Poster
# 22
Poster |
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6th Internet World Congress for Biomedical Sciences |
José Manuel Martínez-Martos(1), María Jesús Ramírez-Expósito(2), María Dolores Mayas-Torres(3), Isabel Prieto-Gómez(4), Manuel Ramírez-Sánchez(5)
(1)(3)(4)(5)Unit of Physiology. University of Jaén - Jaén. Spain
(2)Unit of Physiology. University of Jaen - Jaén. Spain
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[Neuroscience] |
[Physiology] |
Frontal cortex synaptosomes from eight male Wistar rats (body weight 265±15 g) and eight male Balb/C mice (body weigh 28.59±0.6 g) were prepared as described previously (Whittaker, Michaelson & Kirkland, 1964) with slight modifications. Briefly, animals were sacrificed by decapitation; their brains were removed and immediately rinsed in cold isotonic saline. The frontal cortex was dissected and maintained on ice until it was homogenized in cold 0.32 M sucrose (approximately 1:20 original tissue weight to volume (g/ml)). All next steps were performed at 4 C. The homogenate was centrifuged at 2000 xg for 12 min, and the resulting supernatant was then centrifuged at 30000 xg for 27 min. The supernatant was discarded and the pellet was resuspended in 0.32 M sucrose. A three layer sucrose gradient was prepared by layering the membrane preparation on top of 1.4 M sucrose plus 0.8 M sucrose for rats, or 1.2 M sucrose plus 0.8 M sucrose for mice. The gradient was centrifugated for 30 min at 30000 xg. The 0.8 M layer pellet was collected and resuspended in an incubation buffer containing 116 mM NaCl, 5.4 mM KCl, 0.9 mM MgCl2, 0.9 mM NaH2PO4, 1.8 mM CaCl2, 25 mM NaHCO3, 10 mM glucose and 1 mM 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) , pH 7.2. Total protein content was adjusted to a final concentration of 1, 2 and 4 mg/ml, measured by the method of Bradford (1976), using BSA as standard. Depolarization of synaptosomes was performed by the addition of increased concentrations of KCl (25, 50 and 100 mM) in the incubation buffer. The increase of K+ was compensated by a proportional reduction of Na+ to maintain osmolarity. ATP stimulation of synaptosomes was carried out by the addition of the corresponding amount of a stock solution of ATP 1 mM in incubation buffer, to get a final concentration of 1, 2, 4, 10 and 20 µM. After that, mitochondrial activity was measured by the colorimetric assay previously described by Mosmann (1983). 50 µl of the suspension of synaptosomes were incubated in triplicate in a 96-well plate for 1 or 2 hours at 37°C. Then, 100 µl of HCl in isopropanol (0.04 M) was added to each well and the mixture was shaked vigorously to dissolve the dark blue crystals. Finally, the samples were measured in a microplate reader using a test wavelength of 550 nm and a reference wavelength of 620 nm. The values were expressed in optical density units as mean±SEM. The one-way analysis of variance (ANOVA) with the Newman-Keul´s post-hoc test, was used for comparisons among the different groups. The linear correlation coefficient test was used for relationships between protein content and MTT formazan production. The null hypothesis was rejected when p<0.05.
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[Neuroscience] |
[Physiology] |
