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

描述（由申请人提供）：使用先进显微镜和光遗传学工具绘制全光高通量功能连接图我们提出了一种创新的和可转化的方法来绘制神经元群体中细胞之间的功能连接。连通性图是分析神经网络的基本步骤。传统上，这种图谱是通过利用微移液管对神经元进行电记录来建立的。最近，多补丁协议已被使用，需要复杂的设备和高技能的实验者。高分辨率连接体目前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谱，识别功能连接仍然很困难。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峰值阈值以上进行光学去极化，并且可以在连接的细胞中监测突触后信号。最初，采用混合方法，通过光学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，人们探索了全光学映射方法；结合光激活通道光学唤起活动和光学指标监测活动。然而,

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.