Electron Microscopic Methods for Determining Changes in the Density of Synaptic Input to Neurons in the Aging Brain

Changes in the synaptic input to aging neurons can best be evaluated using electron microscopy (EM). Immunocytochemistry is used to identify neuronal populations and to distinguish the chemical identity of their synaptic input, using a double-label protocol (e.g., diaminobenzidine for the sites of the first antigen/antibody complexes and tetramethylbenzidine for the second). In preparing tissue for EM examination, neurons are sectioned in the plane of the nucleus at 70 nm and sections collected on slot hole grids. Photographic montages of neurons are made at a minimum of 3 depths of section, with about 1 μm intervening. The original micrographs are taken at 10,000× and printed at 25,000×. Morphometric analyses are performed using the Bloquant program (R&M Biometrics) and an IBM computer. The perikaryal membrane is outlined on an X-Y digitizing pad, and regions along which there is synaptic modification are measured. These synaptic regions are expressed as a percentage of the perikaryal membrane measured. Data are tested using a non-parametric statistic (Mann-Whitney U, P < 0.05). In some cases, the entire neuronal soma is serially sectioned in order to determine whether synapses are randomly distributed over the neuronal surface.