Adult, male Sprague–Dawley rats (Beijing HFK Bioscience Co., Ltd., China) weighing 230–250 g (7 weeks old) were housed with free access to food and water, 12 h light/dark cycle, constant temperature and humidity. Animal use and care were conformed to the Guidelines of Laboratory Animals Ethics of Tongji Medical College, Huazhong University of Science and Technology. The protocol was approved by the Ethics Committee of Huazhong University of Science and Technology.

6-OHDA (2 μg/μl) and apomorphine hydrochloride (0.1 μg/μl) were dissolved in saline with 0.02% ascorbic acid, and L-DOPA methyl ester and benserazide (12 and 6 mg/kg) were dissolved in saline immediately before use (Sigma-Aldrich). PD98059 (Calbiochem) was dissolved in 20% DMSO, 10% Tween80, and diluted to 0.4 μg/μl with saline (Miller and Marshall, 2005). Both isoflurane and pentobarbital were purchased from Sigma-Aldrich.

Rats were deeply anesthetized with isoflurane (induction 3%, maintenance 1.5%) in oxygen and mounted on the stereotaxic frame. A temperature controller system was used to maintain body temperature at 37°C. 6-OHDA (2 μg/μl, 4 μl) was injected into the right medial forebrain bundle through a 10-μl microsyringe at a rate of 0.5 μl/min at the following stereotactic coordinates: relative to bregma, −4.4 mm anterior (A), +1.5 mm lateral (R), and 7.8 mm deepness (D) (Lindgren et al., 2009). In 33 rats, a guide cannula connected to a microinjection system (RWD Life Science Co., Ltd) was implanted to the right lateral ventricle (A −0.7 mm, R +1.5 mm, D 4.0 mm) according to atlas of Paxinos and Watson (2005) and secured to the skull with dental cement. The efficacy of the dopaminergic lesion was tested by measuring contralateral turning behavior with an acute subthreshold dose of apomorphine (0.05 mg/kg s.c.) 2 weeks post-surgery. Only rats exhibiting more than 200 turns contralateral to the lesion side in 30 min were considered to be compatible with the model of full lesion, and were chosen for further study (Boldry et al., 1995). TH staining revealed also a complete lesion of substantia nigra (SN) ipsilateral to the lesion (see Figure 1).

Three days after apomorphine screening (Figure 2A), 45 successful hemiparkinsonian rats received intraperitoneal injections of L-DOPA plus benserazide (12/6 mg/kg) once daily. L-DOPA-induced AIMs were recorded every other day. Rats were observed for 1 min every 35 min intervals for a total of 140 min following L-DOPA treatment. AIMs were evaluated using the validated AIMs scale. Orofacial, limb, and axial dyskinesia were graded from score 0 to 4: 0 = absent; 1 = occasional, present during less than half min; 2 = frequent, present during more than half min; 3 = continuous but interrupted by strong sensory distraction; 4 = continuous, not interrupted by strong sensory distraction (Winkler et al., 2002; Lundblad et al., 2004). The total of axial, limb and orofacial dyskinesia was also named ALO dyskinesia and the maximum ALO dyskinesia score in each session was 48. Rotation (contralateral turns) test was performed as before (Breger et al., 2013) at day 1, 4, 12, 16, and 20, and only turns of completed 360° were counted.

From day 10, 42 rats with complete dyskinesia (ALO AIMs score = 12 at 70 min after L-DOPA) were divided into 3 groups: LID group, LID + PD98059 group, and LID + vehicle group. There was cannula implantation for each rat in the latter two groups. A volume of 3 μl PD98059 solution (0.4 μg/μl) or vehicle was infused into the right lateral ventricle over 6 min through the cannula connected to the microinjection system half hour before L-DOPA administration from day 10 to 21 (Figure 2A). The infusion needle was left in place for 2 min at the end of infusion for drug diffusion. Normal rats with sham operation and hemiparkinsonian rats were chosen as normal group and PD group, respectively. There were 14 rats in each group.

The stepping test was carried out 15 min before (pre-) and after (post-) L-DOPA treatment as previously described (Pinna et al., 2007, 2010) on days 2, 10, and 18. Rats were moved on the surface of the table for 0.7 m in 4 s by the experimenter. The number of adjusting steps of left and right forelimbs in the forward directions was counted.

All behavioral tests were performed by a blinded examiner.

At day 21, 4 h after the last L-DOPA treatment, 4 rats of each group (normal, PD, LID, LID + Vehicle, and LID + PD98059) were anesthetized with an overdose of pentobarbital and then perfused transcardially with saline followed by 4% ice-cold paraformaldehyde (PFA) in phosphate buffer (pH 7.4). Brain tissues were removed, post-fixed with 4% PFA overnight and 25% sucrose for 24 h. Then the fixed samples were embedded in paraffin for immunohistochemistry and immunofluorescence. Another 10 rats of each group were sacrificed by decapitation. Brain tissues were rapidly removed, dissected for striatum on powdered dry ice. Striatum from 4 of the 10 rats were immediately stored at −80°C until protein extraction for Western blotting analysis, and 6 of the 10 were incubated in RNAlater (Qiagen) at 4°C overnight and then stored at −80°C for further Microarray analysis (3 rats) and real-time quantitative PCR (3 rats).

Brain tissues were homogenized with glass homogenizers in ice-cold enhanced RIPA lysis buffer, containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 2 mM Na3VO4, 1 mM PMSF, 10 mM NaF, and a complete set of protease inhibitors (Roche, USA). Lysates were centrifuged at 12,000 g at 4°C for 15 min and the protein concentrations were determined by a BCA assay kit (Pierce, Rockford). The resulting supernatant was added with 1% SDS loading buffer, and boiled for 10 min. Equal amounts of protein (some 40 μg) of each sample were separated on 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE), transferred for 90 min onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), blocked for 1 h at 25°C in 5% non-fat powdered milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBST), and immunoblotted overnight at 4°C with primary antibodies. The following primary antibodies were deployed: rabbit polyclonal antibody anti-ΔFosB (1:500; Cell Signaling Technology; #9890), rabbit monoclonal antibody against p44/42 MAPK (ERK1/2; 1:1,000; Cell Signaling Technology; #4695), rabbit monoclonal antibody against phospho-p44/42 MAPK (p-ERK1/2; Thr202/Tyr204; 1:1,000; Cell Signaling Technology; #4370). The membranes were washed in TBST, incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; GeneTex: GTX213110-01), and visualized with an ECL detection kit (Thermo Scientific). The membranes were stripped and incubated with rabbit polyclonal antibody against β-actin (1:1,000; AntGene; ANT010) as a loading control. Bands intensities were analyzed quantitatively by Gel Pro Analyzer version 6.0 (Media Cybernetics, Bethesda, MD, USA). Densitometry was represented as relative optical density. All Western blots were repeated not less than three times.

The paraffin-embedded brain tissues were sectioned at thickness of 4 μm. SN and striatum sections were mounted on glass slides, deparaffinized by xylene for 15 min (2 times), dehydrated in graded ethanol solutions, baked in the basic antigen retrieval buffer (pH = 6.0), and washed with phosphate buffer (pH 7.4) for 5 min (3 times). After washing, sections were blocked with 3% Bovine Serum Albumin for 30 min at room temperature (RT), then incubated with diluted primary antibody in a humidified chamber overnight at 4°C overnight. The following primary antibodies were used: rabbit polyclonal antibody against FosB (1:100, sc-48, Santa Cruz, detecting FosB-ΔFosB) for the immunohistochemistry staining in striatum, rabbit polyclonal antibody against TH (1:750, ab112, Abcam) for the immunohistochemistry staining in SN. Then all sections were washed with phosphate buffer (pH 7.4) for 5 min (3 times), subsequently incubated with biotinylated goat anti-rabbit IgG at 37°C for 50 min, washed again as above, incubated with Horseradish peroxidase labeled streptavidin fluid at 37°C for 30 min, washed, followed by DAB solutions for 5 min, washed, counterstained with Harris hematoxylin for 3 min, dehydrated in graded ethanol solutions, and eventually cover slipped. Images were collected through an Olympus camera connected to the microscope at the same light intensity, and analyzed using Image-Pro Plus software by an independent experimenter blinded to the sections by counting the number of positive cells in the SN or dorsal striatum of the lesioned side (relative to bregma, A −4.5 mm for SN and A 0.6 mm for striatum sections, N = 4 sections for counting). TH staining in SN was used to estimate the extent of the dopaminergic lesion (shown in Figure 1).

Immunofluorescence staining shared a same procedure with immunohistochemistry staining before secondary antibodies incubation. Primary antibodies used in the double staining contained rabbit polyclonal antibody against FosB-ΔFosB (1:100, sc-48, Santa Cruz, detecting FosB-ΔFosB) and mouse monoclonal against p-H3 (1:200, Ab14955, Abcam). Primary antibodies used in the single staining contained mouse monoclonal antibody against HIF-1β (1:200, ab2771, Abcam, detecting ARNT) and rabbit polyclonal antibody against TH (1:750, ab112, Abcam). After being washed, sections were incubated in dark with an appropriately diluted Alexa 488- or Cyanine 3-coupled secondary antibodies for 50 min followed by DAPI (4′, 6-diamidino-2-phenylindole) dyeing nucleus for 10 min. Images were collected using laser confocal microscopy marked with image manipulation software, and analyzed using Image-Pro Plus software by an independent experimenter blinded to the sections by counting the number of positive cells in the dorsal striatum of the lesioned side (relative to bregma, A 0.6 mm for striatum sections, N = 4 sections for counting).

Samples (striatum of lesioned side) were sent to Shanghai biotechnology Corporation for whole transcriptome analysis using microarray. Total mRNA was extracted using TRIZOL Reagent (Cat#15596-018, Life technologies, Carlsbad, CA, US) according to the manufacturer's instructions. RNA integrity was evaluated with the Agilent Bioanalyzer 2,100 and RNA 6,000 Nano/Pico Kit (Agilent Technologies, Santa Clara, CA, US). Qualified total RNA was further purified by RNeasy micro kit (Cat#74004, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany). Concentrations of extracted RNA were assessed with the Nanodrop spectrophotometer (Nanodrop Technologies). A total of 100–150 ng RNA per sample was reverse transcribed to double stranded cDNA and then transcribed into cRNA using the Genechip WT Expression Kit (Affymetrix). Second cycle was carried out following generation of cRNA in order to transform the cRNA into single-strand cDNA. The cDNA was fragmented and the Genechip WT Terminal Labeling Kit (Affymetrix) was used to label the single-stranded DNA with biotin. Samples were hybridized to an Affymetrix Genechip Rat Gene 2.0 ST Array Platform. Array hybridization and wash was performed using GeneChip® Hybridization, Wash and Stain Kit (Cat#900720, Affymetrix, Santa Clara, CA, US) in Hybridization Oven 645 (Cat#00-0331-220V, Affymetrix, Santa Clara, CA, US), and Fluidics Station 450 (Cat#00-0079, Affymetrix, Santa Clara, CA, US) according to the manufacturer's instructions. Slides were scanned by GeneChip® Scanner 3000 (Cat#00-00212, Affymetrix, Santa Clara, CA, US).

Total mRNA was extracted and reversely transcribed using the same method as in microarray. Quantification of mRNAs was performed by real-time PCR using Agilent-Stratagene Mx3000P Q-PCR System. The following primers (Invitrogen) were used: Th forward, 5′-GACATTGGACTTGCATCTCTG-3′, and Th reverse, 5′-GCTGGTAGGTTTGATCTTGGT-3′; Arnt forward, 5′-GAACCGAGAATGGCTGTGGATG-3′, and Arnt reverse, 5′-GCTGTGACCTCTGGATTGTGTTAG-3′; FosB forward, 5′-GTGAGAGATTTGCCAGGGTC-3′, and FosB reverse, 5′-GTGAGAGATTTGCCAGGGTC-3′; beta-actin forward, 5′-GGAGATTACTGCCCTGGCTCCTA-3′, and beta-actin reverse, 5′-GACTCATCGTACTCCTGCTTGCTG-3′. The SYBR Green Premix Ex Taq™ GC (Takara, RR420A) was employed. The PCR started with 94°C for 5 min, and then continued with 40 cycles of 10 s at 94°C, 20 s at 60°C, and 15 s at 72°C followed by 1 cycle of 30 s at 94°C, 30 s at 55°C, and 30 s at 94°C. Amplification plots and dissociation curves were obtained to analyze PCRs product and confirm amplification specificity. Expression levels of mRNA were determined using the ΔΔCT method. Each sample was tested in triplicate.

The Expression Console software (Affymetrix, Santa Clara, CA, US) was used to format the raw microarray data. Data pre-processing, including background adjustment, log fold transformation and normalization was completed using the “exon level” option in the software. Moreover, the dataset was normalized using the Robust Multi Array Average (RMA) method to control the inter-array variability. Normalized signal intensities of probes which belong to one transcript of each sample were processed by median for further data analysis. Gene chip and RNA quality were assessed by examining total mRNA expression for each striatum. We have submitted the microarray data to the GEO repository. The GEO accession numbers is GSE93695.

R software was used to screen the differential gene expression transcripts (DETs) among these samples. The adjusted P < 0.05 and fold change (FC) ≥ 1.2 or ≤ 0.8333 were used as the cut-off criteria. After getting the DETs, we proceeded with the functional analysis, searching the function and signaling pathway of the genes. In this step, we used the known databases mainly, including DAVID Gene Ontology (http://david.abcc.ncifcrf.gov/). Considering the large amount and complex branch structure of Gene ontology (GO) biological processes, we used a significance threshold P < 0.05 for biological process terms.

The co-expression analysis starts by constructing a matrix of pairwise correlations between all pairs of transcripts across samples of LID + PD98059, LID + Vehicle and LID group. We built an unsigned co-expression network with all the 37,177 transcripts in microarray using the Weighted Gene Correlational Network Analysis (WGCNA) package [PMID: 19114008]. GO term enrichment tests were performed for individual gene co-expression modules compared to a background set of all genes expressed in these brain samples using the R packages GOstats (version 2.26.0), biomaRt version (2.14.0), AnnotationDbi (version 1.20.7), and org.Hs.eg.db (version 2.8.0).

To specialize LID + PD98059 from other treatment, unsupervised hierarchical clustering of the candidates in every trait module was performed by bootstrapping analysis using MeV software (http://www.tm4.org/). Bootstrapping analysis provides confidence values for the stability of each cluster derived by hierarchical clustering.

RT-qPCR data were expressed as fold changes in relative gene expression compared with the Normal group using beta-actin levels as an endogenous control. The significance level was set at p < 0.05. Data are presented as mean ± SEM.

Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey HSD or LSD post-hoc tests for multiple comparisons between groups, and Student's t-test for comparing TH positive neurons in the intact and lesioned side of SN. All statistical analyses were performed in SPSS 21.0 software. The significance level was set at p < 0.05. Data are presented as mean ± SEM for behavioral assessments, immunohistochemical count, and blot quantifications.

TH immunostaining was used to determine the DA neurons loss in SN following 6-OHDA lesions (Figure 1). DA neurons significantly decreased in the lesioned side (t = 23.432, p < 0.001 Figure 1D). Chronic L-DOPA administration (12 mg/kg, s.c., once daily for 21 days, Figure 2A) to rats with unilateral nigrostriatal 6-OHDA lesion led to the development of increasingly severe AIMs (axial, orofacial, limb AIMs) and rotation, all reaching a plateau after day 9 (Figure 2B, LID and LID + Vehicle groups). MEK inhibitor PD98059 (1.2 μg, injected into the right lateral ventricle 30 min before L-DOPA on days 10–21) clearly attenuated L-DOPA-induced AIMs after day 13 (Figure 2B). The total ALO (axial, limb and orofacial) scores of each group (LID, LID + Vehicle, LID + PD98059) were 34.40 ± 0.90, 34.30 ± 0.90, 19.80 ± 1.33 on day 17 [Figure 2B, F_{(2, 39)} = 62.247, p < 0.01], respectively. ALO scores at day 20 were decreased by PD98059 at all time points (35, 70, and 105 min after L-DOPA; Figures 2C,D). In contrast, rotation (contralateral turns) showed little difference among groups (Figure 2E).

The Stepping test showed that L-DOPA could significantly improve motor function of the contralateral forelimb in rats with unilateral nigrostriatal 6-OHDA-lesion (Figures 2F,G). PD98059 did not affect the stepping test scores (Figures 2F,G).

L-DOPA administration induced a significant increase in p-ERK1/2 level in the striatum of unilaterally 6-OHDA-lesioned rats (Figures 3A,B, Supplementary Figure 1). And this effect was prevented by the inhibitor of MEK PD98059 [Figures 3A,B, Supplementary Figure 1; p-ERK1/2 vs. ERK1/2, F_{(4, 15)} = 6.244 one-way ANOVA followed by Tukey HSD and LSD post-hoc test, N = 4/group]. The increase of p-ERK1/2 was accompanied by overexpression of ΔFosB that represents a net increase of ΔFosB (Figures 3A,C, Supplementary Figure 1). PD98059 also decreased ΔFosB levels [Figures 3A,C, Supplementary Figure 1; ΔFosB vs. β-actin, F_{(4, 15)} = 15.543 one-way ANOVA followed by Tukey HSD and LSD post-hoc test, N = 4/group]. Furthermore, PD98059 reduced FosB-ΔFosB immunoreactive neurons in the dorsolateral striatum on the lesioned side [Figures 3D,E; F_{(4, 15)} = 989.202, one-way ANOVA followed by Tukey HSD post-hoc test, N = 4/group]. The number of FosB-ΔFosB immunoreactive neurons of LID + Vehicle and LID + PD98059 groups was 176.5 ± 4.3/section and 90.3 ± 2.9/section, respectively (Figures 3D,E). No difference was found between LID + Vehicle and LID groups. Double immunolabeling images of the two molecules exhibited colocalization between FosB-ΔFosB and p-H3 in rats with dyskinesia (Figure 4A). PD98059 reduced the striatal co-expression of FosB-ΔFosB and p-H3 positive cell count [Figure 4B; co-expression of FosB-ΔFosB and p-H3, F_{(3, 12)} = 154.413, one-way ANOVA followed by Tukey HSD and LSD post-hoc test, N = 4/group]. The quantification of neurons with colocalization in LID + Vehicle group and LID + PD98059 group was 49.25 ± 2.136, 21.75 ± 1.493, respectively (Figure 4B). Quantification demonstrated that about 50 and 25% of striatal p-H3 positive neurons co-expressed with FosB-ΔFosB in LID + Vehicle group and LID + PD98059 group, respectively (Figure 4C). P-H3 negative neurons didn't express FosB-ΔFosB (data not shown).

The expression of TH and ARNT was also analyzed using Immunofluorescence (Figure 8). Chronic L-DOPA administration increased the number of TH and ARNT positive neurons in the striatum of 6-OHDA-lesioned rats (Figure 9). This effect was reversed by the inhibitor of MEK PD98059 [Figure 9; TH, F_{(3, 12)} = 34.268; HIF-1 β (ARNT), F_{(3, 12)} = 28.696; one-way ANOVA followed by Tukey HSD and LSD post-hoc test, N = 4/group]. No difference was found between LID + Vehicle and LID groups.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.