Changes in Extracellular Space of the Mouse Cerebral Cortex During Hydroxyadipaldehyde Fixation and Osmium Tetroxide Post-Fixation

Perfusion of the cerebral cortex of mice with a 4.5 and 12.5% hydroxyadipaldehyde (HAA) solution in a cacodylate buffer caused a biphasic change in the tissue conductivity. After a latency of a fraction of a minute the cortical conductivity dropped markedly, reaching a minimum in 1.5-2 min. Then the conductivity increased again. Electron micrographs (EMs) of material perfused with HAA for 15-20 min and post-fixed with osmium tetroxide showed electron-transparent swollen structures, some of which could be identified as dendritic. The extracellular space consisted of 100-200 Å slits between the tissue elements and larger spaces in bundles of small profiles (unmyelinated axons). Cortex frozen after 2 min perfusion with HAA and subjected to substitution in acetone containing 2 % OsO₄ at -85 °C showed swollen (dendritic) structures and a paucity of extracellular material in accordance with the conductivity drop. Often tight junctions between the tissue elements were present. Tissue frozen after 15-20 min of HAA perfusion when the conductivity had increased again yielded EMs which were characterized by an abundance of extracellular space between the small profiles. The mitochondria in the swollen (dendritic) structures were enormously enlarged. Cortex perfused for 15-20 min with HAA, post-fixed with OsO₄ and then freeze substituted produced EMs resembling those of tissue fixed in the same way but not subjected to freeze substitution. The examination of the fixation process by freeze substitution demonstrated a sequence of major changes in the fluid distribution of the tissue which precludes any direct relationship between the spaces in the normal and fixed tissue.