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Effect of anxiety on rat brain mitochondrial function The present study explores the role of mitochondria in anxiety responses in rats following exposure to novelty and open spaces that induces fear but also a conflict between the drive to explore and the drive to escape/avoid. Our experiments were conducted in an open space test (OST) proposed by Ennaceur et al. (2006) where animals are forced to explore and cannot escape or avoid. The OST consists of a platform (80 cm width, 80 cm length) with no surrounding ledges and elevated by 120 cm from the ground. The test is identical to that carried out in an open-field. Rats are individually placed on the platform and allowed to explore for 12 minutes. The platform is divided in outer area (15 cm wide from the edge of the field) and centre area (10 cm x 10 cm). The number and latency of crossings between areas as well the time spent in each area are recorded. After 12 min exposure to the OST animals were returned to their home cage and sacrificed immediately, 2hrs, 24hrs or 74hrs later. Forebrains from female Sprague-Dawley (SD) or Wistar (250-300g) were removed and placed in isolation buffer containing 220mM mannitol, 60mM sucrose, 5mM Tris-HCl, 0.5mM EGTA and 1mg ml -1 bovine serum albumin (BSA; fatty acid free) pH 7.4. Homogenates were centrifuged at 2,000 rpm for 6 min at 0-4 ° C and the resulting supernatant decanted off and spun for 8 min at 10,000 rpm. The pellet produced was resuspended in 9 ml of buffer and aliquots layered onto 10 ml ice-cold Percoll solution containing 250mM sucrose, 5mM Tris-HCl, 0.1mM EGTA and 18% ( w/ v) Percoll, pH 7.4. The resulting density gradient was centrifuged at 10,000 rpm for 45 min. A loose mitochondrial pellet, free from most contamination, was formed at the bottom of the tube. This pellet was isolated and centrifuged at 10,000 rpm in isolation buffer minus EGTA (incubation buffer) to remove the Percoll. Oxygen consumption was measured polarographically (Sweetman & Weetman, 1972) using a Clark-type oxygen electrode (Rank Bros, Bottisham, UK). Respiratory studies were performed after the method of Markham et al. (2004) and recorded on a Kipp and Zonen Flat bed recorder. Calculating the RCI (Respiratory Control Index; a measure of the efficiency of respiratory coupling) assessed mitochondrial integrity. Data were analysed using a one-way ANOVA followed by Dunnett’s post-test. A significant decrease in the RCI of 5mM glutamate plus 5mM malate or 5mM succinate is observed in naïve Wistar rats (8.96 ± 0.44, n=7 or 4.66 ± 0.45, n=8, respectively) compared to naive SD rats (13.22 ± 1.205, n=6, P<0.01 or 5.97 ± 0.32, n=6, P< 0.05, respectively). A time-dependant decrease in the RCI of glutamate and malate or succinate is also observed immediately (0 hours) (10.73 ± 1.26, n=6, P <0.05 or 4.69 ± 0.54, n=6, P <0.05, respectively), 2 hours (11.94 ± 0.62, n=5 or 4.81 ± 0.54, n=5, respectively), 24 hours (10.11 ± 0.68, n=5, P <0.01 or 4.28 ± 0.24, n=5, P <0.01, respectively), and 72 hours (8.38 ± 0.53, n=6, P <0.01 or 4.26 ± 0.18, n=6, P <0.01, respectively) after a unique exposure to the test. RCI of glutamate plus malate or succinate was significantly decreased (9.41 ± 0.55, P <0.01 or 4.16 ± 0.39, P <0.01, respectively) in animals subjected to repeated exposure to the test. No significance was displayed between acutely anxious rats sacrificed 24 hours post-OST and chronically exposed rats. Current studies show that Wistar rats appear more anxious than SD rats. In acutely anxious SD rats Complex I or II activity was reduced in a time-dependent manner in SD rats after a unique exposure to OST. In chronically anxious SD rats, no significant difference in complex I or II activity was shown between acutely or chronically anxious SD rats; however a reduction was seen compared to non exposed SD.
Ennaceur, A., et al.., (2006). Behavioural Brain Res., 171, 26-49. |
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