RII Track-2 FEC: The Creation of Next-Generation Tools for Neuroscience - Noninvasive Radioluminescence Approaches to Optogenetics

非技术性描述本研究基础设施改进轨道-2重点EPSCoR协作（RII轨道-2 FEC）提案是南卡罗来纳州、亚拉巴马州和新墨西哥州的四个机构之间的协作，即克莱姆森大学、亚拉巴马大学伯明翰分校、新墨西哥大学和南卡罗来纳州大学。该项目的目的是扩大光遗传学实验方法的使用，自2005年引入以来，该方法对神经生物学产生了变革性的影响。这种方法允许实验者通过闪光来激活单个神经元或神经元组，具有高水平的空间和时间控制。标准光遗传学的主要限制之一是可见光无法深入活体组织。在该项目中，将开发一种系统，允许使用低剂量X射线而不是可见光作为激活信号。该项目包括让学生，特别是代表性不足的少数群体成员参与的多种机会。在研究密集型大学现有的暑期研究项目中，温斯洛普大学和北方新墨西哥州学院的学生将获得接待协议。该项目还包括指导初级教师的计划，特别是在建议的发展。技术说明这个多学科的项目涉及逐步发展的新的实验方法。首先，将产生辐射发光纳米颗粒（RLP），当暴露于X射线时会发光。RLP将被化学修饰，以允许特异性共价连接到神经元中表达的基因工程膜结合视蛋白，确保两种组分的紧密接近，以有效转移光刺激。将在培养的细胞、脑切片制备物和最后在完整动物（大鼠和小鼠）中测试该系统（有效性和无不良副作用）。RLP将通过注射到脑脊液中引入动物体内，颗粒可通过脑脊液扩散到大脑中。然后将测试X射线照射对固定和自由移动动物行为的影响，以验证运动和听觉皮层中神经细胞的成功激活。

Non-Technical DescriptionThis Research Infrastructure Improvement Track-2 Focused EPSCoR Collaboration (RII Track-2 FEC) proposal is a collaboration between four institutions in South Carolina, Alabama, and New Mexico, namely Clemson University, the University of Alabama Birmingham, the University of New Mexico, and the University of South Carolina. The aim of the project is to extend the uses of the experimental method of optogenetics, which, since its introduction in 2005, has had a transformative impact on neurobiology. This method allows experimenters to activate individual neurons or groups of neurons, with high levels of spatial and temporal control, by flashing light on them. One of the main limitations of standard optogenetics is the inability of visible light to penetrate deep within living tissues. In this project, a system will be developed to allow the use of low-dosage X-rays, rather than visible light, as the activating signal. The project includes multiple opportunities to involve students, especially members of under-represented minority groups. Agreements are in place to host students from Winthrop University and Northern New Mexico College, which serve highly diverse student populations, in existing summer research programs at the research-intensive universities. The project also includes plans for mentoring junior faculty, especially in proposal development.Technical DescriptionThis multi-disciplinary project involves step-wise development of novel experimental methods. First, radioluminescent nanoparticles (RLPs) will be produced that emit light when exposed to X-rays. The RLPs will be chemically modified to allow specific covalent attachment to a genetically engineered membrane-bound opsin protein expressed in neurons, ensuring close proximity of the two components for efficient transfer of the light stimulus. The system will be tested (for efficacy and the absence of undesired side effects) in cultured cells, then brain slice preparations, and finally in intact animals (rats and mice). RLPs will be introduced into animals by injection into the cerebrospinal fluid, through which the particles may diffuse into the brain. The effects of X-ray exposure on the behavior of immobilized and freely moving animals will then be tested to verify successful activation of neural cells in the motor and auditory cortexes.