Proliferating cells can differentiate into neurons in the striatum of normal adult monkey

Neuroscience Letters 328 (2002) 213–216 www.elsevier.com/locate/neulet

Proliferating cells can differentiate into neurons in the striatum of normal adult monkey Andre´anne Be´dard, Martine Cossette, Martin Le´vesque, Andre´ Parent* Centre de Recherche Universite´ Laval-Robert-Giffard 2601, Chemin de la Canardie`re, Local F-6500, Beauport, Que´bec, G1J 2G3 Canada Received 13 March 2002; received in revised form 29 April 2002; accepted 2 May 2002

Abstract In this study we used bromodeoxyuridine (BrdU), a thymidine analogue that is incorporated into the DNA of mitotic cells, to study the cytogenesis status of the striatum in normal, adult, squirrel monkeys (Saimiri sciureus). Three weeks following BrdU injection, numerous BrdU-labeled (1) cells were encountered within both the dorsal and the ventral striatum, including the nucleus accumbens. Their number ranged from 5 to 50 per 40 mm-thick section. These BrdU 1 cells were more abundant medially than laterally and displayed a rostrocaudal-decreasing gradient in the caudate nucleus and putamen. Double-immunofluorescence confocal studies have revealed that about 5–10% of the BrdU 1 striatal cells expressed the neuronal nuclear antigen (NeuN), a marker for mature neurons. These findings suggest that new neurons are produced throughout adult life in the striatum of normal, adult primates. This result raises the possibility of experimentally enhancing the recruitment of these newborn neurons as a means to alleviate the symptoms of neurodegenerative diseases that affect the striatum. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Basal ganglia; Postnatal neurogenesis; Primate brain; Striatum; Neurodegenerative diseases; Bromodeoxyuridine labeling

There exist two major areas in the mammalian brain—the subventricular zone (SVZ) and the hippocampal dentate gyrus (DG)—where neurogenesis occurs throughout adulthood. Studies undertaken in rodents with the thymidine analog 5-bromo-2 0 -deoxyuridine (BrdU), a marker of DNA synthesis that labels proliferating cells, have shown that newborn neurons in the DG become incorporated into the normal hippocampal circuitry [4], whereas neuroblasts generated in the SVZ migrate toward the olfactory bulb, where they differentiate into local circuit neurons [13]. Similar studies have established that neurogenesis also occurs in the DG and SVZ of adult primates, including humans [7,9]. Recently, the intraventricular administration of epidermal growth factor (EGF), transforming growth factor alpha or brain-derived neurotrophic factor (BDNF) in rodents was found to markedly augment the neuronal recruitment in the olfactory bulb. Furthermore, this treatment induced neuronal addition to the mature striatum [1,6,8,15], a brain structure where the proliferative activity of mitotically active neural progenitors is normally maintained at a low level [12,14]. * Corresponding author. Tel.: 11-418-663-5747; fax: 11-418663-8756. E-mail address: [email protected] (A. Parent).

As the possibility of recruiting new neurons in the adult striatum has profound implications for neurodegenerative diseases that affect the basal ganglia and because there is actually no information on the status of adult neurogenesis in primate striatum, we used the BrdU approach to test the hypothesis that new neurons are being produced throughout life in untreated, normal, adult monkeys. Three male, adult (4–6 years of age), squirrel monkeys (Saimiri sciureus), with a body weight ranging from 800 to 1200 g, were used in this study. The animals were raised in a markedly enriched environment for about three years before being used for experimentation. They lived in a colony of 25–30 individuals housed in a single large animal facility room in which they could play with various toys and used a complex system of ropes and shelves to move around and rest. All the experimental procedures followed the guidelines of the Canadian Council on Animal Care and the Laval University Committee on Animal Use and Housing approved our experimental protocol. The monkeys were first lightly anesthetized with isoflurane and then received once a day for three consecutive days an intravenous injection of BrdU (150 mg/kg, Sigma, St. Louis, MO) dissolved in 0.9% NaCl with 0.007 N NaOH. At such doses, BrdU is known to act as a specific and non-toxic marker of dividing cells in adult rodents, whereas lower doses label only a

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fraction of the S-phase cells [3]. The animals were allowed to survive for three weeks after the last BrdU injection principally to avoid the possibility of visualizing cells that may have incorporated BrdU while undergoing apoptosis. Three weeks after the last BrdU injection, the animals were deeply anesthetized with a mixture of ketamine–xylazine and perfused transcardially with 500 ml of 0.9% saline solution, followed by 2 l of 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). The brains were then removed from the skull and sectioned along the sagittal plane at 40 mm with a freezing microtome. BrdU was detected on free floating sections that were incubated for 2 h in 50% formamide (VWR)/standard saline citrate (SSC) 2 £ at 65 8C, rinsed for 5 min in SSC 2 £ , reincubated for 30 min in 2 N HCl at 37 8C, and rinsed for 10 min in 0.1 M boric acid. The sections were then blocked 30 min in a phosphate-buffered saline solution (0.1 M, pH 7.4) containing 0.1% Triton X-100 (Sigma) and 2% normal goat serum. They were incubated for 48 h at 4 8C in the same solution containing rat anti-BrdU (Accurate Scientific, Westbury, NY, 1:200) plus an antibody raised in mouse against the neuronal nuclear protein (NeuN, Chemicon, Temecula, CA, 1:100), which served as a marker of mature neurons. The two markers were revealed simultaneously with a double-immunofluorescence approach using Alexa 488-conjugated goat anti-rat IgG for BrdU and Alexa 568conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR, 1:200) for NeuN. Sections incubated without the primary antibodies remain unstained and served as controls. Fluorescent signals were imaged by using a Zeiss (Oberkochen, Germany) LSM 510 confocal laserscanning microscope. To ensure that a cell that appeared to be doubled-labeled was not, instead, two closely apposed, single-labeled cells [11], a Z-stack analysis was performed at 1-mm intervals. The emission signals of Alexa 488 and 568 were assigned to green and red channels, respectively. Numerous BrdU-labeled (1) cells were found in the primate homologue of the rodent anterior portion of the SVZ, adjacent to the parenchyma of the head of the caudate nucleus. A significant number of BrdU 1 cells also occurred in the striatum itself and their number and distribution were similar in the three animals. These BrdU 1 cells were more numerous in the caudate nucleus than in the putamen and more abundant in the dorsomedial than ventrolateral part of both the caudate nucleus and putamen (Fig. 1A). Their distribution displayed clear rostrocaudaland mediolateral-decreasing gradients. Some BrdU 1 cells were also present in the ventral striatum, which includes the nucleus accumbens, the most ventral portions of both caudate nucleus and putamen, and the deeper layers of the olfactory tubercle. Most BrdU 1 nuclei were round to oval and displayed a moderately intense fluorescence when viewed under the confocal microscope (Fig. 1C,D). Many BrdU 1 cells appeared in pairs (‘doublets’) and were likely daughter cells of a mitotic event in the striatum [11]. The number of BrdU 1 cells ranged from about 25–50/

section for sections located medially compared with 5–10/ section for sections located more laterally. The number of double-labeled (BrdU 1 /NeuN 1 ) cells varied from 2–5/ section medially to 1–3/section laterally. However, the latter figures might be an underestimate of the true number of BrdU 1 /NeuN 1 striatal cells, because highly stringent criteria were applied for the identification of double-labeled cells. Of all the BrdU 1 cells, only those that were deemed double-labeled with NeuN at all observation angles and in each merged and orthogonal images obtained with the confocal microscope, were considered to display a neuronal phenotype (Fig. 1B–D). Our data reveal that cells are newly generated in the striatum of normal, adult, squirrel monkeys and that a distinct subset of these cells develop a mature neuronal phenotype. Although not particularly robust, the neuronal proliferative activity that occurs under normal circumstances at striatal level may provide a basis for the much more prominent neuronal recruitment that results from exposure to different growth promoting molecules. In rodents, intraventricularly injected EGF and BDNF were shown to act as proliferation, survival, and migration factors for SVZ precursor cells and to lead to the expansion of these populations into normal brain parenchyma, including the striatum [1,6,12,15]. A large proportion of these newly generated striatal cells displayed a neuronal phenotype [1,15]. The presence of newborn striatal neurons in growth factor-treated animals indicates that the DG and SVZ are not the only forebrain areas that generate new neurons throughout adult life [10]. Furthermore, these findings have important functional implications, particularly in respect to neurodegenerative diseases that affect the striatum, such as Huntington’s and Parkinson’s disease (PD). A small population of dopaminergic neurons intrinsic to the striatum was detected in primates, including humans [2,5]. Furthermore, the number of these striatal neurons was shown to increase markedly in dopamine-denervated (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-intoxicated) monkeys and in patients with advanced PD [2,16], presumably to compensate for the loss of extrinsic dopaminergic innervation. The absence of the aging pigment lipofuschin in these striatal dopaminergic cells has lead to the hypothesis that proliferative progenitor cells in the striatum might contribute to the increase in the dopaminergic cells in response to dopamine lesion [2]. However, this increase may also result from the triggering of the dopaminergic machinery in pre-existing, striatal cells that were undetectable in normal animals [14]. The latter possibility might indeed be a more rapid and effective means than neurogenesis to compensate for an early dopamine loss. However, it would be important to characterize the effect of chronic dopamine-depletion on striatal neurogenesis by using the BrdU approach in old PD monkeys or by studying the expression of various molecular markers of neurogenesis in postmortem material from advanced PD patients. In our study, the abundance of BrdU 1 cells in the SVZ

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adjoining the caudate nucleus and the progressive decrease in the number of such cells along the rostrocaudal axis of the striatum raise the possibility that newborn striatal cells were

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generated in the SVZ and migrated afterward into the striatal parenchyma. However, it is also possible that newly generated striatal cells result from division of in situ

Fig. 1. (A) Camera lucida drawing of a parasagittal section through the squirrel monkey brain showing the distribution and relative number of the BrdU 1 cells in the striatum. This section was located at about 0.9 mm lateral to the midline and each symbol represents one labeled cell. The green dots represent BrdU 1 nuclei, whereas the green dots surrounded by a red circle indicate double-labeled (BrdU 1 /NeuN 1 ) cells. (B) Confocal microscope images of a double-labeled (BrdU 1 /NeuN 1 ) newborn neuron in the head of the caudate nucleus. The lower panel shows single labeling figures of this neuron, with NeuN (red) on the left and BrdU (green) on the right. The central panel displays a reconstructed orthogonal image viewed from the sides in x–z (top) and y–z (right) planes. (C,D) Confocal stacked z-dimension images of two double-labeled (BrdU 1 /NeuN 1 ) newborn neurons in the rostroventral part of the putamen. This series of single optical sections taken at regular intervals (1 and 2 mm) confirms that the BrdU 1 nucleus (green) belongs to the NeuN 1 (red) neuron. ac, anterior commissure; cc, corpus callosum; CD, caudate nucleus; ic, internal capsule; LV, lateral ventricle; PUT, putamen; SVZ, subventricular zone. Scale bars, 20 mm.

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progenitor cells in the striatal parenchyma, because multipotent progenitor cells have been shown to reside in the striatum of adult rodents [17]. The fact that the three monkeys used in this study were sacrificed at the same time after the last BrdU injection does no allow us to address the issues of the source and migration routes of the newborn cells. Our data nevertheless clearly demonstrate that a subset of the newly generated striatal cells develop a mature neuronal phenotype in the striatum of squirrel monkeys. Future studies with different molecular markers of neurogenesis and gliogenesis should help determine the fate of the majority of the newborn cells in the striatum of adult primates. This research was supported by grant NRF-5781 of the Canadian Institutes for Health Research. [1] Benraiss, A., Chmielnicki, E., Lerner, K., Roh, D. and Goldman, S.A., Adenoviral brain-derived neurotrophic factor induces both neostriatal and olfactory neuronal recruitment from endogenous progenitor cells in the adult forebrain, J. Neurosci., 21 (2001) 6718–6731. [2] Betarbet, R., Turner, R., Chockan, V., DeLong, M.R., Allers, K.A., Walters, J., Levey, A.I. and Greenamyre, J.T., Dopaminergic neurons intrinsic to the primate striatum, J. Neurosci., 17 (1997) 6761–6768. [3] Cameron, H.A. and McKay, R.D.G., Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus, J. Comp. Neurol., 435 (2001) 406–417. [4] Cameron, H.A., Wooley, C.S., McEwen, B.S. and Gould, E., Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat, Neuroscience, 565 (1993) 337–344. [5] Cossette, M., Le´ vesque, M. and Parent, A., Extrastriatal dopaminergic innervation of the human basal ganglia, Neurosci. Res., 34 (1999) 51–54. [6] Craig, C.G., Troppe, V., Morshead, C.M., Reynolds, B.A., Weiss, S. and van der Kooy, D., In vivo growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain, J. Neurosci., 15 (1996) 2649–2658.

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