Optogenetic mapping of synaptic activity and control of intracellular signaling

 描述(申请人提供)：这项建议旨在开发新的分子技术来绘制神经元的活动图，操纵神经元之间的通信强度，并干扰细胞内信号。这些“光遗传学”方法将被用来加深我们对大脑行为功能的理解，并对我们对神经疾病和神经退行性疾病的理解具有重要意义。第一个目标是开发一种技术，研究人员可以使用光学方法来识别在行为任务执行期间活跃的突触连接。这个报告系统可以用光打开，这定义了活动报告的窗口，如果在两个定义的细胞组之间存在显著的活动，则可以检测到荧光信号。许多现有的方法只能用来映射兴奋性连接，而所提出的方法可以用来识别利用任何神经递质的突触之间的活动。该方法将利用分裂荧光蛋白方法，其中其互补和荧光信号的产生依赖于活性。这种方法将测试确定的突触连接是否与行为的执行有关。第二个目标是开发一种技术，研究人员可以利用光来调节神经元之间突触通信的强度。突触强度的增加被认为是记忆和学习的基础，它的破坏与药物成瘾和许多神经疾病有关。具有调节突触强度的实验能力可以用来询问突触强度的变化是如何改变学习和记忆的，从而导致动物在正常和病理条件下观察到的适应行为。许多小的蛋白质片段可以改变神经元之间的突触强度。光响应蛋白质可以用来在黑暗中功能性地掩盖这些蛋白质片段，而光可以用来功能性地释放这些蛋白质片段。这将使突触强度的快速实验控制成为可能，它们的功能效应可以在行为动物身上进行研究。此工具 可以用来理解学习和适应过程中突触强度的变化。该项目的第三个目标是开发一种技术，在这种技术中，G蛋白偶联受体介导的第二信使途径被光抑制。G蛋白偶联受体在神经系统中介导神经调节剂和神经肽的作用，在调节和/或调节行为方面具有重要作用。使用如上所述的类似方法，破坏G-蛋白偶联受体-G蛋白相互作用的竞争性结合肽或直接抑制G-蛋白通路效应器的多肽可以用光响应蛋白和光照明来掩蔽和揭开。通过这种方法，光将快速关闭G蛋白激活或G蛋白途径的效应器，以确定的时间分辨率询问特定细胞中神经调节剂或神经肽的行为效应。

 DESCRIPTION (provided by applicant): This proposal aims to develop new molecular techniques to map activities of neurons, manipulate the strength of communication between neurons and disrupt intracellular signaling. These 'optogenetic' approaches will be used to further our understandings of brain function on behavior and have important implications in our understandings of neurological conditions and neurodegenerative diseases. The first goal is to develop a technique where the researchers can use optical approach to identify synaptic connections that were active during the performance of a behavior task. This reporter system can be turned on with light, which defines the window of activity reporting, and fluorescence signal can be detected if there is significant activity between two defined cell groups. Many existing approaches can only be used to map excitatory connections, whereas the proposed approach can be used to identify activities between synapses utilizing any neurotransmitters. The approach will utilize a split fluorescent protein approach where its complementation and the generation of fluorescent signal is activity dependent. This approach will test whether a defined synaptic connection is involved in the performance of a behavior. The second goal is to develop a technique where the researchers can use light to modulate the strength of synaptic communication between neurons. Increasing synaptic strength is believed to underlie memory and learning, and its disruption has been implicated in drug addiction and many neurological conditions. Having the ability to modulate the synaptic strength experimentally can be used to interrogate how changes in synaptic strength alter learning and memory, leading to the observed adaptive behavior in the animals in both normal and pathological conditions. Many small protein fragments can alter synaptic strengths between neurons. A light-responsive protein can be used to functionally mask these protein fragments in the dark and light can be used to functionally release these protein fragments. This will permit rapid experimental control of synaptic strength and their functional effects can be studied in the behaving animals. This tool can be used to understand how alteration in synaptic strength changes during learning and adaption. The third goal of the project is to develop a technique where G-protein coupled receptor mediated second messenger pathway is inhibited by light. G-protein coupled receptors mediate the effects of neuromodulator and neuropeptides in the nervous system and they have great importance in modulating and/or mediating behaviors. Using a similar approach as described above, competitive binding peptides that disrupt G-protein coupled receptor-G protein interactions or peptides that directly inhibit the effectors of G- protein pathways can be masked and unmasked with light-responsive protein and light illumination. With this approach, light will turn off G protein activation or effectors of G-protein pathway rapidly to interrogate the behavioral effects of neuromodulators or neuropeptides in specific cells with defined temporal resolution.