MITOCHONDRIA SUPPORT INOSITOL 1,4,5-TRISPHOSPHATE-MEDIATED CA2+ WAVESIN CULTURED OLIGODENDROCYTES

We have examined the spatial and temporal nature of Ca2+ signals activ ated via the phosphoinositide pathway in oligodendrocytes and the cell ular specializations underlying oligodendrocyte Ca2+ response characte ristics. Cultured cortical oligodendrocytes were incubated with fluo 3 or fura 2, and digital video fluorescence microscopy was used to stud y the effect of methacholine on [Ca2+](i). Single peaks, oscillations, and steady-state plateau [Ca2+](i) elevations were evoked by increasi ng agonist concentration. The peaks and oscillations were found to be Ca2+ wave fronts, which propagate via distinct amplification regions i n the cell where the kinetics of Ca2+ release (amplitude and rate of r ise of response) are elevated. Staining with loro-1,1',3,3'-tetraethyl benzimidazolecarbocyanine iodide (JC-1) and 3,3'-dihexyloxacarbocyanin e iodide revealed that mitochondria are found in groups of three or mo re in oligodendrocyte processes and that the groups are distributed wi th considerable distance separating them. Cross-correlation analysis s howed a high degree of correlation between sites where mitochondria ar e present and peaks in the amplitude and rate of rise of the Ca2+ resp onse. Intramitochondrial Ca2+ concentration, measured using rhod 2, in creased upon treatment with methacholine. Methacholine also evoked a r apid change in mitochondrial membrane potential as measured by the J-a ggregate fluorescence of JC-1. Pretreatment with the mitochondrial inh ibitors carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (1 mu M, 2 min) or antimycin (2 mu g/ml, 2 min) altered the methacholine-evoked Ca2+ response in most cells studied, responses being either markedly potentiated or inhibited. The results of this study demonstrate that s timulation of phosphoinositide-coupled muscarinic acetylcholinoceptors activates propagating Ca2+ wave fronts in oligodendrocytes and that t he characteristics of these waves are dependent on mitochondrial locat ion and function.