Isolation of cell nuclei from the mammalian cerebral cortex and their assortment on a morphological basis.
An aqueous method is described for the isolation of highly purified nuclei from the cerebral cortex of adult guinea pigs. Erythrocytes were removed by a short-time perfusion of the brain, myelin fragments by a rapid mechanical method, and blood capillaries by a centrifugal sieving through dense sucrose solutions. The nuclear preparation retained the activity of ATP:NMN adenylyltransferase. Recoveries of DNA in the P4I, P4II, P(L) and P(S) preparations were 30, 43, 8, and 7%, respectively. Microscopy and phase contrast microscopy showed a satisfactory removal of erythrocytes, myelin fragments, capillaries, and cytoplasmic elements. Biochemical purity of samples was verified by the absence of several cytoplasmic enzyme activities. In the electron microscope, the majority of nuclei showed well-preserved nuclear membranes, with nuclear pores, and were provided with a finely textured nucleoplasm. Occasional contaminants were elements of endoplasmic reticulum and of the endothelium. Assortment of nuclei on a morphological basis showed that 55-65% and 47-53% of nuclei in the P4I and P4II preparations, respectively, consisted of neuronal nuclei. In the P(L) preparation, the population of neuronal nuclei ranged between 72 and 83%, while 94-99% of the nuclei in the P(S) preparation consisted of smaller nuclei, most likely of oligodendroglial origin.
D000110:Acetylcholinesterase / Q000032:analysis; D000251:Adenosine Triphosphatases / Q000032:analysis; D000818:Animals; D001681:Biological Assay; D002467:Cell Nucleus / Q000032:analysis* / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002540:Cerebral Cortex / Q000032:analysis / Q000166:cytology*; D002802:Cholinesterases / Q000032:analysis; D004247:DNA / Q000032:analysis; D004721:Endoplasmic Reticulum; D005260:Female; D005969:Glutamate Dehydrogenase / Q000032:analysis; D006168:Guinea Pigs; D008297:Male; D008854:Microscopy, Electron; D008858:Microscopy, Phase-Contrast; D009713:Nucleotidyltransferases / Q000032:analysis; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis; D013385:Succinate Dehydrogenase / Q000032:analysis; D014461:Ultracentrifugation
D011506:Proteins; D012313:RNA; D004247:DNA; D013385:Succinate Dehydrogenase; D005969:Glutamate Dehydrogenase; D009713:Nucleotidyltransferases; D000110:Acetylcholinesterase; D002802:Cholinesterases; D000251:Adenosine Triphosphatases