Brain-derived neurotrophic factor (BDNF) governs both the selective survival of neurons during development and the experience-based regulation of synaptic strength throughout life (Black, 1998).

Forebrains from female Wistar rats (250-300g) were removed and placed in isolation buffer containing 220mM mannitol, 60mM sucrose, 5mM Tris-HCl, 5mM Tris-base, 0.5mM EGTA and 1mg ml^-1 bovine serum albumin (BSA; fatty acid free) pH 7.4. All homogenates were centrifuged at 2,000 rpm for 6 min at 0-4°C, 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 with a synaptosomal fraction forming a separate layer at the top of the tube. The two layers were then isolated and centrifuged at 10,000 rpm in isolation buffer minus EGTA to remove the Percoll.

Oxygen consumption was measured polarographically using a Clark-type oxygen electrode (Rank Bros, Bottisham, UK). The in vitro model of anoxia-reoxygenation used was described by Morin et al., 2002. Anaerobic conditions were performed with ~0.3mg mitochondria plus ~0.5mg synaptosomes, which consume the oxygen content of 0.2ml respiratory buffer (300mM mannitol, 10mM KH₂PO₄, 10mM KCl and 5mM MgCl₂, pH 7.2) at 20°C. 10mM glutamate plus 10 mM malate were used as substrates and anaerobic conditions obtained after addition of 0.2mM ADP to a closed chamber. Anoxia was kept for 6 min followed by 6 min reoxygenation after addition of 0.3 ml respiratory buffer. Then the chamber was closed and the RCI (Respiratory Control Index; a measure of the efficiency of respiratory coupling) measured after the addition of 0.2 mM ADP. BDNF (333 ng ml^-1) and other drugs were added either before the anoxia or at reoxygenation. A control (open chamber) was performed under the same conditions but without the anoxia. Data were analysed using a one-way ANOVA followed by Dunnett's post-test.

Anoxia significantly decreased the RCI to 73.33 ± 0.88 % of control (P<0.01; n = 4). BDNF (333 ng ml^-1) significantly increased the RCI from the anoxia to 94 ± 2.5 % control. 10µM PD98059, 1µM U0126 (inhibitors of MEK; Tocris) and 1.33µg ml^-1 Ab-1 (an anti-BDNF antibody, Calbiochem) abolished this effect. BDNF (333 ng ml^-1) also significantly increased the RCI from an anoxia value of 64.8 ± 3.99 % control to 77.6 ± 3.67 % control (P<0.05; n = 5) when added at reoxygenation. Again 10µM PD98059, 1µM U0126 and 1.33mg µl^-1 Ab-1 abolished this effect.

BDNF protects rat brain mitochondria from experimental stresses induced by in vitro anoxia and reoxygenation.

Black, I.B. (1998) In Brain and Mind: evolutionary perspectives. M.S. Gazzaniga & J.S. Strasbourg Eds.
Morin, C. et al., (2002). Neuroscience, 115 (2), 415-424.