Endogenous hydrogen peroxide regulates the excitability of midbrain dopamine neurons via ATP-sensitive potassium channels

Abstract

ATP-sensitive K+ (K(ATP)) channels link metabolic state to cell excitability. Here, we examined regulation of K(ATP) channels in substantia nigra dopamine neurons by hydrogen peroxide (H2O2), which is produced in all cells during aerobic metabolism. Blockade of K(ATP) channels by glibenclamide (100 nM) or depletion of intracellular H2O2 by including catalase, a peroxidase enzyme, in the patch pipette increased the spontaneous firing rate of all dopamine neurons tested in guinea pig midbrain slices. Using fluorescence imaging with dichlorofluorescein to visualize intracellular H2O2, we found that moderate increases in H2O2 during partial inhibition of glutathione (GSH) peroxidase by mercaptosuccinate (0.1-0.3 mM) had no effect on dopamine neuron firing rate. However, with greater GSH inhibition (1 mM mercaptosuccinate) or application of exogenous H2O2, 50% of recorded cells showed K(ATP) channel-dependent hyperpolarization. Responsive cells also hyperpolarized with diazoxide, a selective opener for K(ATP) channels containing sulfonylurea receptor SUR1 subunits, but not with cromakalim, a selective opener for SUR2-based channels, indicating that SUR1-based K(ATP) channels conveyed enhanced sensitivity to elevated H2O2. In contrast, when endogenous H2O2 levels were increased after inhibition of catalase, the predominant peroxidase in the substantia nigra, with 3-amino-1,2,4-triazole (1 mM), all dopamine neurons responded with glibenclamide-reversible hyperpolarization. Fluorescence imaging of H2O2 indicated that catalase inhibition rapidly amplified intracellular H2O2, whereas inhibition of GSH peroxidase, a predominantly glial enzyme, caused a slower, smaller increase, especially in nonresponsive cells. Thus, endogenous H2O2 modulates neuronal activity via K(ATP) channel opening, thereby enhancing the reciprocal relationship between metabolism and excitability.

Figure 2.

Regulation of spontaneous activity in DA cells by H₂O₂ and K_ATP channels. a, In all DA neurons tested (n = 17), depolarizing current injection (0.2 nA, 350 ms) induced an increase in the firing rate (top) that was accompanied by elevated H₂O₂ levels [DCF fluorescence intensity (FI); shown as pseudocolored images; p < 0.01 vs basal FI (bottom)]. The dashed vertical line and arrow indicate the onset of current injection. Stim, Stimulated. Scale bars, 20 μm. b, Pacemaker activity in a representative DA neuron under control conditions and after 15 min in glibenclamide (100 nm; Glib); K_ATP channel blockade by glibenclamide increased the firing rate (n = 5; p < 0.01). c, The firing rate also increased progressively when catalase (Cat; 500 IU/ml) was included in the patch pipette backfill solution [n = 7; ^**p < 0.01 and ^***p < 0.001 vs respective control frequency after 10 min of recording (ANOVA)]; heat-inactivated catalase (I-Cat) did not alter spontaneous activity (n = 6). Glib (100 nm) applied 15 min before patching prevented the Cat-induced increase in the firing rate (n = 5). Data are means ± SEM.

Figure 5.

Activity-dependent H₂O₂ generation in DA neurons. The average time course and amplitude of MCS-induced increases in intracellular H₂O₂ in responders (Resp; n = 7) and nonresponders (Nonresp; n = 7) is shown. FI, DCF fluorescence intensity. Intracellular levels of H₂O₂ did not change when MCS was applied in the presence of TTX (1 μm; n = 3), indicating that MCS-induced increases in H₂O₂ require action potentials. Data are means ± SEM. ^**p < 0.01 and ^***p < 0.001 versus basal fluorescence; neither time course nor amplitude differed significantly between responders and nonresponders.

Figure 8.

Effect of SUR1- and SUR2-selective K_ATP channel openers on H₂O₂ responders and nonresponders. a, Representative continuous current-clamp recording in a responder: H₂O₂ (1.5 mm) caused membrane hyperpolarization and inhibition of spontaneous firing activity in this DA neuron; cromakalim (60 μm; 20 min) had no effect, whereas subsequently applied diazoxide (60 μm; 20 min) caused hyperpolarization and loss of spontaneous activity. b, Representative current-clamp recording in a nonresponder: H₂O₂ (1.5 mm) applied for 10 min did not alter membrane properties; diazoxide had no effect, whereas subsequently applied cromakalim caused membrane hyperpolarization and loss of spontaneous activity.

Figure 1.

Identification of DA neurons. a, Representative current-clamp record from a neuron in the SNc showing spontaneous “pacemaker” firing activity. b, Voltage responses to hyperpolarizing current injection in this cell; note the prominent time-dependent rectification (sag). c, This cell was filled with Lucifer yellow during recording (left), and then the slice was processed for TH immunochemistry (TH-ir; middle). Double labeling (right) confirmed that the recorded cell was a DA neuron. Scale bars, 20 μm.

Figure 3.

Concentration-dependent effect of GSH peroxidase inhibition by MCS on H₂O₂ amplification. Inhibition of GSH peroxidase by MCS (0.1-1 mm) caused a progressive increase in H₂O₂ levels [DCF fluorescence intensity (FI); R² = 0.946]. All concentrations were tested in each cell (n = 7; ^***p < 0.001 vs basal). Images are representative examples of DCF fluorescence for each concentration of MCS tested in one cell. Scale bars, 20 μm.

Figure 4.

Effects of GSH peroxidase inhibition on membrane properties and H₂O₂. a, Inhibition of GSH peroxidase by MCS (1 mm) caused hyperpolarization and cessation of spontaneous activity in one population of DA neurons (responders; n = 20 of 38). b, Simultaneously recorded DCF fluorescence in the responder in a before (Basal) and during MCS exposure. c, Time course of H₂O₂ elevation [fluorescence intensity (FI)] in this cell. d, MCS did not affect spontaneous activity in a second population of DA neurons (nonresponders; n = 18 of 38). e, Simultaneously recorded DCF fluorescence images before and during MCS in the nonresponder in d. f, Time course of H₂O₂ elevation in this cell. Scale bars, 20 μm.

Figure 6.

Endogenous H₂O₂ activates K_ATP channels. a, Representative voltage-clamp record from a DA neuron during MCS application showing activation of an outward current. b, Glibenclamide (Glib; 3 μm) reversed the MCS-induced membrane hyperpolarization and loss of spontaneous firing in the presence of MCS. c, Voltage responses to hyperpolarizing current recorded in this cell under control conditions, in MCS, and in MCS plus Glib.

Figure 7.

Effect of exogenous H₂O₂ on physiological properties of DA neurons. a, Exogenous H₂O₂ (1.5 mm) applied for 10 min caused a reversible membrane hyperpolarization and loss of spontaneous firing activity in one population of identified DA neurons (responders; n = 15 of 28) but not in a second population of DA cells (b; nonresponders; n = 13 of 28). c, Current-clamp recording from a responder during application of exogenous H₂O₂; glibenclamide (Glib; 3 μm) reversed the H₂O₂-induced membrane hyperpolarization and restored spontaneous activity in the continuous presence of H₂O₂ (n = 7).

Figure 9.

Effects of catalase inhibition on membrane properties and H₂O₂. a, Current-clamp record showing a reversible membrane hyperpolarization and loss of spontaneous firing in a DA neuron by catalase inhibition with ATZ (1 mm); all DA neurons responded to ATZ (n = 12). b, Simultaneously recorded DCF fluorescence in this cell before (Basal) and during ATZ exposure confirmed H₂O₂ elevation. Scale bar, 20 μm. c, Time course of H₂O₂ increase. FI, Fluorescence intensity. d, e, Average time course and amplitude of H₂O₂ increases induced by ATZ versus those in MCS responders (d; MCS_Resp) and MCS nonresponders (e; MCS_Nonresp). Data are means ± SEM. ^**p < 0.01 and ^***p < 0.001 versus MCS. f, Glibenclamide (Glib; 10 μm) reversed ATZ-induced membrane hyperpolarization and loss of cell firing in the continued presence of ATZ (n = 4).