Dopamine D3 receptor agonist delivery to a model of Parkinson's disease restores the nigrostriatal pathway and improves locomotor behavior

Abstract

The presence of endogenous stem cell populations in the adult mammalian CNS suggests an innate potential for regeneration and represents a potential resource for neuroregenerative therapy aimed at the treatment of neurodegenerative disorders, such as Parkinson's disease. However, it is first necessary to examine the microenvironmental signals required to activate these innate reparative mechanisms. The small molecule neurotransmitter dopamine has been shown to regulate cell cycle in developing and adult brain, and the D3 receptor is known to play an important role in dopaminergic development. Pharmacological activation of the dopamine D3 receptor has been shown to trigger neurogenesis in the substantia nigra of the adult rat brain. Here, we examined the cell proliferative, neurogenic, and behavioral effects of the dopamine D3 receptor agonist 7-OH-DPAT (7-hydroxy-N,N-di-n-propyl-2-aminotetralin) in a 6-hydroxydopamine model of Parkinson's disease. Consistent with previous findings, we observed a significant induction of cell proliferation in the substantia nigra pars compacta (SN(C)) with a time-dependent adoption of a neuronal dopaminergic phenotype in many of these cells. Indices of nigrostriatal integrity were also affected. Dopaminergic cell counts in the lesioned SN(C) recovered substantially in a time-dependent manner. Similarly, retrograde tracing revealed a restoration of striatal innervation from these cells, with evidence for projections arising from newly generated cells. Finally, we observed a substantial and persistent recovery of locomotor function in these animals. The results of these studies will further our understanding of the environmental signals regulating neurogenesis in the adult brain and could have significant implications for the design of novel treatment strategies for Parkinson's disease.

Figure 1.

7-OH-DPAT stimulates cell proliferation in the ipsilateral SN_C. A, B, Representative fluorescent photomicrographs depicting BrdU immunolabeling in a coronal section through the ipsilateral SN in animals treated with saline (A) or 7-OH-DPAT (B) for 2 weeks. C, Quantitative analysis of BrdU-positive cells in the ipsilateral SN_C. Each bar represents the mean (±SEM, n = 6) number of BrdU-positive cells counted in the ipsilateral SN_C after 2, 4, or 8 weeks of treatment. Significant difference from saline-treated controls, ∗∗p < 0.001.

Figure 2.

7-OH-DPAT stimulates cell proliferation in the ipsilateral SVZ. A, B, Representative fluorescent photomicrographs depicting BrdU immunolabeling in a coronal section through the ipsilateral SVZ in animals treated with saline (A) or 7-OH-DPAT (B) for 2 weeks. The asterisk indicates the lateral ventricle.

Figure 3.

7-OH-DPAT stimulates expression of endogenous markers of cell proliferation in the ipsilateral SN_C. A, B, Representative fluorescent photomicrographs depicting PCNA immunolabeling in a coronal section through the ipsilateral SN in animals treated with saline (A) or 7-OH-DPAT (B) for 2 weeks. C, Quantitative analysis of PCNA-positive cells in the ipsilateral SN_C. Each bar represents the mean (±SEM, n = 6) number of PCNA-positive cells counted in the ipsilateral SN_C after 2, 4, or 8 weeks of treatment. Significant difference from saline-treated controls, ∗∗p < 0.001.

Figure 4.

7-OH-DPAT increases the number of PCNA-positive mitotic figures in the ipsilateral SN_C. A, Representative fluorescent photomicrograph depicting PCNA immunolabeling in a pair of proliferating cells appearing to be undergoing mitosis in the ipsilateral SN_C. B, Quantitative analysis of PCNA-positive mitotic figures in the ipsilateral SN_C. Each bar represents the mean (±SEM, n = 6) number of PCNA-positive mitotic pairs counted in the ipsilateral SN_C after 2, 4, or 8 weeks of treatment. Significant difference from saline-treated controls, ∗∗p < 0.001. Scale bar, 20 μm.

Figure 5.

7-OH-DPAT promotes the adoption of a neuronal phenotype in newly generated cells of the SN_C. A–C, Representative fluorescent photomicrographs depicting serial sections through a single cell immunolabeled for TH (A), BrdU (B), and NeuN (C). D, Merged image depicting immunolabeling for all three markers. E, Images were rotated in orthogonal planes to confirm the presence of all three markers within a single cell rather than individual cells in close proximity. F, Counts of BrdU-positive cells also labeled for either NeuN or GFAP in the ipsilateral SNc. Each bar represents the mean (±SEM, n = 6) number of BrdU-positive cells immunolabeled for GFAP or NeuN after 2, 4, or 8 weeks of treatment. Cells were counted in the ipsilateral SN_C. The asterisk indicates significant difference from saline-treated controls. Scale bar, 10 μm.

Figure 6.

7-OH-DPAT treatment partially restores TH immunolabeling in the ipsilateral SN_C. A–C, Photomicrographs depicting TH immunolabeling in the ipsilateral SN_C after 2, 4, and 8 weeks of 7-OH-DPAT treatment. D, TH⁺ cell counts in the ipsilateral SN_C. Each bar represents the mean percentage (±SEM, n = 6) of TH⁺ cells remaining in the ipsilateral SN_C relative to the contralateral, intact hemisphere based on total cell counts. Significant difference from saline-treated controls, ∗∗p < 0.001; ∗p < 0.02.

Figure 7.

7-OH-DPAT partially restores retrograde FG labeling in the SN_C. A–F, Representative photomicrographs depicting FG labeling in the ipsilateral SN after 2 (A, B), 4 (C, D), and 8 (E, F) weeks of saline (A, C, E) or 7-OH-DPAT (B, D, F) treatment. G, Quantitative analysis of FG-positive cells counted across four sections through the ipsilateral SN. Each bar represents the mean percentage (±SEM, n = 6) of FG-labeled cells remaining in the ipsilateral SN_C relative to the contralateral, intact hemisphere based on total cell counts. Significant difference from saline-treated controls, ∗∗p < 0.001.

Figure 8.

Newly generated cells in the ipsilateral SN_C have projections in the striatum. Representative fluorescent photomicrographs depicting double immunolabeling for FG (A) and BrdU (B) in the ipsilateral SN_C after 8 weeks of 7-OH-DPAT treatment. C, Merged images indicate colocalization of both markers within cells of the ipsilateral SN_C. D, Images were rotated in orthogonal planes to confirm the presence of both markers within a single cell rather than individual cells in close proximity. Scale bar, 20 μm.

Figure 9.

7-OH-DPAT promotes functional recovery after denervation in a rodent model of PD. Functional asymmetry was assessed before and after 7-OH-DPAT treatment using two standard behavioral measures. A, Counts of amphetamine-induced rotations 3 d after 0, 2, 4, or 8 weeks of treatment. Each point represents the mean (±SEM, n = 6) number of ipsilateral rotations expressed as a percentage of pretreatment values. Significant difference from saline-treated controls, ∗∗p < 0.001. B, Counts of amphetamine-induced rotations at various time points after the termination of 8 weeks of treatment. Each bar represents the mean (±SEM, n = 3–6) number of ipsilateral rotations expressed as a percentage of pretreatment values. Significant difference from saline-treated controls, ∗∗p < 0.001. C, Counts of pellets eaten in the staircase test. D, Counts of the number of steps reached in the staircase test. Each point represents the mean (±SEM, n = 6) number of pellets eaten or steps reached. Significant difference from ipsilateral paw, ∗∗p < 0.001. Squares, Ipsilateral paw; triangles, contralateral paw; open symbol, saline treatment; filled symbol, 7-OH-DPAT treatment.