Neuronal and neuropil fractions from developing rat brain.

Abstract

Abstract— A method is described for the preparation of enriched fractions containing isolated neuronal and glial cells from brains derived from 1 to 20‐day‐old rats. The method is based on mechanical disaggregation in a medium containing Ficoll‐PVP followed by centrifugation on a single‐stage two‐step gradient at 13,000g for 30min. The neuronal and neuropil (glial) fractions are approx 70–80% pure in cellular terms.The cells showed well‐preserved cytoplasmic and nuclear morphology at the light and electron microscope level and between 70 and 80% excluded trypan blue. Despite changes in the total cell population with age due to glial proliferation, the proportionate recovery of cells in the separated fractions was fairly constant: based on DNA determination, 23 and 29% of all neurons and 15 and 17% of glia were recovered in the purified fractions from Day 1 and Day 20 animals respectively.Changes in neuronal cell size with age were reflected in a 2.5‐fold increase in protein recovered in the neuronal fraction per mg DNA. Protein and RNA levels/mg DNA in the neuropil fraction reached a maximum at Day 10. It is concluded that the method produces a defined and reliable purification of cells in the separated fractions throughout the studied age range and therefore provides a sound basis for studies on the distribution of biochemical systems between cell types during post‐natal development.