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

描述(申请人提供)：全光高通量功能连接图谱使用先进的显微镜和光遗传学工具我们提出了一种创新的和翻译的方法来绘制神经元群体中细胞之间的功能连接图谱。连通性图是分析神经元网络的基本步骤。传统上，这种图谱是通过微吸管从成对的神经元进行电记录来建立的。最近，已经使用了多补丁协议，需要复杂的设备和高技能的实验人员。目前，高分辨率的连接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，这种方法主要是产生解剖图，识别功能连接仍然困难。光遗传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高于其尖峰阈值的情况下被光学去极化，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；结合光激活的通道来光学地唤起活动，并结合光学指示器来监控活动。然而， 当使用全光学方法来生成功能连接图时，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到的光信号，因为这些亚阈值突触后电位不会产生峰电位。其次，同时进行光刺激和记录通常需要两个独立的激发波长，因此需要两个昂贵的激光器。幸运的是，这两个挑战都可以应对。基于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的并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，我们将在激发阈值时光学激活假定的突触后细胞，并假定突触前细胞远高于阈值。将突触后细胞的放电概率保持在50%将导致容易检测到的光信号，从而最大限度地提高区分兴奋性和抑制性联系的灵敏度。对于同步刺激和记录，我们将利用较小的双光子激发体积。虽然这一效应被视为为以上工作招募足够的光激活通道的障碍 阈值刺激，我们将利用它来利用单个波长独立地通过扫描照射细胞体来刺激，并通过单点照射来记录。总体而言，所提出的用于通过纯光学手段确定功能连接的协议非常适合于高通量功能连接。多光子激发和3D激光扫描的结合使用使得从大脑切片到活体皮质的转换变得简单。

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.