New approaches for better protein voltage sensors

该提案旨在开发更好的工具来分析脑细胞和电路， 大脑活动的大规模记录。目前可用的工具在灵敏度方面相对原始 和速度.神经元的一个主要功能是处理电信号。因此，一个工具，特别是 重要的是高速膜电位成像。遗传编码荧光蛋白电压 指标（GEVI）是一个明显的战略方法“可视化大脑的行动”。遗传编码 传感器对神经科学家来说特别有趣，因为作为蛋白质，它们可以在个体中表达 哺乳动物大脑中的细胞类型。因为每个大脑区域有多达100种不同的细胞类型， 在特定细胞类型中的表达对于成像该细胞类型的活性是必需的。 最近，GEVI的信号大小有了显著的改进（即，ArcLight，40%/100 mv），但ArcLight具有相对较慢的响应时间常数（τ=10 msec）。现在有几个更快的GEVI （τ=0.3至2.0毫秒），但它们具有较小的信号大小（~10%/100 mv）。该提案的一个目标是制定一个 GEVI具有对膜电位变化的大而快的响应。 除尺寸和速度外，几个探头特性也至关重要。一是 激发和发射的波长范围。目前，许多传感器和激活剂都是基于GFP及其 类似物因此，具有红色激发和发射光谱的探针将允许同时的双重功能 测量.一个目标是开发有用的红色GEVI。其次，许多GEVI都有一个S形曲线， 荧光-电压关系该关系沿着电压轴的位置可以通过 GEVI的电压敏感域中的突变。因此，GEVI可以是不同范围的选择性报告物 的神经元膜电位，从而选择性的动作电位活动与阈下活动。 这种选择性取决于半最大激活时的电压以及S形曲线的陡度。 曲线最后，重要的是将GEVI靶向神经元的特定区域，包括细胞体， 树突状突触后区和突触前末梢。 该方案中的所有探针都是基于膜蛋白的电压敏感结构域（a 来自玻璃海鞘或斑马鱼的磷酸酶），在N-或C-末端插入一个或两个荧光蛋白 地区改进的GEVI的开发将涉及用于产生新探针的分子生物学（更大， 更快，红色，解剖学和生理学靶向），然后在培养的HEK 293细胞中进行急性 分离的神经元和斑马鱼胚胎。在这些初始屏幕中运行良好的探头将被 用于哺乳动物脑中的体内转染和测量。改进 选择性GEVI将有助于社区进行大脑活动的光学测量。

This proposal aims to develop better tools for analyzing brain cells and circuits and for large-scale recordings of brain activity. The currently available tools are relatively primitive in terms of sensitivity and speed. One major function of a neuron is to process electrical signals. Thus a tool that is of particular significance is high speed membrane potential imaging. Genetically encoded fluorescent protein voltage indicators (GEVI's) are a obvious strategic approach for “visualizing the brain in action”. Genetically encoded sensors are especially interesting to neuroscientists because, as proteins, they can be expressed in individual cell types in the mammalian brain. Because each brain region has up to 100 different cell types, sensor expression in a specific cell type is essential for imaging the activity of that cell type. Recently, there has been a dramatic improvement in the signal size of GEVIs (i.e. ArcLight, 40%/100 mv) but ArcLight has a relatively slow response time constant (τ=10 msec). There are now several faster GEVIs (τ=0.3 to 2.0 msec) but they have smaller signal sizes (~10%/100mv). One goal of this proposal is developing a GEVI with both large and fast responses to membrane potential changes. Several probe characteristics other than size and speed are also critically important. One is the wavelength range of excitation and emission. At present many sensors and activators are based on GFP and its analogues. Thus, probes with red excitation and emission spectra would allow simultaneous dual function measurements. One aim is to develop useful red GEVIs. Second, many GEVIs have a sigmoidal fluorescence-voltage relationship. The position of this relationship along the voltage axis can be adjusted via mutations in the voltage sensitive domain of the GEVI. Thus GEVIs can be selective reporters of different ranges of the neuron membrane potential and thereby selective for action potential activity versus subthreshold activity. This selectivity depends on both the voltage at half-maximal activation as well as the steepness of the sigmoidal curve. Lastly, it will be important to target the GEVIs to specific regions of the neuron including cell body, dendritic post-synaptic zones, and presynaptic terminals. All of the probes in the proposal are based on the voltage sensitive domain of a membrane protein (a phosphatase from Ciona or zebrafish) with one or two fluorescent proteins inserted into the N- or C-terminal region. The development of improved GEVIs will involve molecular biology for generating novel probes (bigger, faster, red, targeted anatomically and physiologically) followed by testing in cultured HEK293 cells, acutely dissociated neurons, and zebrafish embryos. Probes that function well in these initial screens will then be incorporated into virus particles for in vivo transfection and measurements in the mammalian brain. Improved and selective GEVIs would be useful to the community carrying out optical measurements of brain activity.