Revealing circuit control of neuronal excitation with next-generation voltage indicators

摘要 实时记录完整大脑回路中单个神经元的电脉冲一直是一个长期的目标 神经科学。电压记录的一个潜在的广泛应用将是测试突触后的反应 突触前伙伴的生理或光基因激活。在神经元的输入受到控制的情况下记录它 使我们能够详细了解单个神经元是如何处理信息的。这一理解变得很重要 当环路在疾病中改变时，例如在帕金森氏病或亨廷顿病的纹状体中，这将有助于解释 突变表型的发病机制，并提出可能的治疗方法。然而，现在唯一可靠地测量 在体膜电压是进行电生理的，其难度大且通过量低阻碍了广泛的应用 领养。 我们的目标是开发一种新的范例，利用遗传编码来确定神经元的输入输出关系 电压指示器(GEVI)和双光子成像。GEVI可以提供关于亚阈值电压变化的信息， 它们构成了神经元计算的基础，并调节兴奋性，以及神经元动作的时间和顺序 潜力。这些构成了理解大脑回路中信息处理所需的基本基本信息。 然而，活体电压成像目前是有限的。没有已发表的GEVI以足够的速度和幅度做出响应 用于双光子激发下的单次试验中的尖峰检测。 在这个项目中，我们将开发方法来记录大脑中个别神经元的深度电活动，使用两种- 光子显微镜。我们的方法将能够响应双光子照明的GEVI工程与 建立在脑切片和活脑中使用GEVI的条件。具体来说，我们将开展以下工作 目标：(1)产生更明亮、反应更灵敏的ASAP2s变体，这是双光子激发下表现最好的变体，以及 在单光子激发下表现领先的Ace-mNeonGreen；(2)验证GEVI变体的报告能力 特定输入对苍蝇视觉系统中各种神经元的亚阈值和动作电位反应的贡献， 在单项试验中，在活体内；以及(3)系统地测试双光子激发下小鼠纹状体棘的GEVI表现 在体外急性脑片和活体小鼠中的投射神经元，使用GEVI来确定细胞类型- 对最近发现的持续树枝状电压平台现象的特定输入。 该项目将整合蛋白质工程、光学方法开发和三个小组的专业知识 系统神经科学改进GEVI的双光子成像，以便它们可以用于成像单个 审判。如果成功，该项目将向许多感兴趣的研究人员开放GEVI的活体双光子成像， 潜在地催化了我们测量活着的大脑中神经元反应的方式的转变。

ABSTRACT Recording the electrical impulses of individual neurons in intact brain circuits in real time has been a longstanding goal in neuroscience. One potentially widely applicable use of voltage recording would be to test postsynaptic responses upon physiological or optogenetic activation of presynaptic partners. Recording a neuron while its inputs are controlled would enable a detailed understanding of how individual neurons process information. This understanding becomes important when circuity is altered in disease, e.g. in the striatum with Parkinson's or Huntington's diseases, as it would help explain the pathogenesis of the mutant phenotype and suggest possible therapies. However, the only way now to reliably measure membrane voltage in vivo is to perform electrophysiology, whose difficulty and low throughput hamper widespread adoption. We aim to develop a new paradigm for determining the input-output relationshps of neurons using genetically encoded voltage indicators (GEVIs) and two-photon imaging. GEVIs can provide information on subthreshold voltage changes, which form the basis of neuronal computation and modulate excitability, and on timing and order of neuronal action potentials. These constitute basic essential information required for understanding information processing in brain circuits. However, in vivo voltage imaging is currently limited. No published GEVIs respond with sufficient speed and amplitude for spike detection in single trials under two-photon excitation. In this project, we will develop methods to record electrical activity from individual neurons in the brain at depth using two- photon microscopy. Our approach combines engineering of GEVIs that can respond to two-photon illumination with the establishment of conditions for using GEVIs in brain slices and living brains. Specifically, we will carry out the following aims: (1) Generate brighter and more responsive variants of ASAP2s, the best performer under two-photon excitation, and of Ace-mNeonGreen, a leading performer under one-photon excitation; (2) validate GEVI variants for their ability to report contributions of specific inputs to subthreshold and action potential responses in a variety of neurons of the fly visual system, in single trials, in vivo; and (3) systematically test GEVI performance under two-photon excitation in mouse striatal spiny projection neurons in ex vivo acute brain slices and in living mice in vivo, using GEVIs to determine the role of cell type- specific inputs to a recently discovered phenomenon of long-lasting dendritic voltage plateaus. This project will integrate the expertise of three groups spanning protein engineering, optical method development, and systems neuroscience to improve two-photon imaging of GEVIs so they can be used to image voltage transients in single trials. If successful, this project will open up in vivo two-photon imaging of GEVIs to many interested researchers, potentially catalyzing a transformation in how we measure neuronal responses in living brains.