Acute action of rotenone on nigral dopaminergic neurons – involvement of reactive oxygen species and disruption of Ca²⁺ homeostasis

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 microm) 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 [Ca(2+)] ([Ca(2+)](i)) (73.8 +/- 7.7 nm) and intracellular [Na(+)] (3.1 +/- 0.6 mm) (all with 1 microm). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca(2+)](i) rise was abolished by removing extracellular Ca(2+), 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 [Ca(2+)](i) rise resulted in a large (46.6 +/- 25.3 nm) Ca(2+) response when baseline [Ca(2+)](i) was increased by a 'priming' protocol that activated voltage-gated Ca(2+) channels. There was also a positive correlation between 'naturally' occurring variations in baseline [Ca(2+)](i) and the rotenone-induced [Ca(2+)](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 [Ca(2+)](i) rise by a small increase in baseline [Ca(2+)](i).

Quantitation of Hydrogen Peroxide Fluctuations and Their Modulation of Dopamine Dynamics in the Rat Dorsal Striatum Using Fast-Scan Cyclic Voltammetry

“…Endogenous H 2 O 2 is formed upon mitochondrial dysfunction 9 and elicited by rotenone administration. 21 It can also be amplified by irreversible inhibition of peroxidase enzymes, and can lead to K ATP channel opening and suppression of axonal DA release. 22 Furthermore, in vivo studies investigating acute exposure to rotenone have shown increased generation of ROS during DA metabolism in nigrostriatal neurons.…”

“…22 Furthermore, in vivo studies investigating acute exposure to rotenone have shown increased generation of ROS during DA metabolism in nigrostriatal neurons. 21 32 In this work, the carbon-fiber electrode was positioned in the dorsal striatum of an anesthetized rat and ramped from −0.4 to 1.4 V in a triangular waveform every 100 ms at a scan rate of 400 V/s. This produces a large and stable nonfaradaic background current that can be subtracted to reveal smaller faradaic currents produced by electroactive analytes in the immediate vicinity of the electrode.…”

Molecular role of catalase on oxidative stress-induced Ca2+signaling and TRP cation channel activation in nervous system

“…Fenamates like FFA are antiinflammatory drugs known to alter ion fluxes through the plasma membrane. They are for instance, potent blockers of cation and anion channels, and FFA at ≥50 μM is now commonly used to block currents through TRP channels and receptor-operated channels (40). Previous experiments have shown that tonic activation of TRP channels maintains basal firing rate, as well as the regularity of firing, in SNr neurons (47).…”

“…Rotenone decreases intracellular ATP levels and increases the production of ROS including superoxide anion, H 2 O 2 and singlet oxygen (39). It also releases glutamate presynaptic terminals, leading to an additional increase in ROS production (40). Mitochondrial derived ROS can also activate TRPM2 channels (32).…”

“…Mitochondrial derived ROS can also activate TRPM2 channels (32). As TRPM2 channels are permeable to Ca 2+ and have previously been implicated in other neurodegenerative disorders (41), activation of these channels is a potentially important mechanism that may contribute to the pathogenesis of DRG and SNc neuron dependent pain and disorders (40).…”