Engineering an activity-dependent transcription factor for mapping neural circuits

 描述（由申请人提供）：神经回路被组织起来，以引起对环境刺激的适当行为反应。技术的进步允许可视化和操纵神经活动，使人们有可能建立电路功能和行为之间的因果关系。该提案试图通过利用NFAT分子的独特特征-钙/钙调神经磷酸酶依赖性核定位来扩展现有技术的全部功能。在命名为Ca莱克萨的新型活性报告系统中，含有必需的核定位信号的截短形式的NFAT与莱克萨融合以产生合成转录因子莱克萨- NFAT。由此产生的活动报告系统将不仅检测持续的神经活动，但阶段性活动，以及在自由移动，行为动物的高灵敏度。我们已经确定了几种最先进的分子技术，这些技术将增加CaLexA活性报告系统的信噪比，并增加区分阶段性和持续性神经活动的能力。在目标1中，我们将设计新的LexA-NFAT结构来抑制不需要的背景并增强行为相关信号。在目标2中，我们将设计新的LexA-NFAT构建体，其将允许检测瞬时钙活性。在目标3中，我们将使用光遗传学来生成精确控制的神经活动模式，以便可以比较钙成像和CaLexA报告系统的灵敏度和动态范围，以获得所有LexA-NFAT变体的完整表征。这些新的CaLexA活性报告系统的创建和表征将为果蝇神经生物学社区提供一套新的工具，用于研究神经回路和行为，以及可以容易地修改为其他遗传模式生物的分子设计。

 DESCRIPTION (provided by applicant): Neural circuits are organized to elicit appropriate behavioral responses to environmental stimuli. Advancement in technologies allowing the visualization and manipulation of neural activity has made it possible to establish a causal relationship between circuit function and behavior. This proposal attempts to expand the repertoire of existing technologies by exploiting a unique feature of the NFAT molecule - calcium/calcineurin-dependent nuclear localization. In the novel activity reporter system named CaLexA, a truncated version of NFAT containing the necessary nuclear localization signal is fused to LexA to make the synthetic transcription factor LexA- NFAT. The resulting activity reporter system will detect not only persistent neural activity but phasic activity as well with hih sensitivity in freely moving, behaving animals. We have identified several state-of-the-art molecular technologies that will increase the signal- to-noise ratio of the CaLexA activity reporter system as well as add the ability to discriminate between phasic and sustained neural activity. In Aim 1, we will engineer new LexA-NFAT constructs to suppress unwanted background and enhance behaviorally relevant signal. In Aim 2, we will design new LexA-NFAT constructs that will allow the detection of transient calcium activity. In Aim 3, we will use optogenetics to generate precisely controlled patterns of neural activity so that calcium imaging and the CaLexA reporter system can be compared for their sensitivity and dynamic range to gain a full characterization of all the LexA-NFAT variants. The creation and characterization of these new CaLexA activity reporter systems will make available to the Drosophila neurobiology community a new set of tools for the study of neural circuits and behavior, and molecular designs that can be readily modified for other genetic model organisms.