Morphological and biochemical studies on isolated molecular and granular layers from bovine cerebellum

Molecular layer, granular layer and white matter are dissected from bovine cerebellum under optical microscope and without freezing under conditions which preserve their main anatomical features. Polyacrylamide gel electrophoresis in sodium dodecyl sulphate of the isolated layers reveal that the P400 protein characteristic of the Purkinje cells is found in the isolated molecular layer and in the molecular layer free from Purkinje cell soma, but not in the granular layer or white matter. The histones (F1, F2A1,2, F2B, F3) are abundant in the granular layer and myelin proteins in the white matter. The DNA content per wet weight is 10 times greater in the isolated granular layer than in the other layers and the RNA content twice as great in the granular layer than in any other layer. The specific activity of acetylcholinesterase is 4 times greater in the granular layer than in the other layers. Homogenates of the isolated layers take up labeled amino-acids and the velocity of glutamate incorporation is 9 times greater in the molecular layer than in the granular layer, while GABA incorporation is about twice as great in the granular layer than in the molecular layer. Homogenates of isolated molecular layer are centrifuged on discontinuous Ficoll gradient after incubation with L[3H]glutamate and [14C]GABA. The analysis of the distribution of glutamate and GABA after centrifugation reveals that the particles which incorporate glutamate can be separated from those which take up GABA.