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Keywords:

  • Enzyme Cytochemistry;
  • Glycogen;
  • Glucose-6-phosphatase;
  • Central nervous system;
  • Neurons;
  • Glia

Abstract

Reliable ultrastructural techniques are applied for cytochemical identification of glycogen and localization of glucose-6-phosphatase (G6Pase) activity within neurons and glia of the adult mammalian CNS. Modulations in the cerebral localizations of glycogen and G6Pase activity are identified during various experimental conditions (i.e., salt-stress, fasting, and trauma). The cytochemical reaction for demonstration of G6Pase activity implies that the enzyme acts as a phosphohydrolase to convert glucose-6-phosphate to glucose. The degradation of glycogen in vivo is one source of glucose-6-phosphate as a substrate for G6Pase. Glycogen is preserved by perfusion-fixation of the brain with 2% glutaraldehyde-2% formaldehyde. Chopper sections of this material are postfixed in buffered 1% osmium tetroxide-1.5% potassium ferrocyanide, which serves as a contrast stain for glycogen, or in buffered 1% osmium tetroxide. Plastic-embedded ultrathin sections of CNS tissue postfixed in 1% osmium tetroxide are stained for glycogen with periodic acid–thiocarbohydrazide-silver protein. Intracellular glycogen appears as electron-dense isodiametric particles and, under normal and experimental conditions, is most abundant within astrocytes. Neuronal glycogen is sparse to negligible normally but appears increased within specific neuronal populations during stressful states.

Optimal preservation of G6Pase activity in the brain is obtained by brief perfusion-fixation with 2% glutaraldehyde. Tissue sections are incubated in a modified Leskes medium containing glucose-6-phosphate or mannose-6-phosphate as substrate and lead nitrate. Utilizing the Gomori lead capture technique, G6Pase reaction product is localized within the lumen of the endoplasmic reticulum (ER) and related organelles (i.e., nuclear envelope, Golgi complex) of perikarya, dendrites, and glia. The ER in axons and axon terminals fails to express G6Pase activity under normal conditions but does so in some neurons exhibiting a degenerating appearance. A transient, cytochemical decrease in G6Pase activity may occur within some perikarya during stressed conditions.

The results indicate that within neurons and glia of the adult CNS cytochemical stains are well suited for ultrastructural identification of glycogen and localization of G6Pase activity. Modulations in glycogen particle concentration and in localization of G6Pase activity in the neuron can occur in response to conditions that influence the energy metabolism of the cell. These modulations may reflect differences in the regional utilization of glucose as an energy-producing substrate and as a derivative of glycogenolysis within the CNS.