Novel molecular genetic tool for large-scale labeling and modulating activity of neurons associated with particular physiological processes and behaviors

我们建议开发一种大规模标记和调节神经元活动的新技术 与特定的生理过程和行为相关的。这项技术将不同于所有 目前用于生物研究的其他技术，因为它基于这样一个新想法 特定的神经元组装可以通过驱动报告蛋白的表达而成为靶点 既活跃又暴露在近红外光下的神经元。我们将产生载体，在其中 报告蛋白(如荧光蛋白mCherry)的表达受两种活性敏感蛋白控制 启动子和细菌光敏色素-1(BphP1)光受体。因此，该蛋白将不会在 活动但不暴露于近红外光或暴露于近红外光但 未激活。只有当神经元既活跃又暴露在近距离辐射中时，蛋白质的表达才会发生。 红外线。由于灯可以打开和关闭，我们提出的工具将允许精确的时间 光照和行为之间的对应关系。这种对应关系对于标注是至关重要的 神经元在没有及时维持的行为中。通过将mCherry的图案可视化 表达，我们将能够识别在特定行为期间活跃的神经元(例如，探索新的 物体、修饰)或生理状态(例如，快速眼动睡眠)。我们计划将这项技术应用于 睡眠研究，但它也可以应用于广泛的神经生物学研究。

We propose to develop a novel technology for large-scale labeling and modulating activity of neurons associated with particular physiological processes and behaviors. This technology will be different from all other technologies that are currently used in biological research because it is based on the novel idea that particular neuronal assembles can be targeted by driving the expression of a reporter protein specifically in the neurons that are both active and exposed to the near-infrared light. We will produce vectors in which expression of a reporter protein (such as fluorescent protein mCherry) is controlled by both an activity-sensitive promoter and bacterial phytochrome-1 (BphP1) photoreceptor. Therefore, this protein will not be expressed in neurons that are either active but not exposed to the near-infrared light or exposed to the near-infrared light but not active. Expression of the protein will occur only if the neuron is both active and exposed to the near- infrared light. Because the light can be turned on and off, our proposed tool will allow precise temporal correspondence between the light exposure and the behavior. Such correspondence is critical for labeling neurons during the behaviors that are not sustained in time. By visualizing the pattern of the mCherry expression, we will be able to identify the neurons that are active during specific behaviors (e.g., exploring new object, grooming) or physiological states (e.g., rapid eye movement sleep). We plan to apply this technology in sleep research studies, but it can be also applied in a wide range of neurobiology studies.