Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells.
1. A procedure has been developed for the separation of intact metabolically active neuronal and glial cells in bulk from rat cerebral cortex. Separation depended on dispersion of the tissue in a Ficoll medium followed by centrifugation on a discontinuous Ficoll gradient. Up to 1.5x10(7) neuronal cells could be collected from 12 brains within 3hr. The morphological appearance of these cells seemed good, and the fraction was 8.5-fold purified in terms of dry weight. Average dry weight per neuron was 2300mumug. Maximum glial contamination of the neuronal fraction was 11% as determined by carbonic anhydrase measurements. The glial fraction was free from neurons but contained various subcellular contaminants. 2. Concentrations of nucleic acids, phospholipid, protein and phosphoprotein were determined in the separated fractions. The neuronal fraction was richer than the glial in all except phospholipid. Succinate dehydrogenase was equally distributed between neurons and glia but the neuronal fraction was 1.8-fold enriched in cytochrome oxidase. 3. Measurement of respiration by the cells showed an endogenous uptake of 117mmumoles of oxygen/mg./hr. in neurons, and 173mmumoles of oxygen/mg./hr. in glia. Addition of substrate at 10mm stimulated uptake to similar values in both fractions. With glucose it was 390, with pyruvate 355, and with glutamate 215mmumoles of oxygen/mg./hr. This represented a larger stimulation of neuronal than of glial respiration compared with the basal level. 4. Respiration in cell suspensions was 70-80% of that of slices, whereas fractionated tissue homogenates had respiratory rates of only one-third those of the cell suspensions. Lactate dehydrogenase content of cell suspensions was maintained during gradient centrifugation and washing. 5. The possible uses of isolated cell preparations are discussed.
D000818:Animals; D002540:Cerebral Cortex / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008858:Microscopy, Phase-Contrast; D009457:Neuroglia / Q000378:metabolism*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D010101:Oxygen Consumption; D011773:Pyruvates / Q000378:metabolism; D051381:Rats; D013385:Succinate Dehydrogenase / Q000378:metabolism; D014461:Ultracentrifugation
D011773:Pyruvates; D007770:L-Lactate Dehydrogenase; D013385:Succinate Dehydrogenase; D003576:Electron Transport Complex IV; D005947:Glucose