The DNA content of cerebral cortex neurons. Determinations by cytophotometry and high performance liquid chromatography

Abstract

Previous work from our laboratories has indicated that the DNA content of rat cerebral cortex neurons increases postnatally to a level of slightly above 3c, where 2c denotes the diploid DNA complement. We have re-evaluated this concept by using various cytophotometric assays and a novel high performance liquid chromatography (HPLC) technique. The latter consists of digesting the DNA in isolated neuronal nuclei by a mixture of DNA-degrading enzymes followed by analysis of the resulting deoxynucleosides by HPLC. We find that the various methods fall into two groups. The first gives evidence of a postnatal DNA (or histone) increase, while the second does not. The first group (DNA increase) comprises cytofluorometry for DNA following Schiff-type staining with fluorochromes 2,5- bis-(4-aminophenyl)-1,3,4-oxadiazole (BAO) and pararosaniline, ultraviolet absorption scanning for DNA and cytofluorometry for histones following staining with sulfaflavine at pH 8. The second group (no DNA increase) consists of cytofluorometry for DNA following staining with the DNA-complexing agents mithramycin, chromomycin A 3, 4′, 6-diamidino-2-phenylindole (DAPI) and bisbenzimide (Hoechst 33258), as well as the newly developed HPLC technique. Since the HPLC technique measures DNA by a direct chemical approach without interference from other nuclear constituents or from higher order packaging in the chromatin, and detects at least 94–95% of the total DNA contained in neuronal nuclei independent of the developmental stage, we infer that the HPLC technique and, by consequence, the cytochemical assays of the second group reflect true DNA values. Therefore, we propose that cerebral cortex neurons retain a diploid DNA level throughout development.