Combined Atomic Force Microscopy/Fluorescence Spectroscopy approach for measuring adhesion, connectivity and electrical activity of neurons patterned on 2-dimensional protein subst

原子力显微镜/荧光光谱相结合的方法用于测量二维蛋白质基质上神经元的粘附、连接和电活动

• 批准号: 1067093
• 负责人: Cristian Staii
• 金额: $ 33万
• 依托单位: Tufts University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2014-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1067093&HistoricalAwards=false
• 关键词: Combined; Atomic; Force; Microscopy; Fluorescence

1067093StaiiIntellectual merit: The objective of this proposal is to gain a deeper understanding of the basic rules that neuronal cells use to form functional connections with one another. Understanding the brain is of tremendous fundamental importance, but it is immensely challenging because of the complexity of both its architecture and function. The central nervous system consists of many different spatially localized and yet highly interconnected regions. To date the processes involved in forming functional neuronal connections, the mechanisms of axonal navigation to their target region and their specific interactions with guidance factors such as chemical gradients and mechanical cues are still largely unknown. The scientific goal of the current project is to understand the fundamental processes governing the development of connections and communications between neurons in living systems by studying the growth and interconnectivity of small numbers of neurons patterned in simplified, well-controlled geometries. The central hypothesis is that simplifying the neuronal growth environment by creating highly controlled neuronal circuits in vitro will allow the basic rules that underlie neuronal development and the formation of neural connections to be elucidated.Simple neuronal networks will be created on two dimensional substrates, guiding the formation of synapses and measuring their electrical activity using a) atomic force microscope nanolithography; b) atomic force imaging and atomic force based electrical force microscopy; c) fluorescence spectroscopy. Specifically, one aims to: 1) pattern different types of proteins/growth factors at precise locations on surfaces and use them as growth templates for fluorescently labeled neurons; 2) guide the formation of neuronal synapses by controlling the type and geometry of the underlying protein patterns; 3) systematically investigate the adhesion and growth of neuronal processes using both atomic force and fluorescence spectroscopy measurements; 4) map the electrical activity of the network by combined electrical force microscopy and fluorescence microscopy. The crucial aspect for this last step is the use of a voltage-biased atomic force tip as a movable electrode to both stimulate and record the electrical activity of patterned neurons, both at the synapse level and along the neuronal pathway. Simultaneous fluorescence monitoring will identify the specific signaling molecules released during synapse formation as well as during the propagation of the electrical signal. By performing these experiments one seeks to a) quantify the role that different types of biochemical and geometrical cues play in neuronal growth and development; b) to measure under what conditions synaptic junctions are functional and c) to learn to control the formation of functional synapses in neuronal circuits having well-defined geometries. Broader Impacts: The proposed research may lead to great insights into diseases that result when the growth of neuronal processes fails, including birth defects, mental disorders, and sensory-motor deficits. Further, options to direct nerve-material interfaces have broad applicability for prosthetic devices to better mimic human functions. A specific goal for broader impact will be to use the research in the grant as a focused teaching tool for the undergraduates. Specifically, the investigators will establish a Research Mentorship Team which will provide undergraduate students with: a) research intensive experience b) multidisciplinary teams and projects (integration between physics, biology and engineering) such that the students gain exposure to broader thinking outside of their own discipline; c) mentorship experience at the undergraduate level, as senior students will serve as the upper class mentors to the second and third year undergraduate students helping to prepare them for their senior year. The postdoctoral researcher and graduate student involved in the grant will be part of the mentorship team. As part of this activity the investigators will also work directly with Tufts Center for Engineering Education Outreach to explore how to modularize the tools and teaching for use in the broader outreach activities.

1067093智力优点：本提案的目的是更深入地了解神经元细胞用于形成彼此功能连接的基本规则。了解大脑具有巨大的根本重要性，但由于其结构和功能的复杂性，它具有极大的挑战性。中枢神经系统由许多不同的空间定位，但高度互连的区域。迄今为止，参与形成功能性神经元连接的过程，轴突导航到其靶区域的机制以及它们与引导因子（如化学梯度和机械提示）的特定相互作用在很大程度上仍然未知。当前项目的科学目标是通过研究以简化，良好控制的几何形状图案化的少量神经元的生长和相互连接来了解生命系统中神经元之间连接和通信发展的基本过程。核心假设是通过在体外建立高度受控的神经元回路来简化神经元生长环境，将允许阐明神经元发育和神经连接形成的基本规则。简单的神经元网络将在二维基底上建立，引导突触的形成并使用a）原子力显微镜纳米光刻测量它们的电活动; B）原子力成像和基于原子力的电力显微镜; c）荧光光谱法。具体而言，目标之一是：1）在表面上的精确位置对不同类型的蛋白质/生长因子进行图案化，并将它们用作荧光标记神经元的生长模板; 2）通过控制底层蛋白质图案的类型和几何形状来引导神经元突触的形成; 3）使用原子力和荧光光谱测量系统地研究神经元过程的粘附和生长; 4）通过结合电力显微镜和荧光显微镜绘制网络的电活性。这最后一步的关键方面是使用电压偏置原子力尖端作为可移动电极来刺激和记录模式神经元的电活动，无论是在突触水平还是沿着神经元通路。同步荧光监测将识别突触形成期间以及电信号传播期间释放的特定信号分子。通过进行这些实验，人们试图a）量化不同类型的生物化学和几何线索在神经元生长和发育中所起的作用; B）测量突触连接在什么条件下是功能性的;以及c）学习控制具有明确几何形状的神经元回路中功能性突触的形成。更广泛的影响：这项拟议中的研究可能会导致对神经元过程生长失败导致的疾病的深刻见解，包括出生缺陷，精神障碍和感觉运动缺陷。此外，引导神经-材料界面的选择对于假体装置具有广泛的适用性，以更好地模拟人类功能。更广泛影响的一个具体目标将是使用赠款中的研究作为本科生的重点教学工具。具体而言，研究者将建立一个研究导师团队，为本科生提供：a）研究密集型经验B）多学科团队和项目（物理学，生物学和工程学之间的融合），使学生获得接触到自己的学科以外的更广泛的思考;（c）本科生的导师经验，因为高年级学生将担任本科二年级和三年级学生的高年级导师，帮助他们为高年级做好准备。参与资助的博士后研究员和研究生将成为导师团队的一部分。作为这项活动的一部分，调查人员还将直接与塔夫茨工程教育外展中心合作，探讨如何将工具和教学模块化，以便在更广泛的外展活动中使用。