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

 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定还远未完全理解。了解神经回路中每个神经元的神经递质使用情况以及潜在的功能容量将极大地促进神经回路分析，并将提高神经亚群功能操作的空间精确度。这一建议旨在为每一种果蝇神经递质开发一套神经递质特异性的果蝇品系，该品系利用交叉遗传方法使：1)所有果蝇神经元的神经递质使用的完整定义；以及2)神经递质特异性沉默和转基因靶向，以在GAL4表达模式中交叉神经元亚群。该方法包括将B3重组酶靶点(B3RT)插入到神经递质特异性基因中，从而维持基因的功能并在必要的编码序列两侧进行编码。随后使用由GAL4驱动器控制的UAS-B3重组酶转基因表达B3重组酶会导致在表达GAL4驱动器的所有神经元内的B3RT之间的DNA被切除。这种切除会在神经递质特异性基因中产生一种完全丧失功能的突变。此外，LexA转录因子的编码序列被插入，从而在B3重组酶介导的切除之后，创建了LexA驱动程序，该驱动程序概括了神经递质特异性基因的内源表达模式。尽管B3重组酶介导的切除将发生在给定GAL4驱动程序表达的所有神经元中，但由此产生的LexA驱动程序将仅在利用特定神经递质的神经元子集中表达。这些神经元很容易使用荧光报告程序可视化，但任何转基因都可以使用LexA驱动程序表达，包括操纵神经元活动的转基因，如通道视紫红质(用于光遗传兴奋)或温度敏感Shibire(用于神经元沉默)。在特定神经递质特定基因的遗传背景中，利用该神经递质的GAL4表达模式中的神经元在B3重组酶介导的切除后将被沉默。定义感兴趣的神经回路中每个神经元的神经递质身份，以及增强了操纵小部分神经元的活动的能力 基于神经递质使用和GAL4表达模式的交集，将使比现有方法更精细的神经电路分析成为可能，从而有可能更深入地了解神经电路的功能特性。

 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.