Stimulation by neurotransmitters activates propagating Ca²⁺ waves in cultured astrocytes and oligodendrocytes. Our experiments showed that locally discrete cellular sites with elevated Ca²⁺ release kinetics (increased amplitude and rate of rise of response) which are typically found along processes provide regenerative Ca²⁺ release necessary for propagation. These specialized elementary Ca²⁺ release sites act as wave amplification points. We have characterized the possible involvement of mitochondria and endoplasmic reticulum specializations in achieving such locally elevated Ca²⁺ release kinetics. Cells were loaded with fluo 3 and fluorescence images were analyzed at high resolution (0.8 µm-wide slices along the cell axis, 1 image every 66 ms), followed by immunocytochemistry or direct organellar staining while cells remained on the microscope stage. Using the dyes JC-1 and DiOC6(3), mitochondria were found to be located singly or in convoluted groups along oligodendrocyte processes. Cross-correlation analysis revealed that these mitochondrial groups were closely associated with sites of elevated Ca²⁺ release kinetics. Ca²⁺ uptake into rhod 2-loaded mitochondria occurred during the cytosolic Ca²⁺ wave, and pretreatment with FCCP (1 µM, 2 min) greatly altered response amplitude. In addition, changes in mitochondrial membrane potential were also recorded during the Ca²⁺ wave. Immunocytochemistry revealed that these Ca²⁺ release sites were endowed with higher density of type 2 inositol trisphosphate receptors (InsP3R2), SERCA pumps as shown by staining with fluorescently tagged thapsigargin and bead like concentration of intraluminal calreticulin. It therefore appears that multiple specializations underlie domains of elevated Ca²⁺ release in oligodendrocyte processes and these include the presence of mitochondria, which may modulate the level of Ca²⁺ near release sites, and by elevated levels of proteins involved in Ca²⁺ signaling such as calreticulin, InsP3R2 Ca²⁺ release channels and SERCA pumps. Two-dimensional images of intracellular Ca²⁺ flux during a propagating wave were developed using a computational method.

Back to Workshop Schedule