pH Changes in the Mitochondrial Matrix and Cytosol during Glutamate Deregulation of Ca2+ Homeostasis in Cultured Rat Hippocampal Neurons

Application of glutamate (Glu) in high concentrations to the rat brain primary neuronal cultures leads to a secondary rise in the intracellular free Ca2+ concentration (delayed calcium deregulation, DCD), which develops synchronously with a strong mitochondrial depolarization. In this work, pH in the mitochondrial matrix (pHm) and cytosol (pHc) was measured simultaneously with the intracellular concentration of free Ca2+ ([Ca2+](i)) in the soma of cultured neurons exposed to a toxic concentration of Glu (100 mu M). To this end, we performed the expression of the pH-sensitive fluorescence proteins, green mtYFP in the mitochondria and red mKate in the cytosol, in primary cultures from the hippocampus of newborn rats. To measure [Ca2+](i) simultaneously with pHm, and pHc neuronal cultures were loaded with low affinity Ca2+-sensitive indicator Fura-FF. It was found that during the first phase of the Ca2+-response to Glu, when only partial depolarization of mitochondria occurs, there is an increase in the pH gradient between the mitochondrial matrix and cytosol (Delta pH). The increase in.pH compensates for the decrease in the electrical mitochondrial potential (Delta Psi m), maintaining a relatively constant electrochemical mitochondrial potential and maintaining ATP synthesis at least until DCD begins. The development of DCD led to a sharp decrease both in Delta Psi m and Delta pH in the soma of neurons; however, a complete collapse of Delta pH was not observed. This probably means that DCD (1) is not caused by a nonspecific pore in the mitochondrial inner membrane (mPTP), as it is commonly thought, or (2) some of the mitochondria in the soma of neurons retain the barrier properties of the inner membrane and do not form mPTP even at high [Ca2+](i) plateau.