CRCNS: Transmitter Release Site Organization in Plasticity and Disease at the NMJ

描述（由申请人提供）：神经系统中细胞之间的交流是所有复杂行为的基础，发生在神经细胞被称为突触的特殊区域。突触的工作原理是从一个叫做活跃区的区域释放化学递质，激活邻近的细胞。我们提出表征活动区功能和结构组织之间的关系，在青蛙和老鼠神经肌肉突触。我们假设神经肌肉活动区是由一个基本的递质释放构建块组装而成的：由停靠的突触囊泡及其相关的Ca2+通道组成的不可靠的单囊泡释放位点。我们进一步假设突触功能和突触前稳态可塑性的主要方面可以通过活性区内这些单囊泡释放位点的数量和组织的变化来解释。我们的方法的特点是三个实验室之间的无缝协作，具有细胞生理学（Dittrich实验室），突触解剖学，生理学和Ca2+成像（Meriney实验室）的计算机模拟专业知识，以及突触蛋白数量和空间分布的超分辨率成像（Blanpied实验室）。重要的是，作为该提案的一部分，来自所有三个实验室的学员将在每个实验室接受交叉训练。我们将使用这种合作的方法来开发一个突触前递质释放位点的综合mccell计算机模型，这将大大增加我们对活跃区组织和突触功能之间关系的理解。这种洞察力不仅会带来更好的

DESCRIPTION (provided by applicant): Communication between cells in the nervous system underlies all complex behaviors, and occurs at specialized regions of the nerve cell called synapses. Synapses work by releasing chemical transmitter from a region called the active zone, which activates a neighboring cell. We propose to characterize the relationship between active zone function and structural organization within frog and mouse neuromuscular synapses. We hypothesize that neuromuscular active zones are assembled from a basic transmitter release building block: the unreliable single-vesicle release site consisting of a docked synaptic vesicle and its associated Ca2+ channels. We further hypothesize that major aspects of synaptic function and presynaptic homeostatic plasticity can be explained by changes in the number and organization of these single-vesicle release sites within active zones. Our approach is characterized by a seamless collaboration between three labs with expertise in computer simulations of cellular physiology (Dittrich lab), synaptic anatomy, physiology, and Ca2+ imaging (Meriney lab), and super-resolution imaging of the number and spatial distribution of synaptic proteins (Blanpied lab). Importantly, as part of this proposal, trainees from all three laboratories will receive crosstraining in each lab. We will use this collaborative approach to develop a comprehensive MCell computer model of the presynaptic transmitter release site that will significantly increase our understanding of the relationship between active zone organization and synaptic function. This insight will not only lead to a better understanding of presynaptic mechanisms of homeostatic plasticity but also aid in our understanding of synaptic diseases, which are known to underlie a large number of neurological disorders. Intellectual Merit: A significant number of neurological diseases are known to affect the synapse by targeting synaptic organization and function. While most research on this important topic has to date focused on postsynaptic adaptations, it has become increasingly clear that presynaptic homeostatic changes are likely to be just as important. Thus, a better understanding of the role of presynaptic structure and organization in synaptic function under both control and disease conditions is needed. Broader Impacts: The MCell model that we will develop will enhance our teaching mission in many ways. It will provide an example of unprecedented scale and realism for the illustration of nerve terminal structure and function. This material will be used in courses and programs at the University of Pittsburgh, the University of Maryland, and Carnegie Mellon University. These include undergraduate and graduate Neuroscience courses, a Computational Biology PhD program that spans PITT and Carnegie Mellon University, summer workshops, and web-based tutorials (www.mcell.org). These simulations will expand previous models that already have been converted into instructive 3D movies, which are routinely shown to a broad range of audiences during open houses, student visits or classroom teaching. This work will also provide source material for teaching examples tailored to high school outreach programs at the Pittsburgh Supercomputing Center, particularly the CMIST program (Computational Modules in Science Teaching, www.cmist.org) of the National Resource for Biomedical Supercomputing (NRBSC) directed by Dr. Dittrich. Our proposed work will have a broad impact on K-12 education, undergraduate teaching and training, graduate and post-graduate training, community outreach, STEM teaching, training at underrepresented minority institutions, and knowledge of synaptic function in the field. Dr. Meriney is a member of the Neuroscience outreach committee at the University of Pittsburgh (PITT), which organizes a variety of community events. Dr. Meriney's laboratory is in the Arts and Sciences College, so the proposed research would contribute to undergraduate teaching via undergraduate research participation in the proposed work, and changes to content for undergraduate courses based on new research insights. Dr. Dittrich will also train undergraduate students in his laboratory as participants in the proposed work. He is training faculty in the NSF funded TECBio REU program at the PITT and typically mentors 1-2 students in computational projects as part of the program. In addition, Dr. Dittrich is a training faculty in the PA Governors School for the Sciences, an intense summer program for talented high school students in Pennsylvania. Drs. Dittrich, Meriney, and Blanpied will bring graduate researchers and postdoctoral fellows into their labs who will directly participate in the proposed experiments, receive cross training in all three laboratories, and receive career training. Lastly, Dr. Ulises Ricoy (an under-represented minority faculty member) from Northern New Mexico College will visit during each summer to learn new research, teaching, and training tools to bring back to underrepresented minority undergraduates at Northern New Mexico College. This will expose these underrepresented minority students to an intense academic research environment and aid in their training and career planning.