Regulation of Substantia Nigra Pars Reticulata GABAergic Neuron Activity by H₂O₂ via Flufenamic Acid-Sensitive Channels and KATP Channels

Abstract

Substantia nigra pars reticulata (SNr) GABAergic neurons are key output neurons of the basal ganglia. Given the role of these neurons in motor control, it is important to understand factors that regulate their firing rate and pattern. One potential regulator is hydrogen peroxide (H₂O₂), a reactive oxygen species that is increasingly recognized as a neuromodulator. We used whole-cell current clamp recordings of SNr GABAergic neurons in guinea-pig midbrain slices to determine how H₂O₂ affects the activity of these neurons and to explore the classes of ion channels underlying those effects. Elevation of H₂O₂ levels caused an increase in the spontaneous firing rate of SNr GABAergic neurons, whether by application of exogenous H₂O₂ or amplification of endogenous H₂O₂ through inhibition of glutathione peroxidase with mercaptosuccinate. This effect was reversed by flufenamic acid (FFA), implicating transient receptor potential (TRP) channels. Conversely, depletion of endogenous H₂O₂ by catalase, a peroxidase enzyme, decreased spontaneous firing rate and firing precision of SNr neurons, demonstrating tonic control of firing rate by H₂O₂. Elevation of H₂O₂ in the presence of FFA revealed an inhibition of tonic firing that was prevented by blockade of ATP-sensitive K(+) (K(ATP)) channels with glibenclamide. In contrast to guinea-pig SNr neurons, the dominant effect of H₂O₂ elevation in mouse SNr GABAergic neurons was hyperpolarization, indicating a species difference in H₂O₂-dependent regulation. Thus, H₂O₂ is an endogenous modulator of SNr GABAergic neurons, acting primarily through presumed TRP channels in guinea-pig SNr, with additional modulation via K(ATP) channels to regulate SNr output.

Keywords: GABA; TRP channels; basal ganglia; diffusible messenger; hydrogen peroxide; reactive oxygen species.

Figure 1

Exogenous H₂O₂ increases the firing rate of SNr GABAergic neurons from guinea pig recorded in vitro. (A) Whole-cell current clamp recording from a SNr GABAergic neuron under control conditions and (B) following application of H₂O₂ (1.5 mM). (C) H₂O₂ caused an increase in firing rate without affecting the regularity of firing as measured by the coefficient of variation in (D). (E) The voltage deflection caused by a hyperpolarizing current pulse under control conditions (black trace) was attenuated in the presence of H₂O₂ (red trace) indicating that H₂O₂ decreased input resistance, consistent with ion-channel opening. (***p < 0.001).

Figure 2

Glutathione (GSH) peroxidase inhibition increases levels of endogenously produced H₂O₂ in SNr GABAergic neurons. (A) Pseudocolored photomicrograph of basal DCF fluorescence in a SNr GABAergic neuron. (B) DCF fluorescence intensity (FI) increased following inhibition of GSH peroxidase with mercaptosuccinate (MCS; 1 mM). Scale bar = 20 μm. (C) Plot of the DCF FI increase caused by MCS alone (black), by MCS in the presence of catalase (500 U/mL; green), and by MCS in the presence of TTX (2 μM; red). The MCS-induced increase in DCF FI was strongly attenuated when MCS was applied in the presence of catalase as well as when spontaneous activity was silenced with TTX when measured at the same duration of MCS exposure. These data indicate that GSH peroxidase inhibition by MCS increases intracellular H₂O₂ concentration in SNr neurons and that spontaneous activity contributes to endogenous H₂O₂ production. (***p < 0.001 basal vs. MCS; ++p < 0.01 MCS vs. MCS + catalase; *p < 0.05 MCS vs. MCS + TTX).

Figure 3

Amplifying endogenous H₂O₂ levels increases SNr GABAergic neuron firing rate. (A) Spontaneous firing from a SNr GABAergic neuron under control conditions and (B) after inhibition of GSH peroxidase with mercaptosuccinate (MCS; 1 mM). (C) Amplifying endogenous H₂O₂ with MCS caused a significant increase in the firing rate of SNr GABAergic neurons. (D) The regularity of firing as measured by the coefficient of variation was unaffected by increasing endogenous H₂O₂. (***p < 0.001).

Figure 4

Depletion of endogenous H₂O₂ with catalase slows the firing rate of SNr GABAergic neurons and decreases the regularity of their spontaneous activity. (A) Spontaneous activity of a SNr GABAergic neuron under control conditions and (B) after depletion of endogenous H₂O₂ with catalase (500 U/mL). (C) Catalase (Cat) caused a decrease in the spontaneous firing rate of SNr GABAergic neurons. (D) In addition, spontaneous activity became more irregular in catalase, reflected in an increase in the coefficient of variation. (**p < 0.01; ***p < 0.001).

Figure 5

Flufenamic acid (FFA) reverses H₂O₂-induced increases in firing rate. (A) Spontaneous activity of a SNr GABAergic neuron under control conditions, (B) following H₂O₂ (1.5 mM) application, and (C) with FFA (20 μM) applied in the continued presence of H₂O₂. (D) The H₂O₂-induced increase in firing rate was reversed by FFA and the resulting firing rate suppressed below control. (E) Activity of another SNr GABAergic neuron under control conditions, (F) following amplification of endogenous H₂O₂ with MCS (1 mM), and (G) in FFA (20 μM) in the continued presence of MCS. (H) Increases in firing rate induced by amplified endogenous H₂O₂ were similarly reversed and suppressed below control levels by FFA. (*p < 0.05; **p < 0.01; ***p < 0.001).

Figure 6

FFA-sensitive channels are tonically activated by H₂O₂ in SNr GABAergic neurons. (A) Spontaneous firing of a SNr GABAergic neuron under control conditions, (B) following depletion of basal H₂O₂ with catalase (500 U/mL), and (C) in FFA (20 μM) in the continued presence of catalase. (D) Catalase (Cat) caused a significant decrease in firing rate which was unchanged when FFA was added in the continued presence of catalase. (NS not significant; *p < 0.05).

Figure 7

H₂O₂ can alter SNr GABAergic neuron activity via both TRP and K_ATP channels. (A) Spontaneous firing of a SNr GABAergic neuron under control conditions, (B) with TRP channels blocked by FFA (20 μM), and (C) with H₂O₂ (1.5 mM) in the continued presence of FFA. (D) Following blockade of TRP channels, exogenous H₂O₂ suppressed SNr neuron firing. In some cases [as in (C)] a marked hyperpolarization was seen that was sufficient to silence the neuron. (E) This suppression of firing was prevented by the K_ATP channel blocker glibenclamide (Glib; 3 μM). (F) Recording from another SNr GABAergic neuron under control conditions, (G) with FFA (20 μM), and (H) with MCS (1 mM) in the continued presence of FFA. (I) Amplifying endogenous H₂O₂ levels with MCS caused a suppression of firing rate when TRP channels were blocked with FFA. (J) The MCS-induced suppression of neuronal activity was also prevented by glibenclamide. (NS not significant; *p < 0.05; **p < 0.01).

Figure 8

Exogenous H₂O₂ suppresses the firing of SNr GABAergic neurons in mouse slices. (A) Whole-cell current clamp recording from a mouse SNr GABAergic neuron recorded in vitro under control conditions. (B) The same neuron recorded in the presence of exogenous H₂O₂ (750 μM) exhibited decreased firing rate and eventual hyperpolarization leading to cessation of spontaneous firing. (C) H₂O₂ caused a significant decrease in firing rate in SNr GABAergic neurons from mouse. (**p < 0.01).