Dopamine D3 Receptor Modulates l-DOPA-Induced Dyskinesia by Targeting D1 Receptor-Mediated Striatal Signaling

Abstract

The dopamine D3 receptor (D3R) belongs to the dopamine D2-like receptor family and is principally located in the ventral striatum. However, previous studies reported D3R overexpression in the dorsal striatum following l-DOPA treatment in parkinsonian animals. This fact has drawn attention in the importance of D3R in l-DOPA-induced dyskinesia (LID). Here, we used D3R knockout mice to assess the role of D3R in LID and rotational sensitization in the hemiparkinsonian model. Mice lacking D3R presented a reduction in dyskinesia without interfering with the antiparkinsonian l-DOPA effect and were accompanied by a reduction in the l-DOPA-induced rotations. Interestingly, deleting D3R attenuated important molecular markers in the D1R-neurons such as FosB, extracellular signal-regulated kinase, and histone-3 (H3)-activation. Colocalization studies in D1R-tomato and D2R-green fluorescent protein BAC-transgenic mice indicated that l-DOPA-induced D3R overexpression principally occurs in D1R-containing neurons although it is also present in the D2R-neurons. Moreover, D3R pharmacological blockade with PG01037 reduced dyskinesia and the molecular markers expressed in D1R-neurons. In addition, this antagonist further reduced dyskinetic symptoms in D1R heterozygous mice, indicating a direct interaction between D1R and D3R. Together, our results demonstrate that D3R modulates the development of dyskinesia by targeting D1R-mediated intracellular signaling and suggest that decreasing D3R activity may help to ameliorate LID.

Keywords: Parkinson disease; abnormal involuntary movements; basal ganglia; behavioral sensitization; striatonigral.

Figure 1.

Effect of genetic deletion of D3R on LID and behavioral sensitization. (A) Schematic view of the experimental design. The D3^−/− mice exhibited a reduction in axial (B), limb (C), and orolingual (D), as well as the cumulated total score (E) compared with littermate controls. Dyskinetic movements were evaluated 40 min after l-DOPA. Two-way ANOVA with repeated measures followed by a Bonferroni post hoc test showed significant differences for genotype (F_3,150 = 414.3, P < 0.001 for axial; F_3,150 = 212.6, P < 0.001 for limb; F_3,150 = 187.9, P < 0.001 for orolingual; F_3,150 = 375.5, P < 0.001 for total score) and day (F_5,150 = 39.5, P < 0.001 for axial; F_5,150 = 44.1, P < 0.001 for limb; F_5,150 = 40.4, P < 0.001 for orolingual; F_5,150 = 75.1, P < 0.001 for total score). The kinetic profile of dyskinetic symptoms was evaluated once every 20 min over 180 min on Day 13 of the l-DOPA treatment (F). Two-way ANOVA followed by a Bonferroni test showed significant differences for genotype (F_3,270 = 294.4, P < 0.001) and time (F_5,270 = 39.07, P < 0.001). Contralateral turns in hemiparkinsonian mice evaluated for 15 min on the indicated day (F). Two-way ANOVA with repeated measures followed by a Bonferroni test showed significant differences for genotype (F_3,150 = 68.6, P < 0.001) and day (F_5,150 = 5.6, P < 0.001). Data are expressed as the mean ± S.E.M. *P < 0.05 and **P < 0.01 versus WT + l-DOPA. n = 10 for each group, except in the D3^−/− saline group, n = 3.

Figure 2.

Genetic deletion of D3R does not affect the therapeutic effect of l-DOPA. (A) Motor coordination on the rotarod was evaluated in D3^−/− and WT mice before 6-OHDA lesion (prelesion), 3 weeks after lesion (pre-l-DOPA), and on Day 14 of the chronic treatment, 90 min after the l-DOPA injection (post-l-DOPA). Two-way ANOVA, followed by Bonferroni's test. **P < 0.01 versus D3^−/− (pre-l-DOPA) mice; ^#P < 0.05 versus WT (pre-l-DOPA) mice. (B) Total distance traveled (cm) during 30 min was measured in a multicage activity meter system. Two-way ANOVA, followed by Bonferroni's test. ****P < 0.0001 versus D3^−/− (pre-l-DOPA) mice; ^####P < 0.0001 versus WT (pre-l-DOPA) mice. (C) Scatter dot plot of the extent of striatal lesions assessed by percentage of striatal volume completely denervated. (D) Coronal section from a dyskinetic D3^−/− mouse stained for TH. n = 10 for each group. Scale bar = 500 µm.

Figure 3.

Genetic inactivation of D3R attenuates molecular markers of LID. Photomicrographs of l-DOPA-induced FosB, pERK, and pAcH3 expression in the dopamine-denervated dorsal striatum (A). Immunohistochemical analysis shows that the D3R deletion decreases FosB- (B), pERK- (C), and pAcH3- (D) positive cells induced by l-DOPA. *P < 0.05 and **P < 0.01 versus WT (unpaired t-test). n = 10 for each group. Scale bar = 50 µm.

Figure 4.

Chronic l-DOPA increases D3R expression in the DA-depleted dorsal striatum. (A) Confocal images of the dorsal striatum of intact, parkinsonian, and dyskinetic D1R-BAC-transgenic mice illustrating D3R expression (green) in D1R-positive neurons (red-tomato) and in D1R-negative neurons. Nuclei are visualized via Hoechst staining. Arrows indicate examples of D3R-positive and D1R-positive neurons; arrow-heads point to D3R-positive and D1R-negative neurons. (A′) Graph showing the percentage of D3R-positive cells in D1R-positive or -negative cells. Data are expressed as the mean ± SEM. Two-way ANOVA, followed by Bonferroni's test. ***P < 0.001 versus intact D1R-positive neurons; ^###P < 0.001 versus intact D1R-positive neurons. (B) Confocal images of the dorsal striatum of dyskinetic D2R-BAC-transgenic mice illustrating D3R expression (red) in D2R-positive neurons (green-GFP) and in D2R-negative neurons. Nuclei are visualized via Hoechst staining. Arrows indicate examples of D3R-positive and D2R-negative neurons; arrow-heads point to D3R-positive and D2R-positive neurons. (B′) Graph showing the percentage of D3R-positive cells in D2R-positive or negative cells. n = 3–4 for each group. Scale bar = 25 µm.

Figure 5.

l-DOPA-induced FosB expression occurs in striatonigral projection neurons. (A) Confocal images of the dorsal striatum of parkinsonian and dyskinetic D1R-BAC-transgenic mice illustrating FosB expression (green) in D1R-positive neurons (red-tomato). Nuclei are visualized via Hoechst staining. (B) Graph showing the percentage of D3R-positive cells in D1R-positive or -negative cells. Data are expressed as the mean ± SEM. n = 3–4 for each group. Scale bar = 40 µm.

Figure 6.

Effect of D3R-preferring antagonist PG01037 on the development of LID. (A) Schematic view of the experimental design. (B and C) The chronic coadministration of l-DOPA with PG01037 decreased the development of LID. **P < 0.01 and ***P < 0.001 versus l-DOPA plus saline, two-way repeated-measures ANOVA followed by Bonferroni post hoc test. (D) PG01037 cotreatment did not affect the latency to fall from the rotarod. P = 0.17 versus l-DOPA plus saline, unpaired t-test. Immunohistochemical analysis shows that PG01037 decreases FosB- (E) and pAcH3- (F) positive cells induced by l-DOPA. *P < 0.05 versus WT, unpaired t-test. All data are expressed as mean ± S.E.M. n = 9 for each group. Scale bar = 50 µm.

Figure 7.

Effect of D3R-preferring antagonist PG01037 on the expression of established LID. (A) The time line shows experimental design. (B and C) The administration of PG01037 diminished the expression of established LID. *P < 0.05 and **P < 0.01 versus l-DOPA plus saline, two-way repeated-measures ANOVA followed by Bonferroni post hoc test. (D) PG01037 cotreatment did not affect the latency to fall from the rotarod on established LID. P = 0.08 versus l-DOPA plus saline, unpaired t-test. Immunohistochemical analysis showed that coadministration of PG01037 does not significantly decrease FosB- (E) and pAcH3- (F) positive cells in mice with established LID (P = 0.08 and P = 0.22, respectively), unpaired t-test. All data are expressed as mean ± S.E.M. n = 8–9 for each group. Scale bar = 50 µm.

Figure 8.

Effect of coadministration of PG01037 plus l-DOPA in D1^+/− mice. (A) The time line shows experimental design. (B) Total dyskinetic score was evaluated 40 min after l-DOPA administration at the indicated days. Two-way ANOVA followed by a Bonferroni test showed significant differences for genotype (F_3,234 = 165.5, P < 0.001) and day (F_8,234 = 108.6, P < 0.001). (C) Time course of dyskinetic symptoms evaluated once every 20 min during 180 min on Day 13 of l-DOPA treatment. Two-way ANOVA followed by a Bonferroni test showed significant differences for genotype (F_3,156 = 71.04, P < 0.001) and time (F_5,156 = 13.86, P < 0.001). (D) Immunostaining for pAcH3. Photomicrographs at high and low magnifications of coronal sections from the dopamine-denervated striatum of L-DOPA-treated WT, D1^+/− mice, and L-DOPA plus PG01037-treated mice. Scale bar = 500 µm for low-magnification and 100 µm for high-magnification images. (E,F) Immunohistochemical analysis shows that the D3R blockade decreases FosB- and pAcH3-positive cells induced by l-DOPA in D1^+/− mice. *P < 0.05, **P < 0.01, and ***P < 0.001 versus D1^+/− + l-DOPA; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001. All data are expressed as mean ± S.E.M. n = 6–10 for each group.