Attenuated AIMs and striatal cellular responses in Ras-GRF1–KO mice after l-dopa treatment. (A) The success of the lesion was evaluated 2 wk after 6-OHDA injection by counting spontaneous ipsilateral rotations in a squared open arena over a 10-min session. WT (n = 16) and mutants (n = 16) displayed an equivalent turning score. (B) Temporal profile of axial, limb, and orolingual AIMs induced by an increasing l-dopa regimen (1.5, 3, and 6 mg/kg, twice a day) administered for 9 consecutive days. The AIM scores were reduced significantly in Ras-GRF1–KO mice (Ras-GRF1–KO l-dopa, dashed line, open circles, n = 9) in comparison with their littermate controls (WT l-dopa, solid line, closed circles, n = 9). Saline treatment (dashed and dotted line marked “x”) did not induce involuntary movements (Ras-GRF1–KO saline, n = 7; WT saline, n = 7). Repeated measures and post hoc Tukey's honestly significant difference (HSD) tests showed a genotype effect (^#P < 0.001) and a genotype–time interaction effect (P < 0.001). (C) No difference in the locomotive AIMs in WT and Ras-GRF1–KO animals were found in response to l-dopa. (D) Abnormal levels of pERK activation were observed in dyskinetic WT animals, whereas a pronounced reduction was seen in Ras-GRF1–KO animals. The total number of pERK-immunopositive cells/mm² was counted in the dorsolateral striatum (intact and lesion sides) in saline- and l-dopa–treated groups. Two-way ANOVA followed by a Tukey's HSD test indicated a significant genotype difference between WT and Ras-GRF1–KO lesioned lateral striatum (^★P < 0.0001). (E) FosB/ΔFosB expression is severely attenuated in the lesioned striata of Ras-GRF1–KO animals (black bars) in comparison with littermate controls (white bars). The total number of FosB/ΔFosB-positive cells/mm² was counted in the dorsolateral striatum of all experimental groups. Two-way ANOVA followed by Tukey's HSD test showed a significant genotype difference between WT mice and Ras-GRF1–KO mice treated with l-dopa (^★P < 0.0001 difference).

Treatment of Ras-GRF1–KO mice with a normally ineffective dose of SL327 caused a further reduction of the AIMs. (A) Sum of axial, limb, and orolingual scores (ALO AIMs) after 9 d of escalating doses of l-dopa expressed as means of 3-d doses showed a stronger attenuation of dyskinesia in Ras-GRF1–KO animals pretreated with a low dose (10 mg/kg, i.p.) of SL327 (hatched bars) than in Ras-GRF1–KO mice treated with l-dopa only (black bars). Repeated measures and post hoc Tukey's HSD test: ^#P < 0.001 WT l-dopa (white bar, n = 10) vs. Ras-GRF1–KO l-dopa (n = 9); ^✩P < 0.01 Ras-GRF1–KO l-dopa vs. Ras-GRF1–KO SL327 + l-dopa (n = 10). (B) Locomotive AIMs expressed as means of 3-d doses were not affected by Ras-GRF1 ablation and SL327 treatment. (C) Total numbers of contralateral turns (mean ± SEM) induced by l-dopa were comparable among groups. (D) Representative photomicrographs of pERK1/2 immunoreactive cells (Upper) and FosB/ΔFosB expression (Lower) in the dorsolateral part of the striatum after 9 d of l-dopa treatment in combination with SL327 (10 mg/kg). (E) Quantification of pERK1/2-positive cells (mean ± SEM) in the intact and lesioned dorsolateral striatum of Ras-GRF1 mutants and control animals. Two-way ANOVA revealed a significant reduction (genotype effect) in the number of pERK-positive cells in lesioned striatum of Ras-GRF1 mutants in comparison with littermate controls (Tukey's HSD test, ^#P < 0.001) and a greater treatment effect in lesioned striatum of Ras-GRF1–KO mice pretreated with SL327 (Tukey's HSD test, ^✩P < 0.001). (F) Quantification of FosB/ΔFosB-positive cells (mean ± SEM) in the intact and lesioned striata of Ras-GRF1–KO mice. Two-way ANOVA showed significant differences (genotype effect) between WT mice and Ras-GRF1–KO mice treated with l-dopa (Tukey's HSD test, ^#P < 0.001). A significant reduction in the number of FosB//ΔFosB immunoreactive cells (treatment effect) also was observed in Ras-GRF1–KO mice treated with SL327 in comparison with Ras-GRF1–KO mice injected with l-dopa only (Tukey's HSD test, ^✩P < 0.001).

Expression of Ras-GRF1 in MSN subpopulations and its protein levels in dyskinetic animals. Immunofluorescence of Ras-GRF1 (red), EGFP (green), and nuclear labeling with DAPI (blue) of striatonigral neurons (direct pathway) of M4-EGFP mice (A) and striatopallidal neurons (indirect pathway) of A2A-EGFP mice (C). (Scale bars in A and C: 20 μm.) (E) The graph shows the percentage of the total Ras-GRF1–positive neurons that are GFP-positive, indicating that Ras-GRF1 is expressed equally in each subpopulation. As expected, Ras-GRF1 was not expressed either in the direct pathway of M4-EGFP Ras-GRF1–KO mice (B) or in the indirect pathway of A2A EGFP Ras-GRF1–KO mice (D). (F) Protein levels of Ras-GRF1, Ras-GRF2, and pERK1/2 in intact (I) and lesioned (L) striata of WT mice after 9 d of l-dopa treatment were determined by Western blot analysis. p140 ^Ras-GRF1 (G) and p135 ^Ras-GRF2 (H) levels were not altered in dopamine-denervated striata after saline or l-dopa treatment, whereas phosphorylation of ERK1/2 is enhanced only in l-dopa–treated striata (I) (one-way ANOVA, ^#P < 0.01).

LV-Mix-GFP expression in the macaque motor striatum reduces the dyskinesia elicited by l-dopa (A–F) and D1 and D2 agonists (G–O). Time-course panels reporting clinical ratings feature median scores ± SEM, but those reporting activity counts are shown without SEM for readability. Administration of l-dopa (levodopa/carbidopa 4:1, see Materials and Methods) or dopamine agonists starts at t = 0 min. (A) LV-Mix-GFP expression had no impact on the l-dopa–induced PD score at any time point. (B) The analysis of the area under the curve (AUC) of PD scores confirms this lack of effect (mean ± SEM). (C) LV-Mix-GFP expression reduced LID from t = 40 to t =160 min (but note peak at t = 50 min) in comparison with the LV-GFP animals (median scores; Mann–Whitney test, *P < 0.05). (D) The overall positive effect on LID severity is demonstrated further by the AUC data (mean ± SEM; unpaired t test; *P < 0.05). (E and F) Consequently, locomotor activity was lower in LV-Mix-GFP animals (mean ± SEM; unpaired t test; *P < 0.05). (G–O) Effects of D1 and D2 agonists in LV-Mix-GFP and LV-GFP monkeys were analyzed separately. (G–I) As with l-dopa, the antiparkinsonian effect of both (G) SKF-38393 (D1 agonist; 1.5 mg/kg, s.c.) and (H) quinpirole (D2 agonist; 1.5 mg/kg, s.c.) was comparable in both groups. (J and K) Although the severity of dyskinesia was comparable in both groups after administration of the D2 agonist (K), the LV-Mix-GFP animals displayed a reduced severity of D1 agonist-induced dyskinesia from t = 40 min to t = 160 min (J) (median scores; Mann–Whitney test; *P < 0.05). (l) AUC analysis supports this conclusion, showing that only the severity of dyskinesia induced by the D1 agonist is reduced more significantly in LV-Mix-GFP animals than in GFP animals (unpaired t test; *P < 0.05). (M–O) Analysis of the AUC of activity counts further supports the D1 agonist-mediated reduction in dyskinesia severity, because activity counts are significantly reduced in the LV-Mix-GFP animals compared with the GFP animals only after D1 agonist administration (unpaired t test; *P < 0.05).