AbstractThe volume of the extracellular space, which contributes to the microenvironment of neurons, is diminished in the brains of senescent (as compared to adult) rats and an age‐related change in its composition has been hypothesized. To test this hypothesis we have compared the penetration of ruthenium red, a polyanion selectively distributed in the extracellular space, into the dentate gyri of young adult and senescent Fischer 344 rats.Slices of hippocampal formation were fixed by immersion, first in a glutaraldehyde solution containing ruthenium red, then in a solution ofosmium tetroxide containing ruthenium red. A specific area of the dentate gyrus was identified, sectioned and examined by electron microscopy. Dense particles of ruthenium red reaction product were readily localized in intercellular channels and synaptic clefts and the depth of penetration was measured in a standardized manner so as to derive a mean depth of penetration for each of the 11 animals studied. A 28% decrease in the mean depth of penetration of ruthenium red in 25‐month‐old rats, as compared with 3‐month‐old animals, was found.These data indicate an age‐related change in the charge density of the intercellular channels in the dentate gyrus of 25‐month‐old rats. They suggest a primary age‐related change in the charge density of extracellular macromolecules, presumed to be primarily glycosaminoglycans, with a consequent change in water binding capacity and volume of the ex