The distribution of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) in the rat cerebellar cortex has been studied at the electron microscopic level using the peroxidase-antiperoxidase procedure. Extensive staining of cytoplasmic regions, with some increased staining at mitochondrial profiles, was seen in the cell bodies of both neurons (basket, stellate, Lugaro, Golgi, and granule cells) and astrocytes. Oligodendrocytes showed little or no detectable staining. Purkinje cell perikarya were much less intensely stained than were the perikarya of other neurons. The initial portion of the Purkinje dendrite was, like the perikaryon from which it emerged, lightly stained. More intense staining was seen in the secondary and tertiary branches of the Purkinje dendrite, but the terminal branches were devoid of stain. Granule cell dendrites were well stained in their initial portions but devoid of stain in their terminal dendritic digits which form part of the cerebellar glomeruli. In contrast to the unstained granule cell dendritic digits, the central mossy fiber nerve terminal of the glomerulus exhibited intense staining of the mitochondrial profiles and of synaptic vesicles adjacent to the mitochondria. Axons of basket cells showed intense staining in the segments adjacent to the Purkinje cell soma, while terminal twigs of the basket axons in the pinceau surrounding the (unstained) initial segment of the Purkinje axon showed markedly decreased staining intensity. These results indicate that there may be substantial variation in hexokinase levels between the various regions of neuronal processes. Hexokinase was seen at both cytoplasmic and mitochondrial locations in a variety of cells. It does not appear likely that location of hexokinase can be directly correlated with cell type, i.e., with neurons versus glia.

Astrocytes have been cultured from neonatal rat brain according to the flask culture procedure of Booher and Sensenbrenner. Approximately 80% of the hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) activity is found in the soluble fraction in homogenates of these cells, in contrast to only 20% of the total activity in the soluble fraction of whole brain homogenates. The hexokinase from the cultured astrocytes has been compared with the cytoplasmic and glucose-6-P-solubilized mitochondrial enzymes from whole brain. In kinetic properties and pH-activity relationships, the glial hexokinase was similar to the cytoplasmic enzyme but different from the mitochondrial enzyme of whole brain. Using immunohistochemical methods for detecting hexokinase localization at the electron microscopic level, most of the cells showed prominent staining of cytoplasmic areas. If the cultured astrocytes are accepted as valid models for astrocytes in situ, these results support the suggestion of Bigl and co-workers that the predominant form of hexokinase in glial cells is the cytoplasmic enzyme.