An ultrastructural study of nerve and glial cells by freeze-substitution

The ultrastructure of nerve and glial cells in the cerebral and cerebellar cortices of mice was studied after rapid freezing followed by substitution fixation. The cerebral and cerebellar cortices were frozen by bringing them into contact with a polished pure copper block cooled at a temperature of about -196 degrees C. The tissues were fixed and substituted in acetone containing 2-4% OsO4 at -78 degrees C for 2-3 days and then prepared for electron microscopy. Tissue fixed by this method displayed the following characteristics. (1) The contour of cells, processes and intracellular membrane systems was smooth. (2) The extracellular spaces were of variable widths. (3) Microtubules were well preserved and were often observed to extend into nerve terminals and to run close to presynaptic membranes. (4) The matrix of cytoplasm and mitochondria was electron dense. Dense granules, possibly binding sites of divalent cations, were often found in the mitochondrial matrix. (5) The plasma membrane of neuronal processes was thicker than that of glial processes. (6) The plasma membranes of nerve fibres and glial processes appeared asymmetrical, the inner leaflet being slightly thicker than the outer leaflet, whereas membranes of cell organelles such as smooth endoplasmic reticulum. Golgi bodies, lysosomes, multivesicular bodies, mitochondria and synaptic vesicles, were symmetrical.