EAGER: Nano-resolution microscopy studies of signaling in presynaptic terminals

神经细胞的突触前末端是神经递质（如谷氨酸、乙酰胆碱和多巴胺）在突触传递过程中释放的部位。突触功能对所有形式的行为、正常的学习和记忆、动机和情绪都至关重要。理解突触功能是理解和干预包括成瘾行为和痴呆在内的许多疾病的关键。可以说，突触前末端有几十种不同的蛋白质，是神经细胞中最复杂的区域，而大脑是人体最复杂的器官。在突触处，神经递质被包含在直径为30纳米（3/100,000毫米）的小袋或囊泡中。这些囊泡在末端被不同的蛋白质转运，并停靠在释放点，在那里它们可以与膜融合，释放神经递质。复杂多样的蛋白质群，突触末端的小尺寸和突触位点形态的不规则性使得在分子水平上分析突触末端功能变得困难。此外，突触囊泡的分辨率低于传统光或共聚焦显微镜。在过去的5年中，在以单个囊泡分辨率成像活细胞中单个分子的新策略方面取得了巨大进展。在大脑中甚至在细胞培养中突触的复杂几何形状现在是限制因素。本项目的目的是开发一种新技术，在培养中诱导神经细胞在亚微米的膜蛋白神经素岛上形成突触终端。利用计算机芯片光刻技术将这些神经素纳米点固定在玻璃盖上生成的铬金点上。利用一种称为PALM的技术，在盖片表面诱导人工突触前释放位点，将为实时成像单个光激活分子的行为创造最佳条件。这种人工突触方法的发展将利用海蜗牛的高鲁棒性神经元来完成，海蜗牛是分析突触机制的一个非常有利的模型。一旦这项技术得到验证，它将在哺乳动物神经元上进行测试。即将开发的通用NLG纳米点技术将促进工程师和神经科学家之间的未来互动。艾布拉姆斯博士在引导本科生进行研究方面有着悠久的历史，有几位本科生将参与这个项目，其中包括一名已经在马里兰纳米中心（Maryland NanoCenter）生产亚微米铬金点方面发挥重要作用的学生。

The presynaptic terminals of nerve cells are the sites where neurotransmitters (such as glutamate, acetylcholine and dopamine) are released during synaptic transmission. Synaptic function is critical for all forms of behavior, for normal learning and memory and for motivation and mood. Understanding synaptic function is key to understanding and intervening in numerous disorders including addictive behavior and dementia. Arguably, the presynaptic terminal, with a few dozen different proteins, is the most complex region of nerve cells within what is the most complex organ of the body, the brain. At synapses, neurotransmitter is contained in small sacks or vesicles 30 nanometers (3/100,000 of a millimeter) in diameter. These vesicles are translocated within the terminal by different proteins and docked at release sites, where they can fuse with the membrane, releasing neurotransmitter. The complex, diverse group of proteins, the small size of synaptic terminals and the irregular morphologies of synaptic sites has made the analysis of synaptic terminal function at the molecular level difficult to analyze. Moreover, synaptic vesicles are below the resolution of traditional light or confocal microscopy. In the past 5 years, there have been dramatic advances in novel strategies for imaging single molecules in living cells at the resolution of single vesicles. The complex geometry of synapses in the brain or even in cell culture is now the limiting factor. The objective of this project is to develop a novel technology for inducing nerve cells in culture to form synaptic terminals onto submicron islands of the membrane protein neuroligin. These neuroligin nanospots will be immobilized on chromium-gold spots generated on glass coverslips using computer chip photolithography technology. Inducing artificial presynaptic release sites on the surface of cover slips will create an optimal situation for imaging the behavior of single photoactivatable molecules in real time, using a technique called PALM. The development of this artificial synapse approach will be done using the highly robust neurons of the marine snail Aplysia, which has been a highly favorable model for analysis of synaptic mechanisms. Once this technique is validated, it will be tested with mammalian neurons.The general NLG nanospot technology that will be developed will promote future interactions between engineers and neuroscientists. Dr. Abrams has a long history of introducing undergraduate students to research, and several will be involved in this project, including a student who is already playing a major role in producing submicron chromium-gold spots at the Maryland NanoCenter.