Parallel dopamine D1 receptor activity dependence of l-Dopa-induced normal movement and dyskinesia in mice

Abstract

l-3,4-Dihydroxyphenylalanine (l-Dopa)-induced dyskinesia (LID) in Parkinson's disease (PD) is a major clinical problem. The prevailing view is that in PD patients and animal PD models dyskinesia develops after repeated l-dopa use or priming, independent of l-dopa's anti-PD therapeutic effect that occurs immediately. Here we show that in mice with severe and consistent dopamine (DA) loss in the dorsal striatum, rendered by transcription factor Pitx3 null mutation, the very first injection of l-dopa or D1-like agonist SKF81297 induced both normal ambulatory and dyskinetic movements. Furthermore, the robust stimulating effects on normal and dyskinetic movements had an identical time course and parallel dose-response curves. In contrast, D2-like agonist ropinirole stimulated normal and dyskinetic movements relatively modestly. These results demonstrate that severe DA loss in the dorsal striatum sets the stage for dyskinesia to occur on the first exposure to l-dopa or a D1 agonist without any priming. These results also indicate that l-dopa stimulated both normal and dyskinetic movements primarily via D1 receptor activation and that proper D1 agonism is potentially an efficacious therapy for PD motor deficits.

Fig. 1

Severe DA loss in the dorsal striatum in PixtHomo mice. (A) A confocal image of 3 μm optical section showing the typical intense DA axons as labeled by tyrosine hydroxylase (TH) immunostain in the entire striatum in a PitxWT mouse. The box area is expanded and displayed in A1. (B) A confocal image, obtained under identical conditions as in A, evidenced by the identical background signal in the non-striatal areas, showing the typical gradient pattern of DA axon loss in the striatum in PitxHomo mice. The dorsal striatum is largely void of DA axons, the middle striatum retains a significant number of DA axons, whereas the ventral striatum or the nucleus accumbens retains substantial amounts of DA axons. The box area is expanded and displayed in B1. (C) The intensity of DA axons in the striatal subregions was quantified by calculating the fractional area occupied by DA axons in 4 PitxWT mice (16 sections) and 4 PitxHomo mice (16 sections). All sections were from the middle part of the striatum on the anterior-posterior axis. AC, anterior commissure. OT, olfactory tubercle. **, p<0.01, t-test.

Fig. 2

A small dose of AMPT renders PitxHomo mice hypokinetic. (A) IP injection of 30 mg/kg AMPT had no overt motor effect in PitxWT mice but induced a clear hypokinesia in PitxHomo mice. Measurements were taken 1 hour after saline or AMPT injection. Five mice in each group. **, p<0.01, t-test. (B) Example FCV records (B1) and summary FCV data (B2) showing the moderate reduction in DA release caused by 30 mg/kg AMPT pretreatment in 4 PitxWT (3 slices from each mouse) and 4 PitxHomo mice (3 slices from each mouse). All slices were from the middle part of the striatum on the anterior-posterior axis. The residual DA signal was still substantial in the striatal subregions in PitxWT mice. In contrast, in the striatal subregions in PitxHomo mice, the already diminished DA signal was further reduced by AMPT such the residual DA signal became very small. *, p<0.05, t-test.

Fig. 3

Motor effects of the first and subsequent injections of L-dopa in AMPT-PitxHomo mice. (A) The first IP injection of 6 mg/kg L-dopa with 5 mg/kg benserazide or 1 mg/kg SKF81297 induced strong normal horizontal movements in AMPT-PitxHomo mice. The first IP injection of 0.1 mg/kg ropinirole induced only weak responses in AMPT-PitxHomo mice. Note the increase in the responses during the first 3 days. Horizontal speed was measured during 30^th and 39^th min after injection. The horizontal speeds under L-dopa and SKF81297 were strongly correlated with a correlation coefficient of 1, but were weakly correlated with the horizontal speeds under ropinirole with a correlation coefficient of 0.61, Pearson test. The weak correlation is likely due to the coincidental drug injection and coincidental D1 and D2 activation and also indicates that L-dopa’s stimulating effect on horizontal motor activity was primarily mediated by D1Rs, not D2Rs. (B) In separate, fresh groups of AMPT-PitxHomo mice, the first IP injection of 6 mg/kg L-dopa with 5 mg/kg benserazide or 1 mg/kg SKF81297 induced dyskinetic movements. The first IP injection of 0.1 mg/kg ropinirole induced relatively weak responses in AMPT-PitxHomo mice. Dyskinesia duration was manually measured during 30^th and 39^th min after injection. N=5 mice for each data point. The 2 arrows indicate the first injections. Note dyskinetic movements increased during the first 3 days. The dyskinesia durations under L-dopa and SKF81297 were correlated with a strong correlation coefficient of 1, indicating a common D1R activation besides coincidental drug injection during these days. However, the dyskinesia duration under ropinirole was weakly correlated with the dyskinesia durations under L-dopa and SKF81297 with a correlation coefficient of 0.66, Pearson test. The weak correlation was likely due to coincidental drug injection during these days and also indicates that L-dopa’s effect was primarily mediated by D1Rs, not D2Rs.

Fig. 4

Identical time course and parallel dose-response curves of L-dopa-stimulated normal and dyskinetic movements in AMPT-PitxHomo mice. (A1, B1, C1) L-dopa (6 mg/kg, A1), SKF81297 (1 mg/kg, B1) and ropinirole (0.1 mg/kg, C1) stimulated normal and dyskinetic movements with identical time courses for each drug. The normal horizontal speed was measured, using a Locomotor Monitor, during the 9^th injection of L-dopa on treatment day 5 when the responses had plateaued and were stable. Dyskinesia duration was measured manually in separate groups of mice. N=5–6 mice for each data point. The correlation coefficient for the 3 pairs of curves was 1, 1 and 0.87, respectively (Pearson test). (A2, B2, C2) L-dopa (A2), SKF81297 (B2) and ropinirole (C2) stimulated normal and dyskinetic movements with parallel dose-response curves. The horizontal movement speed and the dyskinesia duration were measured during the 9^th injection of L-dopa on treatment day 5 in separate groups of PitxHomo mice. N=4–6 mice for each data point. The correlation coefficient for the 3 pairs of curves was 0.98, 0.98 and 0.93, respectively, Pearson test.

Fig. 5

Effects of L-dopa, SKF81297 and ropinirole on challenging beam traversal time. Each data point is the average of the measurements from 5 male AMPT-PitxHomo mice, while the measurement from an individual mouse was the average of 5 consecutive runs to minimize variation. The baseline traversal time in 11–12 week old male PitxHomo mice before any drug treatment (i.e. before AMPT treatment) was around 22 s. (A and B) L-dopa and SKF81297 substantially shortened the time to traverse the beam. One-way ANOVA for L-dopa effects: F(2, 12) = 91.2, p < 0.01. Post hoc Turkey test: p < 0.01 for 0 mg/kg L-dopa vs. 6 mg/kg L-dopa and 0 mg/kg L-dopa vs. 20 mg/kg L-dopa, p < 0.05 for 6 mg/kg L-dopa vs. 20 mg/kg L-dopa. One-way ANOVA for SKF81297 effects: F(2, 12) = 185.5, p < 0.01. Post hoc Turkey test: p < 0.01 for 0 mg/kg SKF81297 vs. 0.5 mg/kg SKF81297 and 0 mg/kg SKF81297 vs. 2 mg/kg SKF81297, p < 0.01 for 0.5 mg/kg SKF81297 vs. 2 mg/kg SKF81297. The dose of 0 mg/kg was saline control. (B*) AMPT-PitxHomo mice injected with 2 mg/kg SKF81297 were able to stand and walk on the narrowest part of the beam. The beam does not show well since it is made of transparent plastic. (C) Ropinirole at 0.05 mg/kg, 0.1 mg/kg and 0.2 mg/kg (IP) reduced the beam traversal time while a higher dose (0.8 mg/kg) prolonged the traversal time. One-way ANOVA: F(4, 20) = 65.6, p < 0.01. Post hoc Turkey test: p < 0.05 for all pairs except p > 0.05 for 0.05 mg/kg and 0.2 mg/kg Ropinirole. (C*) AMPT-PitxHomo mice injected with 1 mg/kg ropinirole often straddled the narrowest part of the beam. The beam shows better here than in B* because the mouse moved slowly in C* and the camera was set for the best picture while it was set for speed in B* for the fast moving mouse.