DEFINING, STIMULATING AND RECORDING NEURAL CIRCUITS USING LIGHT

描述(由申请人提供)：我们的目标是开发一种基于光学方法的系统，用于在体外创建和测试神经电路。我们开发了一种多光子激光技术(活细胞衬底图案化或LCSP)，允许在细胞电镀后对衬底进行图案化。根据新的生长动态改变衬底的能力使我们能够创建定义良好的简单(少数单元)电路。使用传统的电生理学方法对这些电路进行了功能测试。我们现在计划使用钙成像来记录活动的变化。细胞将被负载钙指示剂或被转染以表达钙指示剂蛋白。我们还将使用光刺激来激活或沉默分别表达通道视紫红质-2或卤视紫质的细胞。这种全光学方法将允许细胞的重复激活和成像，这对于研究突触形成过程中的缓慢变化事件是必要的。例如，将有可能解决诸如突触活动何时开始起作用以及突触形成如何随着活动的变化而改变等问题。它还将提供一种测试广泛的细胞和分子机制的手段，这些机制可能是电路结构或功能的基础。 公共卫生相关性：需要新的方法来创建和测试神经回路，以便对神经功能进行建模。使用微创光学方法，我们将能够控制电路的形成，并随着时间的推移以高分辨率刺激和记录活动。

DESCRIPTION (provided by applicant): Our goal is to develop a system based on optical methods for creating and testing neural circuits in vitro. We have developed a multiphoton laser technique (Live Cell Substrate Patterning or LCSP) that allows substrates to be patterned after cells have been plated. The ability to alter substrates dynamically in response to new growth enables us to create well-defined simple (few-cell) circuits. Functional tests of these circuits have been made using conventional electrophysiological methods. We now plan to record changes in activity using calcium imaging. Cells will be loaded with a calcium indicator dye or transfected to express a calcium indicator protein. We will also use light stimulation to activate or silence cells expressing channelrhodopsin-2 or halorhodopsin, respectively. This all-optical approach will allow repeated activation and imaging of cells as will be necessary for studies of slow-changing events during synapse formation. For example, it will be possible to address questions such as when synaptic activity first becomes functional and how synapse formation may be altered in response to changes in activity. It will also provide a means for testing a wide range of cellular and molecular mechanisms that may underlie structure or function of circuits. PUBLIC HEALTH RELEVANCE: New approaches are needed to create and test neural circuits in order to model neural function. Using minimally invasive optical methods we will be able to control circuit formation and also stimulate and record activity at high resolution over time.