Our current understanding of brain function is limited by available experimental techniques. In particular, methods for measuring in vivo neurochemical dynamics are lacking. This is critical considering that it is these dynamics that are the primary mechanism of the neuronal communication that gives rise to brain function. Microdialysis is currently the dominant technique for monitoring in vivo chemical dynamics. Unfortunately, microdialysis suffers from some significant drawbacks. Temporal resolution is often on the order of 10-20 minutes. Sampling regions are large (1-5 mm) making it difficult to measure activity in specific brain regions. Damage during implantation is significant and rigorous calibration is not possible. Introduction of online microdialysis - capillary electrophoresis (MD-CE) assays have improved temporal resolution to 10- 30 s but many issues remain. We propose to systematically overhaul existing online MD-CE instrumentation with the goal of achieving significant gains in temporal and spatial resolution. Micro-free flow electrophoresis (u-FFE) will be introduced as a separation technique capable of continuously analyzing sampled neurotransmitters. The sample stream will be compartmentalized using an immiscible fluorous solvent to prevent loss of temporal information as neurotransmitters travel from the probe, through the online labeling reaction and to the separation interface. An electrically actuated, inline micropump will be developed to directly sample fluid from the rat brain at very low flow rates (<50 nL/min). The spatial resolution of this direct sampling technique will only be limited by the outlet size of the sampling capillary (40 um). Combined we expect an online direct sampling - uFFE instrument will be capable of monitoring the in vivo dynamics of a number of important neurotransmitters, including glutamate, gaba, glycine, D-serine and taurine, on a -100 ms time scale in <100 um brain regions. Measurements on this time and size scale for a range of neurotransmitters would impact all areas of neuroscience research including stroke, epilepsy, memory, pain, addiction, etc. As an added benefit the instrumentation described in this proposal is much simpler than existing high temporal resolution microdialysis assays, suggesting that it may be more easily adopted by non-specialists in traditional neuroscience laboratories.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS043304-08
Application #
7800889
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Talley, Edmund M
Project Start
2002-04-01
Project End
2012-04-30
Budget Start
2010-05-01
Budget End
2012-04-30
Support Year
8
Fiscal Year
2010
Total Cost
$280,001
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Graf, Neil J; Bowser, Michael T (2013) Effect of cross sectional geometry on PDMS micro peristaltic pump performance: comparison of SU-8 replica molding vs. micro injection molding. Analyst 138:5791-800
Turgeon, Ryan T; Fonslow, Bryan R; Jing, Meng et al. (2010) Measuring aptamer equilibria using gradient micro free flow electrophoresis. Anal Chem 82:3636-41
Frost, Nicholas W; Bowser, Michael T (2010) Using buffer additives to improve analyte stream stability in micro free flow electrophoresis. Lab Chip 10:1231-6
Rosenberg, Dina; Kartvelishvily, Elena; Shleper, Maria et al. (2010) Neuronal release of D-serine: a physiological pathway controlling extracellular D-serine concentration. FASEB J 24:2951-61
Turgeon, Ryan T; Bowser, Michael T (2009) Improving sensitivity in micro-free flow electrophoresis using signal averaging. Electrophoresis 30:1342-8
Turgeon, Ryan T; Bowser, Michael T (2009) Micro free-flow electrophoresis: theory and applications. Anal Bioanal Chem 394:187-98
Fonslow, Bryan R; Bowser, Michael T (2008) Fast electrophoretic separation optimization using gradient micro free-flow electrophoresis. Anal Chem 80:3182-9
Graf, Neil J; Bowser, Michael T (2008) A soft-polymer piezoelectric bimorph cantilever-actuated peristaltic micropump. Lab Chip 8:1664-70
Klinker, Chanda Ciriacks; Bowser, Michael T (2007) 4-fluoro-7-nitro-2,1,3-benzoxadiazole as a fluorogenic labeling reagent for the in vivo analysis of amino acid neurotransmitters using online microdialysis-capillary electrophoresis. Anal Chem 79:8747-54
O'Brien, Kylie B; Bowser, Michael T (2006) Measuring D-serine efflux from mouse cortical brain slices using online microdialysis-capillary electrophoresis. Electrophoresis 27:1949-56

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