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

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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.