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

• 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 similar to 100 mu m spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus.

Ämnesord

NATURVETENSKAP  -- Biologi -- Cellbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Cell Biology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Organotypic hippocampal slice cultures

membrane-bound enzymes

enzyme activity

electroosmotic

PUSH-PULL PERFUSION

HORMONE-DEGRADING ECTOENZYME

CONVECTION-ENHANCED

DELIVERY

ELECTROLYTE-FILLED CAPILLARY

SINGLE-CELL ELECTROPORATION

IN-VIVO

COENZYME-A

ELECTROCHEMICAL DETECTION

AMINOPEPTIDASE-N

BRAIN-TISSUE

Biochemical Research Methods

Chemistry

Analytical

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)