Cytochrome oxidase activity in hippocampal synaptic mitochondria during aging: a quantitative cytochemical investigation
C. Bertoni-Freddari, P. Fattoretti, B. Giorgetti, M. Solazzi, M. Balietti
Neurobiology of Aging Laboratory, INRCA Research Department, Via Birarelli 8, 60121 Ancona, Italy
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.
Key words:
Mitochondrial metabolic competence, synaptic mitochondria, cytochrome oxidase activity, hippocampus, morphometry
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