Article Figures & Data
Figures
- Figure 1.
Rotenone-induced nigrostriatal degeneration causes motor deficits in adult rats. A, Representative images of Nissl-stained midbrain from rotenone- or vehicle-treated rats, indicating substantial neuronal cell body loss in the SNc in response to rotenone; 20× magnification. Dashed lines indicate SNc. Scale bar, 100 µm. B, Representative images of TH-immunostained DA neurons (red) from the SNc (left) and striatum (right) from vehicle- or rotenone-treated rats; 20× magnification. Dashed lines indicate SNc. Scale bars, 100 µm. C, Quantification of immunostained TH+ DA neurons in the SNc (Mann–Whitney U test, p = 0.0025) and (D) TH fluorescence intensity from TH+ nerve terminals in the dorsolateral striatum showed significant loss in response to rotenone compared with vehicle (Mann–Whitney U test, p = 0.0022). Striatal TH fluorescence intensity is normalized to vehicle condition. E, PIT of vehicle (black)- or rotenone (red)- treated rats shows impairment in rotenone-treated rats (H14 = 41.8, p < 0.001). Data are mean ± SEM. N = 5-7 per group for stereology and immunohistochemistry. N = 3 per group for PIT. *p < 0.05. **p < 0.01.
- Figure 2.
VGLUT2-expressing DA neurons are more resistant to rotenone-induced cell loss. Representative images of TH and VGLUT2 mRNA in vehicle- and rotenone-treated rats. A, 4× and (B) 60× magnification images of VTA/SNc of vehicle-treated rats showed a high level of TH expression (red) and a subset of TH+ cells that coexpress VGLUT2 (green). Yellow arrows indicate TH+/VGLUT2+-coexpressing cells. Scale bars: 4× image, 2 mm; 60× image, 25 µm. Lipofuscin depicted in 60× images (white). C–I, Quantification of TH and VGLUT2 mRNA expression in rotenone- and vehicle-treated rats. C, D, Although TH mRNA grains per average cell volume were significantly decreased in rotenone-treated rats compared with vehicle (Mann–Whitney U test, p = 0.032; C), there was no change in VGLUT2 grains per average cell volume in rotenone-treated rats (Mann–Whitney U test, p = 0.99; D). E, There was a significant decrease in number of DAPI-stained nuclei in VTA and SNc in response to rotenone treatment. F, Rotenone diminished the number of TH+ cells in both VTA and SNc compared with controls. G, In contrast, TH+/VGLUT2+ cell numbers were not significantly changed with rotenone treatment. H, The percentage of TH+ cells that expressed VGLUT2 increased after rotenone treatment, indicating that TH/VGLUT2-coexpressing neurons were more resistant to rotenone-induced neurodegeneration. I, There was no increase in VGLUT2 expression within TH+/VGLUT2+ cells after rotenone. Data are mean ± SEM. N = 4 or 5 per group. *p < 0.05 compared with vehicle. #p < 0.05 compared with other regions.
- Figure 3.
Rotenone increases density of VGLUT2+ neurons. A, Rotenone treatment increased the total number of VGLUT2+ cells that did not also coexpress TH mRNA (TH–/VGLUT2+) (H1 = 4.6, p = 0.033). B, There was no change in VGLUT2 mRNA grain expression within TH–/VGLUT2+ cells in either VTA or SNc of rotenone-treated rats compared with the vehicle control. Data are mean ± SEM. N = 4 or 5 per group, *p < 0.05.
- Figure 4.
Rotenone upregulates VGLUT2 protein expression in surviving DA neurons. A, Representative 20× and (B) 100× images showing an upregulation of VGLUT2 in the VTA and SNc of rotenone-treated rats. Scale bar, 30 µm. C, While there was no significant change in relative intensity of VGLUT2+ puncta in either the VTA or SNc, there was a significant increase in (D) VGLUT2+ puncta density and (E) the number of VGLUT2-immunoreactive puncta within TH+ cells. Data are mean ± SEM. N = 3-8 per group. *p < 0.05. **p < 0.01.
- Figure 5.
Rotenone treatment alters actin cytoskeleton and VGLUT2 distribution in N27 cells. Representative images of the rat dopaminergic neuron N27 cell line showed increased VGLUT2 signal (green) in the cell body with accompanying retraction of cell processes and reorganization of cortical β-actin (red) after 100 nm rotenone treatment. Scale bar, 20 µm.
- Figure 6.
Rotenone leads to region-specific loss of TH protein and increased density of TH and VGLUT2-colocalizing puncta in the NAc. A, Representative 60× images showing loss of TH signal but no change in VGLUT2 in the CPu of rotenone-treated rats. Scale bar, 20 µm. B, There was a significantly lower TH signal intensity in the CPu (Dunn's post hoc p = 0.048), but not the NAc core (post hoc p > 0.99) or shell (post hoc p > 0.99), after rotenone treatment. Similarly, there was a significantly lower TH puncta density in the CPu (post hoc p = 0.048), but not in the NAc core (post hoc p > 0.99) or shell (post hoc p > 0.99), after rotenone treatment. C, There was no change in the intensity or density of VGLUT2+ puncta in nerve terminals within the CPu or NAc of rotenone-treated rats compared with vehicle (Kruskal–Wallis H test, all p > 0.06). D, Representative 60× images of TH (red) and VGLUT2 (green) puncta in the NAc core and shell in vehicle- and rotenone-treated rats. White circles represent colocalizing TH and VGLUT2 puncta. Scale bar, 10 µm. E, Within the NAc, a greater density of TH puncta colocalized with VGLUT2 in the NAc shell compared with the NAc core (Kruskal–Wallis H test, p = 0.031), but there was no significant effect of rotenone (p = 0.061). Data are mean ± SEM. N = 3-6 per group. *p < 0.05 compared with vehicle. ##p < 0.05 compared with CPu. See Extended Data Figure 6-1.
Tables
Antigen Antibody catalog information Company Host species Immunohistochemistry/immunocytochemistry concentration TH AB1542 EMD Millipore Sheep 1:2000 VGLUT2 Ab79157 Abcam Mouse 1:500 VGLUT2 135403 Synaptic Systems Rabbit 1:250 β-Actin Ab8226 Abcam Mouse 1:1000
Figure 6-1
TH and VGLUT2 pixel overlap in the NAc is greater than overlap that occurs by randomly distributed fluorescent pixels (supporting Fig. 6). Measurement of Pearson's correlation coefficient (Pearson's r) of TH and VGLUT2 pixels in the NAc is greater than correlation of TH and VGLUT2 after the pixels have been randomized in the images. N = 9 per group. **p < 0.01 (Wilcoxon matched pairs signed-rank test). Download Figure 6-1, TIF file.
