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【3rd.Nov.】Combination of NanoSIMS with electrochemical methods to understand the chemical structure of neurotransmitter vesicles and neurotransmission

2018-11-03


题目:Combination of NanoSIMS with electrochemical methods to understand the chemical structure of neurotransmitter vesicles and neurotransmission 
报告人: Professor Andrew Ewing, Royal Swedish Academy of Sciences, University of Gothenburg, Sweden
时间:11月3日(周 六),上午10:00
地点: 化学楼五楼演讲厅(528)
邀请人:任吉存 教授


报告人介绍
Andrew Ewing received his BS degree from St. Lawrence University and a PhD from Indiana University. After a postdoc at the University of North Carolina he joined the faculty at Penn State University for 25 years. 


He is now Professor at the University of Gothenburg, Sweden, and Honorary Professor at both Nanjing University of Science and Technology and Beijing University of Science and Technology. 


Focusing on the neuronal process of exocytosis, Ewing and his group have pioneered small-volume chemical measurements at single cells, electrochemical detection for capillary electrophoresis, novel approaches for electrochemical imaging of single cells, and new electrochemical strategies to both measure release during exocytosis and the contents of individual nanometer vesicles in cells. 


They also pioneered the development and application of mass spectrometry imaging for subcellular and neurochemical analysis. 


Ewing has received the Charles N Reilley Award from the Society for Electroanalytical Chemistry (2013), the American Chemical Society Award in Electrochemistry (2013), the Norblad-Ekstrand Medal of the Swedish Chemical Society (2014), and the SACP Analytical Chemistry Award (2015). 


He was made a Knut and Alice Wallenberg Scholar in 2011 and again in 2017. He was elected to the Royal Swedish Academy of Sciences (class 4, chemistry, Nobel Class) in 2012 and to the Gothenburg Academy of Arts and Sciences in 2013.


报告内容介绍
An electrochemical measurement of the content of nanometer transmitter vesicles combined with mass spectrometry imaging is a powerful approach. 


Vesicle impact electrochemical cytometry (VIEC) is a method whereby vesicles filled with electroactive metabolites impact an electrode surface, adsorb, and then are electroporated to expose their contents that are quantified by oxidation. 


This allows accurate determination of the contents of single nanometer neurotransmitter and hormonal vesicles and by comparison to the amount released in exocytosis, we have determined that only a fraction of the transmitter load of a large dense core vesicle is released during an exocytotic event, again experimentally demonstrating partial release. 


We have also developed a method, intracellular vesicle impact electrochemical cytometry (IVIEC), where a nanotip electrode is placed into a cell with the vesicles opening on the electrode tip for quantification of content. Comparing the content of large dense core vesicles (approx. 200 nm) to release shows partial, but greater than half released. 


Comparing content to release for small synaptic vesicles (50-60 nm) in the fruit fly nerve terminals and the fraction released is only 5-20 %! Using IVIEC, we have discovered that several drugs that affect cognition change exocytosis, but not all affect vesicle content. 


All these drugs appear to change the fraction of transmitter that is released. We have examined the effects of cisplatin, tamoxifen, zinc, lidocaine, and barbiturate and found that all these drugs act to changing the partial opening of the pore during exocytosis and the fraction of transmitter released seems to be a key aspect for plasticity. 


Using secondary ion mass spectrometry we have imaged the lipids across model brains after treatment with either cocaine or methylphenidate and we have then subsequently examined the effect of these drugs on exocytosis and partial release. 


From this we have made a model of what might happen in terms of the cognitive effects of these drugs. We have used NanoSIMS imaging to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells 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 and to compare to the amount imaged in vesicles. This allows us to add a novel quantitative aspect to the mass spectrometry imaging experiment. 


These nanoanalytical tools quantitatively reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process, and we have been able to quantitatively image in 3D across the cell and vesicles. 


The combination of these analytical approaches and their application is leading to a unified model of vesicle structure, exocytosis, and the beginning stages of plasticity, memory, at the cellular level.


Background Reference
N.T.N. Phan, X. Li, A.G. Ewing, “Measuring synaptic vesicles using cellular electrochemistryand nanoscale molecular imaging,” Nature Reviews Chemistry, 1, 2017, 1-18; DOI:10.1038/s41570-017-0048.




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