**2.2 Methods**

## *2.2.1 Electron microscopy*

Multiple samples of a small size (2 × 2 × 2 mm) were excised from the hippocampus, the prefrontal area of the cortex, the superior parietal lobe, the occipital pole, the visual cortex, the Hessl gyri of the temporal neocortex, the vermis of the cerebellum and the cerebellar hemispheres, the hypothalamus, the mammillary bodies and the medial geniculate bodies. The samples were selected bilaterally and immersed directly in Sotelo's fixing solution [96], composed of 1% paraformaldehyde, 2.5% glutaraldehyde in cacodylate buffer 0.1 M, adjusted at pH 7.35.

Then all the specimens were post fixed in 1% osmium tetroxide for 30 min at a room temperature of 18°C and dehydrated in graded alcohol solutions and in propylene oxide twice. After dehydration, the specimens were embedded in araldite mixture and cut in ultrathin sections by a Reichert ultratome.

The sections were placed on the grids where they were contrasted with uranyl acetate and lead citrate, and studied in a Zeiss electron microscope of the type 9aS.

The study electron microscopy examination was particularly focused on the morphology of the organelles, mainly on the mitochondria of neurons and astrocytes. In addition, the Golgi complex, the endoplasmic reticulum, the endosomes, the dendritic profiles, the spines, the axons, the axonic collaterals and the synaptic components were studies in all of the sections.

The morphometric estimation was carried out on micrographs of a standard magnification of 56,000×. The analysis of each macrograph was performed with an image analyzer. The surface area of mitochondria as well as the volume and the circularity ratio (CR) were calculated on a total of 8000 mitochondria.

The statistical analysis of the data was evaluated by Student t tests.
