The Mechanism of BDNF Evoked Release of Dopamine from the Rabbit Retina

Abstract

Abstract: : Purpose:In the vertebrate retina, TrkB, the receptor for BDNF is found on ganglion cells and amacrine cells, including dopaminergic amacrine cells. The present study was undertaken to see whether BDNF affected the release of dopamine. Methods:Dopamine release was measured from the retinas of New Zealand white rabbits that were killed with an intravenous injection of pentobarbital. Isolated hemi-retinas were incubated in 300µl Krebs bicarbonate medium containing pargyline (50µM), nomifensine (0.5µM) and BSA (0.1%), bubbled with O₂/CO₂ (95/5%) at 25C. The medium was changed every 10 min, and after five baseline samples were collected, BDNF was added for a further two periods. Dopamine in the resultant samples was measured by HPLC. The BDNF content of the retina was measured using an ELISA assay. Results:The concentration of BDNF in the retina was 5.940.45pg/mg protein (n=7). Exposure of the retina to BDNF caused a concentration dependent increase in the release of dopamine. The maximal increase, produced by 150ng/ml BDNF, was 18010.3% (n=20, P<0.001) above the resting release. This release was abolished in a calcium free medium. The tyrosine kinase inhibitors k252a (5µM) and genistein (100µM) blocked the effect of BDNF on dopamine release (P<0.01, n=4). BDNF (150ng/ml) did not increase dopamine release by inhibiting GABA release because its action was unaffected by picrotoxin (20µM). Activation of TrkB receptors sometimes causes the activation of phospholipase-Cγ and the production of inositoltrisphosphate (InsP₃). To see whether this mechanism was involved in the BDNF evoked release of dopamine, we tested the effects of the phospholipase inhibitor U73122 (5µM) and the sarco/endoplasmic reticulum Ca₂₊-ATPase (SERCA) inhibitors thapsigargin (5µM) and cyclopiazonic acid (5µM). All three drugs completely inhibited the release of dopamine caused by BDNF (150ng/ml). Conclusion:BDNF can, within minutes, increase the release of dopamine from amacrine cells. The mechanism seems to involve activation of phospholipase-C, the production of InsP₃ and an increase in intracellular [Ca₂₊] that arises partly from the release of intracellular stores but also from a "capacitive" entry of extracellular calcium.

Keywords: 389 dopamine • 312 amacrine cells