Liang Conrad W, Mohammadi Michael, Santos M Daniel, Tang Cha-Min
Department of Neurology, Baltimore VA Medical Center, University of Maryland School of Medicine, USA.
J Vis Exp. 2011 Mar 2(49):2003. doi: 10.3791/2003.
Light is a versatile and precise means to control neuronal excitability. The recent introduction of light sensitive effectors such as channel-rhodopsin and caged neurotransmitters have led to interests in developing better means to control patterns of light in space and time that are useful for experimental neuroscience. One conventional strategy, employed in confocal and 2-photon microscopy, is to focus light to a diffraction limited spot and then scan that single spot sequentially over the region of interest. This approach becomes problematic if large areas have to be stimulated within a brief time window, a problem more applicable to photostimulation than for imaging. An alternate strategy is to project the complete spatial pattern on the target with the aid of a digital micromirror device (DMD). The DMD approach is appealing because the hardware components are relatively inexpensive and is supported by commercial interests. Because such a system is not available for upright microscopes, we will discuss the critical issues in the construction and operations of such a DMD system. Even though we will be primarily describing the construction of the system for UV photolysis, the modifications for building the much simpler visible light system for optogenetic experiments will also be provided. The UV photolysis system was used to carryout experiments to study a fundamental question in neuroscience, how are spatially distributed inputs integrated across distal dendritic branch points. The results suggest that integration can be non-linear across branch points and the supralinearity is largely mediated by NMDA receptors.
光作为一种控制神经元兴奋性的手段，具有通用性和精确性。最近引入的诸如通道视紫红质和笼锁神经递质等光敏感效应器，引发了人们对开发更好的方法来控制光在空间和时间上的模式的兴趣，这对于实验神经科学很有用。共聚焦显微镜和双光子显微镜中采用的一种传统策略是将光聚焦到一个受衍射限制的点上，然后在感兴趣的区域上依次扫描该单个点。如果必须在短时间窗口内刺激大面积区域，这种方法就会出现问题，这个问题在光刺激中比在成像中更适用。另一种策略是借助数字微镜器件（DMD）将完整的空间模式投射到目标上。DMD方法很有吸引力，因为硬件组件相对便宜，并且有商业利益的支持。由于这种系统不适用于直立显微镜，我们将讨论这种DMD系统构建和操作中的关键问题。尽管我们将主要描述用于紫外光解的系统的构建，但也将提供构建用于光遗传学实验的更简单的可见光系统的修改方法。紫外光解系统用于进行实验，以研究神经科学中的一个基本问题，即空间分布的输入如何在远端树突分支点上进行整合。结果表明，跨分支点的整合可能是非线性的，并且超线性主要由NMDA受体介导。
