The role of the subthalamic nucleus in L-DOPA induced dyskinesia in 6-hydroxydopamine lesioned rats

Abstract

L-DOPA is the most effective treatment for Parkinson's disease (PD), but prolonged use leads to disabling motor complications including dyskinesia. Strong evidence supports a role of the subthalamic nucleus (STN) in the pathophysiology of PD whereas its role in dyskinesia is a matter of controversy. Here, we investigated the involvement of STN in dyskinesia, using single-unit extracellular recording, behavioural and molecular approaches in hemi-parkinsonian rats rendered dyskinetic by chronic L-DOPA administration. Our results show that chronic L-DOPA treatment does not modify the abnormal STN activity induced by the 6-hydroxydopamine lesion of the nigrostriatal pathway in this model. Likewise, we observed a loss of STN responsiveness to a single L-DOPA dose both in lesioned and sham animals that received daily L-DOPA treatment. We did not find any correlation between the abnormal involuntary movement (AIM) scores and the electrophysiological parameters of STN neurons recorded 24 h or 20-120 min after the last L-DOPA injection, except for the axial subscores. Nonetheless, unilateral chemical ablation of the STN with ibotenic acid resulted in a reduction in global AIM scores and peak-severity of dyskinesia. In addition, STN lesion decreased the anti-dyskinetogenic effect of buspirone in a reciprocal manner. Striatal protein expression was altered in dyskinetic animals with increases in ΔFosB, phosphoDARPP-32, dopamine receptor (DR) D3 and DRD2/DRD1 ratio. The STN lesion attenuated the striatal molecular changes and normalized the DRD2/DRD1 ratio. Taken together, our results show that the STN plays a role, if modest, in the physiopathology of dyskinesias.

Figure 1. Schematic representation of experimental design and groups.

At the beginning of the study, animals received a vehicle or 6-OHDA injection into the right medial forebrain bundle and were screened by amphetamine-induced rotations 2 weeks later. Rats were treated daily with saline or L-DOPA injections (6 mg/kg plus 12 mg/kg benserazide) for 21 days. AIMs were rated 2–3 days per week (testing sessions are marked in black). Experiment 1: After the chronic treatment was completed, L-DOPA was administered twice per week, and electrophysiological experiments were performed. All animals were perfused transcardially and processed for histology. Experiment 2: After the last testing session (day 21), the animals received a vehicle (in white) or ibotenic acid (in red) injection into the right STN. After a second period of L-DOPA treatment and AIMs rating (in black) animals were killed and the striata were dissected and frozen for western blot, and STN was postfixed for histology. Experiment 3: After chronic treatment and AIMs rating (in black), animals were killed and striata removed and frozen for western blot analysis.

Figure 2. Characterization of hemiparkinsonian rats with L-DOPA induced dyskinesia.

Evolution of dykinesia scores showing ( A ) the sum of AIM scores for axial, limb and orolingual ratings and ( B ) locomotive score, during L-DOPA chronic treatment. Time course of ( C ) axial, limb and orolingual AIMs score and ( D ) locomotive scores evaluated after a single injection of L-DOPA on the last testing session (day 21st). Groups: sham saline (n = 9), sham L-DOPA (n = 12), 6-OHDA saline (n = 12) and 6-OHDA L-DOPA (n = 13). Note that animals in the sham saline, sham L-DOPA and 6-OHDA saline groups did not develop any AIMs. ( E ) Photomicrographs showing the extensive loss of TH positive fibers in the striatum (upper right panel) and TH positive cells in the substantia nigra (lower right panel) ipsilateral to 6-OHDA lesion. All 6-OHDA lesioned animals included in this experiment showed >95% reduction in TH-fiber optical density in the lesioned striatum. Groups: sham saline (n = 9), sham L-DOPA (n = 12), 6-OHDA saline (n = 12) and 6-OHDA L-DOPA (n = 13). ( F ) Western blot results showing a significant reduction of TH expression in all the 6-OHDA lesioned groups. Groups: sham L-DOPA (n = 7), 6-OHDA saline (n = 9), 6-OHDA L-DOPA (n = 10) and 6-OHDA L-DOPA+STN lesion (n = 8). R = right, ipsilateral to sham or 6-OHDA injection; L = left, contralateral to sham or 6-OHDA injection. Data are expressed as mean ± S.E.M. *** p<0.001 (one-way ANOVA, followed by Bonferroni post-hoc).

Figure 3. Electrophysiological characterization of neurons in the subthalamic nucleus.

( A ) A single spike from an STN neuron recorded in vivo. ( B ) Histological verification of the recording site in the STN, counterstained with neutral red. Scale bar: 200 µm. Examples of action potential traces, showing the three characteristic firing patterns of STN neurons. ( C ) Tonic firing pattern, in which the density histogram follows a Gaussian distribution. ( D ) Random firing pattern, in which the density histogram represents a Poisson distribution. ( E ) Bursting firing pattern, in which the density histogram represents a distribution significantly different from a Poisson distribution, with a significantly positive skewness of the density discharge distribution histogram.

Figure 4. Electrophysiological characterization of the subthalamic nucleus.

( A ) Firing rate of STN glutamatergic neurons. Baseline firing rate (white bars) was significantly different between the groups (F_(4,307) = 15.25, p<0.0001, one-way ANOVA). The acute L-DOPA challenge (grey bars), induced an increase in firing rate in sham saline animals only. ( B ) Coefficient of variation (%). Baseline activity recording (white bars) showed significant differences between groups (F_(4,307) = 11.85, p<0.0001, one-way ANOVA) and acute L-DOPA treatment produced a significant reduction in the sham saline group. ( C ) Percentage of bursting, random and tonic neurons. Baseline burst firing pattern (white bars) was significantly increased in 6-OHDA lesioned groups (p<0.05, Fisher's test). Both sham groups showed a trend to increase the number of bursting neurons after an acute L-DOPA dose (sham saline, p = 0.08, and sham L-DOPA, p = 0.06 vs its baseline). Data are expressed as mean ± S.E.M. All data included in the acute L-DOPA challenge were obtained between 20 and 120 min after L-DOPA injection (time period corresponding to high dyskinetic behaviour). * p<0.05 vs sham saline and sham L-DOPA (one-way ANOVA, followed by Bonferroni post-hoc). ^& p<0.05 vs before L-DOPA administration (unpaired t test). ^# p<0.05 vs sham saline and sham L-DOPA (Fisher's exact test for firing pattern).

Figure 5. Effect of subthalamic nucleus lesion on the severity of L-DOPA-induced dyskinesia.

Evolution of ( A ) AIM scores/session, showing significant reduction after STN lesion in the sum of axial, limb and orolingual AIMs scored per session, whereas ( B ) locomotive AIMs scored per session were similar in both groups. The arrow marks the time of STN injection at day 22. Graphical representation of AIM scores/time point (AUC) comparing the day before STN injection (day 21^st, black triangle) and the last testing session after STN injection (day 48^th, black square). Time course of axial, limb and orolingual AIMs scored in ( C ) STN-lesion and ( D ) STN-sham animal groups. The results show a significant decrease in AIMs scores after STN lesion. Groups: 6-OHDA L-DOPA+STN-lesion (n = 14) and 6-OHDA L-DOPA+STN-sham (n = 5). Data are expressed as mean ± S.E.M. ^& p<0.05 vs day 21st (one-way RM ANOVA, followed by Bonferroni post-hoc). * p<0.05 vs STN-sham (two-way RM ANOVA, followed by Bonferroni post-hoc). ^# p<0.05 (two-way RM ANOVA, followed by Bonferroni post-hoc).

Figure 6. Effect of subthalamic nucleus lesion on the severity of the different subtypes of L-DOPA-induced dyskinesia.

Evolution of AIM scores/session of the ( A ) axial, ( C ) limb and ( E ) orolingual AIMs pre- and post-STN injection, comparing STN-lesion and STN-sham groups. Time course of score for each subtype of AIM pre- and post-STN injection; there were significant differences between pre- and post- injection in the STN-lesion in ( B ) axial, ( D ) limb and ( F ) oral subtypes. Groups: 6-OHDA L-DOPA+STN-lesion (n = 14) and 6-OHDA L-DOPA+STN-sham (n = 5). ( G ) The response to buspirone was maintained after the STN lesion, but the effect was significantly smaller than in the ( H ) STN-sham group. Groups: 6-OHDA L-DOPA+STN-lesion (n = 6) and 6-OHDA L-DOPA+STN-sham (n = 5). ( I ) Thionine-staining of the STN (upper panel). The bottom panels correspond to higher magnification of the ipsilateral (right) and contralateral (left) sides. Scale bar: 400 µm (upper panel) and 200 µm (lower panel). Data are expressed as mean ± S.E.M. ^& p<0.05 vs day 21st (one-way RM ANOVA, followed by Bonferroni). * p<0.05 vs STN-sham (two-way RM ANOVA, followed by Bonferroni post-hoc). ^# p<0.05 (two-way RM ANOVA, followed by Bonferroni post-hoc).

Figure 7. Effect of subthalamic nucleus lesion in protein expression in the striatum.

( A ) Western blot representative images (upper panel) and quantification (lower panel) of cFos protein showing no significant differences between the groups. ( B ) Quantification of ΔFosB/FosB western blot showed significant increased in 6-OHDA L-DOPA group versus 6-OHDA saline group ratio, indicating that L-DOPA treatment increase ΔFosB expression in the lesioned striatum. ( C ) pDARPP32/DARPP ratio was significantly higher in lesioned animal treated chronically with L-DOPA. ( D ) Results of the analysis of DRD1 and ( E ) DRD2 expression showing no significant differences between the groups. ( F ) Analysis of DRD2/DRD1 ratio showed a significant increase in 6-OHDA L-DOPA group. ( G ) DRD3 was significantly higher in 6-OHDA treated with L-DOPA compared to the animals that received saline. ( H ) mGluR5 protein expression quantification revealed a significant reduction in 6-OHDA L-DOPA group versus 6-OHDA saline group. Groups: sham L-DOPA (n = 7), 6-OHDA saline (n = 9), 6-OHDA L-DOPA (n = 10) and 6-OHDA L-DOPA+STN-lesion (n = 8). R = right, ipsilateral to sham or 6-OHDA injection; L = left, contralateral to sham or 6-OHDA injection. Data are expressed as mean ± S.E.M. * p<0.05 and ** p<0.01 (one-way ANOVA, followed by Bonferroni post-hoc).