Electron Microscope Histochemical Evidence for a Partial or Total Block of the Tricarboxylic Acid Cycle in the Mitochondria of Presynaptic Axon Terminals

Abstract

Respiration-linked, massive accumulation of Sr2+ is used to reveal the coupled oxidation of pyruvate, α-oxoglutarate, succinate, and malate by in situ mitochondria. All of these substrates were actively oxidized in the dendritic and perikaryal mitochondria, but no α-oxoglutarate or succinate utilization could be demonstrated in the mitochondria of the presynaptic axon terminals. A block at an early step of α-oxoglutarate and succinate oxidation is proposed to account for the negative histochemical results, since the positive reaction with pyruvate and malate proves that these mitochondria possess an intact respiratory chain and energy-coupling mechanism essential for Sr2+ accumulation. This indicates that the mitochondria in the axon terminals would be able to generate energy for synaptic function with at least some of the respiratory substrates. With regard to the block in the tricarboxylic acid cycle, the oxaloacetate necessary for citrate formation is suggested to be provided by fixation of CO2 into some of the pyruvate.

Mitochondrial accumulation of Sr2+ supported by pyruvate in the cerebellar cortex of the rat. Cellular variations during aging.

“…The two different types of reaction products, needles and granulations, are proof of the existence of strong and weak reactions respectively (12). The use of various substrates, including pyruvate, according to the results obtained by Greenawalt and Carafoli, has demonstrated blockage of the tricarboxylic acid cycle in axonal terminals (15). We have previously studied the metabolic differences between succinate and glutamate in cells of the cerebellar cortex in adult, growing and senile rats (2,33,34,36).…”

“…media with copper-ferricyanide), 100-500 pm sections were employed (Vibratome-Oxford, TC2-Sorvall), obtaining similar results. Nevertheless, since the diffusion of the products contained in the incubation medium with this technique is very good (15,17) and the reaction is constant throughout the thickness of the sections, the use of small blocks permits the study of a larger area (300 pm) with a better conservation of the structure after disregarding the first superficial 50 pm.…”

“…17, 18). The accumulation of Sr2+ produced by the aerobic mitochondrial oxidation of diverse metabolites using the Kerpel-Fronius' technique (17) has proven to be a good method for the demonstration of metabolic differences in distinct cellular regions of neurons and glial cells of the cerebellar cortex (15). The two different types of reaction products, needles and granulations, are proof of the existence of strong and weak reactions respectively (12).…”

“…They were killed by decapitation and their cerebelli rapidly extracted and transfered to ice. Blocks 0.5 mm in diamter were obtained from the superior vermis (Neocerebellum), rinsed in a 0.32 M sucrose solution for 30 min at 0-4°C and then incubated at 37°C for 60 or 90 min in a medium containing 10 mM pyruvate and 4 mM Sr(N03)2, 80 mM NaCI, 4 mM sodium-potassium phosphate bufffer (pH 7.0) and 20 mM Tris-HC1 buffer (pH 7.0) (15,17). Incubations were carried out in open glass dishes with constant stirring (32).…”

“…Nevertheless, the nervous system displays specific mechanisms, such as membrane depolarization (28). Biochemical activities of synaptic mitochondria are inferior to those of non-synaptic ones (4); histochemical deposits are therefore less intense in the first type of mitochondria (15,32). The activity and regulative mechanism in the brain of senile animals are similar to those of other tissues (4), exhibiting nevertheless some differences in the regulation of its activity (28).…”