Quantitation of hydrogen peroxide fluctuations and their modulation of dopamine dynamics in the rat dorsal striatum using fast-scan cyclic voltammetry

Abstract

The dopaminergic neurons of the nigrostriatal dopamine (DA) projection from the substantia nigra to the dorsal striatum become dysfunctional and slowly degenerate in Parkinson's disease, a neurodegenerative disorder that afflicts more than one million Americans. There is no specific known cause for idiopathic Parkinson's disease; however, multiple lines of evidence implicate oxidative stress as an underlying factor in both the initiation and progression of the disease. This involves the enhanced generation of reactive oxygen species, including hydrogen peroxide (H2O2), whose role in complex biological processes is not well understood. Using fast-scan cyclic voltammetry at bare carbon-fiber microelectrodes, we have simultaneously monitored and quantified H2O2 and DA fluctuations in intact striatal tissue under basal conditions and in response to the initiation of oxidative stress. Furthermore, we have assessed the effect of acute increases in local H2O2 concentration on both electrically evoked DA release and basal DA levels. Increases in endogenous H2O2 in the dorsal striatum attenuated electrically evoked DA release, and also decreased basal DA levels in this brain region. These novel results will help to disambiguate the chemical mechanisms underlying the progression of neurodegenerative disease states, such as Parkinson's disease, that involve oxidative stress.

Figure 1

Rapid H₂O₂ fluctuations in the dorsal striatum. H₂O₂ is generated at a robust striatal recording site (right) compared with H₂O₂ detection in vitro (left). (A) Color plots each containing 150 background-subtracted voltammograms. The ordinate is potential applied to the carbon-fiber electrode, the abscissa is time, and the current is depicted in false color. (B) Concentration vs time traces extracted from the data at 1.2 V; the peak oxidation potential for H₂O₂. The current is converted to concentration upon electrode calibration. (C) The cyclic voltammogram for a H₂O₂ transient collected in vivo is presented as a black line, and that collected in vitro is presented as a red line for comparison.

Figure 2

Detection of exogenous (left) and endogenous (right) H₂O₂. (A) Left: 1 M H₂O₂ was locally microinfused into the striatum, at a location proximal to the microelectrode. Right: averaged color plots (n = 4) collected ∼4.7 min after the onset of a local microinfusion of 100 mM MCS. (B) Concentration vs time traces extracted from the data at 1.2 V, the peak oxidation potential for H₂O₂. (C) Left: cyclic voltammograms extracted from the data. The cyclic voltammogram for endogenous H₂O₂ is presented as a black line, and that for exogenous H₂O₂ is presented as a red line for comparison (r = 0.98). Right: bar graph quantifying the increase in H₂O₂ elicited by microinfusion of MCS compared to saline (n = 4, p < 0.01, Student’s t test).

Figure 3

H₂O₂ modulates striatal dopamine. (A) Color plots depicting striatal DA dynamics. Left: DA release evoked by electrical stimulation (arrow) of the SN at the start of the experiment. Center: averaged color plots (n = 4) depicting a decrease in basal DA levels ∼6 min after the onset of MCS microinfusion. Right: color plot of DA release electrically evoked (arrow) at the end of the experiment. (B) Concentration vs time traces extracted from the data at 0.6 V, the peak oxidation potential for DA. The bar graph (inset) quantifies the decrease in basal DA levels evoked by the MCS microinfusion, compared to a saline microinfusion (n = 4, p < 0.001, Student’s t test). (C) Cyclic voltammograms extracted from the data qualitatively identifying the analyte as DA.

Figure 4

MCS microinfusion increases the extracellular concentration of dopamine-o-quinone. (A) Cyclic voltammograms extracted from the data and normalized by their peak anodic current. Increased cathodic current is evident in the DA voltammograms collected following MCS microinfusion into the striatum (red line). Postcalibration of the electrodes suggests that oxidative stress chemically generated ∼20 nM DAQ. (B) A bar graph quantifying the significant effect of drug treatment on the anodic/cathodic current ratios [F (3, 11) = 44.87] p < 0.0001, one-way ANOVA]. Posthoc Tukey’s comparisons reveal that the current ratio is significantly decreased compared to both pre- (***) and post- (###) MCS conditions (n = 4, p < 0.0001).

Figure 5

Locally elevated H₂O₂ concentrations attenuate electrically evoked DA release in the striatum, regardless of the pharmacological strategy used to elicit the H₂O₂ increase. Increased levels of endogenous H₂O₂ were elicited by local administration of either rotenone or MCS, allowing us to investigate multiple means of inducing a locally elevated H₂O₂ concentration. Both treatments elicited a significant effect on evoked DA release in the striatum, [F (3, 16) = 30.08], p < 0.0001, one-way ANOVA]. Posthoc Tukey’s comparisons indicate that both 100 mM MCS and 500 nM rotenone significantly attenuated electrically evoked DA release in the striatum (n = 4, ***p < 0.001 vs saline, ^##P < 0.01 vs 100 nM rotenone, ^###p < 0.001 vs 100 nM rotenone).

Figure 6

Distribution of carbon-fiber microelectrode placements in dorsal striatum. Coronal diagram shows recording and microinfusion sites for four subjects used in this study (gray dots). Coordinates and drawings were taken from a stereotaxic atlas.