title: Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures.
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.
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, 15260, USA.
authors.forename: Yangguang
authors.initials: Y
authors.lastname: Ou
authors.forename: Juanfang
authors.initials: J
authors.lastname: Wu
authors.forename: Mats
authors.initials: M
authors.lastname: Sandberg
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Weber
mesh_terms: D000818:Animals; D003065:Coenzyme A / Q000378:metabolism; D053841:Electroosmosis / Q000295:instrumentation*; D004867:Equipment Design; D005110:Extracellular Space / Q000378:metabolism*; D006624:Hippocampus / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism; D010477:Perfusion / Q000295:instrumentation*
chemical_list: D008565:Membrane Proteins; D009479:Neuropeptides; D003065:Coenzyme A