A comparison of enzyme patterns in the granular and molecular layers of the rabbit cerebellar cortex

Abstract

1. (1) Homogenates of hand-dissected granular and molecular layers of the cerebellar cortex of the rabbit were prepared and the following enzymes assayed: acetylcholinesterase, succinate-INT-reductase, particulate [alpha]-glycerophosphate dehydrogenase, arylsulfatase, glutamine synthetase and glutamic decarboxylase. Except for [alpha]-glycerophosphate dehydrogenase which was higher in the granular layer, all of the other activities showed higher values in the molecular layer.2. (2) Differential centrifugation of the homogenates into the nuclear, heavy particulate, microsomal and soluble fractions yielded peaks of relative specific activity of acetylcholinesterase in the microsomal, of glutamic decarboxylase in the soluble and the heavy particulate, of succinate-INT-reductase, [alpha]-glycerophosphate dehydrogenase and arylsulfatase in the heavy particulate and of glutamine synthetase in no single fraction.3. (3) Further separation of the heavy particulate fraction was accomplished by discontinuous gradient density centrifugation which yielded 5 fractions including a pellet. The distribution of glutamic decarboxylase and morphological evidence adduced previously20 make it possible to state that the system of excitatory synapses sedimented predominantly into fractions 0.8 M and 1.0 M of the molecular layer and fractions 1.2 M and 1.4 M of the granular layer. The distribution of [alpha]-glycerophosphate dehydrogenase, an enzyme predominantly associated with glial mitochondria29, further suggested an equal extent of contamination of the molecular and granular layers by glial cells.4. (4) This study also provides a finding of general interest, inasmuch as it reveals that, by prior physical separation of the granular and molecular layers followed by their differential and density gradient centrifugation, it is possible to achieve a partial separation, although by no means a purification, of their excitatory and inhibitory synaptic complexes.