The ratios were then compared between control and PD173074-treated animals

TMRE fluorescence of individually imaged cell bodies proportionally decreases with mitochondrial membrane depolarization. When DL-threo-b-benzyloxyaspartic acid or CGP-37157 were used, they were added starting from the permeabilization step throughout the end of the experiments. The Mg2 concentration in the “intracellular”medium is expected to block the mitochondrial uniporter. Glutamate and/or added drugs were diluted in the perfusion medium and applied by switching the reservoirs of the perfusion system. Analysis of Namit. Namit changes were monitored in intact cells as described previously. Briefly, SH-SY5Y and C6 cells were loaded with 1 mM CoroNa Red in serumfree culture medium for 15 min at 37uC. The pinhole was set to give an optical slice of,1 mm. CoroNa Red was excited at 543 nm and fluorescence emission was measured from 560 nm to 615 nm. ROIs excluding nuclei with a high density of mitochondria were BCTC biological activity selected in individual cells and the average fluorescence signal within these regions was analyzed over time. For each data point we obtained SEM values that were smaller than 7% of the corresponding fluorescence mean values. Analysis of mitochondrial calcium. Ca2mit changes were monitored in cells permeabilized as 24220009 described above. Briefly, SH-SY5Y and C6 cells were loaded with 5 mM Rhod-2 AM in culture medium for 60 min at 37uC. To remove the fluorescence contribution of the cytosolic component, cells were permeabilized with digitonin 9600591 5 mM in intracellular buffer containing 300 nM or 400 nM CaCl2. Rhod-2 was excited at 543 nm and fluorescence emission was measured from 560 nm to 600 nm. Real-time confocal imaging Analysis of mitochondrial inner membrane potential. SH-SY5Y and C6 cells grown for 18 h on poly-L-lysine-coated glass coverslips were loaded at 37uC with 150 nM tetramethylrhodamine ethyl ester . In these conditions, after mitochondrial depolarization, TMRE is released from the quenched matrix to the cytoplasm, resulting in an increase in cytoplasmic fluorescence. After 20 min cells were washed and transferred to a microscopy chamber in standard buffer solution in the presence of 150 nM TMRE. Confocal images were obtained using the 510 LSM microscope equipped with a META detection system and a 406oil immersion objective. Illumination intensity was kept to a minimum to avoid phototoxicity; the pinhole was set to give an optical slice of,1 mm. TMRE was excited at 543 nm and fluorescence was measured from 580 nm to 700 nm in cytoplasmic regions of interest. For data analysis fluorescence was expressed as ratios of fluorescence counts relative to Analysis of ATP production ATP production was evaluated using a commercially available luciferase-luciferin system. Isolated mitochondria. Mitochondria were incubated in a solution containing: KCl, 100; NaCl, 5; CaCl2, 0.0001; mannitol, 75; sucrose, 25; KH2PO4, 10; Tris-HCl, 10; and ADP, 0.1, with or without 0.51 mM glutamate. In preliminary experiments these glutamate concentrations gave the maximal response in terms of stimulation of ATP synthesis without toxic effects in our systems. The tested drugs or the respective vehicles were added to mitochondrial suspensions 15 min before glutamate stimulation and throughout the experiments. Luminescence was measured with a luminescence counter. All experiments were performed using,60 mg of mitochondria, an amount that in preliminary tests ensured strong and reproducible ATP signal, and 1 h incubation, which in the same preliminary tests