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Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

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Abstract

This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push–pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push–pull perfusion can distinguish ectoenzyme activity with a ~100 μm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus.

Schematics for the one-probe electroosmotic sampling (EO sampling, left) and two-probe electroosmotic push-pull perfusion techniques (EOPPP, right). An external electric field drives fluid flow in the direction indicated by the arrows. HBSS - Hanks Balanced Salt Solution.

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Acknowledgements

This work has been funded by the National Institutes of Health (Grants R01 GM044842 and GM066018) and a Dietrich School of Arts and Sciences Fellowship to Y.O. from the Kenneth P. Dietrich School of Arts and Sciences at the University of Pittsburgh.

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Authors and Affiliations

  1. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, 15260, USA

    Yangguang Ou, Juanfang Wu & Stephen G. Weber

  2. Department of Medical Biochemistry and Cell Biology, University of Gothenburg, 40530, Gothenburg, Sweden

    Mats Sandberg

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  1. Yangguang Ou
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  2. Juanfang Wu
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  3. Mats Sandberg
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  4. Stephen G. Weber
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Correspondence to Stephen G. Weber.

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Ou, Y., Wu, J., Sandberg, M. et al. Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures. Anal Bioanal Chem 406, 6455–6468 (2014). https://doi.org/10.1007/s00216-014-8067-2

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  • DOI: https://doi.org/10.1007/s00216-014-8067-2

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