C. Bertoni-Freddari, P. Fattoretti, B. Giorgetti, M. Solazzi, M. Balietti

An impaired energy metabolism is reported to represent a critical condition contributing to physiological aging and predisposing to age-related pathologies. With specific reference to nerve cells, actual and adequate energy provision is a necessary prerequisite for brain performances, thus any alteration affecting the neuronal energy supply machinery may constitute a potential threat for derangements in cell-to-cell communication. To asses the mitochondrial metabolic competence, i.e. the capacity of old organelles to provide proper amounts of adenosinetriphosphate, synaptic mitochondria positive to cytochrome oxidase (COX) activity were quantitatively investigated by computer-assisted morphometric methods at the distal dendrites of the dentate gyrus granule cells in adult (12 months) and old (26-28 months) female Wistar rats. COX activity was preferentially evidenced as a sharp and dark cytochemical precipitate at the inner mitochondrial membranes and cristae by the diaminobenzidine technique. The animals were perfused with 2.5% paraformaldehyde, 1.5% glutaraldehyde in 0.1M Sorensen buffer at pH 7.4 plus 4% sucrose. The hippocampal tissue samples (40-60 mm thickness) were rinsed overnight in buffer and sucrose and then incubated (2 - 4 hours at 37 °C in the dark) in the following solution (100 ml): 50 mg diaminobenzidine (DAB), 27 mg cytochrome C, 4 gr. sucrose. Postfixation in osmium tetroxide was followed by conventional inclusion, sectioning and contrasting procedures. The following morphometric parameters were calculated on COX-positive organelles: the number of mitochondria/mm3 of tissue (numeric density: Nv), the volume fraction occupied by mitochondria/mm3 of tissue (volume density: Vv), the average mitochondrial volume (V), the longer mitochondrial diameter (Fmax) and the ratio (R: mitochondrial area/overall area of the cytochemical precipitate due to COX activity). In old animals, Nv, Vv, V and Fmax were increased at a not significant extent, while R was not significantly decreased. The percent distribution of the Fmax values of the single mitochondria showed that the complement of longer organelles is higher in old animals. While Nv, Vv, V and Fmax report on the ultrastructural features of COX-positive mitochondria, R refers to the functional bearing of the population of organelles selected for sampling. By considering these two aspects of the parameters measured in our study, the present findings first confirm the many data of the current literature documenting that mitochondria show a clear trend to increase in size during aging. Second, they lend further support to the concept that mitochondrial size inversely correlates with mitochondrial metabolic competence: in old rats V and Fmax increased by 16.5 and 12.2%, respectively, but R decreased by 1.5%. COX (complex IV of the respiratory chain) is an integral transmembrane protein synthesised by mitochondrial DNA and nuclear genome. As the terminal molecule of the electron transport chain, COX has been considered as an endogenous marker of neuronal oxidative metabolism, thus, although our findings refer to the discrete subpopulation of COX-positive organelles located at synaptic terminals, they support that alterations of mitochondrial ultrastructure and function may contribute to the age-related decay of neuronal performances.

Keywords (Optional): 
mitochondrial metabolic competence
synaptic mitochondria
cytochrome oxidase activity