Next generation high-throughput random access imaging, in vivo

 描述（由申请人提供）：本提案的目标是开发新方法，用于在完整大脑皮层的背景下高速监测单个活神经元的所有输入中的感觉驱动的突触活动。虽然我们的重点是了解突触输入是如何集成在一个完整的电路嵌入一个单一的神经元，我们提出的下一代随机存取成像技术是更广泛地适用于监测多细胞活动代表大型区域内和区域间神经元网络。该方法将当前随机访问技术的速度和灵敏度提高了近2个数量级，从而能够在几分之一毫秒内对多达104个独立位置进行高通量询问，并且与使用下一代电压敏感指示器的成像兼容。在目的1中，我们提出了一个全面的结构图，将允许随机访问扫描的所有兴奋性和抑制性突触的功能定义的锥体细胞类型表达的遗传编码的Ca+2指标。本目标中生成的数据将用于开发图像分割算法，以使用目标2中描述的快速随机访问成像方法，将树突树和相关突触的结构图像快速转换为具有网格坐标的3D位置图，用于已知位置处的活动模式的稀疏采样。在目标2中，我们将构建和开发一种成像系统，该系统允许在10-100 ms内对大约10，000个位置进行高通量随机寻址，这些位置对应于单个神经元上的所有兴奋性突触和其他功能相关的树突和体位点。在目标3中，我们将测试和验证我们的方法的实用性。

 DESCRIPTION (provided by applicant): The goal of this proposal is to develop new methods for high speed monitoring of sensory-driven synaptic activity across all inputs to single living neurons in the context of the intact cerebral cortex. Although our focus is on understanding how synaptic inputs are integrated across a single neuron embedded in an intact circuit, the next generation random access imaging technology we propose is more broadly applicable for monitoring multi-cellular activity representing large intra-and inter areal neuronal networks. The approach improves on the speed and sensitivity of current random-access technology by nearly 2 orders of magnitude, enabling high- throughput interrogation of up to 104 independent locations within a fraction of a millisecond and compatible with imaging using next generation voltage sensitive indicators. In Aim 1 we propose to generate a comprehensive structural map that will allow random access scanning of all excitatory and inhibitory synapses on functionally defined pyramidal cell types expressing a genetically encoded Ca+2 indicator. The data generated in this Aim will be used to develop image segmentation algorithms to quickly convert structural images of the dendritic tree and the associated synapses into a 3D location map with grid coordinates for sparse sampling of activity patterns at known locations using a fast random access imaging approach described in Aim 2. In Aim 2 we will construct and develop an imaging system allowing high throughput, random addressing within 10-100 ms of approximately 10,000 locations corresponding to all excitatory synapses and other functionally relevant dendritic and somal sites on a single neuron. In Aim 3 we will test and validate the utility of our approach.