Preparation of cell bodies from the developing cerebellum: structural and metabolic integrity of the isolated cells.

A method is described for the isolation from the developing rat cerebellum of cell bodies which display a high level of ultrastructural organization. The procedure, which utilizes isotonic conditions throughout, begins with a brief trypsinisation at low enzyme concentration (0.025%). Proteolysis is terminated by trypsin inhibitor and followed by short exposure to EDTA. The technique is effective with cerebella from rats up to 2 weeks after birth. Recoveries of cell bodies vary from 130-410 million/g wet weight of tissue, depending on age: this represents, in terms of recovered DNA, a mean value for yield of 33%. Suspensions contain little debris, free nuclei are rare and about 80% of the perikarya excludes trypan blue. Survey electron micrographs show that most cell bodies possess uninterrupted plasma membrane profiles and retain highly organised cytoplasmic and nuclear ultrastructure. Structural preservation is highlighted in the case of Purkinje cell bodies in which may characteristic features survive including, most notably, perisomatic spines. Metabolic integrity appears to parallel morphological preservation as judged by several functional criteria, including the ability to metabolise glucose, accumulate K+ ions and synthesize proteins. SDS-polyacrylamide gel electrophoresis shows that the tissue dossociation technique does not lead to major deletions of cell proteins and that the pattern of perikaryal protein synthesis in vitro closely resembles that in vivo. These perikaryal preparations therefore hold out great promise as a simplified system for metabolic studies and as a starting material for the derivation of purified sub-populations of cell bodies from developing cerebellum.