PREPARATION OF CELL BODIES FROM DEVELOPING CEREBELLUM - STRUCTURAL AND METABOLIC INTEGRITY OF ISOLATED CELLS

The isolation from the developing rat cerebellum of cell bodies which display a high level of ultrastructural organization was described. The procedure, which utilizes isotonic conditions throughout, began with a brief trypsinization at low enzyme concentration (0.025%). Proteolysis was terminated by trypsin inhibitor and followed by short exposure to EDTA. The technique was effective with cerebella from rats up to 2 wk after birth. Recoveries of cell bodies varied from 130-140 million/g wet weight of tissue, depending on age; this represented, in terms of recovered DNA, a mean value for yield of 33%. Suspensions contained little debris, free nuclei were rare and about 80% of the perikarya excluded trypan blue. Survey electron micrographs showed that most cell bodies possessed uninterrupted plasma membrane profiles and retained highly organized cytoplasmic and nuclear ultrastructure. Structural preservation was highlighted in the case of Purkinje cell bodies in which many characteristic features survive including, most notably, perisomatic spines. Metabolic integrity appeared to parallel morphological preservation as judged by severval functional criteria, including the ability to metabolize glucose, accumulate K+ ions and synthesize proteins. SDS[sodium dodecylsulfate]-polyacrylamide gel electrophoresis showed that the tissue dissociation technique did not lead to major deletions of cell proteins and that the pattern of perikaryal protein synthesis in vitro closely resembled that in vivo. These perikaryal preparations are important as a simplified system for metabolic studies and as starting material for the derivation of purified sub-populations of cell bodies from developing cerebellum.