FIGURE LEGENDS
Figure 1. 6-OHDA nigrostriatal lesion characterization and AIMs scores. Panel A shows the distribution of TH fibers in the striatum (CPu) of controls (No-lesion- A and the inset) shows intense TH-immunoreactive fiber terminals. Panel B shows the effect of 6-OHDA on TH-immunoreactive fiber terminals. Likewise in the SNc, TH-immunoreactive neurons were greatly reduced (D ) compared to controls (C ). Panels E and Frepresent the effects of doxycycline on the development of abnormal involuntary movements (AIMs - E ) and time course of AIMs during 180 min (F ) induced by chronic administration of L-DOPA to 6-OHDA-lesioned rats. *P <0.001, Mann-Whitney, comparing Vehicle+ L-DOPA (dyskinetic) with doxycycline-L-DOPA treated rats. Data are mean ± SEM. DOXY: doxycycline; L-DOPA, L-3, 4-dihydroxyphenylalanine; 6-OHDA, 6-hydroxydopamine; VEH: vehicle; ALO AIMs: global AIMs score including all AIM categories (axial, forelimb, and orofacial movements).
Figure 2. NG2 distribution in striatum. A:Experimental design line. The striata was divided in dorsomedial (DM), dorsolateral (DL), ventromedial (VM) and ventrolateral (VL) for quantification analysis. B-E : Representative sections showing NG2-glia immunostaining in the DL striatum from rats without lesion (B- No lesion); lesioned with 6-OHDA toxin (C- 6OHDA); lesioned with 6-OHDA+Vehicle+ L-DOPA treatment (D- 6OHDA+Vehicle +L-DOPA); dyskinetic rats treated with doxycycline (E-6OHDA+L-DOPA+DOXY). Bar represents 20µm. F-I : Quantification of NG2-glia by optic density (One-Way ANOVA) in the DM, DL, VM, VL striatum of No lesion, 6OHDA, 6OHDA+DOXY, 6OHDA+Vehicle+L-DOPA and 6OHDA+DOXY+L-DOPA experimental groups. J-M : Spearman’s rank correlation coefficient of NG2-glia with global AIMs scores axial, forelimb, and orofacial dyskinesia scores in the DM, DL, VM, VL parts of striatum. N : Relative NG2-glia protein expression level (One-Way ANOVA) in DL striatum measured by Western blot (NG2: 26kDa; β actin: 54kDa) in No lesion, 6OHDA, 6OHDA+DOXY, 6OHDA+Vehicle+L-DOPA and 6OHDA+DOXY+L-DOPA experimental groups of rats. *P <0.05, 6OHDA+LDOPA vs. all other groups. **P<0.001, 6OHDA+DOXY+L-DOPA vs. 6OHDA+Vehicle+LOPA. #P<0.05, vs. No lesion. Data are mean ± SEM. DOXY: doxycycline; L-DOPA, L-3, 4-dihydroxyphenylalanine; 6-OHDA, 6-hydroxydopamine. DL= dorsolateral, DM= dorsomedial, VM= ventromedial; VL= ventrolateral.
Figure 3. NG2-glia undergoes morphological changes in the striata of rats presenting AIMs. A: Ramification analysis figures; A´: Representative image of a NG2-glia cell (green) used for the morphological analysis. Bar represents 5µm. A´´:Illustration of skeletonized cell; used for analysis to get information of the cell morphology complexity. A´´´: Illustration of Sholl analysis used to quantify process branching. For quantification, the ramification index (RI) was calculated using the following formula: RI = convex area/cell area (Heppner et al., 1998), where convex area is the area of a polygonal object that is defined by the cells’ most prominent projections and cell area is the area from soma. B-E: Data from the ramification index of rats with L-DOPA-induced dyskinesia (6OHDA+Vehicle+L-DOPA) and treated with Doxycycline (6OHDA+DOXY+L-DOPA) in dorsomedial (DM), dorsolateral (DL), ventromedial (VM) and ventrolateral (VL) striata areas. F: Morphometry analysis data summary for DM, DL, VM and VL striatal areas including number of branches, number of junctions, average branch length and average soma area analyzed. *P<0.001 (One-Way ANOVA), vs. 6OHDA+Vehicle+LOPA. #P<0.001, vs. 6OHDA. Data are mean ± SEM. DOXY: doxycycline; L-DOPA, L-3, 4-dihydroxyphenylalanine; 6-OHDA, 6-hydroxydopamine. DL= dorsolateral, DM= dorsomedial, VM= ventromedial; VL= ventrolateral.
Figure 4. NG2-glia and GFAP double labeling in the striatum. A-D : Graphs show the percentage of NG2-glia co-labeling with GFAP in the total of NG2-glia in dorsomedial (DM), dorsolateral (DL), ventromedial (VM) and ventrolateral (VL) striatum.E-I : Representative sections showing NG2-glia (green), GFAP (red) staining and a merged image in DL striatum from No lesion, 6OHDA, 6OHDA+DOXY, 6OHDA+Vehicle+L-DOPA and 6OHDA+DOXY+L-DOPA experimental groups of rats. Bar represents 20µm. Data are mean ± SEM. DOXY: doxycycline; L-DOPA, L-3, 4-dihydroxyphenylalanine; 6-OHDA, 6-hydroxydopamine.
Figure 5. NG2-glia and OX-42 double labeling in the striatum. A-D : Graphs show the percentage of NG2-glia co-labeling with OX-42 in the total of NG2-glia in dorsomedial (DM), dorsolateral (DL), ventromedial (VM) and ventrolateral (VL) striatum.E-I : Representative sections showing NG2-glia (green), OX-42 (red) staining and a merged image in DL striatum from No lesion, 6OHDA, 6OHDA+DOXY, 6OHDA+Vehicle+L-DOPA and 6OHDA+DOXY+ L-DOPA groups of rats. Bar represents 20µm. Data are mean ± SEM. DOXY: doxycycline; L-DOPA, L-3, 4-dihydroxyphenylalanine; 6-OHDA, 6-hydroxydopamine.