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

该研究基础设施改进轨道2重点EPSCoR合作（RII轨道2 FEC）提案是南卡罗来纳州、阿拉巴马州和新墨西哥州四个机构之间的合作，即克莱姆森大学、阿拉巴马大学伯明翰分校、新墨西哥大学和南卡罗来纳大学。该项目的目的是扩展光遗传学实验方法的应用，自2005年引入以来，光遗传学对神经生物学产生了变革性的影响。这种方法允许实验人员通过闪光来激活单个神经元或神经元群，这些神经元具有高度的空间和时间控制能力。标准光遗传学的主要限制之一是可见光无法穿透活体组织的深处。在这个项目中，将开发一个系统，允许使用低剂量的x射线，而不是可见光，作为激活信号。该项目包括让学生参与的多种机会，特别是代表性不足的少数群体的成员。温斯洛普大学（Winthrop University）和北新墨西哥学院（Northern New Mexico College）的学生就读于这两所研究型大学现有的暑期研究项目。这两所大学的学生群体非常多样化。该项目还包括指导初级教师的计划，特别是在提案制定方面。技术描述这个多学科项目涉及逐步发展新的实验方法。首先，将生产出放射发光纳米粒子（rlp），当暴露在x射线下时会发光。rlp将经过化学修饰，使其能够与神经元中表达的基因工程膜结合视蛋白特异性共价附着，从而确保两种成分的紧密结合，从而有效地传递光刺激。该系统将在培养细胞中进行测试（测试有效性和无不良副作用），然后是脑切片制剂，最后是完整的动物（大鼠和小鼠）。rlp将通过注射脑脊液进入动物体内，颗粒可能通过脑脊液扩散到大脑。x射线暴露对固定和自由运动动物行为的影响将被测试，以验证运动和听觉皮层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.