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Ultrasmall Chemical Imaging of Cells and Vesicular Release

Final Report Summary - NANOCELLIMAGE (Ultrasmall Chemical Imaging of Cells and Vesicular Release)

The team has developed a new method to measure the contents of nanometer neurotransmitter vesicles and used this to demonstrate that the main form of transmitter release (exocytosis) is in fact open and closed and not all or none. The work for the mechanism of vesicle opening during this process was given the Nobel Proze in Physiology and Medicine in 2013 and yet, the closing portion has not been understood. The discovery that exocytosis is generally open and closed is incredibly important as it provides models for understanding learning and memory and is a new unconventional pharmaceutical target. Again at the microscopic level, we have carried out measurements of vesicle contents in situ with a nanoelectrode in a single biological cell. Still another breakthrough has been the measurement of single exocytosis events from varicosities in the fly, and the discovery of how the process works at real nerve cells. This is leading to fascinating new ideas about how the release of transmitters from vesicles is regulated and will likely allow us to unravel the learning and memory process in the near future.

We developed unique MS imaging capabilities to image a number of samples of neuroscience importance. A new method of SIMS imaging using cluster ions to detach intact ions has been developed and used to examine the fly brain and to demonstrate that the drug methylphenidate alters the lipid composition in a way that might be important in diet and in memory. The fly brain is a model used in our laboratory and we have used a new, one of a kind, 40 keV argon cluster source to obtain the best images to date anywhere of the fly brain. We have focused on lipids of cell membranes. A major finding has been that when flies are fed methylphenidate, the lipids across the brain change spatially and different lipids change differentially. Lipids that favor high curvature portions of membranes are enhanced and those that favor low curvature sections of membranes are diminished with this drug. As this drug enhances attention and cognition, it is possible this indicates a lipid-based mechanism as part of short-term memory formation, a truly astounding discovery!

Finally, we have developed protocols with a high technology method, called NanoSIMS, to obtain 50 nm spatial resolution and to examine the inner chemical distribution of 200 nm transmitter vesicles. We have used this approach to measure the transmitter dopamine inside a single nanometer vesicle.

There are many other somewhat smaller, but all important, contributions and in total the grant supported 52 publications.