Independence of protein synthesis and drug uptake in nerve cell bodies and glial cells isolated by a new technique.

Abstract

A severe handicap in any study of the cellular biochemistry of the brain is the unavailability of sufficient amounts of pure neurones and glial cells. Many efforts to separate one from the other have been made1, but only four basic types of isolation procedures have been described for routine use: (1) brain is treated with a mixture of acetone-glycerol and water before mincing and centrifugation3–5; (2) brain mince is sieved and subjected to gradient centrifugation1,6–8—this separates neuronal cell bodies from neuropil6 or from intact but rather contaminated glial cells8; (3) whole brain is disrupted in a tissue press and zonal centrifugation is used to separate cellular and subcellular components as they leave the zonal centrifuge rotor9; and (4) chopped brain is incubated in oxygen at 37° C in the presence of 1 % (w/v) trypsin10, a treatment which, in our estimation, severely prejudices this technique's general usefulness, because it precludes meaningful comparative investigations of enzymes and non-enzymatic structural and/or soluble cell proteins. Since investigations of the latter type are the aim in our laboratory, we needed a technique in which brain is not subjected to deleterious treatments such as immersion in acetone-glycerol-water mixtures3–5 or trypsinization10 and in which no special instrumentation9, tissue presses9 or homogenizers8 are required. We now report a technique which successfully accomplishes this objective, for it makes possible the isolation of highly purified neuronal perikarya and of intact glial cells from a few grams of brain, in yields which permit the subsequent subcellular fractionation of the isolated cells. We also report two applications of the new technique which have enabled us to determine the in vivo time course of protein synthesis in and the uptake of the radioactive convulsant agent methionine sulphoximine11,12 by, the neurones and the glial cells of the immature rat brain cortex.