Quantifying Synaptic Reorganization in the Developing Cerebellum Using Serial-Section Scanning Electron Microscopy Data

 描述（由申请人提供）：精神和其他神经系统疾病经常在发育期间或接近发育结束时出现，并且与发育中发生的变化一样，其症状通常是永久性的。这提供了强有力的证据表明，发育病理学可能与这些疾病的发病率有关。为了了解发育异常的影响，它们如何引起精神或神经障碍的症状，因此什么样的治疗可能是适当的，对正常发育原则的基本理解是必不可少的。发育在神经系统的某些部分中表现为神经元类别与其目标之间的突触重新布线。在大多数情况下，对指导这一进程的机制的了解很少。在周围神经系统的容易接近的部分，如神经肌肉接头，体内荧光成像和电生理学等技术已经揭示了一些关于发生在那里的重新布线过程的信息：一般来说，神经元的类别最初是高度互连的，随着发育，许多连接被修剪，而幸存的突触被加强，导致神经布线精细化。这个过程被称为“突触消除”，被认为是由突触活动驱动的，因此是经验。然而，类似的技术在中枢神经系统中的信息量要少得多。为了克服这一障碍，以了解突触重新布线在高等教育中心，该项目提出使用连续切片扫描电子显微镜，以产生高分辨率图像的野生型小脑组织的小鼠在出生后早期发育的三维体积。与其他方法不同，电子显微镜的分辨率足以清楚地识别组织样本中的所有突触。连续切片扫描电子显微镜是该技术的最新改进，其中切割一长串薄切片，收集在胶带上，并自动成像，能够以最小的损失对100微米厚的组织块进行成像，并且相当快。小脑是一个很好的研究系统，因为它本质上是简单和紧凑的。[In这个项目将重建浦肯野细胞和它们的攀爬纤维输入，并测量它们的突触的数量、位置和强度，以建立这些信息的基础事实。然后，这将被用来提供初步了解小脑重新布线的机制，通过确定，第一，这个过程是一个小的改进或主要的重新布线;第二，小脑重新布线的介质是否类似于外周突触消除。这项工作是理解中枢神经系统正常发育基础的重要一步，最终将为研究发育障碍的根本原因奠定基础。为了研究者的利益，本培训中有一个教学神经科学部分。]

 DESCRIPTION (provided by applicant): Mental and other neurological disorders frequently surface during or near the end of development and, like the changes that occur in development, their symptoms are typically permanent. This provides strong evidence that developmental pathologies may be related to incidence of these disorders. In order to understand the effects of abnormalities in development, how they may give rise to symptoms of a mental or neurological disorder, and therefore what therapies might be appropriate, a baseline understanding of the principles that govern normal development is essential. Development is physically manifested in parts of the nervous system as a rewiring of synapses between classes of neurons and their targets. For the most part, the knowledge of the mechanisms that guide this process is poor. In easily accessible parts of the peripheral nervous system like the neuromuscular junction, techniques like in vivo fluorescence imaging and electrophysiology have revealed some information about the rewiring process that occurs there: in general, classes of neurons are initially highly interconnected and over development many of the connections are pruned, while surviving synapses are strengthened, resulting in a refined neural wiring. This process is called "synapse elimination" and is thought to be driven by synaptic activity and therefore experience. Similar techniques have been much less informative in the central nervous system, however. In order to overcome this barrier to understanding synaptic rewiring in higher learning centers, this project proposes to use serial section scanning electron microscopy to produce 3D volumes of high-resolution images of wild-type cerebellum tissue from mice in early postnatal development. Unlike other methods, the resolution of electron microscopy is sufficient to clearly identify all synapses in a tissue sample. Serial section scanning electron microscopy, a recent adaptation of this technique in which a long series of thin sections is cut, collected on tape, and automatically imaged, is capable of imaging blocks of tissue 100s of microns thick with minimal loss, and reasonably quickly. The cerebellum is a good system to investigate because it is intrinsically simple and compact. [In this project Purkinje cells and their climbing fiber inputs wll be reconstructed and the numbers, positions, and strengths of their synapses will be measured to establish a ground truth for this information. This will then be used to provide initial insight about the mechanisms underlying cerebellar rewiring by determining, first, whether this process is a minor refinement or a major rewiring; and second, whether or not the mediators of cerebellar rewiring are similar to those of peripheral synapse elimination. This work is an essential step toward understanding the underpinnings of normal development in the central nervous system, which will eventually lay the groundwork for investigations into the underlying causes of developmental disorders. A didactic neuroscience component is present in this training for the benefit of the investigator.]