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A possible role for dopamine D3 receptor stimulation in the induction of neurogenesis in the adult rat substantia nigra
Neuroscience 136 (2005) 381–386
A POSSIBLE ROLE FOR DOPAMINE D3 RECEPTOR STIMULATION IN THE INDUCTION OF NEUROGENESIS IN THE ADULT RAT SUBSTANTIA NIGRA J. M. VAN KAMPEN1 AND H. A. ROBERTSON*
FH (2002) The adult substantia nigra contains progenitor cells with neurogenic potential. J Neurosci 22:6639 – 6649; Zhao M, Momma S, Delfani K, Carlen M, Cassidy RM, Johansson CB, Brismar H, Shupliakov O, Frisen J, Janson AM (2003) Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci U S A 100:7925–7930]. We have found that chronic intraventricular administration of 7-hydroxy-N,N-di-n-propyl-2-aminotetralin triggers a profound induction of cell proliferation in the rat substantia nigra and promotes the adoption of a neuronal phenotype in a proportion of these newly generated cells. © 2005 Published by Elsevier Ltd on behalf of IBRO.
Department of Pharmacology, Dalhousie University, Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5
Abstract—Small molecule neurotransmitters, such as dopamine, have been shown to regulate cell cycles in the developing brain [Spencer GE, Klumperman J, Syed NI (1998) Neurotransmitters and neurodevelopment: Role of dopamine in neurite outgrowth, target selection and specific synapse formation. Perspect Dev Neurobiol 5:451– 467; Ohtani N, Goto T, Waeber C, Bhide PG (2003) Dopamine modulates cell cycle in the lateral ganglionic eminence. J Neurosci 23:2840 –2850] and may provide an alternative to traditional growth factors for the regulation of neurogenesis. Specifically, the dopamine D3 receptor appears to play an important role in neural development, and shows a persistent expression through adulthood in the proliferative subventricular zone [Diaz J, Ridray S, Mignon V, Griffon N, Schwartz JC, Sokoloff P (1997) Selective expression of dopamine D3 receptor mRNA in proliferative zones during embryonic development of the rat brain. J Neurosci 17:4282– 4292]. Furthermore, pharmacological stimulation of D3 receptors promotes proliferation of adult subventricular zone cells, both in vitro [Coronas V, Bantubungi K, Fombonne J, Krantic S, Schiffmann SN, Roger M (2004) Dopamine D3 receptor stimulation promotes the proliferation of cells derived from the post-natal subventricular zone. J Neurochem 91:1292–1301] and in vivo [Van Kampen JM, Hagg T, Robertson HA (2004) Induction of neurogenesis in the adult rat subventricular zone and neostriatum following dopamine D3 receptor stimulation. Eur J Neurosci 19:2377–2387]. In earlier work, we have demonstrated the induction of cell proliferation in the subventricular zone of the adult rat brain accompanied by a dramatic 10-fold induction of neurogenesis in the neighboring neostriatum, following administration of the preferential D3 receptor agonist, 7-hydroxy-N,N-di-n-propyl-2-aminotetralin [Van Kampen JM, Hagg T, Robertson HA (2004) Induction of neurogenesis in the adult rat subventricular zone and neostriatum following dopamine D3 receptor stimulation. Eur J Neurosci 19:2377– 2387]. Dopamine D3 receptors have also been found in the substantia nigra [Diaz J, Pilon C, Le Foll B, Gross C, Triller A, Schwartz JC, Sokoloff P (2000) Dopamine D3 receptors expressed by all mesencephalic dopamine neurons. J Neurosci 20:8677– 8684], a region of the adult brain shown to exhibit ongoing cytogenesis and neurogenic potential [Lie DC, Dziewczapolski G, Willhoite AR, Kaspar BK, Shults CW, Gage
Parkinson’s disease (PD) is a neurodegenerative disorder involving the gradual and near total to total loss of striatal dopamine resulting from the progressive degeneration of dopaminergic projection neurons in the substantia nigra pars compacta (SNC). While dopamine replacement therapy in the form of levodopa remains the primary mode of treatment for PD, long-term exposure to L-DOPA is complicated by the emergence of fluctuations in motor function and a variety of involuntary movements, termed dyskinesias (Marsden, 1984). These complications may result, at least in part, from ongoing cell loss. Thus, cell-replacement therapies may provide a more effective alternative. However, the effectiveness of fetal tissue or stem cell transplants has been varied, and issues of tissue availability, and ethical concerns limit such transplantation procedures. Endogenous adult neural stem/progenitor cells may provide an alternative source of tissue for neuroregenerative therapy, bypassing the need for transplantation. While the neuroregenerative capacity of the adult CNS is limited, there is evidence for ongoing neurogenesis in various discrete regions. The most active neurogenic regions include the subventricular zone/rostral migratory stream system and the dentate gyrus of the hippocampus (McKay, 1997; Kempermann and Gage, 2000). However, it is becoming increasingly apparent that low levels of neurogenesis also occur in various other regions of the adult CNS including the SNC (Lie et al., 2002; Zhao et al., 2003), a finding, which may have profound implications for the treatment of PD. However, these findings are controversial and not all studies have been able to corroborate these findings (Frielingsdorf et al., 2004). In order to capitalize on this neurogenic potential for future therapeutic application, however, the pharmacological signals regulating adult neurogenesis must first be identified. One such signal may involve dopamine D3 receptor activation. Indeed, the dopamine D3 receptor appears to play an important neurogenic modulatory role in CNS de-
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Present address: Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, USA. *Corresponding author. Tel: ⫹1-902-494-2563; fax: ⫹1-902-494-8008. E-mail address: [email protected] (H. A. Robertson). Abbreviations: BrdU, bromodeoxyuridine; GFAP, glial fibrillary acidic protein; PCNA, proliferating cell nuclear antigen; PD, Parkinson’s disease; SNC, substantia nigra pars compacta; TBS, Tris-buffered saline; TH, tyrosine hydroxylase; 7-OH-DPAT, 7-hydroxy-N,N-di-n-propyl-2aminotetralin. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.07.054
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velopment. Expression of this receptor in the CNS occurs quite early in development and is almost exclusively found in the proliferative neuroepithelium (Diaz et al., 1997; Ohtani et al., 2003). Although dopamine D3 receptor expression is significantly more abundant during pre- and early postnatal ontogeny, a restricted D3 receptor expression persists in the striatal SVZ through adulthood, coincident with continued proliferation in this region (Diaz et al., 1997). In cells transfected with the human D3 receptor, dopamine increases [3H]thymidine incorporation (a marker of cell proliferation) by 50 –100% (Pilon et al., 1994). Dopamine D3 receptor activation has also been shown to significantly increase neurogenesis in the SVZ and striatum of the adult rat brain in vivo (Van Kampen et al., 2004). Moreover, D3 receptor stimulation also increases proliferation of cells derived from adult SVZ (Coronas et al., 2004). In the present report, we examine the effects of dopamine D3 receptor activation on neurogenesis in the SNC of the adult rat brain. This is the first study to describe pharmacological regulation of neurogenesis in the SNC.
EXPERIMENTAL PROCEDURES All studies used female Sprague–Dawley rats (Charles River, Montreal), weighing approximately 250 g at the start of the experiment. Animals were housed in a temperature-controlled environment with a 12-h light/dark cycle (lights on at 07:00 h) and ad libitum access to standard rat chow and water. All procedures used in this study were approved by the Dalhousie University
Committee on Laboratory Animals Animal Care in accordance with the Canadian Council on Animal Care. All efforts were made to minimize animal suffering and reduce the number of animals used. Animals were anesthetized using halothane (0.1%) and placed in a Kopf stereotaxic frame. Stainless steel indwelling cannulae (30 Ga, Plastics ONE, Roanoke, VA, USA) were placed into either the ventral third ventricle (antero-poserior (AP) ⫺2.00, medio-lateral (ML) 0.00, dorso-ventral (DV) 8.00) (Walls and Wishart, 1977; Paxinos and Watson, 1986). The cannula was fixed to the skull using dental acrylic and jeweler’s screws. Each cannula was attached, by 50 PE polyethylene tubing, to an osmotic minipump (model 2002; 0.5 l/h, 2 weeks; Alza, CA, USA), which was placed under the skin at the base of the neck. Each pump was filled with either the preferential dopamine D3 receptor agonist, 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) (Sigma, St. Louis, MO, USA) (2 g/l) or its vehicle, 0.9% saline. During the 2 weeks of drug infusion, animals received daily i.p. injections of bromodeoxyuridine (BrdU) (50 mg/kg; Sigma), a marker of cell proliferation. Following 2 weeks of drug treatment, animals were killed by transcardial perfusion with 4% paraformaldehyde. Brains were removed and postfixed for 24 h in 4% paraformaldehyde followed by cryoprotection in 30% sucrose for a minimum of 24 h. Symmetrical 30 m-thick sections were cut on a freezing microtome and stored in a Millonig’s solution. Every twelfth section was processed for immunohistochemistry. Free-floating sections were pretreated with 50% formamide/280 mM, incubated in 2 M HCl at 37 °C for 30 min, and rinsed in 0.1 M boric acid (pH 8.5) at room temperature for 10 min. Sections were incubated in 1% H2O2 in phosphate-buffered saline for 15 min, in blocking solution (3% goat or donkey serum/0.3% Triton X-100/Tris-buffered saline
Fig. 1. BrdU labeling following 7-OH-DPAT infusion into the third ventricle. Following 2 weeks’ 7-OH-DPAT infusion into the third ventricle, BrdU labeling was significantly elevated in the ventricular lining and SNC. (A, B) Representative photomicrographs depicting BrdU labeling in the area surrounding the third ventricle of a coronal section following (A) saline versus (B) 7-OH-DPAT infusion. (C, D) Representative photomicrographs depicting BrdU labeling in the substantia nigra following (C) saline versus (D) 7-OH-DPAT infusion. Total BrdU-positive cell counts in the (E) ventricular lining, (F) posterior hypothalamus, and (G) substantia nigra were significantly elevated following 7-OH-DPAT infusion. Each bar represents the mean (⫾S.E.M.) (n⫽6) BrdU-positive cell counts. * Indicates third ventricle; arrows indicate the SNC; scale bars ⫽ 50 m; ** significantly different from saline-treated controls, P⬍0.001.
J. M. Van Kampen and H. A. Robertson / Neuroscience 136 (2005) 381–386 [TBS]) for 1 h at room temperature, followed by the appropriate antibody at 4 °C overnight. Anti-BrdU antibodies were mouse monoclonal anti-BrdU (1:250, Chemicon, Temecula, CA, USA) or sheep polyclonal anti-BrdU (1:10,000, RDI, Concord, MA, USA). The other primary antibodies were monoclonal mouse anti-glial fibrillary acidic protein (GFAP) (1:1000, Chemicon), monoclonal mouse anti-NeuN (1:1000; Chemicon), and proliferating cell nuclear antigen (PCNA) (1:1000, Santa Cruz). For proper identification of the SNC, all sections were incubated with either polyclonal mouse anti-tyrosine hydroxylase (TH) (1:10,000; Chemicon) or monoclonal mouse anti-TH (1:1,000; Chemicon). For fluorescent visualization, sections were incubated with the respective secondary antibody conjugated to either rhodamine, fluorescein, or BODIPY TR (Vector, Burlingame, CA, USA). In between steps, sections were washed for 3⫻10 min in TBS. Sections were mounted on unsubbed glass slides and coverslipped in Citifluor. Fluorescence signals were detected with a Zeiss axiophot confocal microscope at excitation/emission wavelengths of 535/565 nm (red), 470/505 nm (green), and 585/615 nm (blue). BrdU-positive and double-labeled cells were counted blindly in the SNC and predefined regions of the ventricular wall and surrounding hypothalamus in four sections per animal (3V: ⫺2.12, ⫺2.80, ⫺3.60, ⫺4.16 mm; SNC: ⫺4.80, ⫺5.30, ⫺5.60, ⫺6.04 mm), using unbiased stereology. BrdU- and PCNA-positive cells were counted using a 20⫻ objective (sampling frame area, 90,000 m2) containing an optical grid. Fluorescent double-labeling was observed in microfine slices (1.5 m) using a laser scanning microscope (Zeiss, LSM 510) under a 40⫻ objective. All double-labeling was confirmed by rotating the image along each axis. For the counting of substantia nigra neurons, the compacta regions were defined by the distribution of the TH-positive neurons and a set of clear anatomical landmarks/boundaries. The number of TH-immunopositive compacta neurons having a maximal cell body diameter
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10 m was determined. Data were analyzed using a one-way analysis of variance.
RESULTS In order to determine the effects of chronic, intraventricular infusion of the preferential dopamine D3 receptor agonist, 7-OH-DPAT, on proliferation in the adult rat brain, we analyzed the distribution of BrdU- and PCNA-positive cells surrounding the ventral third ventricle, a region previously associated with adult neurogenesis (Alonso, 1999; Pencea et al., 2001). Consistent with the literature, our analysis revealed newly generated cells concentrated along the ventricular surface of the third ventricle. Significantly more BrdU-positive cells were observed following chronic infusion of 7-OH-DPAT into the third ventricle (F1,10⫽23.50, P⫽0.0007), when compared with saline infusion (Fig. 1a, b, e). Few BrdU-positive cells were found dispersed throughout the surrounding hypothalamic parenchyma of saline-treated controls. However, following 7-OH-DPAT infusion, numerous BrdU-positive cells were evident in this region (F1,10⫽25.73, P⫽0.0005) (Fig. 1f). It is becoming increasingly apparent that adult neurogenesis is not limited to the subependyma of periventricular regions. Restricted progenitor cells have been identified in various “quiescent” regions of the adult brain including the subgranular layer of the dentate gyrus in the hippocampus (Seaberg and van der Kooy, 2002), the neocortex (Palmer et al., 1999; Magavi et al., 2000), striatum (Palmer et al., 1995; Pencea et
Fig. 2. PCNA labeling following 7-OH-DPAT infusion into the third ventricle. Following 2 weeks’ 7-OH-DPAT infusion into the third ventricle, PCNA labeling was significantly elevated in the ventricular lining and SNC. (A, B) Representative photomicrographs depicting PCNA labeling in the area surrounding the third ventricle of a coronal section following (A) saline versus (B) 7-OH-DPAT infusion. (C, D) Representative photomicrographs depicting PCNA labeling in the substantia nigra following (C) saline versus (D) 7-OH-DPAT infusion. Total PCNA-positive cell counts in the (E) ventricular lining and (F) substantia nigra were significantly elevated following 7-OH-DPAT infusion. Each bar represents the mean (⫾S.E.M.) (n⫽6) BrdU-positive cell counts. * Indicates third ventricle; fat arrows indicate doublets; scale bars ⫽ 50 m; ** significantly different from saline-treated controls, P⬍0.001.
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al., 2001), and most recently, the SN (Lie et al., 2002). Here, we observed the appearance of a number of BrdUpositive cells in the SNC and these numbers significantly increased in response to chronic intraventricular 7-OHDPAT infusion (F1,10⫽8.65, P⫽0.0148) (Fig. 1c, d, g). Immunolabeling for PCNA, an endogenous marker of cell proliferation, was also significantly elevated both in the ventricular lining (Fig. 2) (F1,10⫽31.22, P⫽0.0002) and the SNC (F1,10⫽6.63, P⫽0.0277). In order to determine the ultimate phenotype of these newly generated cells, sections were stained with glia- and neuron-associated markers. Within the SNC, despite significant elevations in BrdU labeling in response to 7-OHDPAT, no corresponding increase in labeling for GFAP, an astroglial marker, was observed (Fig. 3c). By contrast, there was a significant increase in the proportion of BrdU-positive cells coexpressing the mature neuronal marker, NeuN (Fig. 3a, b) and the dopaminergic marker, TH (Fig. 3d, e) (F1,10⫽ 9.51, P⫽0.0116, TREATMENT main effect; F1,12⫽10.54, P⫽0.0088, MARKER main effect; F1,12⫽25.69, P⫽0.0005, TREATMENT⫻MARKER interaction effect). Thus, 7-OHDPAT treatment not only enhanced differentiation in general, but appeared to promote the adoption of a neuronal phenotype.
DISCUSSION Here we demonstrate that intraventricular administration of the preferential dopamine D3 receptor agonist, 7-OHDPAT, significantly increased proliferation in the lining of the ventral third ventricle and in the neighboring SNC. Not only was there evidence of increased cell proliferation in these regions but a proportion of these newly generated cells appeared to adopt a neuronal phenotype following 7-OH-DPAT treatment, suggesting a potential neurogenic role for this compound. While low doses of 7-OH-DPAT, similar to those used here, are thought to preferentially activate dopamine D3 receptors (Khroyan et al., 1995), the potential involvement of D2 receptors, which have a lower affinity for the compound, cannot be completely ruled out. These findings are consistent with earlier reports of enhanced neurogenesis in the SVZ and striatum of the adult rat brain following similar treatment, an effect blocked by a selective dopamine D3 receptor antagonist (Van Kampen et al., 2004). Thus, dopamine D3 receptor stimulation may provide a useful means of triggering neurogenesis in the adult brain. Dopamine D3 receptor activation has also been associated with neuroprotective effects. The dopamine D3 receptor agonist, pramipexole, attenuates levodopa-induced dopamine cell loss in mesencephalic cultures (Carvey et al., 2001) and rodent models of PD (Vu et al., 2000; Anderson et al., 2001). As well, Zitter rats, which display a progressive age-related loss of dopaminergic innervation to the striatum and nucleus accumbens, show a significant loss of dopamine D3 receptor expression preceding the massive loss of dopamine innervation (Joyce et al., 2000) suggesting that a loss of trophic support is incurred subsequent to the loss of D3 receptors.
In the SN, the total number of neurons remains constant despite ongoing apoptosis, suggesting that new cells must be continually added (Zhao et al., 2003). Thus, any shift in the balance between neurogenesis and apoptosis may affect nigral cell counts. It is possible that potential neuroprotective actions by 7-OH-DPAT may simply have promoted the survival of BrdU-labeled cells in the SNC. However, treatment with the preferential dopamine D3 agonist also significantly increased the endogenous marker, PCNA. PCNA is an endogenous marker of cell proliferation. Thus, PCNA antibodies label only cells proliferating at the time of death as opposed to BrdU, which is taken within the cell and maintained through migration and differentiation. Here, the increase in PCNA labeling within the lining of the third ventricle and the SNC following 7-OH-DPAT suggests that increases in BrdU-positive cell counts reflect a true effect on cell proliferation rather than cell survival. The appearance of PCNA-positive cells within the SNC and the dramatic increase in their numbers following 7-OHDPAT treatment, also suggest that proliferation is occurring within this structure. Indeed, a population of actively dividing progenitor cells has, recently, been described in the SN (Lie et al., 2002). This does not preclude, however, the possibility of progeny migration from the lining of the third ventricle. Indeed, dopamine D3 receptors are located both in the regions surrounding the ventral third ventricle and in the SN (Diaz et al., 2000). The ability of 7-OH-DPAT treatments to significantly increase the proportion of newly generated cells expressing a neuronal marker is of particular importance for the development of potential neuroregenerative strategies. In studies of injury-induced proliferation, newly generated cells within the SN adopt either a glial phenotype or remain uncommitted (Kay and Blum, 2000). More recently, Lie et al. (2002) found that progenitor cells in the SN give rise primarily to mature glial cells in situ. However, when these cells were transplanted to the hippocampus, they assumed a neuronal phenotype. Thus, these cells do appear to have the capacity to generate new neurons when exposed to appropriate environmental signals. Perhaps dopamine D3 receptor activation plays a role in providing the signals necessary to promote a neuronal phenotype. Although our results are in substantial agreement with those of Lie et al. (2002), Zhao et al. (2003) and Coronas et al. (2004), they stand in apparent contrast to the findings of Frielingsdorf et al. (2004). However, unlike these other studies, the focus of our study is the effects of dopamine D3 receptor activation on cell proliferation and differentiation. The findings reported here could have profound implications for the treatment of PD. Indeed, the loss of primarily one discrete population of cells makes PD a prime candidate for such neuroregenerative therapy. Recent identification of ongoing neurogenesis in the adult mammalian CNS presents the intriguing possibility of utilizing endogenous progenitor cells as a novel source of tissue for cell replacement strategies.
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Fig. 3. Phenotypic analysis of newly generated cells in the substantia nigra following 7-OH-DPAT infusion. Following 2 weeks’ 7-OH-DPAT infusion, newly formed BrdU-positive cells in the substantia nigra appear to adopt primarily a neuronal phenotype. (A, B) Representative fluorescent photomicrographs depicting immunolabeling for BrdU (red) and the neuronal marker, NeuN (green), in a 30 m coronal section through the substantia nigra. Although some cells did not double-label (BrdU⫹/NeuN⫺, BrdU-/NeuN⫹), the majority of BrdU-positive cells were also NeuN-positive (e.g. arrows) following 7-OH-DPAT treatment, suggesting the adoption of a neuronal phenotype by these newly-formed cells. (C) Representative fluorescent photomicrograph depicting double immunolabeling for BrdU (red) and the astrocyte marker, GFAP (green). Although GFAP-positive cells are intermingled with BrdU-positive cells, they do not overlap. Counterstaining for the dopaminergic marker, TH, revealed elevations in co-labeling for this marker in the SNC. (D) Representative fluorescent photomicrograph depicting BrdU (blue), NeuN (green), and TH (red) immunolabeling in the SNC following 7-OH-DPAT. Arrows indicate cells labeled with all three markers. (E) Representative fluorescent photomicrographs depicting serial sections (1.5–1.8 m) through a cell double-labeled for BrdU (red) and TH (green). Two of these cells were selected from (D), as indicated by asterisks. (F) Histogram depicting the percentage of BrdU-positive cells co-labeled for the markers GFAP, NeuN, or TH. Each bar represents the mean (⫾S.E.M.) (n⫽6) percentage of BrdU-positive cells co-expressing the markers GFAP, NeuN, or TH.
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Acknowledgments—These studies were funded by the Canadian Institutes of Health Research. J.V.K. was a post-doctoral fellow of the Parkinson Society Canada.
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Marsden CD (1984) The pathophysiology of movement disorders. Neurol Clin 2:435– 459. McKay RD (1997) Stem cells in the central nervous system. Science 276:66 –71. Ohtani N, Goto T, Waeber C, Bhide PG (2003) Dopamine modulates cell cycle in the lateral ganglionic eminence. J Neurosci 23: 2840 –2850. Palmer TD, Markakis EA, Willhoite AR, Safar F, Gage FH (1999) Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci 19:8487– 8497. Palmer TD, Ray J, Gage FH (1995) FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol Cell Neurosci 6:474 – 486. Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates. New York: Academic Press. Pencea V, Bingaman KD, Weigand SJ, Luskin MB (2001) Infusion of brain-derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J Neurosci 21:6706 – 6717. Pilon C, Levesque D, Dimitriatdou V, Griffon N, Martres MP, Schwartz JC, Sokoloff P (1994) Functional coupling of the human dopamine D3 receptor in a transfected NG 108 –15 neuroblastoma-glioma hybrid cell line. Eur J Pharmacol 268:129 –139. Seaberg RM, van der Kooy D (2002) Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors. J Neurosci 22: 1784 –1793. Spencer GE, Klumperman J, Syed NI (1998) Neurotransmitters and neurodevelopment: Role of dopamine in neurite outgrowth, target selection and specific synapse formation. Per Dev Neurobiol 5: 451– 467. Van Kampen JM, Hagg T, Robertson HA (2004) Induction of neurogenesis in the adult rat subventricular zone and neostriatum following dopamine D3 receptor stimulation. Eur J Neurosci 19: 2377–2387. Vu TQ, Ling ZD, Ma SY, Robie CW, Tong CW, Chen EY, Lipton JW, Carvey PM (2000) Pramipexole attenuates the dopaminergic cell loss induced by intraventricular 6-hydroxydopamine. J Neural Transm 107:159 –176. Walls EK, Wishart TB (1977) Reliable method for cannulation of the third ventricle of the rat. Physiol Behav 19:171–173. Zhao M, Momma S, Delfani K, Carlen M, Cassidy RM, Johansson CB, Brismar H, Shupliakov O, Frisen J, Janson AM (2003) Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci U S A 100:7925–7930.
(Accepted 20 July 2005) (Available online 10 October 2005)
A POSSIBLE ROLE FOR DOPAMINE D3 RECEPTOR STIMULATION IN THE INDUCTION OF NEUROGENESIS IN THE ADULT RAT SUBSTANTIA NIGRA J. M. VAN KAMPEN1 AND H. A. ROBERTSON*
FH (2002) The adult substantia nigra contains progenitor cells with neurogenic potential. J Neurosci 22:6639 – 6649; Zhao M, Momma S, Delfani K, Carlen M, Cassidy RM, Johansson CB, Brismar H, Shupliakov O, Frisen J, Janson AM (2003) Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci U S A 100:7925–7930]. We have found that chronic intraventricular administration of 7-hydroxy-N,N-di-n-propyl-2-aminotetralin triggers a profound induction of cell proliferation in the rat substantia nigra and promotes the adoption of a neuronal phenotype in a proportion of these newly generated cells. © 2005 Published by Elsevier Ltd on behalf of IBRO.
Department of Pharmacology, Dalhousie University, Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5
Abstract—Small molecule neurotransmitters, such as dopamine, have been shown to regulate cell cycles in the developing brain [Spencer GE, Klumperman J, Syed NI (1998) Neurotransmitters and neurodevelopment: Role of dopamine in neurite outgrowth, target selection and specific synapse formation. Perspect Dev Neurobiol 5:451– 467; Ohtani N, Goto T, Waeber C, Bhide PG (2003) Dopamine modulates cell cycle in the lateral ganglionic eminence. J Neurosci 23:2840 –2850] and may provide an alternative to traditional growth factors for the regulation of neurogenesis. Specifically, the dopamine D3 receptor appears to play an important role in neural development, and shows a persistent expression through adulthood in the proliferative subventricular zone [Diaz J, Ridray S, Mignon V, Griffon N, Schwartz JC, Sokoloff P (1997) Selective expression of dopamine D3 receptor mRNA in proliferative zones during embryonic development of the rat brain. J Neurosci 17:4282– 4292]. Furthermore, pharmacological stimulation of D3 receptors promotes proliferation of adult subventricular zone cells, both in vitro [Coronas V, Bantubungi K, Fombonne J, Krantic S, Schiffmann SN, Roger M (2004) Dopamine D3 receptor stimulation promotes the proliferation of cells derived from the post-natal subventricular zone. J Neurochem 91:1292–1301] and in vivo [Van Kampen JM, Hagg T, Robertson HA (2004) Induction of neurogenesis in the adult rat subventricular zone and neostriatum following dopamine D3 receptor stimulation. Eur J Neurosci 19:2377–2387]. In earlier work, we have demonstrated the induction of cell proliferation in the subventricular zone of the adult rat brain accompanied by a dramatic 10-fold induction of neurogenesis in the neighboring neostriatum, following administration of the preferential D3 receptor agonist, 7-hydroxy-N,N-di-n-propyl-2-aminotetralin [Van Kampen JM, Hagg T, Robertson HA (2004) Induction of neurogenesis in the adult rat subventricular zone and neostriatum following dopamine D3 receptor stimulation. Eur J Neurosci 19:2377– 2387]. Dopamine D3 receptors have also been found in the substantia nigra [Diaz J, Pilon C, Le Foll B, Gross C, Triller A, Schwartz JC, Sokoloff P (2000) Dopamine D3 receptors expressed by all mesencephalic dopamine neurons. J Neurosci 20:8677– 8684], a region of the adult brain shown to exhibit ongoing cytogenesis and neurogenic potential [Lie DC, Dziewczapolski G, Willhoite AR, Kaspar BK, Shults CW, Gage
Parkinson’s disease (PD) is a neurodegenerative disorder involving the gradual and near total to total loss of striatal dopamine resulting from the progressive degeneration of dopaminergic projection neurons in the substantia nigra pars compacta (SNC). While dopamine replacement therapy in the form of levodopa remains the primary mode of treatment for PD, long-term exposure to L-DOPA is complicated by the emergence of fluctuations in motor function and a variety of involuntary movements, termed dyskinesias (Marsden, 1984). These complications may result, at least in part, from ongoing cell loss. Thus, cell-replacement therapies may provide a more effective alternative. However, the effectiveness of fetal tissue or stem cell transplants has been varied, and issues of tissue availability, and ethical concerns limit such transplantation procedures. Endogenous adult neural stem/progenitor cells may provide an alternative source of tissue for neuroregenerative therapy, bypassing the need for transplantation. While the neuroregenerative capacity of the adult CNS is limited, there is evidence for ongoing neurogenesis in various discrete regions. The most active neurogenic regions include the subventricular zone/rostral migratory stream system and the dentate gyrus of the hippocampus (McKay, 1997; Kempermann and Gage, 2000). However, it is becoming increasingly apparent that low levels of neurogenesis also occur in various other regions of the adult CNS including the SNC (Lie et al., 2002; Zhao et al., 2003), a finding, which may have profound implications for the treatment of PD. However, these findings are controversial and not all studies have been able to corroborate these findings (Frielingsdorf et al., 2004). In order to capitalize on this neurogenic potential for future therapeutic application, however, the pharmacological signals regulating adult neurogenesis must first be identified. One such signal may involve dopamine D3 receptor activation. Indeed, the dopamine D3 receptor appears to play an important neurogenic modulatory role in CNS de-
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Present address: Mayo Clinic Jacksonville, 4500 San Pablo Road, Jacksonville, FL 32224, USA. *Corresponding author. Tel: ⫹1-902-494-2563; fax: ⫹1-902-494-8008. E-mail address: [email protected] (H. A. Robertson). Abbreviations: BrdU, bromodeoxyuridine; GFAP, glial fibrillary acidic protein; PCNA, proliferating cell nuclear antigen; PD, Parkinson’s disease; SNC, substantia nigra pars compacta; TBS, Tris-buffered saline; TH, tyrosine hydroxylase; 7-OH-DPAT, 7-hydroxy-N,N-di-n-propyl-2aminotetralin. 0306-4522/05$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.07.054
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velopment. Expression of this receptor in the CNS occurs quite early in development and is almost exclusively found in the proliferative neuroepithelium (Diaz et al., 1997; Ohtani et al., 2003). Although dopamine D3 receptor expression is significantly more abundant during pre- and early postnatal ontogeny, a restricted D3 receptor expression persists in the striatal SVZ through adulthood, coincident with continued proliferation in this region (Diaz et al., 1997). In cells transfected with the human D3 receptor, dopamine increases [3H]thymidine incorporation (a marker of cell proliferation) by 50 –100% (Pilon et al., 1994). Dopamine D3 receptor activation has also been shown to significantly increase neurogenesis in the SVZ and striatum of the adult rat brain in vivo (Van Kampen et al., 2004). Moreover, D3 receptor stimulation also increases proliferation of cells derived from adult SVZ (Coronas et al., 2004). In the present report, we examine the effects of dopamine D3 receptor activation on neurogenesis in the SNC of the adult rat brain. This is the first study to describe pharmacological regulation of neurogenesis in the SNC.
EXPERIMENTAL PROCEDURES All studies used female Sprague–Dawley rats (Charles River, Montreal), weighing approximately 250 g at the start of the experiment. Animals were housed in a temperature-controlled environment with a 12-h light/dark cycle (lights on at 07:00 h) and ad libitum access to standard rat chow and water. All procedures used in this study were approved by the Dalhousie University
Committee on Laboratory Animals Animal Care in accordance with the Canadian Council on Animal Care. All efforts were made to minimize animal suffering and reduce the number of animals used. Animals were anesthetized using halothane (0.1%) and placed in a Kopf stereotaxic frame. Stainless steel indwelling cannulae (30 Ga, Plastics ONE, Roanoke, VA, USA) were placed into either the ventral third ventricle (antero-poserior (AP) ⫺2.00, medio-lateral (ML) 0.00, dorso-ventral (DV) 8.00) (Walls and Wishart, 1977; Paxinos and Watson, 1986). The cannula was fixed to the skull using dental acrylic and jeweler’s screws. Each cannula was attached, by 50 PE polyethylene tubing, to an osmotic minipump (model 2002; 0.5 l/h, 2 weeks; Alza, CA, USA), which was placed under the skin at the base of the neck. Each pump was filled with either the preferential dopamine D3 receptor agonist, 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) (Sigma, St. Louis, MO, USA) (2 g/l) or its vehicle, 0.9% saline. During the 2 weeks of drug infusion, animals received daily i.p. injections of bromodeoxyuridine (BrdU) (50 mg/kg; Sigma), a marker of cell proliferation. Following 2 weeks of drug treatment, animals were killed by transcardial perfusion with 4% paraformaldehyde. Brains were removed and postfixed for 24 h in 4% paraformaldehyde followed by cryoprotection in 30% sucrose for a minimum of 24 h. Symmetrical 30 m-thick sections were cut on a freezing microtome and stored in a Millonig’s solution. Every twelfth section was processed for immunohistochemistry. Free-floating sections were pretreated with 50% formamide/280 mM, incubated in 2 M HCl at 37 °C for 30 min, and rinsed in 0.1 M boric acid (pH 8.5) at room temperature for 10 min. Sections were incubated in 1% H2O2 in phosphate-buffered saline for 15 min, in blocking solution (3% goat or donkey serum/0.3% Triton X-100/Tris-buffered saline
Fig. 1. BrdU labeling following 7-OH-DPAT infusion into the third ventricle. Following 2 weeks’ 7-OH-DPAT infusion into the third ventricle, BrdU labeling was significantly elevated in the ventricular lining and SNC. (A, B) Representative photomicrographs depicting BrdU labeling in the area surrounding the third ventricle of a coronal section following (A) saline versus (B) 7-OH-DPAT infusion. (C, D) Representative photomicrographs depicting BrdU labeling in the substantia nigra following (C) saline versus (D) 7-OH-DPAT infusion. Total BrdU-positive cell counts in the (E) ventricular lining, (F) posterior hypothalamus, and (G) substantia nigra were significantly elevated following 7-OH-DPAT infusion. Each bar represents the mean (⫾S.E.M.) (n⫽6) BrdU-positive cell counts. * Indicates third ventricle; arrows indicate the SNC; scale bars ⫽ 50 m; ** significantly different from saline-treated controls, P⬍0.001.
J. M. Van Kampen and H. A. Robertson / Neuroscience 136 (2005) 381–386 [TBS]) for 1 h at room temperature, followed by the appropriate antibody at 4 °C overnight. Anti-BrdU antibodies were mouse monoclonal anti-BrdU (1:250, Chemicon, Temecula, CA, USA) or sheep polyclonal anti-BrdU (1:10,000, RDI, Concord, MA, USA). The other primary antibodies were monoclonal mouse anti-glial fibrillary acidic protein (GFAP) (1:1000, Chemicon), monoclonal mouse anti-NeuN (1:1000; Chemicon), and proliferating cell nuclear antigen (PCNA) (1:1000, Santa Cruz). For proper identification of the SNC, all sections were incubated with either polyclonal mouse anti-tyrosine hydroxylase (TH) (1:10,000; Chemicon) or monoclonal mouse anti-TH (1:1,000; Chemicon). For fluorescent visualization, sections were incubated with the respective secondary antibody conjugated to either rhodamine, fluorescein, or BODIPY TR (Vector, Burlingame, CA, USA). In between steps, sections were washed for 3⫻10 min in TBS. Sections were mounted on unsubbed glass slides and coverslipped in Citifluor. Fluorescence signals were detected with a Zeiss axiophot confocal microscope at excitation/emission wavelengths of 535/565 nm (red), 470/505 nm (green), and 585/615 nm (blue). BrdU-positive and double-labeled cells were counted blindly in the SNC and predefined regions of the ventricular wall and surrounding hypothalamus in four sections per animal (3V: ⫺2.12, ⫺2.80, ⫺3.60, ⫺4.16 mm; SNC: ⫺4.80, ⫺5.30, ⫺5.60, ⫺6.04 mm), using unbiased stereology. BrdU- and PCNA-positive cells were counted using a 20⫻ objective (sampling frame area, 90,000 m2) containing an optical grid. Fluorescent double-labeling was observed in microfine slices (1.5 m) using a laser scanning microscope (Zeiss, LSM 510) under a 40⫻ objective. All double-labeling was confirmed by rotating the image along each axis. For the counting of substantia nigra neurons, the compacta regions were defined by the distribution of the TH-positive neurons and a set of clear anatomical landmarks/boundaries. The number of TH-immunopositive compacta neurons having a maximal cell body diameter
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10 m was determined. Data were analyzed using a one-way analysis of variance.
RESULTS In order to determine the effects of chronic, intraventricular infusion of the preferential dopamine D3 receptor agonist, 7-OH-DPAT, on proliferation in the adult rat brain, we analyzed the distribution of BrdU- and PCNA-positive cells surrounding the ventral third ventricle, a region previously associated with adult neurogenesis (Alonso, 1999; Pencea et al., 2001). Consistent with the literature, our analysis revealed newly generated cells concentrated along the ventricular surface of the third ventricle. Significantly more BrdU-positive cells were observed following chronic infusion of 7-OH-DPAT into the third ventricle (F1,10⫽23.50, P⫽0.0007), when compared with saline infusion (Fig. 1a, b, e). Few BrdU-positive cells were found dispersed throughout the surrounding hypothalamic parenchyma of saline-treated controls. However, following 7-OH-DPAT infusion, numerous BrdU-positive cells were evident in this region (F1,10⫽25.73, P⫽0.0005) (Fig. 1f). It is becoming increasingly apparent that adult neurogenesis is not limited to the subependyma of periventricular regions. Restricted progenitor cells have been identified in various “quiescent” regions of the adult brain including the subgranular layer of the dentate gyrus in the hippocampus (Seaberg and van der Kooy, 2002), the neocortex (Palmer et al., 1999; Magavi et al., 2000), striatum (Palmer et al., 1995; Pencea et
Fig. 2. PCNA labeling following 7-OH-DPAT infusion into the third ventricle. Following 2 weeks’ 7-OH-DPAT infusion into the third ventricle, PCNA labeling was significantly elevated in the ventricular lining and SNC. (A, B) Representative photomicrographs depicting PCNA labeling in the area surrounding the third ventricle of a coronal section following (A) saline versus (B) 7-OH-DPAT infusion. (C, D) Representative photomicrographs depicting PCNA labeling in the substantia nigra following (C) saline versus (D) 7-OH-DPAT infusion. Total PCNA-positive cell counts in the (E) ventricular lining and (F) substantia nigra were significantly elevated following 7-OH-DPAT infusion. Each bar represents the mean (⫾S.E.M.) (n⫽6) BrdU-positive cell counts. * Indicates third ventricle; fat arrows indicate doublets; scale bars ⫽ 50 m; ** significantly different from saline-treated controls, P⬍0.001.
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al., 2001), and most recently, the SN (Lie et al., 2002). Here, we observed the appearance of a number of BrdUpositive cells in the SNC and these numbers significantly increased in response to chronic intraventricular 7-OHDPAT infusion (F1,10⫽8.65, P⫽0.0148) (Fig. 1c, d, g). Immunolabeling for PCNA, an endogenous marker of cell proliferation, was also significantly elevated both in the ventricular lining (Fig. 2) (F1,10⫽31.22, P⫽0.0002) and the SNC (F1,10⫽6.63, P⫽0.0277). In order to determine the ultimate phenotype of these newly generated cells, sections were stained with glia- and neuron-associated markers. Within the SNC, despite significant elevations in BrdU labeling in response to 7-OHDPAT, no corresponding increase in labeling for GFAP, an astroglial marker, was observed (Fig. 3c). By contrast, there was a significant increase in the proportion of BrdU-positive cells coexpressing the mature neuronal marker, NeuN (Fig. 3a, b) and the dopaminergic marker, TH (Fig. 3d, e) (F1,10⫽ 9.51, P⫽0.0116, TREATMENT main effect; F1,12⫽10.54, P⫽0.0088, MARKER main effect; F1,12⫽25.69, P⫽0.0005, TREATMENT⫻MARKER interaction effect). Thus, 7-OHDPAT treatment not only enhanced differentiation in general, but appeared to promote the adoption of a neuronal phenotype.
DISCUSSION Here we demonstrate that intraventricular administration of the preferential dopamine D3 receptor agonist, 7-OHDPAT, significantly increased proliferation in the lining of the ventral third ventricle and in the neighboring SNC. Not only was there evidence of increased cell proliferation in these regions but a proportion of these newly generated cells appeared to adopt a neuronal phenotype following 7-OH-DPAT treatment, suggesting a potential neurogenic role for this compound. While low doses of 7-OH-DPAT, similar to those used here, are thought to preferentially activate dopamine D3 receptors (Khroyan et al., 1995), the potential involvement of D2 receptors, which have a lower affinity for the compound, cannot be completely ruled out. These findings are consistent with earlier reports of enhanced neurogenesis in the SVZ and striatum of the adult rat brain following similar treatment, an effect blocked by a selective dopamine D3 receptor antagonist (Van Kampen et al., 2004). Thus, dopamine D3 receptor stimulation may provide a useful means of triggering neurogenesis in the adult brain. Dopamine D3 receptor activation has also been associated with neuroprotective effects. The dopamine D3 receptor agonist, pramipexole, attenuates levodopa-induced dopamine cell loss in mesencephalic cultures (Carvey et al., 2001) and rodent models of PD (Vu et al., 2000; Anderson et al., 2001). As well, Zitter rats, which display a progressive age-related loss of dopaminergic innervation to the striatum and nucleus accumbens, show a significant loss of dopamine D3 receptor expression preceding the massive loss of dopamine innervation (Joyce et al., 2000) suggesting that a loss of trophic support is incurred subsequent to the loss of D3 receptors.
In the SN, the total number of neurons remains constant despite ongoing apoptosis, suggesting that new cells must be continually added (Zhao et al., 2003). Thus, any shift in the balance between neurogenesis and apoptosis may affect nigral cell counts. It is possible that potential neuroprotective actions by 7-OH-DPAT may simply have promoted the survival of BrdU-labeled cells in the SNC. However, treatment with the preferential dopamine D3 agonist also significantly increased the endogenous marker, PCNA. PCNA is an endogenous marker of cell proliferation. Thus, PCNA antibodies label only cells proliferating at the time of death as opposed to BrdU, which is taken within the cell and maintained through migration and differentiation. Here, the increase in PCNA labeling within the lining of the third ventricle and the SNC following 7-OH-DPAT suggests that increases in BrdU-positive cell counts reflect a true effect on cell proliferation rather than cell survival. The appearance of PCNA-positive cells within the SNC and the dramatic increase in their numbers following 7-OHDPAT treatment, also suggest that proliferation is occurring within this structure. Indeed, a population of actively dividing progenitor cells has, recently, been described in the SN (Lie et al., 2002). This does not preclude, however, the possibility of progeny migration from the lining of the third ventricle. Indeed, dopamine D3 receptors are located both in the regions surrounding the ventral third ventricle and in the SN (Diaz et al., 2000). The ability of 7-OH-DPAT treatments to significantly increase the proportion of newly generated cells expressing a neuronal marker is of particular importance for the development of potential neuroregenerative strategies. In studies of injury-induced proliferation, newly generated cells within the SN adopt either a glial phenotype or remain uncommitted (Kay and Blum, 2000). More recently, Lie et al. (2002) found that progenitor cells in the SN give rise primarily to mature glial cells in situ. However, when these cells were transplanted to the hippocampus, they assumed a neuronal phenotype. Thus, these cells do appear to have the capacity to generate new neurons when exposed to appropriate environmental signals. Perhaps dopamine D3 receptor activation plays a role in providing the signals necessary to promote a neuronal phenotype. Although our results are in substantial agreement with those of Lie et al. (2002), Zhao et al. (2003) and Coronas et al. (2004), they stand in apparent contrast to the findings of Frielingsdorf et al. (2004). However, unlike these other studies, the focus of our study is the effects of dopamine D3 receptor activation on cell proliferation and differentiation. The findings reported here could have profound implications for the treatment of PD. Indeed, the loss of primarily one discrete population of cells makes PD a prime candidate for such neuroregenerative therapy. Recent identification of ongoing neurogenesis in the adult mammalian CNS presents the intriguing possibility of utilizing endogenous progenitor cells as a novel source of tissue for cell replacement strategies.
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Fig. 3. Phenotypic analysis of newly generated cells in the substantia nigra following 7-OH-DPAT infusion. Following 2 weeks’ 7-OH-DPAT infusion, newly formed BrdU-positive cells in the substantia nigra appear to adopt primarily a neuronal phenotype. (A, B) Representative fluorescent photomicrographs depicting immunolabeling for BrdU (red) and the neuronal marker, NeuN (green), in a 30 m coronal section through the substantia nigra. Although some cells did not double-label (BrdU⫹/NeuN⫺, BrdU-/NeuN⫹), the majority of BrdU-positive cells were also NeuN-positive (e.g. arrows) following 7-OH-DPAT treatment, suggesting the adoption of a neuronal phenotype by these newly-formed cells. (C) Representative fluorescent photomicrograph depicting double immunolabeling for BrdU (red) and the astrocyte marker, GFAP (green). Although GFAP-positive cells are intermingled with BrdU-positive cells, they do not overlap. Counterstaining for the dopaminergic marker, TH, revealed elevations in co-labeling for this marker in the SNC. (D) Representative fluorescent photomicrograph depicting BrdU (blue), NeuN (green), and TH (red) immunolabeling in the SNC following 7-OH-DPAT. Arrows indicate cells labeled with all three markers. (E) Representative fluorescent photomicrographs depicting serial sections (1.5–1.8 m) through a cell double-labeled for BrdU (red) and TH (green). Two of these cells were selected from (D), as indicated by asterisks. (F) Histogram depicting the percentage of BrdU-positive cells co-labeled for the markers GFAP, NeuN, or TH. Each bar represents the mean (⫾S.E.M.) (n⫽6) percentage of BrdU-positive cells co-expressing the markers GFAP, NeuN, or TH.
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Acknowledgments—These studies were funded by the Canadian Institutes of Health Research. J.V.K. was a post-doctoral fellow of the Parkinson Society Canada.
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(Accepted 20 July 2005) (Available online 10 October 2005)