Abstract:
Rotenone is a toxin used to generate animal models of Parkinson's disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05-1 mu m) effects on SNc neurons in acute rat midbrain slices, using whole-cell patch-clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide-sensitive outward current (94 +/- 15 pA) associated with increases in intracellular [Ca2+] ([Ca2+](i)) (73.8 +/- 7.7 nm) and intracellular [Na+] (3.1 +/- 0.6 mm) (all with 1 mu m). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca2+](i) rise was abolished by removing extracellular Ca2+, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N-(p-amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine-123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca2+](i) rise resulted in a large (46.6 +/- 25.3 nm) Ca2+ response when baseline [Ca2+](i) was increased by a 'priming' protocol that activated voltage-gated Ca2+ channels. There was also a positive correlation between 'naturally' occurring variations in baseline [Ca2+](i) and the rotenone-induced [Ca2+](i) rise. This correlation was not seen in non-dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP-gated K+ channels and TRPM2-like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone-induced [Ca2+](i) rise by a small increase in baseline [Ca2+](i).
