BRIEF REPORT |
Correspondence to: Carlo BertoniFreddari, Neurobiology of Aging Laboratory, INRCA Research Dept., Via Birarelli 8, Ancona AN 60121, Italy.
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Summary |
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To map the mitochondrial capacity to provide adenosine triphosphate (ATP), the activities of cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were respectively evidenced by diaminobenzidine (DAB) and copper ferrocyanide cytochemical techniques in the cerebellar cortex of adult rats. Sampling of the positive mitochondria was carried out by the disector procedure. The ratio (R) overall area of the precipitates due to COX activity within the single mitochondrion/area of the same organelle was automatically calculated to estimate enzyme activity vs mitochondrial size. The number of SDH-positive mitochondria/µm3 of tissue (numeric density, Nv) was morphometrically calculated. Cytochemistry of key enzymes of the respiratory chain enables measurement of the actual capacity of individual mitochondria to provide ATP. This quantitative estimation allows morphofunctional mapping of the mitochondrial metabolic competence in discrete tissue and/or cellular compartments. (J Histochem Cytochem 49:11911192, 2001)
Key Words: mitochondrial mapping, adenosine triphosphate, cytochrome oxidase, succinic dehydrogenase, cytochemistry
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Introduction |
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The mitochondrial capacity to provide actual and adequate amounts of adenosine triphosphate (ATP), defined as the organelle's metabolic competence, constitutes an important determinant in several biological processes and pathological conditions. According to energy requests, the cellular oxidative phosphorylation potential for ATP undergoes continuous adaptation by regulation of mitochondrial biogenesis and respiratory chain enzyme activities. In this context, assessment of the morphofunctional mitochondrial capacity to supply ATP may enable identification of changes in cell metabolism as early signs of a progressing dysfunction. We carried out quantitative cytochemical studies on the activities of COX and SDH, two key enzymes of the respiratory chain considered as reliable markers of mitochondrial efficiency.The cerebellar cortex of adult rats (12 months of age) was adopted as a tissue model for our experimental procedures. In the cerebellar glomeruli, COX activity was evidenced by the DAB procedure (
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In Purkinje cell perikarya, SDH activity was preferentially revealed by the copper ferrocyanide reaction (
where h is the disector height (in our case h = 0.2 µm, i.e., each disector consisted of four sections of 0.05- µm thickness), and aDis is the disector area, directly measured by our image analysis system. The number of mitochondria/µm3 (Nv) was calculated by applying the following formula:
where Q- is the total number of counted mitochondria. The examined area of the reference section (aDis) was 19.35 µm2 and each disector volume (VDis) measured by us was 3.87 µm3 (
An example of the data that can be obtained on COX- and SDH-positive mitochondrial populations is shown in Fig 2. The value of R was calculated by sampling 330 COX-positive organelles and can be presumed to account for the percent of mitochondrial surface area involved in actual COX activity. The Nv value of SDH-positive mitochondria was obtained by sampling 12 Purkinje cell bodies from a single animal. Referring this datum to the results of numeric density estimated in tissue samples prepared according to conventional electron microscopic procedures, information on the fraction of SDH-active mitochondria can be obtained.
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Heteroplasmy, i.e., the mixture of normal and mutated mitochondrial genomes, is a well-documented feature of the mitochondrial population of postmitotic cells, particularly of old individuals. As a consequence, the potential for energy production in these cells is dependent on a mosaic of organelles with different functional capacities (
Received for publication December 12, 2000; accepted February 16, 2001.
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Literature Cited |
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BertoniFreddari C, Fattoretti P, Casoli T, Di Stefano G, Solazzi M, MeierRuge W (2001) Quantitative cytochemical mapping of mitochondrial enzymes in rat cerebella. Micron 32:405-410[Medline]
Hajos F, KerpelFronius S (1971) Electron microscope histochemical evidence for a partial or total block of the tricarboxylic acid cycle in the mitochondria of presynaptic axon terminals. J Cell Biol 5:216-222
Seligman AM, Karnovsky MJ, Wasserkrug HL, Hanker JS (1968) Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymeric osmiophilic reagent, diaminobenzidine (DAB). J Cell Biol 38:1-14
Sterio DC (1984) The unbiased estimation of number and size of arbitrary particles using the disector. J Microsc 134:127-136[Medline]