The Journal of Neuroscience, February 22, 2006 • 26(8):2321–2325 • 2321 Brief Communication Dopaminergic Substantia Nigra Neurons Project Topographically Organized to the Subventricular Zone and Stimulate Precursor Cell Proliferation in Aged Primates Nils Freundlieb,1,2* Chantal Franc¸ois,2* Dominique Tande´,2 Wolfgang H. Oertel,1 Etienne C. Hirsch,2 and Gu¨nter U. Ho¨glinger1 1Experimental Neurology, Philipps University, D-35033 Marburg, Germany, and 2Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ Mixte de Recherche 679, Experimental Neurology and Therapeutics, Hoˆpital de la Salpetrie`re, Universite´ Pierre et Marie Curie Paris 6, F-75651 Paris, France The subventricular zone of the adult primate brain contains neural stem cells that can produce new neurons. Endogenous neurogenesis might therefore be used to replace lost neurons in neurodegenerative diseases. This would require, however, a precise understanding of the molecular regulation of stem cell proliferation and differentiation in vivo. Several regulatory factors, including dopamine, have been identified in rodents, but none in primates. We have, therefore, studied the origin and function of the dopaminergic innervation of the subventricular zone in nonhuman primates. Tracing experiments in three macaques revealed a topographically organized projection from the substantia nigra pars compacta (SNpc), but not the adjacent retrorubral field, to the subventricular zone: the anteromedial SNpc projects to the anteroventral subventricular zone, the posterolateral SNpc to the posterodorsal subventricular zone. Double immunola- beling for tyrosine hydroxylase and BrdU (5-bromo-2Јdeoxyuridine) incorporated into the DNA of proliferating cells showed that dopaminergic fibers approach proliferating cells in the subventricular zone. We investigated the effect of this nigro-subventricular projection on cell proliferation in six aged macaques, because the rate of neurogenesis differs between young adult and aged primates and because neurodegenerative diseases mainly affect aged humans. Three macaques were treated with MPTP (1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine) to decrease dopaminergic innervation of the subventricular zone. A significant decrease in the number of PCNA ϩ (proliferating cell nuclear antigen-positive) proliferating cells (Ϫ44%) and PSA-NCAM ϩ (polysialylated neural cell adhesion molecule- positive) neuroblasts (Ϫ59%) was found in the denervated regions of the subventricular zone, suggesting that an intact dopaminergic nigro-subventricular innervation is crucial for sustained neurogenesis in aged primates. Key words: nonhuman primate; aging; dopamine; adult neurogenesis; neural precursor cells; substantia nigra; Parkinson’s disease Introduction therefore to understand how the proliferation, migration, and The subventricular zone (SVZ) in the adult primate brain con- differentiation of endogenous neural stem and precursor cells are tains neural stem cells that have the potential to produce new regulated. However, up to now, nothing has been known about neurons, astrocytes, and oligodendrocytes (Kornack and Rakic, the molecular signals that govern neurogenesis in adult primates. 2001; Sanai et al., 2004). Under physiological conditions, most of The neurotransmitter dopamine is an important factor to the cells born in the SVZ migrate to the olfactory bulb, where they stimulate precursor cell proliferation in the SVZ of adult rodents differentiate and integrate as interneurons (Kornack and Rakic, (Baker et al., 2004; Coronas et al., 2004; Ho¨glinger et al., 2004; 2001). Under some pathological conditions, such as cerebral Van Kampen et al., 2004; Winner et al., 2006). Consistently, we ischemia, Huntington’s disease, or demyelination, SVZ-derived found a reduced rate of cell proliferation in the SVZ of patients neural cells appear to contribute to brain repair (Arvidsson et al., with Parkinson’s disease, a condition characterized by forebrain 2002; Picard-Riera et al., 2002; Curtis et al., 2003). Controlled dopamine depletion secondary to degeneration of the dopami- stimulation of these spontaneous repair processes might be an nergic neurons in the substantia nigra pars compacta (SNpc) elegant way to treat neurodegenerative diseases. It is important (Ho¨glinger et al., 2004). The dopaminergic regulation of SVZ precursor cell proliferation appears therefore to be conserved in humans, but the present evidence is weak, because it is difficult to Received Nov. 11, 2005; revised Jan. 10, 2006; accepted Jan. 14, 2006. control for all potentially confounding factors such as concomi- This work was supported by the German Federal Ministry of Education and Research Network “Stem Cells in PD,” the Peter Hofmann Research Project, a Boehringer Ingelheim research fellowship to N.F., the Fo¨rderverein Neurolo- tant disease or medication that might influence the evaluation of gieMarburg,theFrenchNetwork“NeuralStemCellsforCellTherapyinPDandMS,”theInstitutNationaldelaSante´ neurogenesis in studies on human postmortem material. Fur- et de la Recherche Me´dicale, and Fondation de l’Avenir. We thank Merle Ruberg for revising this manuscript. thermore, it is not known whether the dopaminergic innervation *N.F. and C.F. contributed equally to this work. of the SVZ actually degenerates in Parkinson’s disease, because Correspondence should be addressed to Gu¨nter U. Ho¨glinger, Experimental Neurology, Philipps University, D-35033 Marburg, Germany. E-mail: [email protected]. not all dopaminergic cell populations are equally vulnerable to DOI:10.1523/JNEUROSCI.4859-05.2006 the disease process (Hirsch et al., 1988). We have therefore exam- Copyright © 2006 Society for Neuroscience 0270-6474/06/262321-05$15.00/0 ined in adult primates whether, first, there is an axonal projection 2322 • J. Neurosci., February 22, 2006 • 26(8):2321–2325 Freundlieb et al. • Nigro-Subventricular Projection in Primates from the SNpc to the SVZ and whether, second, a destruction of ethanol and 0.3% hydrogen peroxide in PBS (20°C; 30 min) to inhibit the dopaminergic neurons in the SNpc results in reduced precur- endogenous peroxidases, in PBS containing 10% normal goat serum and sor cell proliferation in the SVZ. 0.3% Triton X-100 to block nonspecific binding sites (20°C; 30 min), and then in the following antibodies (4°C; 2 d): mouse IgM anti- polysialylated neural cell adhesion molecule (PSA-NCAM) (1:400; Materials and Methods Chemicon, Temecula, CA), rabbit anti-tyrosine hydroxylase (TH) (1: Animals. All experiments were performed in accordance with the Euro- 500; Pel-Freez, Compiegne, France), rabbit anti-dopamine ␤ pean Communities Council Directive of 1986 (86/609/EEC). The mon- -hydroxylase (1:500; Chemicon), rabbit anti-dopamine transporter keys were housed in individual cages under standard conditions (12 h (DAT) (1:500; Chemicon), rat anti-BrdU (1:200; ImmunologicalsDirect, light cycles; 23°C; 50% humidity). They had not been used previously for Oxfordshire, UK), and mouse anti-proliferating cell nuclear antigen experimentation. For tracing studies, we used three Cercopithecus ae- (PCNA) (1:500; DakoCytomation, High Wycombe, UK; Glostrup, Den- thiops monkeys, 4–6 years of age, weighing 5.5–6.5 kg. To study the mark). Samples were pretreated with 2N HCl (37°C; 30 min) and rinsed Ϫ effect of dopamine depletion, we used six aged macaques (Macaca mu- in 0.1 M boric acid for BrdU detection and in 70% ethanol ( 20°C; 30 latta), 20–25 years of age, weighing 8–15 kg. Two young macaques (Ma- min) for PCNA detection. The antibodies were visualized by peroxidase caca fascicularis), 4–6 years of age, weighing 3.5–4 kg, were used to histochemistry with diaminobenzidine (DAB) as substrate (Vector Lab- compare the SVZ of young adult and aged macaques. Both species used oratories, Burlingame, CA) or with fluorescent secondary antibodies (Ceropithecus, Macaca) belong to the same Old World monkey subfamily (FITC- or Cy3-conjugated goat anti-rat or rabbit IgG; 1/200; Jackson (Cercopithecine) and both have a comparable body size and brain ImmunoResearch, West Grove, PA). Nuclei were stained with methyl Ј Ј development. green or 4 ,6 -diamidino-2-phenylindole (DAPI). BrdU labeling. We injected unlesioned aged macaques under anesthe- Tracer visualization. Free-floating sections were incubated with an avi- sia (10 mg ketamine/kg body weight, i.m.) with the thymidine analog din–biotin complex (ABC) (Elite; Vector Laboratories) diluted 1:100 in 5-bromo-2Јdeoxyuridine (BrdU) (Sigma, St. Louis, MO) to label prolif- 0.1 M PBS containing 1% Triton X-100 (20°C; 24 h). After washing in erating cells. So that infrequently dividing precursors would be labeled, Tris-HCl buffer, the ABC incubation was repeated. The BDA tracer was BrdU (40 mg BrdU/kg body weight, 5 mg/ml in 0.9% NaCl with 7 mM then visualized by incubation in a solution containing 0.06% hydrogen NaOH, i.p.) was injected 10 times at 2 d intervals over a 3 week period peroxide, 0.2% nickel ammonium sulfate, and 0.05% DAB. The reaction (cumulative dose per animal, 400 mg BrdU/kg body weight). Animals was stopped in Tris buffer. were killed 3 weeks after the last BrdU injection to allow the majority of Image analysis. Cell counts were performed on four regularly spaced restricted precursors to migrate out of the SVZ (Kornack and Rakic, coronal sections along the rostrocaudal extent of the SVZ from 7 mm 2001). The BrdU ϩ cells remaining in the SVZ are therefore likely to be anterior to 1 mm posterior to the anterior commissure and