3 RT）插入神经递质特异性基因中，使得基因的功能得以维持并且必需的编码序列侧接。使用由GAL 4驱动控制的UAS-B3重组酶转基因的B3重组酶的后续表达导致GAL 4驱动表达的所有神经元内的B3 RT之间的DNA切除。切除在神经递质特异性基因中产生完全丧失功能的突变。此外，插入莱克萨转录因子的编码序列，使得在B3重组酶介导的切除后，产生再现神经递质特异性基因的内源表达模式的莱克萨驱动子。尽管B3重组酶介导的切除将发生在其中表达给定GAL 4驱动物的所有神经元中，但所得莱克萨驱动物将仅在其中利用特定神经递质的神经元子集中表达。这些神经元容易使用荧光报告子可视化，但是任何转基因都可以使用莱克萨驱动器表达，包括用于操纵神经元活性的转基因，例如通道视紫红质（用于光遗传学激发）或温度敏感性shibire（用于神经元沉默）。在遗传背景中，对于给定的神经递质特异性基因的突变体，利用该神经递质的GAL 4表达模式内的神经元将在B3重组酶介导的切除后沉默。定义感兴趣的神经回路内每个神经元的神经递质身份，以及增强操纵小神经元子集活动的能力 基于神经递质使用和GAL 4表达模式的交叉，将使比目前现有方法更精细的神经回路分析成为可能，从而有可能更深入地了解神经回路的功能特性。