Optimization of permeabilized brain tissue preparation to improve the analysis of mitochondrial oxidative capacities in specific subregions of the rat brain

Abstract

Background

As the major energy producer of cerebral tissue, mitochondria play key roles in brain physiology and physiopathology. Yet, the fine details of the functioning of mitochondrial oxidative phosphorylation in this organ are still scattered with grey area. This is partly due to the heterogeneity of this tissue that challenges our abilities to study specific cerebral subregions. In the last decades, cerebral mitochondria have largely been studied as a single entity by isolating mitochondria from large sections of brain. Given the evidence that these organelles must adapt to brain areas functions, it seems crucial to develop technologies enabling study of the mitochondria in given subregions.

New method

A few years ago, a method allowing the investigation of mitochondrial functions in permeabilized brain subregions have been proposed by Holloway’s team. Although this protocol represented a significant advance, we propose improvements in the tissue permeabilization procedure and in the conditions for measuring oxidative capacity.

Results and comparison with existing methods

The present study demonstrates that adjustments enabled obtention of higher respiration values than Holloway’s protocol and might allow the detection of slight mitochondrial alterations. In a second part of this study, we showed that cortex, striatum, hippocampus and cerebellum displayed similar maximal oxidative capacities (under pyruvate, malate and succinate) while complex IV-driven respiration is significantly lower in cerebellum compared to cortex. These observations were supported by the measurement of citrate synthase and cytochrome oxidase activities.

Conclusion

The developed procedure improves the investigations of mitochondrial electron transfer chain in specific cerebral regions.