Fig. 5. Stored calcium was necessary for the mitochondrial depolarisations. (A) Astrocytes were exposed to a combination of 10 µM BAPTA-AM and 200 nM thapsigargin in order to empty intracellular Ca²⁺ stores and to chelate released Ca²⁺. The efficacy of this manipulation was demonstrated by application of 100 µM ATP, which mediates Ins(1,4,5)P₃-mediated Ca²⁺ release (left), failed to raise [Ca²⁺]_c in the treated cells (right). (B) The relative occurrence of mitochondrial depolarisations was assayed by calculation of `integrated activity'; digital images of each cell were differentiated, revealing how the signal changed with time. The peak signals in the differentiated series were then plotted against time and integrated in order to measure the number or amplitude of depolarisations (see Image processing and statistical analysis). The peak fluorescence with time over representative single cells is shown. Emptying and chelation of ER Ca²⁺ stores significantly reduced integrated activity: 14.6±5.4 (n=117 cells) versus 10.7±2.4 (n=68) (mean±s.d., P<0.0001, Mann-Whitney U test), confirming that stored Ca²⁺ was necessary for the mitochondrial depolarisations. Line images extracted from TMRE-loaded cells are shown in C. Treatment of cells with thapsigargin and BAPTA-AM effectively emptied ER Ca²⁺ stores. Cells were then loaded with TMRE and imaged as before. Multiple transient mitochondrial depolarisations were revealed along a line drawn through a single control cell, but very few were seen in cells treated with thapsigargin and BAPTA.

Return to article