NOX1/NADPH Oxidase Promotes Synaptic Facilitation Induced by Repeated D2 Receptor Stimulation: Involvement in Behavioral Repetition

Abstract

Repetitive behavior is a widely observed neuropsychiatric symptom. Abnormal dopaminergic signaling in the striatum is one of the factors associated with behavioral repetition; however, the molecular mechanisms underlying the induction of repetitive behavior remain unclear. Here, we demonstrated that the NOX1 isoform of the superoxide-producing enzyme NADPH oxidase regulated repetitive behavior in mice by facilitating excitatory synaptic inputs in the central striatum (CS). In male C57Bl/6J mice, repeated stimulation of D₂ receptors induced abnormal behavioral repetition and perseverative behavior. Nox1 deficiency or acute pharmacological inhibition of NOX1 significantly shortened repeated D₂ receptor stimulation-induced repetitive behavior without affecting motor responses to a single D₂ receptor stimulation. Among brain regions, Nox1 showed enriched expression in the striatum, and repeated dopamine D₂ receptor stimulation further increased Nox1 expression levels in the CS, but not in the dorsal striatum. Electrophysiological analyses revealed that repeated D₂ receptor stimulation facilitated excitatory inputs in the CS indirect pathway medium spiny neurons (iMSNs), and this effect was suppressed by the genetic deletion or pharmacological inhibition of NOX1. Nox1 deficiency potentiated protein tyrosine phosphatase activity and attenuated the accumulation of activated Src kinase, which is required for the synaptic potentiation in CS iMSNs. Inhibition of NOX1 or β-arrestin in the CS was sufficient to ameliorate repetitive behavior. Striatal-specific Nox1 knockdown also ameliorated repetitive and perseverative behavior. Collectively, these results indicate that NOX1 acts as an enhancer of synaptic facilitation in CS iMSNs and plays a key role in the molecular link between abnormal dopamine signaling and behavioral repetition and perseveration.SIGNIFICANCE STATEMENT Behavioral repetition is a form of compulsivity, which is one of the core symptoms of psychiatric disorders, such as obsessive-compulsive disorder. Perseveration is also a hallmark of such disorders. Both clinical and animal studies suggest important roles of abnormal dopaminergic signaling and striatal hyperactivity in compulsivity; however, the precise molecular link between them remains unclear. Here, we demonstrated the contribution of NOX1 to behavioral repetition induced by repeated stimulation of D₂ receptors. Repeated stimulation of D₂ receptors upregulated Nox1 mRNA in a striatal subregion-specific manner. The upregulated NOX1 promoted striatal synaptic facilitation in iMSNs by enhancing phosphorylation signaling. These results provide a novel mechanism for D₂ receptor-mediated excitatory synaptic facilitation and indicate the therapeutic potential of NOX1 inhibition in compulsivity.

Keywords: NADPH oxidase; behavioral repetition; dopamine; obsessive-compulsive disorder; striatum.

Figure 1.

Nox1 deficiency ameliorated behavioral repetition induced by repeated D₂ receptor stimulation. A, Schematic diagram of the experimental protocol. Mice that received 7-12 daily injections of QNP were referred to as sensitized mice (Sens). B, C, Effects of single D₂ receptor stimulation on locomotor activity. n = 6. Two-way repeated-measures ANOVA: F_(1,20) = 1.354, p = 0.2717. D, Schematic diagram of the protocol for behavior monitoring. E, Representative images of chewing behavior. The repeated chewing of cage bedding (wood chips) was observed in sensitized mice. F, Time spent chewing in the 20-30 min after the first (Single) or eighth (Sens) QNP injection (1 mg/kg, i.p.). WT Control: n = 4; KO Control: n = 4; WT Single: n = 4; KO Single: n = 4; WT Sens: n = 9; KO Sens: n = 8. One-way ANOVA: F_(5,27) = 61.71, p < 0.0001. Tukey's multiple comparison test: ***p < 0.001 versus WT Sens. G, Cumulative probability of chewing bout duration. Two-sample Anderson–Darling test: ***p < 0.001. Inset, The number of chewing bouts. Student's t test: t₍₁₅₎ = 1.637, *p = 0.1224. H, Other repetitive behavior during the same recording period as in F and G. I–K, Effects of systemic injection of ML171, a blood–brain barrier-permeable NOX1 inhibitor on repetitive chewing behavior. n = 5. J, Unpaired t test with Welch's correction: t₍₈₎ = 4.280, **p = 0.0079. K, Two-sample Anderson–Darling test: *p = 0.0137. Inset, The number of chewing bouts. Student's t test: t₍₈₎ = 2.659, *p = 0.0288.

Figure 2.

Nox1 deficiency improved cognitive inflexibility induced by a repeated D₂ receptor stimulation. A, Protocol for the spatial discrimination task and reversal learning test combined with repeated D₂ receptor stimulation. B–D, Percentage of correct choices by drug-naive mice (Control) during the training (B), overtraining (C), and reversal learning (D) periods. n = 4. B, Two-way repeated-measures ANOVA: Genotype (F_(1,6) = 0.1017, p = 0.7606), Session number (F_(5,30) = 9.145, p < 0.0001), Interaction (F_(5,30) = 1.572, p = 0.1980). C, Two-way repeated-measures ANOVA: Genotype (F_(1,7) = 0.000, p = 1.0000), Session number (F_(7,49) = 0.866, p = 0.5393), Interaction (F_(7,49) = 1.151, p = 0.3480). D, Two-way repeated-measures ANOVA: Genotype (F_(1,7) = 0.1095, p = 0.7503), Session number (F_(4,28) = 38.95, p < 0.0001), Interaction (F_(4,28) = 1.515, p = 0.2247). E–G, Percentage of correct choices by sensitized mice (Sens) during the training (E), overtraining (F), and reversal learning (G) periods. n = 6 or 7. E, Two-way repeated-measures ANOVA: Genotype (F_(1,11) = 0.6371, p = 0.4417), Session number (F_(5,55) = 9.009, p < 0.0001), Interaction (F_(5,55) = 0.5334, p = 0.7501). F, Two-way repeated-measures ANOVA: Genotype (F_(1,11) = 0.2694, p = 0.6140), Session number (F_(7,77) = 0.6227, p = 0.7356), Interaction (F_(7,77) = 2.077, p = 0.0560). G, Two-way repeated-measures ANOVA: Genotype (F_(1,11) = 15.70, p = 0.0022), Session number (F_(4,44) = 41.85, p < 0.0001), Interaction (F_(4,44) = 1.582, p = 0.1959). Bonferroni post-test: *p < 0.05; **p < 0.01. Lines with and without symbols indicate mean values and individual data points, respectively.

Figure 3.

Repeated D₂ receptor stimulation upregulated Nox1 mRNA expression in a striatal region-specific manner. A–D, Relative expression levels of Nox1 (B), Nox2 (C), and Nox4 (D) mRNAs in the CSTC circuit and related brain regions shown in A. Each sample was obtained from drug-naive mice. n = 3. B, One-way ANOVA: F_(6,14) = 10.52, p = 0.0002; Tukey's multiple comparison test: **p < 0.01 versus OFC. C, One-way ANOVA: F_(6,14) = 0.2269, p = 0.9611. D, One-way ANOVA: F_(6,14) = 2.987, p = 0.0429; Tukey's multiple comparison test: *p < 0.05 versus OFC. E–I, Relative expression levels of Nox1 (F), Nox2 (G), Nox4 (H), and Drd2 (I) mRNAs in the DS from sensitized mice (Sens). n = 4-9. F, Student's t test: t₍₁₄₎ = 1.497, p = 0.1566. G, One-way ANOVA: F_(3,31) = 1.070, p = 0.3776. H, One-way ANOVA: F_(3,21) = 2.156, p = 0.1287. I, One-way ANOVA: F_(3,20) = 0.7442, p = 0.5404. J–N, Relative expression levels of Nox1 (K), Nox2 (L), Nox4 (M), and Drd2 (N) mRNAs in the CS from Sens mice. n = 5-9. K, Unpaired t test with Welch's correction: t₍₁₄₎ = 2.881, p = 0.0205. L, One-way ANOVA: F_(3,31) = 0.9343 p = 0.4372. M, One-way ANOVA: F_(3,18) = 2.000, p = 0.1501. N, One-way ANOVA: F_(3,21) = 0.3562, p = 0.7853.

Figure 4.

Nox1 deficiency did not affect basal electrophysiological properties of both iMSNs and dMSNs in the CS. A–C, Firing activity elicited by current injection in CS iMSNs of drug-naive mice. B, Calibration: 100 ms, 50 mV. C, n = 10 from 5 mice. Two-way repeated-measures ANOVA: Genotype (F_(1,19) = 0.3306, p = 0.5721), Current (F_(5,95) = 97.41, p < 0.0001), Interaction (F_(5,95) = 2.518, p = 0.0347). D, E, Resting membrane potential (D) and input resistance (E) of CS iMSNs from drug-naive mice. n = 10 from 5 mice. D, Student's t test: t₍₁₀₎ = 1.579, p = 0.1318. E, Student's t test: t₍₁₈₎ = 0.5050, p = 0.6197. F, G, Spontaneous EPSC frequency (F) and amplitude (G) in CS iMSNs from drug-naive mice. n = 11-13 from 7 or 8 mice. F, Student's t test: t₍₂₂₎ = 0.6448, p = 0.5257. G, Student's t test: t₍₂₂₎ = 0.2057, p = 0.8389. H–J, Firing activity elicited by the current injection in CS dMSNs of drug-naive mice. I, Calibration: 100 ms, 50 mV. J, n = 13-17 from 5 mice. Two-way repeated-measures ANOVA: Genotype (F_(1,28) = 0.9674, p = 0.3338), Current (F_(5,140) = 156.4, p < 0.0001), Interaction (F_(5,140) = 0.6014, p = 0.6989). K, L, Resting membrane potential (K) and input resistance (L) of CS dMSNs from drug-naive mice. n = 13-17 from 5 mice. K, Student's t test: t₍₂₈₎ = 0.9398, p = 0.3553. L, Student's t test: t₍₂₈₎ = 0.9176, p = 0.3667. M, N, Spontaneous EPSC frequency (M) and amplitude (N) in CS dMSNs from drug-naive mice. n = 9-12 from 8 mice. M, Student's t test: t₍₁₉₎ = 0.6044, p = 0.5527. N, Student's t test: t₍₁₉₎ = 0.8661, p = 0.3972.

Figure 5.

Repeated stimulation of D₂ receptors facilitated excitatory inputs to the CS in a NOX1-dependent manner. A, Schematic diagram of the experimental protocol. B, C, The AMPA/NMDA ratio was recorded from CS iMSNs before and after bath application of QNP (10 μm). Representative traces are shown in B. Calibration: 5 ms, 500 pA. C, n = 11-13 from 6-11 mice. Three-way repeated-measures ANOVA: Pre-Post (F_(1,45) = 0.02340, p = 0.8791), Sensitization (F_(1,45) = 2.983, p = 0.0910), Genotype (F_(1,45) = 0.03614, p = 0.8501), Pre-Post × Sensitization (F_(1,45) = 7.488, p = 0.0089), Pre-Post × Genotype (F_(1,45) = 6.690, p = 0.0130), Sensitization × Genotype (F_(1,45) = 0.2053, p = 0.6527), Pre-Post × Sensitization × Genotype (F_(1,45) = 1.414, p = 0.2407). Sidak's multiple comparisons test: *p < 0.05. D, The AMPA/NMDA ratio was recorded from CS dMSNs before and after bath application of QNP (10 μm). n = 10 or 11 from 7-10 mice. Three-way repeated-measures ANOVA: Pre-Post (F_(1,38) = 0.1673, p = 0.1673), Sensitization (F_(1,38) = 0.4755, p = 0.4947), Genotype (F_(1,38) = 0.6889, p = 0.4117), Pre-Post × Sensitization (F_(1,38) = 0.1492, p = 0.7014), Pre-Post × Genotype (F_(1,38) = 1.596, p = 0.2142), Sensitization × Genotype (F_(1,38) = 1.190, p = 0.2822), Pre-Post × Sensitization × Genotype (F_(1,38) = 1.545, p = 0.2215). E, F, In the presence of a NOX1 inhibitor, ML171 (5 μm), the AMPA/NMDA ratio was recorded from CS iMSNs before and after bath application of QNP (10 μm). F, n = 9 or 10 from 5 or 6 mice. Two-way repeated-measures ANOVA: Pre-Post (F_(1,17) = 2.789, p = 0.1132), Sensitization (F_(1,17) = 0.01199, p = 0.9141), Interaction (F_(1,17) = 0.2266, p = 0.6401). G, H, The AMPA/NMDA ratio was recorded from CS iMSNs before and after bath application of UNC9994 (UNC; 10 μm), an arrestin-biased D₂ receptor agonist. H, n = 9-12 from 5-8 mice. Three-way repeated-measures ANOVA: Pre-Post (F_(1,36) = 1.442, p = 0.2377), Sensitization (F_(1,36) = 3.095, p = 0.0870), Genotype (F_(1,36) = 1.484, p = 0.2310), Pre-Post × Sensitization (F_(1,36) = 8.477, p = 0.0061), Pre-Post × Genotype (F_(1,36) = 1.443, p = 0.2375), Sensitization × Genotype (F_(1,36) = 0.05931, p = 0.8090), Pre-Post × Sensitization × Genotype (F_(1,36) = 2.255, p = 0.1419). Sidak's multiple comparisons test: **p < 0.01. I–K, Following the intracellular application of barbadin, a β-arrestin inhibitor (J; 100 μm) or PP2, a Src kinase inhibitor (K; 1 μm), the AMPA/NMDA ratio was recorded from CS iMSNs before and after bath application of QNP. J, n = 10 from 6 mice. Two-way repeated-measures ANOVA: Pre-Post (F_(1,18) = 3.729, p = 0.0694), Sensitization (F_(1,18) = 0.3481, p = 0.5625), Interaction (F_(1,18) = 1.176, p = 0.2924). K, n = 9 or 10 from 5 mice. Two-way repeated-measures ANOVA: Pre-Post (F_(1,17) = 1.930, p = 0.1827), Sensitization (F_(1,17) = 1.295, p = 0.2708), Interaction (F_(1,17) = 1.272, p = 0.2750).

Figure 6.

Nox1 deficiency did not improve lOFC hyperactivity in sensitized mice. A–C, Current injection-induced firing activity of lOFC pyramidal neurons from WT drug-naive (Control) and sensitized (Sens) mice. B, n = 13 from 3 mice. Two-way repeated-measures ANOVA: Genotype (F_(1,120) = 4.611, p = 0.0421), Current (F_(5,600) = 324.2, p < 0.0001), Interaction (F_(5,600) = 3.697, p = 0.0038). Bonferroni post-test: *p < 0.05; **p < 0.01. C, n = 10-12 from 3 mice. Two-way repeated-measures ANOVA: Genotype (F_(1,100) = 6.359, p = 0.0203), Current (F_(5,500) = 288.5, p < 0.0001), Interaction (F_(5,500) = 5.540, p = 0.0002). Bonferroni post-test: *p < 0.05; **p < 0.01. D, E, Resting membrane potential (D) and input resistance (E) of lOFC pyramidal neurons from WT drug-naive (Control) and sensitized (Sens) mice. n = 10-13 from 3 mice. D, One-way ANOVA: F_(3,44) = 6.351, p = 0.0011. Tukey's multiple comparison test: ***p < 0.001 versus WT Control. E, one-way ANOVA: F_(3,44) = 0.5891, p = 0.6254.

Figure 7.

NOX1 was required for the accumulation of activated Src induced by D₂ receptor stimulation in sensitized mice. A–C, Phosphatase activity of the DS (B) and CS (C) from drug-naive mice. Values are normalized by that of the WT Total group of each region. B, n = 5 or 6. Total: unpaired t test with Welch's correction: t₍₇₎ = 4.368, **p = 0.0033; PTP: unpaired t test with Welch's correction: t₍₉₎ = 2.770, *p = 0.0218; PP: Student's t test: t₍₉₎ = 0.1416, p = 0.1416. C, n = 7 or 8. Total: unpaired t test with Welch's correction: t₍₇₎ = 2.475, *p = 0.0425; PTP: unpaired t test with Welch's correction: t₍₇₎ = 2.850, *p = 0.0247; PP: Student's t test: t₍₁₃₎ = 0.4228, p = 0.6794. D, Regarding Western blotting, Sens mice were divided into two groups and challenged with saline (Sens Sal) or QNP (Sens QNP) 15 min before the dissection of the DS and CS. E–J, Western blot analyses of phosphorylation of Src Y416 in the DS (E–G) and CS (H–J). The values are normalized by that of the respective control mice of each genotype. F, n = 7-9. Two-way ANOVA: Genotype (F_(1,30) = 0.06535, p = 0.8004), Challenge (F_(1,30) = 0.2191, p = 0.6431), Interaction (F_(1,30) = 3.123, p = 0.0874). N.S.: not significant. G, n = 7-9. Two-way ANOVA: Genotype (F_(1,30) = 0.2599, p = 0.6139), Challenge (F_(1,30) = 0.01137, p = 0.9158), Interaction (F_(1,30) = 0.8137, p = 0.3742). I, n = 7-9. Two-way ANOVA: Genotype (F_(1,29) = 4.564, p = 0.0412), Challenge (F_(1,29) = 4.523, p = 0.0421), Interaction (F_(1,29) = 3.532, p = 0.0703). Bonferroni post-test: *p < 0.05. J, n = 7-9. Two-way ANOVA: Genotype (F_(1,29) = 2.462, p = 0.1275), Challenge (F_(1,29) = 0.2305, p = 0.6348), Interaction (F_(1,29) = 0.05911, p = 0.8096).

Figure 8.

NOX1 was required for the accumulation of activated Src induced by D₂ receptor stimulation in primary cultured striatal neurons. A, AAV-miRNA-mediated Nox1 KD in primary cultures of mouse striatal neurons. B, Representative images of immunostaining. Red represents MAP2, a marker for neurons; or GFAP, a marker for astrocytes. Green represents EmGFP, depicting miRNA-expressing cells. Cyan represents DAPI. Scale bar, 50 μm. C–F, Relative expression levels of Nox1 (C), Nox2 (D), Nox4 (E), and Drd2 (F) mRNAs in striatal cultures 7 d after AAV infection; n = 6. C, Student's t test: t₍₁₀₎ = 10.17, p < 0.0001. D, Student's t test: t₍₁₀₎ = 1.558, p = 0.1502. E, Student's t test: t₍₁₀₎ = 0.2264, p = 0.8254. F, Student's t test: t₍₁₀₎ = 0.7681, p = 0.4602. G, Phosphatase activity of AAV-infected neuronal cultures. The values are normalized by that of the Control Total group. n = 9 or 10; Total: Student's t test: t₍₁₈₎ = 3.771, **p = 0.0014; PTP: Student's t test: t₍₁₈₎ = 3.030, **p = 0.0072; PP: Student's t test: t₍₁₅₎ = 0.9569, p = 0.3538. H–L, Western blot analyses of phosphorylation of Src Y416 in neuronal cultures infected with control AAV (I,J) or with AAV-miRNA (K,L) after an incubation in the presence of vehicle (Veh), QNP (1 μm), or UNC (1 μm) for 20 min. The values are normalized by that of the Veh group. I, n = 11. One-way ANOVA: F_(2,32) = 3.445, p = 0.0450; Tukey's multiple comparison test: *p < 0.05 versus Veh. J, n = 11. One-way ANOVA: F_(2,32) = 1.060, p = 0.3591. K, n = 11. One-way ANOVA: F_(2,32) = 0.7293, p = 0.4906. L, n = 11. One-way ANOVA: F_(2,32) = 0.6784, p = 0.5151.

Figure 9.

Overexpression of NOX1 facilitated agonist-induced D₂ receptor internalization and accumulation of activated Src. A, HEK293T cells were transfected with plasmids encoding hNOX1, hNOXO1, hNOXA1, and FLAG-tagged hDRD2. B, Left, Representative images for the immunostaining of hDRD2-expressing HEK293T cells after a 20 min incubation with vehicle (Veh), QNP (1 μm), or QNP + PP2 (1 μm). Green represents PHA-L (plasma membrane). Magenta represents FLAG-DRD2. Scale bar, 10 μm. Right, Quantitative data for internalization of D₂ receptors. Values are normalized by that of the Mock-Veh group. n = 13-17. Two-way ANOVA: Overexpression (F_(1,79) = 4.843, p = 0.0041), Drug (F_(2,79) = 0.6580, p = 0.5207), Interaction (F_(2,79) = 5.192, p = 0.0076). Bonferroni post-test: **p < 0.01 versus Mock-QNP. C–E, Western blot analysis of phosphorylation of Src Y416. Values are normalized by that of the Mock-Veh group. n = 8 or 9. D, Two-way ANOVA: Overexpression (F_(1,31) = 9.618, p = 0.0041), Drug (F_(1,31) = 6.236, p = 0.0180), Interaction (F_(1,31) = 3.974, p = 0.0551). Bonferroni post-test: **p < 0.01. E, Two-way ANOVA: Overexpression (F_(1,31) = 0.01911, p = 0.8909), Drug (F_(1,31) = 0.04204, p = 0.8389), Interaction (F_(1,31) = 0.06882, p = 0.7948).

Figure 10.

Inhibition of NOX1 or β-arrestin in the CS was sufficient to attenuate repetitive behavior. A, Experimental protocol for stereotaxic surgery and the microinjection of inhibitors for behavior analyses. B, Schematic illustration of cannula placements. C, D, Effects of an injection of setanaxib (1 μg/μl/side) in the CS on repetitive chewing behavior in sensitized mice. C, n = 5. Paired t test: t₍₄₎ = 9.131, ***p = 0.0008. D, Two-sample Anderson–Darling test: *p = 0.048. Inset, The number of chewing bouts. Paired t test: t₍₄₎ = 0.5898, p = 0.5870. E, Other repetitive behaviors during the same recording period as in C and D. F, G, Effects of an injection of barbadin (1 μg/μl/side) in the CS on repetitive chewing behavior in sensitized mice. F, n = 5. Paired t test: t₍₄₎ = 5.091, **p = 0.0070. G, Two-sample Anderson–Darling test: ***p < 0.001. Inset, The number of chewing bouts. Paired t test: t₍₄₎ = 2.000, p = 0.1161. H, Other repetitive behaviors during the same recording period as in F and G. I, Experimental protocol for analyzing spontaneous behaviors in striatal neuron-specific Nox1 KD mice. J, Representative image for the AAV-mediated expression of EmGFP in the striatum. Scale bar, 500 μm. K, L, Time spent chewing in control and Nox1 KD-sensitized mice. K, n = 6. Student's t test: t₍₁₀₎ = 2.768, *p = 0.0198. L, Two-sample Anderson–Darling test: ***p < 0.001. Inset, The number of chewing bouts. Student's t test: t₍₁₀₎ = 1.206, p = 0.2557. M, Other repetitive behaviors during the same recording period as in K and L. N, Experimental protocol for spatial discrimination task in striatal neuron-specific Nox1 KD mice. O–Q, Percentage of correct choices by control and Nox1 KD-sensitized mice (Sens) during the training (O), overtraining (P), and reversal learning (Q) periods. Control: n = 9; KD: n = 6. O, Two-way repeated-measures ANOVA: Nox1 KD (F_(1,13) = 0.2582, p = 0.6199), Session number (F_(5,65) = 8.564, p < 0.0001), Interaction (F_(5,65) = 0.08296, p = 0.9947). P, Two-way repeated-measures ANOVA: Nox1 KD (F_(1,13) < 0.001, p > 0.9999), Session number (F_(7,91) = 7.467, p < 0.0001), Interaction (F_(7,91) = 0.3760, p = 0.9141). Q, Two-way repeated-measures ANOVA: Nox1 KD (F_(1,13) = 12.46, p = 0.0037), Session number (F_(4,52) = 12.46, p < 0.0001), Interaction (F_(4,52) = 2.834, p = 0.0335). Bonferroni post-test: *p < 0.05; **p < 0.01. Lines with and without symbols indicate mean values and individual data points, respectively.

Figure 11.

A hypothetical mechanism for the induction of repetitive behavior in QNP-sensitized mice. The acute stimulation of D₂ receptors induces sedation mainly through the G-protein-mediated pathway. After repeated stimulation of D₂ receptors, NOX1 expression was upregulated in the CS. In QNP-sensitized mice, no apparent repetitive behavior, Src activation, and synaptic facilitation were observed before stimulation of D₂ receptors. In contrast, once QNP was injected, activated Src accumulated in a NOX1-dependent manner. Under this condition, β-arrestin-mediated D₂ receptor signaling was increased, resulting in the facilitation of excitatory inputs in CS iMSNs. Such signal modification shifted the behavioral response from sedation to repetitive behavior.