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

基本信息

批准号：
8582420
负责人：
PETER SAGGAU
金额：
$ 22.93万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
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/8582420
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

项目摘要

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）：使用高级显微镜和光遗传学工具的全光通量功能连通性映射我们提出了一种创新和转化的方法，以绘制神经元种群中细胞之间的功能连接。连接图是分析神经元网络的基本步骤。传统上，通过微角对神经元的电记录来确定此类地图。最近，已经使用了多块协议，需要复杂的设备和高技能的实验人员。高分辨率连接组目前是通过先进的组织学技术建立的，涉及串行切片和电子显微镜，但是，这种方法主要产生解剖图，并且功能连接的鉴定仍然很困难。光遗传学工具和两光子显微镜具有巨大变化的功能连接映射。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光学地去极化，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，从光学上激活突触前神经元，并通过全细胞记录测量突触后信号。最近，已经探索了全光学映射方法。结合光激活的通道以光学唤起活动和光学指标以监测活动。然而，当使用全光学方法生成功能连通图时，出现了两个主要挑战：首先，激活单个突触前神经元将难以检测突触后细胞中的光学信号，因为这些亚阈值突触后潜能不会产生尖峰。其次，并发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，可以满足这两个挑战。在我们的高级光学成像方面的专业知识的基础上，我们将利用实验室中开发的3D激光扫描技术，并由许多研究小组成功应用。为了可靠地检测到单个突触连接，我们将在发射阈值和假定的突触前细胞时，光学地激活推测的突触后细胞，远高于阈值。将突触后细胞保持在50％的射击概率将导致容易检测到的光学信号，从而最大程度地提高对区分兴奋性和抑制性连接的敏感性。为了同时进行刺激和记录，我们将利用小的两光激发量。虽然这种效果被视为招募足够的光激活通道的障碍阈值刺激，我们将利用它利用单个波长来独立刺激细胞体照明并通过单点照明记录。总体而言，提出的用于确定纯光学手段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激光扫描的联合使用使人可以直接地翻译到体内皮层。