Preparation and handling of brain mitochondria useful to study uptake and release of calcium

There is increasing evidence for a critical role of mitochondria in calcium homeostasis and neuronal death in excitotoxicity. In spite of much work during the last two decades, the kinetic parameters of Ca(2+) transport in brain mitochondria remain controversial. Analysis of the literature data suggests that these contradictions can be due to differences in the methodology used to prepare or to incubate brain mitochondria. In the present communication, the whole protocol for preparation of non-synaptic rat forebrain mitochondria is described. This report shows that this preparation is well coupled and essentially free of non-mitochondrial contaminants. The mitochondria obtained are useful to study Ca(2+) uptake and release. Both Na(+)-independent, Na(+)-dependent and spontaneous Ca(2+) release may be studied with this preparation. This system is also useful in studies on the role of mitochondria and other intracellular Ca(2+) stores in disturbance of Ca(2+) homeostasis and delayed cell death under excitotoxic conditions.