Lighting up the brain: Optogenetic tools to record, trace, and manipulate brain circuits at cellular resolution

项目总结 大脑回路是神经元的动态网络，以电和化学的形式处理信息 形成记忆和塑造行为的信号。研究大脑回路如何实例化基础 计算潜在的行为，我们需要将它们的接线图与功能分析相结合，位于 细胞分辨率。然而，电(电压)和化学(例如神经肽)信号不是直接的 可见，当前的电路跟踪工具不足以进行有意义的功能分析。使用蛋白质 设计这项提案的目的是开发一种基因编码的荧光记者和示踪剂工具箱 专门为研究神经回路量身定做的。在电气层面上，电压传感器可以成像精确的定时 其他方法检测不到的动作电位和亚阈值电压。然而，即使是最新的电压 传感器在使用双光子照明进行深度成像的高分辨率显微镜上表现不佳 在大脑里。为了克服这些限制，我们正在采取双管齐下的方法，通过在 通过利用光谱学来帮助我们的蛋白质工程工作，我们可以从电压传感器蛋白质的机械心脏入手。 我们相信定向进化将提高电压灵敏度和双光子功能&gt；10倍，使我们能够 成像目前看不见的信号，比如大脑深处的突触电位。在化学层面上， 神经肽在几乎所有的皮质神经元中都高度表达，但它们在动物体内的作用和影响只能 这是因为目前的检测方法，如微透析，是侵入性的，缺乏时空 决议。我们正在利用噬菌体展示技术进化出能够识别神经肽和偶联的纳米体。 从报告分子的构象变化到荧光变化。这些传感器将提供 动物行为过程中神经肽释放在细胞分辨率上的可视化 模仿人类健康和疾病状态的范例。在蜂窝连接层面，当前的电路工具- 测绘就像狂犬病病毒一样，表现出很大的神经毒性，阻碍了有意义的功能分析。我们是 具有天然倾向的工程蛋白质，可以组装成能够传递基因的结构 有效负载到特定的细胞，以产生更有效和毒性更低的工具来绘制和操纵大脑电路。 有效和强大的脑图谱工具将架起功能和结构分析的桥梁，最终允许长期- 基于神经网络连通性的术语研究。总体而言，在这一领域开发的光遗传工具 一项提议将神经回路之间的化学和电信号转化为荧光，可以 很容易测量。因此，它们可以用来解开神经元紊乱的功能基础和原因。 到目前为止还没有达到的详细程度，并使我们能够开发新的治疗方法。

PROJECT SUMMARY Brain circuits are dynamic networks of neurons that process information in the form of electrical and chemical signals to form memories and shape behaviors. To investigate how brain circuits instantiate fundamental computations underlying behaviors, we need to map their wiring diagrams coupled with functional analysis at cellular resolution. However, the electrical (voltage) and chemical (e.g. neuropeptides) signals are not directly visible, and current circuit tracing tools are insufficient for meaningful functional analysis. Using protein engineering this proposal aims to develop a toolbox of genetically-encoded fluorescent reporters and tracers specifically tailored to study neural circuits. At the electrical level, voltage sensors can image the precise timing of action potentials and subthreshold voltage not detectable by other means. However, even the latest voltage sensors do not perform well with high-resolution microscopes that use 2-photon illumination for imaging deep in the brain. To overcome these limitations, we are taking a two-pronged approach by evolving amino acids at the mechanistic heart of voltage sensor proteins and by using spectroscopy to aid our protein engineering efforts. We believe directed evolution will improve voltage sensitivity and 2-photon functionality >10 fold, enabling us to image currently invisible signals, like synaptic potentials, deep inside the brain. At the chemical level, neuropeptides are highly expressed in almost all cortical neurons, but their role and impact in animals can only be inferred because current detection methods, like microdialysis, are invasive and lack spatiotemporal resolution. We are using phage display to evolve nanobodies capable of recognizing neuropeptides and coupling their conformational changes to fluorescence changes from reporter molecules. These sensors will provide visualization of neuropeptide release at cellular resolution throughout an animal’s brain during behavior paradigms that mimic human health and disease states. At the cellular connectivity level, current tools for circuit- mapping, like rabies virus, exhibit substantial neurotoxicity, prohibiting meaningful functional analyses. We are engineering proteins with a natural propensity to assemble into structures capable of delivering a genetic payload to specific cells to produce more effective and less toxic tools to map and manipulate brain circuits. Effective and robust tools to map the brain will bridge functional and structural analysis and finally allow long- term studies of neural networks based on their connectivity. Overall, the optogenetic tools developed in this proposal will translate the chemical and electrical signals between neural circuits into fluorescence that can be easily measured. Consequently, they can be used to unravel the functional basis and causes of neuronal disorders at a level of detail that has not been accessible to date and empower us to develop novel treatments.