Andrew Ewing oral presentation (B&N-Tue1-2-5)
ToF-SIMS imaging, the quest to find and quantify neuro-related lipid and neurotransmitter changes in cells and vesicles
1 Chalmers University of Technology - Department of Chemistry and Chemical Engineering, , SE-412 96 Gothenburg, Sweden
2 University of Gothenburg - Department of Chemistry and Molecular Biology, , SE-412 96 Gothenburg, Sweden
3 National Center for Imaging Mass Spectrometry - Göteborgs universitet, Kemivägen 10, SE-412 96 Göteborg, Sweden
4 Astra Zeneca Company - Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, Pepparedsleden 1, 431 50 Mölndal, Sweden
Nick and I began working to bring ToF SIMS imaging to analysis of lipids in cell membranes to neuroscience 26 years ago, with the ever present theoretical collaborations with Barbara Garrison. Review panels said it couldn’t be done! In some of our most recent work, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to study the effects of cocaine administration on both the localization and abundance of lipids in the brain of Drosophila melanogaster. A J105 ToF-SIMS equipped with a 40 keV Ar4000+ primary ion source enabled us to probe larger fragments and molecular ions of the biomolecules in the fly with a spatial resolution around 3 µm, giving us unique insights into the effect of cocaine on molecular lipids in the nervous system. Significant changes in phospholipid composition were observed in the central brain. Principle component image analysis revealed that changes occurred mainly for phosphatidylcholines, phosphatidylethanolamines, and phosphatidylinositols. Furthermore, the lipid changes caused by cocaine were then compared with those induced by methylphenidate. It was shown that these drugs exert opposite effects on the brain lipid structure and, furthermore we speculate that this might relate to the molecular mechanism of cognition and memory.
We have also used NanoSIMS to develop an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Furthermore, intracellular electrochemical cytometry at nanotip electrodes has been used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. We suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amount of catecholamine from the protein dense core despite its nanometer size.