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

 描述(由申请人提供)：精神和其他神经疾病经常在发育结束期间或接近发育结束时出现，就像发育中发生的变化一样，它们的症状通常是永久性的。这提供了强有力的证据，表明发育病理可能与这些疾病的发生有关。为了了解发育异常的影响，它们可能如何引起精神或神经障碍的症状，因此什么治疗方法可能是合适的，对指导正常发育的原则的基线理解是至关重要的。发育在神经系统的某些部分表现为神经元及其靶点之间的突触重新布线。在很大程度上，人们对指导这一过程的机制知之甚少。在神经肌肉接头等周围神经系统容易接触到的部分，活体荧光成像和电生理学等技术揭示了发生在那里的重新连接过程的一些信息：通常，几类神经元一开始高度相互连接，随着发育，许多连接被修剪，而幸存的突触得到加强，从而产生了精致的神经连接。这一过程被称为“突触消除”，被认为是由突触活动驱动的，因此也就是经验。然而，类似的技术在中枢神经系统中提供的信息要少得多。为了克服这一障碍，了解高等教育中心的突触重新连接，该项目提出使用连续切片扫描电子显微镜来产生3D卷的小鼠出生后早期发育的野生型小脑组织的高分辨率图像。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这种技术的最新改进，它将一长串薄片切割、收集到磁带上并自动成像，能够以最小的损失对数百微米厚的组织块进行成像，并且速度相当快。小脑是一个很好的研究系统，因为它本质上简单而紧凑。在这个项目中，浦肯野细胞及其攀升纤维输入将被重建，并将测量它们突触的数量、位置和强度，以建立这些信息的基本事实。然后，这将被用来提供关于小脑重新连接潜在机制的初步见解，方法是：第一，确定这一过程是微小的改进还是主要的重新连接；第二，小脑重新连接的媒介是否类似于外周突触消除的媒介。这项工作是了解中枢神经系统正常发育的基础的重要一步，最终将为研究发育障碍的根本原因奠定基础。在这次培训中，为了调查人员的利益，提供了神经科学教学内容。]

 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.]