Large-scale connectivity and function in a cortical circuit

描述（由申请人提供）：神经科学中一个基本但尚未解决的问题是神经元之间的特定连接如何在皮层回路中构成信息处理的基础。大脑皮层的局部回路由数万个神经元组成，每个神经元都有数千个连接。也许我们不了解这些电路的最大原因是我们从来没有能够重建它们的实际接线图。但如果我们有一个部分甚至完整的接线图，我们还需要知道回路中的每个神经元在做什么：它的生理机能。在本提案中，我们计划开发和应用大规模电子显微镜的新方法，以实现在功能背景下绘制皮层电路接线图的目标。我们将使用双光子钙成像来观察局部电路中神经元的活动。然后，我们将使用大规模串行切片电子显微镜来追踪同一块皮质中的电路。功能成像和连续切片电子显微镜（EM）的最新进展使我们能够研究这个难题，但在我们能够获得这些方法的好处之前，还需要大量的技术工作。功能成像与双光子显微镜是一个技术成熟的领域，但方法的大规模串行切片EM仍处于起步阶段。我们建议应用功能成像和高分辨率解剖成像的双重方法，我们已经在一个大型试点项目中进行了一次，目的是改进与功能研究相关的大规模EM技术。我们的四年目标是创建一个高通量系统，用于从皮层生成相关的结构/功能数据集。特别是，我们将建立一个新的EM成像系统，第二代传输EM相机阵列（TEMCA），这将使我们能够在一周内捕获非常大的三维数据集（300到500微米），而不是几个月。我们建议解决一类问题：在每个局部皮质回路中是否存在处理不同信息的子网络？但该方法是通用的，可以应用于广泛的问题，包括临床相关的问题。阿尔茨海默病斑块附近的神经连接是否中断？当干细胞整合到一个回路中时，它们会形成起功能作用的连接吗？第一次，这些问题应该触手可及。通过开发用于大规模成像的高通量方法，我们将开始以自己的方式研究神经回路：在所有的复杂性和在许多意义上完整的数据集。

DESCRIPTION (provided by applicant): A fundamental but unsolved question in neuroscience is how specific connections between neurons underlie information processing in the cortical circuits. Local circuits in the cerebral cortex consist of tens of thousands of neurons, each making thousands of connections. Perhaps the biggest reason we don't understand these circuits is that we have never been able to reconstruct their actual wiring diagrams. But if we had a partial or even complete wiring diagram, we would also need to know what each neuron in a circuit is doing: its physiology. In this proposal we plan to develop and apply new approaches for large-scale electron microscopy, towards the goal of mapping wiring diagrams of cortical circuits in a functional context. We will use two-photon calcium imaging to see the activity of neurons in a functioning local circuit. We will then use large-scale serial-section electron microscopy to trace circuits in the same piece of cortex. Recent advances in functional imaging and serial-section electron microscopy (EM) allow us to study this difficult problem, but before we can reap the benefits of these approaches, considerable technical work is necessary. Functional imaging with two-photon microscopy is a technically mature field, but approaches for large-scale serial-section EM are still in their infancy. We propose to apply the dual approach of functional imaging followed by high-resolution anatomical imaging-which we have already performed once in a large pilot project-with the goal of improving the technologies specifically for large-scale EM, correlated with functional studies. Our four-year goal is to create a high-throughput system for generating correlated structure/function data sets from the cortex. In particular, we will build a new EM imaging system, a second-generation Transmission EM Camera Array (TEMCA), that will allow us to capture very large three-dimensional data sets (300 to 500 micrometers on a side) in a week, rather than months. We propose to address one class of question: are there subnetworks within each local cortical circuit that process distinct information? But the approach is general and can be applied to a wide range of questions, including clinically relevant ones. Are neural connections disrupted near plaques in Alzheimer's disease? When stem cells incorporate into a circuit, do they form connections that play a functional role? For the first time, these questions should be within our reach. By developing high-throughput methods for large-scale imaging, we will begin to study neural circuits on their own terms: in all of their complexity and with data sets that are in many senses complete.