Glutamate-dependent inhibition of dopamine release in striatum is mediated by a new diffusible messenger, H2O2

Abstract

How glutamate regulates dopamine (DA) release in striatum has been a controversial issue. Here, we resolve this by showing that glutamate, acting at AMPA receptors, inhibits DA release by a nonclassic mechanism mediated by hydrogen peroxide (H(2)O(2)). Moreover, we show that GABA(A)-receptor activation opposes this process, thereby enhancing DA release. The influence of glutamate and GABA on DA release was assessed in striatal slices using carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Modulation by both transmitters was prevented by H(2)O(2)-metabolizing enzymes. In addition, the influence of GABA(A)-receptor activation was lost when AMPA receptors were blocked with GYKI-52466. Together, these data show that modulation of DA release by glutamate and GABA depends on H(2)O(2) generated downstream from AMPA receptors. This is the first evidence that endogenous glutamate can lead to the generation of reactive oxygen species under physiological conditions. We also show that inhibition of DA release by H(2)O(2) is mediated by sulfonylurea-sensitive K(+) channels: tolbutamide blocked DA modulation by glutamate and by GABA. The absence of ionotropic glutamate or GABA receptors on DA terminals indicates that modulatory H(2)O(2) is generated in non-DA cells. Thus, in addition to its known excitatory actions in striatum, glutamate mediates inhibition by generating H(2)O(2) that must diffuse from postsynaptic sites to inhibit presynaptic DA release via K(+)-channel opening. These findings have significant implications not only for normal striatal function but also for understanding disease states that involve DA and oxidative stress, including disorders as diverse as Parkinson's disease and schizophrenia.

Fig. 1.

Modulation of evoked DA release by glutamate and GABA receptor activation. a, Application of the AMPA-receptor antagonist GYKI-52466 (GYKI; 50 μm) significantly increased evoked [DA]_o in the striatum (p < 0.001;n = 6); mean peak [DA]_o in control records was 1.57 ± 0.12 μm (n = 6). b, The GABA_A antagonist picrotoxin (PTX; 100 μm) caused a significant decrease in evoked [DA]_o (p < 0.001; n = 6), whereas the GABA_Bantagonist saclofen (c, Sac; 50 μm) had no effect (p > 0.05;n = 8). a–c, Data are mean ± SEM, illustrated as percentage of same-site control. DA release was elicited by pulse-train stimulation (10 Hz). Solid barsindicate stimulation period.

Fig. 2.

Modulation of striatal DA release by glutamate depends on the availability of H₂O₂.a, Active catalase (Cat; 500 IU/ml) abolished the effect of GYKI-52466 (50 μm) on evoked [DA]_o (p > 0.05;n = 6); b, in the presence of heat-inactivated catalase (I-Cat), application of GYKI-52466 caused the usual increase of evoked [DA]_o(p < 0.001; n = 6).c, Active GSHPx (3 IU/ml) also prevented the effect of GYKI-52466 on evoked [DA]_o (p> 0.05; n = 8); d, in the presence of heat-inactivated GSHPx (I-GSHPx), GYKI-52466 again caused a significant increase in evoked [DA]_o(p < 0.001; n = 8).a–d, Solid bars indicate stimulation period.

Fig. 3.

Modulation of striatal DA release by endogenous H₂O₂ is AMPA-receptor dependent.a, Average evoked [DA]_o under control conditions and in the presence of the GSHPx inhibitor MCS (1 mm). Application of MCS caused a 40% decrease in evoked [DA]_o from 1.52 ± 0.07 to 0.89 ± 0.04 μm (p < 0.001;n = 7); inset shows identifying DA voltammograms recorded at the peak of the response under control conditions (solid line) and after MCS (dashed line). In the continued presence of MCS, added catalase (Cat; 500 IU/ml) reversed the MCS-induced suppression of evoked [DA]_o (to 1.62 ± 0.12 μm;p > 0.05; MCS plus catalase vs control;n = 7). b, The AMPA-receptor antagonist GYKI-52466 (GYKI; 50 μm) caused a significant increase in evoked [DA]_o(p < 0.001; n = 5). In the continued presence of GYKI-52466, the effect of MCS in evoked [DA]_o was prevented (p > 0.05 vs GYKI-52466 alone; n = 5). c, DA release elicited by single-pulse stimulation (1 p,arrow) was unaffected by GYKI-52466 (50 μm), indicating a lack of glutamate-dependent regulation (p > 0.05 vs control;n = 4). d, Effect of GSHPx inhibition by MCS on DA release evoked by single-pulse and 30-pulse train stimulation (10 Hz). Average maximum [DA]_o evoked by single-pulse stimulation was 1.43 ± 0.08 μm(n = 5) and was not affected by MCS (p > 0.05; n = 5). During pulse-train stimulation, however, MCS caused a decrease in evoked [DA]_o (p < 0.001;n = 5) (indicated by dashed line), which reversed during MCS washout (p > 0.05 wash vs control; n = 5). a,b, d, Solid bar indicates pulse-train stimulation period.

Fig. 4.

The inhibitory effect of H₂O₂ on DA release is direct and species independent. a, H₂O₂ modulates DA release but not uptake. In the presence of the DA transport inhibitor GBR-12909 (GBR; 2 μm), inhibition of GSHPx by MSC (1 mm) decreased maximum evoked [DA]_o from 4.40 ± 0.17 to 2.75 ± 0.08 μm (p < 0.001;n = 4). b, Effect of ascorbate on evoked [DA]_o under control conditions and during GSHPx inhibition. Stable evoked DA release was elicited under control conditions, and then ascorbate (Asc; 400 μm) was applied. The presence of ascorbate did not alter evoked [DA]_o (p > 0.05;n = 5), nor did it interfere with the usual depression of evoked [DA]_o by MCS (1 mm;p < 0.001; n = 5).c, Average evoked [DA]_o in rat striatum in control and in the presence of MCS. Application of MCS caused a reversible 35% decrease in evoked [DA]_o in rat dorsal striatum, from a maximum of 1.80 ± 0.15 to 1.17 ± 0.07 μm (p < 0.001;n = 7). a–c, Solid bars indicate stimulation period.

Fig. 5.

GABA opposes the inhibitory effect of H₂O₂ on synaptic DA release in striatum.a, The decrease in evoked [DA]_o by the GABA_A-receptor antagonist picrotoxin (PTX; 100 μm) was prevented by catalase (Cat) (p > 0.05; n = 6);b, in heat-inactivated catalase (I-Cat), picrotoxin induced a decrease in evoked [DA]_o(p < 0.001; n = 6).c, Application of GYKI-52466 (GYKI; 50 μm) significantly increased evoked [DA]_o(p < 0.001; n = 7).d, In the continued presence of GYKI-52466, the effect of picrotoxin was completely prevented (p> 0.05; n = 7), showing that the influence of GABA on DA release requires AMPA-receptor activation. a–d,Solid bars indicate stimulation period.

Fig. 6.

H₂O₂-dependent modulation of DA release is mediated by sulfonylurea-sensitive K⁺ channels. a, Tolbutamide (Tolb; 200 μm) caused a significant increase in evoked [DA]_o (p< 0.01; n = 8); in the continued presence of tolbutamide, the usual suppression of DA release by MCS (1 mm) was prevented (p > 0.05;n = 8). b, Tolbutamide prevented the usual increase in DA release with GYKI-52466 (GYKI; 50 μm) (p > 0.05;n = 6). c, Tolbutamide prevented the usual decrease in DA release by picrotoxin (PTX; 100 μm) (p > 0.05;n = 8). a–c, Solid bars indicate stimulation period.