ALL-OPTICAL HIGH-THROUGHPUT FUNCTIONAL CONNECTIVITY MAPPING USING ADVANCED MICROS

使用 Advanced Micros 进行全光高通量功能连接映射

• 批准号: 8675233
• 负责人: PETER SAGGAU
• 金额: $ 18.98万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8675233
• 关键词: Biomedical Engineering; Brain; Cells; Computers; Detection; Electron Microscopy; Elements; Equipment; Genetic Engineering; Goals; Histological Techniques; Hybrids; Image; Imaging Device; Imaging Techniques; Individual; Ion Channel; Label; Laser Scanning Microscopy; Lasers; Life; Light; Lighting; Maps; Measures; Methods; Microscopy; Monitor; Nature; Neurons; Neurosciences; Optics; Population; Positioning Attribute; Postdoctoral Fellow; Probability; Property; Protocols documentation; Recruitment Activity; Research; Research Infrastructure; Resolution; Scanning; Signal Transduction; Site; Slice; Source; Speed; Structure; Supervision; Synapses; Technology; Testing; Translations; Whole-Cell Recordings; Work; base; bioimaging; brain tissue; calcium indicator; density; design; experience; flexibility; graduate student; improved; in vivo; information processing; innovation; instrumentation; interest; lens; meetings; multi-photon; neuronal cell body; novel; optical imaging; optogenetics; postsynaptic; presynaptic; protocol development; public health relevance; tool; translational approach; two-photon

DESCRIPTION (provided by applicant): All-Optical High-Throughput Functional Connectivity Mapping using Advanced Microscopy and Optogenetic Tools We propose an innovative and translational approach to map functional connections between cells in neuronal populations. Connectivity maps are the fundamental step to analyze neuronal networks. Traditionally, such maps were established by electrically recording from pairs of neurons by means of micropipettes. More recently, multi-patch protocols have been used, requiring complicated equipment and highly skilled experimenters. High-resolution connectomes are presently established by advanced histological techniques, involving serial sectioning and electron microscopy, however, this approach primarily produces anatomical maps and identification of functional connections remains difficult. Optogenetic tools and two-photon microscopy have dramatically changed functional connectivity mapping. For example, neurons expressing light-activated ion channels can be optically depolarized above their spiking threshold, and postsynaptic signals can be monitored in connected cells. Initially, hybrid approaches were taken, activating presynaptic neurons optically and measuring postsynaptic signals by whole-cell recording. More recently, all-optical mapping methods have been explored; combining light-activated channels to optically evoke activity and optical indicators to monitor activity. However, when using an all-optical approach to generate functional connectivity maps, two main challenges arise: Firstly, activating individual presynaptic neurons will produce hard to detect optical signals in postsynaptic cells as these sub-threshold postsynaptic potentials do not generate spiking. Secondly, concurrent optical stimulation and recording usually requires two separate excitation wavelengths and thus two costly lasers. Fortunately, both challenges can be met. Building on our expertise in advanced optical imaging, we will utilize 3D laser scanning technology developed in our lab and successfully applied by many research groups. To reliably detect single synaptic connections, we will optically activate presumed postsynaptic cells just at firing threshold and presumed pre- synaptic cells well above threshold. Keeping postsynaptic cells at 50% firing probability will result in readily detectable optical signals, maximizing the sensitivity for both discriminating excitatory and inhibitory connections. For concurrent stimulation and recording, we will take advantage of the small two-photon excitation volume. While this effect was seen as an obstacle to recruit sufficient light- activated channels for supra threshold stimulation, we will utilize it to employ a single wavelength to independently stimulate by scanning illumination of cell bodies and record by single-point illumination. Overall, the proposed protocol for determining functional connections by pure optical means is ideally suited for high-throughput functional connectomics. The combined use of multi-photon excitation and 3D laser scanning makes the translation from brain slices to in vivo cortex straightforward.

描述（由申请人提供）：使用先进显微镜和光遗传学工具的全光学高通量功能连接图谱我们提出了一种创新的转化方法来绘制神经元群体中细胞之间的功能连接。连接图是分析神经网络的基本步骤。传统上，这种图谱是通过微量移液管对神经元对进行电记录来建立的。最近，已经使用了多补丁方案，需要复杂的设备和高技能的实验人员。目前，高分辨率连接组是通过先进的组织学技术建立的，包括连续切片和电子显微镜，然而，这种方法主要产生解剖图，功能连接的识别仍然很困难。光遗传学工具和双光子显微镜极大地改变了功能连接图谱。例如，表达光激活离子通道的神经元可以在其尖峰阈值之上进行光学去极化，并且可以在连接的细胞中监测突触后信号。最初，采用了混合方法，以光学方式激活突触前神经元，并通过全细胞记录测量突触后信号。最近，人们探索了全光学测绘方法；结合光激活通道以光学诱发活动和光学指示器以监测活动。然而， 当使用全光学方法生成功能连接图时，出现了两个主要挑战：首先，激活单个突触前神经元将在突触后细胞中产生难以检测的光信号，因为这些亚阈值突触后电位不会产生尖峰。其次，同时进行的光刺激和记录通常需要两个单独的激发波长，因此需要两个昂贵的激光器。幸运的是，这两个挑战都可以得到解决。凭借我们在先进光学成像方面的专业知识，我们将利用我们实验室开发并已被许多研究小组成功应用的 3D 激光扫描技术。为了可靠地检测单个突触连接，我们将在激发阈值处光学激活假定的突触后细胞，并在远高于阈值的情况下激活假定的突触前细胞。将突触后细胞保持在 50% 的放电概率将产生易于检测的光学信号，从而最大限度地提高区分兴奋性和抑制性连接的灵敏度。对于并发刺激和记录，我们将利用小的双光子激发体积。虽然这种效应被视为招募足够的光激活通道以实现上述目的的障碍。 阈值刺激，我们将利用它采用单一波长通过细胞体的扫描照明进行独立刺激并通过单点照明进行记录。总体而言，所提出的通过纯光学手段确定功能连接的协议非常适合高通量功能连接组学。多光子激发和 3D 激光扫描的结合使用使得从大脑切片到体内皮层的转换变得简单。