Heterogeneity of NG2-expressing cells in the newborn mouse cerebellum

Developmental Biology 285 (2005) 409 – 421 www.elsevier.com/locate/ydbio

Heterogeneity of NG2-expressing cells in the newborn mouse cerebellum Lamia Bouslama-Oueghlani*, Rosine Wehrle´, Constantino Sotelo, Isabelle Dusart UMR-7102 NPA, Universite´ Paris VI, Case 12, Bat B, 6e`me e´tage, 9 Quai Saint Bernard, 75005 Paris, France Received for publication 23 May 2005, revised 8 July 2005, accepted 8 July 2005 Available online 9 August 2005

Abstract The function and origin of NG2+ cells in the adult brain are still controversial. A large amount of data is available which strongly indicates that adult NG2-expressing cells form a heterogeneous population, constituted by oligodendrocyte precursor cells (OPCs) and a fourth novel type of glial cells named the synantocytes. Whether these two populations derive from the progressive maturation of perinatal NG2+ OPCs or are generated as separate populations is not known. We used organotypic cultures of newborn mouse cerebellum depleted, by anti-mitotic drug treatment, of their NG2+ cells with perinatal features (high proliferating rate and high oligodendrocytic differentiation ability). In these cultures, despite the lack of myelin after 14 days in vitro, numerous NG2+ cells remained. We show that these BrdUresistant cells were able to slowly divide, as adult NG2+ cells do. Although many of these cells expressed O4, only a very small fraction of them was further engaged in oligodendrocyte lineage, as they had an extremely poor capacity to generate myelin sheaths to the Purkinje cell axons. These results support the view that at least two distinct populations of NG2+ cells coexist in the cerebellum from birth: one with the young OPC characteristics, another with adult NG2+ cell characteristics. Thus, a fraction of adult NG2+ cells do not derive from the maturation of perinatal OPCs. D 2005 Elsevier Inc. All rights reserved. Keywords: Oligodendrocyte depletion; NG2; Oligodendrocyte precursor cell; Myelination; Proliferation; Migration; Cerebellum

Introduction In the adult CNS, a population of immature proliferative cells with some ultrastructural features of oligodendrocytes was described many years ago (Smart, 1961). The identification of these immature proliferative cells at the light microscopic level has been possible in vivo after the generation of an antibody against the NG2 chondroitin sulfate proteoglycan (Levine and Card, 1987; Stallcup and Beasley, 1987). NG2+ cells, with capacity to proliferate, are present during postnatal development and in the adult rat CNS (Levine et al., 1993; for a review, see Nishiyama et al., 2002). In vitro, NG2+ cells, isolated from either perinatal or adult optic nerve or cerebellum, differentiate into either oligodendrocytes in serum-free medium or astrocytes in

* Corresponding author. Present address: Institut Curie, CNRS UMR-144, 26 rue d’Ulm, 75248 Paris Cedex 05, France. E-mail address: [email protected] (L. Bouslama-Oueghlani). 0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.07.003

presence of serum (Levine et al., 1993; Shi et al., 1998; Diers-Fenger et al., 2001). In the absence of serum, as they differentiate in vitro, these cells lose the expression of NG2 proteoglycan and acquire myelin antigens such as galactocerebrosides (Gal-C; Levine et al., 1993; Nishiyama et al., 1996b). NG2+ cells have never been observed to co-express astrocytic markers in vivo, but some can be double stained with markers of the oligodendrocyte lineage such as O4 and Gal-C (Levine et al., 1993; Reynolds and Hardy, 1997). In addition, it has been reported that in the adult CNS remyelination results from proliferation of endogenous precursor cells, corroborating the occurrence of adult OPCs (Gensert and Goldman, 1997). Thus, although many studies showed that perinatal OPCs differ from adult OPCs (the latter divide and differentiate at several times slower pace than perinatal OPCs; FfrenchConstant and Raff, 1986; Wolswijk and Noble, 1989; Wolswijk et al., 1990), NG2+ cells both in the adult and during development have usually been considered immature

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proliferative glial cells identified as oligodendrocyte precursor cells (OPCs; Levine et al., 1993; Shi et al., 1998). However, recent data suggest that not all the adult NG2+ cells are OPCs, but an important fraction of them could represent a novel population of mature glial cells—named synantocytes—with still undefined function (Berry et al., 2002; Butt et al., 2002; Greenwood and Butt, 2003). Synantocytes have a stellate morphology, contact axons at the Ranvier nodes and have a low capacity to myelinate (Berry et al., 2002). Synantocytes monitor neuronal function and react to CNS insults to help form scar tissue (for a review, see Butt et al., 2002). In addition, the use of the CNP-EGFP-transgenic mouse—to visualize all NG2+ cells—has allowed Chittajallu et al. (2004) to analyze the membrane properties of these cells in the mouse white and gray neocortical matter. These authors have disclosed a new functional heterogeneity: a population of gray matter NG2+ cells displays ion channel expression profiles that differ from the bipolar NG2+ cells found in the white matter. Thus, at least three types of NG2+ can currently be distinguished: young OPCs, adult OPCs and synantocytes. The term ‘‘polydendrocytes’’ has been proposed to refer to all NG2+ cells (Nishiyama et al., 2002). The origin of adult NG2+ cells remains an unsolved question. It has been suggested that perinatal OPCs progressively adopt the features of adult OPCs (Wolswijk et al., 1990; Durand et al., 1997; Gao and Raff, 1997). In contrast, Mallon and co-workers, using transgenic mice in which EGFP is driven under the control of the PLP promoter, showed that only a subpopulation of NG2+ cells express EGFP. In this work, it has been suggested that two NG2+ subpopulations are present in the CNS from early stages of development (Mallon et al., 2002). This problem of the origin of adult NG2+ cells is even more complex in view of the results reported by Niehaus et al. (2000). These authors demonstrated that all the precursors of NG2+ cells are not necessarily NG2 positive, because NG2-negative cells are also capable of a fast regeneration of NG2-positive cells in the adult CNS. Thus, the features of the adult NG2+ cells and their origin are still a matter of debate. The present study was undertaken to determine when most of NG2+ cells exhibit adult characteristics in the mouse cerebellum and whether these populations are present from birth or appear later on. We provide evidence that from birth at least two types of NG2+ cells are present, one with a high rate of proliferation and ability to myelinate, and the other with a low rate of proliferation and very limited ability to myelinate.

Materials and methods Animals Newborn (P0), postnatal day 5 (P5), P7, P10, P14, P21 and adult Swiss mice (Janvier, Le Genset St Isle, France)

were used in this study. For each result at least 3 animals and 20 slices were used in three independent experiments. The experimental plan was designed in agreement with European Union Guidelines for care and use of experimental animals. Tissue preparation P0 mice were deeply anaesthetized with ice and P5, P7, P14, P21 and P60 with sodium pentobarbital (200 mg/kg i.p.). All animals were perfused through the ascending aorta with 0.12 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde. Brains were removed, postfixed 4 h and cryoprotected in 30% sucrose for 2 days. The cerebella were cut in the sagittal plane (24-Am-thick free-floating sections) on a freezing microtome. The sections were then treated for immunohistochemistry as described below. Slice culture Cerebellar organotypic cultures of P0 mice were prepared as described previously (Ghoumari et al., 2002). Briefly after decapitation, brains were dissected out into cold Gey’s balanced salt solution (Invitrogen, Cergy Pontoise, France) containing 5 mg/ml glucose. Cerebellar parasagittal slices (350 Am thick) were cut on a McIlwain tissue chopper and transferred onto membranes of 30 mm Millipore culture inserts with 0.4 Am pore size (Millicell, Millipore, Bedford, MA, USA). Slices were maintained in culture in 6-well plates containing 1 ml of nutrient medium at 35-C in an atmosphere of humidified 5% CO2. The nutrient medium was composed of 50% basal medium with Earle’s salts (Invitrogen), 25% Hanks’ balanced salts solution (Invitrogen), 25% horse serum (Invitrogen), l-glutamine (Invitrogen, 1 mM) and 5 mg/ml glucose (Stoppini et al., 1991). Oligodendrocyte depletion Cerebellar slices were exposed to 5V-bromo-2V-deoxyuridine (BrdU; 1.5  10 4 M; Sigma, Saint Louis, MO) dissolved in NaCl (9 g/l) incorporated into the nutrient medium from the time of culture until the third day in vitro (3 DIV) (Bouslama-Oueghlani et al., 2003). Co-culture experiments P0 slices were put in culture and treated with BrdU during the first 3 days. On the seventh day of culture, fresh cerebellar slices from P0 animals were apposed to the P07DIV (days in vitro) slices. We left the co-cultures for 7 additional days in culture before fixation. A thread was put on the P0 slice the day of the co-culture in order to distinguish between P0-14DIV and P0-7DIV slices (Figs. 7A – C). In two experiments, the fresh slices of P0 cerebellum were taken from newborn actin-GFP mice; these mice express GFP (green fluorescent protein) under the

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Rabbit polyclonal antibodies against CaBP (diluted 1/5000; Swant, Bellinzona, Switzerland) were used to visualize Purkinje cells (Dusart et al., 1997). Mouse monoclonal antibodies against MBP (diluted 1/2000; Boehringer Mannheim, Indianapolis, IN) were used to visualize myelin and mature oligodendrocytes (Dusart et al., 1997). Rabbit polyclonal antibodies against NG2 (diluted 1/500; Chemicon, Temecula, CA) were used. These antibodies also stain blood vessels during development (Levine et al., 1993). Rabbit polyclonal antibodies against the cell-cycle-associated antigen Ki67 (diluted 1/1000, NovoCastra, Newcastle, UK) were used to reveal the presence of proliferating cells (Hofstadter et al., 1995; Kee et al., 2002). Mouse monoclonal antibodies against O4 were used to label oligodendrocyte precursor cells (diluted 1/10; a gift of Marie Stephane Aigrot and Bernard Zalc, Sommer and Schachner, 1981). Mouse GFAP-CY3 antibodies were used to label astrocytes (diluted 1/500, Sigma). Isolectin-B4 from Griffonia simplicifolia coupled with FITC (0.02 mg/ ml; Sigma) was applied to visualize microglial cells, macrophages and endothelial cells (Streit and Kreutzberg, 1987). Mouse monoclonal antibodies against GFP (1/200, Qbiogen, NorthAmerica) were used to visualize GFP.

(Calbiochem, France Biochem, Meudon, France) when fluorescent secondary antibodies were used. Alternatively, the avidin –biotin – peroxidase complex (Vector) was used to reveal the biotinylated secondary antibodies. Histochemical detection of peroxidase activity was carried out in 0.1 M Tris buffer containing 0.03% 3,3Vdiaminobenzidine (Sigma) with 0.005% hydrogen peroxidase. For double labeling experiments, the primary antibodies were applied together and revealed with different fluorescent secondary antibodies. We made one exception, with the double NG2/Ki67 immunostaining to determine whether NG2+ cells were able to proliferate. In this specific case, since both antibodies are polyclonal, we first performed the immunostaining with Ki67, a nuclear marker (revealed with the rabbit biotinylated antibody and 3,3Vdiaminobenzidine, DAB), and then the following days the NG2 immunostaining, a cell membrane marker (revealed with anti-rabbit-CY3). The sections were analyzed using a Leica DMR microscope equipped with a coolscan camera (Princeton instruments, Evry, France). Images were captured on a Dell computer using Metaview software (Universal Imaging Corporation, West Chester). In some cases, double staining was confirmed on 1 Am thick confocal sections observed with a Leica confocal microscope (Plateforme d’Imagerie, IFR83). Finally, figure plates were prepared using Adobe Photoshop v.6.0 Software (Adobe system, Inc.). For the NG2+/Ki67 immunostaining, the Ki67 immunostaining images (DAB staining) were first inverted and then copied in green layer; whereas for the NG2 immunostaining, the gray level images were directly copied in the red layer.

Staining procedures

Evaluation of the percentage of dividing NG2+ cells in vivo

The cultures and co-cultures were fixed in 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4) for 1 h at room temperature (RT) at intervals ranging from 7 to 21 days in vitro (DIV). After washing in PBS (Invitrogen), the slices were detached from the Millicell membranes. When antibodies against MBP were used, slices were first immersed in Clark’s solution (95% ethanol/5% acetic acid) for 20 min at 4-C to extract some of the lipids and thus make the MBP antigens accessible for the antibodies, then the slices were washed several times with PBS. In all cases, the slices were incubated for 1 h in PBS containing 0.2% gelatin, 0.1% sodium azide (PBSGA) and lysine (0.1 M), before applying the primary antibodies, diluted in PBSGA, overnight. The primary antibodies were revealed with the following secondary antibodies: goat anti-mouse CY3 (1/200 dilution; Jackson ImmunoResearch Laboratories, Inc., West Baltimore Pike, USA), goat anti-rabbit CY3 (1/ 200 dilution; Jackson ImmunoResearch Laboratories, Inc.), sheep anti-rabbit FITC (1/200 dilution, Eurobio, Les Ulis, France), and goat anti-rabbit biotinylated (1/200 dilution, Vector, Laboratories Burlingame, CA). After 2 h incubation in PBSGA containing the secondary antibodies, the slices were washed several times in PBS and mounted in mowiol

The evaluation was performed on P5 and P7 animals (3 animals per age). In each animal, two to four vermal sections were chosen, and the all the NG2+ cells and the double NG2+/Ki67+ cells present in the Purkinje cell layer and the internal granular cell layer were counted with a 63 oil immersion objective on a Leica microscope. At least 120 NG2+ cells were analyzed per animal. The percentage of NG2+/Ki67+ cells was calculated for each lobule. The means and standard errors of the means were calculated for each time point. Data were statistically analyzed using the Student’s t test.

control of the actin promoter, i.e., in all cell types (Hadjantonakis et al., 1998). Thus, the GFP+ cells observed on the treated slices (BT3-P0-14DIV) have migrated from the actin-GFP P0-7DIV slices (Fig. 7F). Antibodies and lectin

Quantification of NG2+ cells in cerebellar organotypic cultures To determine the effect of BrdU treatment on NG2+ cells, in P0-7DIV, P0-14DIV and P0-21DIV slices (control and treated), the area with the highest number of NG2+ cells was selected and one picture of this area was taken at 40 magnification (93,150 Am2). NG2+ cells were counted in the picture and the cell density (number of NG2+ cells per 100,000 mm2) was calculated for each slice. The means of cell densities and the standard errors of the means (SEMs)

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were then calculated for control and treated slices. Data were statistically analyzed using the Student’s t test. Myelin and oligodendrocyte quantification To assess the state of myelination, internodes and mature MBP-positive oligodendrocytes were quantified under a fluorescence microscope (Leica, DMR) as described previously (Bouslama-Oueghlani et al., 2003). Briefly, three groups of slices were defined. The first group (I) included slices with a small number of oligodendrocytes and of groups of internodes (<15 of each). The second group (II) included slices containing more than 16 oligodendrocytes or 16 –20 internode groups. The third group (III) included slices containing more than 21 groups of internodes. Thus, the group III represents the slices with a maximum amount of myelination.

Results and discussion NG2+ cells in the mouse cerebellum during development In a first part of this study, we investigated when during mouse cerebellar development NG2+ cells exhibit adult characteristics, based on their morphology, distribution and proliferation rate. Distribution of NG2+ cells in the mouse cerebellum in vivo We first studied by immunohistochemistry the temporal distribution of NG2+ cells in the mouse cerebellum during in vivo postnatal development. Currently, to our knowledge, only Diers-Fenger and colleagues have partially described the distribution of NG2+ cells in mouse (Diers-Fenger et al., 2001), since most studies have been carried out in rats (Levine and Stallcup, 1987; Levine et al., 1993). As is already known for other CNS regions (Levine et al., 1993; Diers-Fenger et al., 2001), NG2+ endothelial cells were observed in the developing cerebellum (Fig. 1B). These NG2+ endothelial cells were easily identified and excluded from our study. At birth, NG2+ cells are present throughout the cerebellum with the exception of the external granule cell layer, in which rare NG2+ cells intermingle with other cells in its deepest part (Fig. 1A). At this age, the majority of cerebellar NG2+ cells exhibit a stellate morphology, i.e., a small cell body with many processes radiating in all directions (Fig. 1B). At P7, the density of NG2+ cells increases (compare Fig. 1C with 1A). NG2+ cells are still absent from the external granule cell layer and the nascent molecular layer, where only a few thin processes can be detected (Fig. 1D). The NG2+ cells are widespread in the other regions of the cerebellum. The morphology of NG2+ cells is not the same in the white matter and in the grey matter. In the grey matter, they have the ‘‘classical’’ stellate morphology (Fig. 1D), while in the white matter, although

Fig. 1. Distribution of the NG2+ cells in the mouse cerebellum in vivo during postnatal development. Photomicrographs of parasagittal slices of cerebella at P0 (A, B), P7 (C, D), P14 (E, F) and P60 (G, H) immunostained with an anti-NG2 antibody. (A) Numerous NG2+ cells are present in the white and grey matter of newborn cerebellum, but not in the external granule cell layer. (B) The majority of the NG2+ cells are stellate (arrows). Note that some of the NG2+ cells delineate blood vessels (arrowhead). (C) At P7 the NG2+ cells are present all over the slice except in the external granule layer and the molecular layer. Note that the density of NG2+ cells has increased compared to P0 (compare panels A and C). (D) The NG2+ cells are stellate (arrows). Note the presence of NG2+ processes in the molecular layer (arrowheads). (E) At P14, the NG2+ cells are present in the white matter and the grey matter, but with a lower density than at P7 (compare panels E and C). (F) The NG2+ cells are present in the molecular layer from P14. (G) In the adult cerebellum, NG2+ cells are mainly present in the molecular layer. Note that some regions of the molecular layer are devoid of NG2+ cells. (H) In the adult, NG2+ cells still preserve their stellate morphology. Scale bar is 170 Am in panel A; 350 Am in panels C, E and G; and 28 Am in panels B, D, F and H.

they have numerous processes, the processes are oriented in the direction of the fibers, giving this type of NG2+ cell an elongated appearance (data not shown). At P14, the density of the NG2+ cells decreases drastically in the granular layer and less in the white matter (compare Fig. 1E with 1C). However, at this age stellate NG2+ cells are present in the molecular layer (Fig. 1F). From P21, the distribution of

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NG2+ cells is similar to that observed in the adult cerebellum: NG2+ cells can be observed occasionally in the molecular layer and very rarely in the granular layer and in the white matter (Figs. 1G and 1H). Thus, as partly described in the past (Diers-Fenger et al., 2001), the distribution pattern of the NG2+ cells in the mouse cerebellum during postnatal development is similar to that previously reported in rat (Levine et al., 1993). There are, however, important temporal differences. NG2+ cells are first observed in the molecular layer of the rat cerebellum at the end of the first week, whereas in mouse the NG2+ cells are detected in this layer only from day 14. In addition, since most of these cells have a stellate morphology, neither the location nor the shape are sufficient criteria to characterize adult NG2+ cells. For this reason, we also analyzed their rate of proliferation. Indeed, one important criterion to distinguish adult from perinatal OPCs in dissociated culture is the duration of the cell cycle: adult OPCs have a much longer cell cycle than perinatal OPCs (65 h versus 18 h in culture; Wolswijk and Noble, 1989). Since the proliferation rate is much slower in adult OPCs than in perinatal OPCs, the number of proliferative cells is smaller too. The proliferation rate of NG2+ cells decreases between P5 and P7 An indirect way to evaluate the proliferation rate of NG2+ cells is to double label these cells with a marker of proliferation, and to determine the percentage of NG2+ cells at different developmental periods that are double labeled. We used an anti-Ki67 antibody because Ki67 is a nuclear protein expressed by proliferating cells in all phases of the active cell cycle (Hofstadter et al., 1995; Kee et al., 2002). In vivo, numerous Ki67+ cells are found in the external granular layer and much fewer in the other layers of the cerebellar cortex, because precursors of granule cells are the most abundant proliferating cells in the cerebellum during the first postnatal week (Miale and Sidman, 1961; Fujita, 1967; Fujita et al., 1966). At each time point, NG2+/Ki67+, NG2+/Ki67 and NG2 /Ki67+ cells can be detected (Figs. 2A – 2C), indicating that among the NG2+ cells only some of them divide. In the Purkinje and the granule cell layers, a first broad analysis indicates that the number of Ki67+ cells decreases between P5 and P7. To evaluate the proliferation rate of NG2+ cells at P5 and P7, we counted all the NG2+ cells in lobule 6 and among them the number of NG2+/ Ki67+ cells. The percentage of dividing NG2+ cells decreases very strongly between P5 and P7 (from 27% to 5%, P < 0.001; Fig. 2D). In addition to this rapid decrease in proliferation, we showed that OPCs acquired mature morphology after 14 days, and their adult pattern of distribution after 3 weeks. These progressive maturations follow the myelination process. However, during development myelination occurs in the white matter and in the granule cell layer but not in the molecular layer, but in the molecular layer NG2+ cells

Fig. 2. Proliferation of NG2+ cells in vivo. (A – C) Photomicrographs of a P5 cerebellar sagittal section, double immunostained with anti-NG2 (red, A and C) and anti-Ki67 (green, B and C). NG2+/Ki67+ (arrow in panels A, B, C), NG2+/Ki67 (arrowhead in panels A and C) and NG2 /Ki67+ (arrowhead in panel B) cells can be detected. Note the presence of proliferative granule cells (B, arrowhead). (D) Quantitative analysis of the dividing NG2+ cells in P5 and P7 mice. The percentage of dividing NG2+ cells is lower in P7 than in P5 mice. N is the number of animals, n the number of NG2+ cells analyzed. Scale bar is 10 Am.

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are present as soon as 2 weeks postnatal. From these results, it is not possible to determine whether in the newborn mouse cerebellum the NG2+ cells constitute a homogeneous population of precursor cells from which the adult NG2+ cells will be generated, or a heterogeneous population. We addressed this point using organotypic cultures. Presence of at least two different populations of NG2+ cells in the newborn cerebellum Myelination normally occurs in organotypic cultures (Billings-Gagliardi et al., 1980; Seil et al., 1980; Notterpek et al., 1993) but can be prevented by the addition of antimitotic drugs to the medium to deplete the slices of mature oligodendrocytes (Younkin and Silberberg, 1973; Seil et al., 1980; Stanhope et al., 1986, Bouslama-Oueghlani et al., 2003). The application of an anti-mitotic drug during a defined period of time leads to the destruction of all dividing cells within this period. In our previous work (BouslamaOueghlani et al., 2003), we tested different times of exposure to BrdU and found that treatment during the first 3 days of culture is the shortest BrdU exposure time leading to oligodendrocyte depletion, as defined by the absence of MBP-positive elements (cells and myelin packs) during the first 14 days of the culture. Thus, in mouse organotypic cultures the OPCs that give rise to MBP+ oligodendrocytes within the first 2 weeks in vitro divide during the first 3 days of culture. Based on these results, we assumed that the treatment with BrdU during the first 3 days (BT3) of the culture must provoke the death of all OPCs with perinatal features. With this model in hand, we decided to investigate whether some NG2+ cells with adult characteristics, i.e., slow rate of proliferation (cell cycle more than 3 days) and slow rate of differentiation, are present in the mouse cerebellum from birth. If such NG2+ cells exist, some of them would not divide during the first 3 days of culture and therefore could escape the treatment. To this end we performed NG2 immunohistochemistry on control and BrdU-treated slices. The P0-BT3-7DIV slices still contain abundant NG2+ cells, although their density is much lower than in control slices (compare Figs. 3A and B). In treated Fig. 3. NG2+ cells in control and BrdU-treated cerebellar slices. Photomicrographs of P0-7DIV slices immunostained with anti-NG2 antibody: (A) control (P0-7DIV); (B) treated with BrdU during the first 3 days in vitro (P0-BT3-7DIV; and (C) treated with BrdU during the first 6 days in vitro (P0-BT6-7DIV). (A) In the P0-7DIV control slice, the NG2+ elements form a very dense network in which processes or cell bodies cannot be distinguished with certainty. (B) In the P0-BT3-7DIV slice, many NG2+ cells are present. Note that the cell density of these cells is lower than in the control slice and that the NG2+ cells exhibit a stellate morphology. (C) In the P0-BT6-7DIV slice, there are a few NG2+ cells. Note that the density of these cells diminished in comparison to P0-BT3-7DIV. (D) Quantitative analysis of the NG2+ cell densities in BrdU-treated slices. Note that the density of NG2+ cells is lower in P0-7DIV slices when they are treated during the first 6 DIV, compared to those treated during the first 3 DIV. N is the number of animals and n is the number of slices. Scale bar is 45 Am.

slices, the NG2+ cells can be distinguished from each other and their classical round or stellate morphologies can be easily seen (Fig. 3B). These cells are alive, since numerous NG2+ cells are still present even in P0-BT3-21DIV slices. It is known that a final division is frequently required for the terminal differentiation of progenitor cells. It is therefore possible that BrdU treatment is disabling the cells’ differentiation by preventing their final division prior to differentiation. To test this hypothesis, we have studied whether the NG2+ cells present after 3 days of treatment are still able to proliferate.

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The NG2+ cells present in the BrdU-treated slices are still able to divide To find out if the NG2+ cell subpopulation present after the 3-day BrdU treatment is able to divide, we first prolonged the period of BrdU culture application. When BrdU was applied for 6 instead of 3 DIV, the density of NG2+ cells decreased dramatically at 7 DIV (compare the densities of NG2+ cells in the three and 6 days BrdUtreated slices at 7 DIV; Figs. 3B and C). We counted these cells and showed that more than 50% of the NG2+ cells that resisted the 3-day BrdU treatment disappeared when the treatment lasted for 6 days (Fig. 3D). These observations suggest that at least 50% of the NG2+ cells resistant to the 3-day BrdU treatment divide between the 4th and the 6th days of culture. To confirm this result, slices treated during the first 3 DIV were fixed the 5th day of culture and double immunostained for Ki67 and NG2. Although the majority of the NG2+ cells was not double labeled, a small number of them showed co-localization (Figs. 4A – C). As expected, in control slices there was a much higher number of double-labeled cells, and only a few NG2+ cells remained immunonegative for Ki67 (data not shown). Thus, BrdU treatment does not prevent NG2+ cells from further division once the BrdU is removed. These results show that the inability of the BrdU-resistant NG2+ cells to differentiate within 14 DIV does not result from their inability to undertake a final division prior to differentiation. Furthermore, the fact that the NG2+ cells retain the ability to divide suggests that this cell subpopulation has a progenitor quality (Horner et al., 2000). To determine whether BrdU-resistant NG2+ cells share properties with OPCs, synantocytes or other cell populations, we further characterized this NG2+ cell population in BT3 cerebellar slices. Immunohistochemical features of the BrdU-resistant NG2-positive cells It is known that in dissociated culture NG2+ cells give rise to astrocytes in presence of serum and to oligodendrocytes in its absence (Levine et al., 1993; Shi et al.,

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1998). In addition, it has been described that after spinal cord lesions a small number of reactive macrophages (isolectin-B4 positive) are double stained for anti-NG2 (Jones et al., 2002, Camand et al., 2004). To identify the phenotypes of the BrdU-resistant NG2+ cells, we performed double staining with anti-NG2 and either antiGFAP to label astrocytes, isolectin-B4 to label microglia/ macrophages or O4 to label more differentiated OPCs than only those which were NG2+ (Reynolds and Hardy, 1997). BrdU-resistant NG2+ cells are not astrocytes In our cultures, despite the presence of 25% serum in the medium, and the occurrence of numerous GFAP+ cells (Fig. 5B), we did not detect NG2+/GFAP+ cells (Figs. 5A – C). Furthermore, the morphology of these two types of cells was always different (compare Figs. 5A and B). Our present results corroborate previous studies in vivo and in vitro, in which several antigenic markers for astrocytes were used for double labeling (Nishiyama et al., 1996a,b; Reynolds and Hardy, 1997). These observations do not rule out the possibility that NG2+ cells give rise to astrocytes. Nevertheless, the presence of numerous NG2+/O4+ cells in the BT3 slices (see below) and of MBP+ oligodendrocytes and myelin in control slices (Bouslama-Oueghlani et al., 2003) provides strong evidence that the main differentiation program followed by the P0 NG2+ cerebellar cells is oriented toward the oligodendrocyte lineage. Therefore, if astrocytic differentiation occurs, it would concern only a small fraction of NG2+ cells. Very few BrdU-resistant NG2+ are microglia/macrophage cells Immunostaining for NG2 and isolectin-B4 in P0 slices showed that at 7 DIV there was no co-localization between these two markers. However, as previously described and discussed (Bouslama-Oueghlani et al., 2003), in P0-7DIV slices there were very few isolectin-B4+ cells. The number of isolectin-B4+ cells increased at 14 and 21 DIV, suggesting proliferation and activation of the few resting

Fig. 4. NG2+ cells in the BrdU-treated slices are capable of division. (A – C) Photomicrographs of a P0-BT3-5DIV slice double labeled with NG2 (red, A and C) and Ki67 (green, B and C). NG2+/A – C, in P0-5DIV, some NG2+ cells present in the slice (A, red) are Ki67+ (B, green). NG2+/Ki67+ (arrow in panels A, B, C), NG2+/Ki67 (arrowhead in panels A and C) and NG2 /Ki67+ (double arrowhead in panels B and C) cells can be detected. Note that the NG2 staining is within the cell membrane and that the Ki67 is nuclear. Thus, the double-labeled cells do not appear in yellow but they have a red cell membrane and a green nucleus (C, see arrows). Scale bar is 27 Am.

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Fig. 5. Characteristics of NG2+ cells in the slices treated with BrdU during 3 days (P0-BT3). (A – C) Photomicrographs of a P0-BT3-14DIV slice double labeled with anti-NG2 (red, A and C) and anti-GFAP (green, B and C). No double-labeled cells (NG2+/GFAP+) can be observed. Note that NG2+ cells and GFAP+ cells have different morphologies. (D – F) Photomicrographs of a P0-BT3-14DIV slice double labeled with anti-NG2 (red, D and F) and isolectin-B4 (green, E and F). No double-labeled cells (NG2+/Isolectin-B4+) can be observed. (G – I) Confocal photomicrographs of a P0-BT3-14DIV slice double labeled with anti-NG2 (red, G and I) and isolectin-B4 (green, H and I). Note the presence of an NG2+ cell double labeled with the isolectin-B4 (arrow, G – I). (J – L) Photomicrographs of a P0-BT3-14DIV slice double labeled for NG2 (red, J and L) and O4 (green, K and L). A large number of NG2+ cells are double labeled by the O4 antibody (arrowheads, J – L). Note that some cells are O4+ and NG2 (arrows in panels K and L). Scale bar is 45 Am in panels A – F and J – L and 15 Am in panels G – I.

microglial cells present in the slices at 7 DIV. At these ages, despite the presence of abundant microglial cells, only a very few NG2+ cells are also isolectin-B4+, as shown with confocal microscopy (Figs. 5G – I). In general, almost no double-labeled cells can be observed with these markers (Figs. 5D – F). Thus, similar to what has been reported in the spinal cord after lesion (Jones et al., 2002; Camand et al., 2004), a very small number of reactive macrophages (isolectin-B4 positive) are double stained with anti-NG2 in cerebellar slices. Numerous BrdU-resistant NG2+ cells are O4+ In contrast to GFAP and isolectin-B4 stainings, double labeling with O4 reveals, in P0-BT3-14DIV slices, a large number of NG2- and O4-labeled cells (Figs. 5J –L). In addition, some O4+ cells are NG2 . These results seem to indicate that the fate of BrdU-resistant NG2+ cells is to acquire an oligodendrocyte phenotype. However, in these

slices, we found neither mature oligodendrocytes nor myelin, in agreement with previous results (Fig. 6A; Bouslama-Oueghlani et al., 2003). Thus, the BrdU-resistant NG2+ subpopulation is O4+ but does not pursue its oligodendrocyte differentiation within 14 days. Adult NG2+ cells, including synantocytes, are O4+ (Reynolds and Hardy, 1997, Berry et al., 2002). Thus, the BrdU-resistant NG2+ cells present in the newborn mouse cerebellum may generate NG2+/O4+ cells with adult synantocytic features, i.e., with a slow rate of proliferation and a poor capacity to differentiate into oligodendrocytes. The observed arrest in the oligodendrocyte differentiation process of the BrdU-resistant NG2+ cells could also be due to direct or indirect effects of the BrdU treatment, which could interrupt the differentiation program of the BrdUresistant NG2+ cells without killing them. We therefore undertook experiments to determine whether the BrdU treatment could exert direct or indirect effects on the BrdUresistant NG2+ cell differentiation.

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Fig. 6. Delayed myelination in the treated slices. Photomicrographs of P0-BT3-14DIV (A), P0-BT3-21DIV (B) and P0-BT6-21DIV (C) slices immunostained with MBP antibody. (A) P0-BT3-14DIV slice does not present MBP+ elements. (B) Illustration of a P0-BT3-21DIV slice presenting a high number of MBP+ elements. (C) P0-BT6-21DIV is devoid of MBP+ elements. (D) Quantitative evaluation of myelination. The three groups of slices (I, II and III) were defined according to the number of MBP-positive cells and segments. Group I included the slices with a small number of oligodendrocytes and of packs of internodes (<15 of each). Group II included slices containing more than 16 oligodendrocytes or 16 internode packs but less than 20 packs of internodes. Group III included slices containing more than 21 packs of internodes. Note that all the P0-BT3-14DIV and P0-BT6-21DIV slices are in group I. However, P0-BT3-21DIV slices are distributed between the three groups. Thus, some of the OPCs present in P0-BT3-14DIV were able to differentiate into mature oligodendrocytes. N is the number of animals and n is the number of slices. Scale bar is 300 Am.

A minority of BrdU-resistant NG2+ cells is able to slowly differentiate into oligodendrocytes To further pursue our study of the maturation of the BrdUresistant NG2+ cells, we analyzed their capacity for longterm myelination, as a final proof of their complete functional maturation. In a previous study, we showed that the presence of MBP+ internodes observed at the light microscopic level was correlated with the presence of compact myelin at the electron microscopic level (Bouslama-Oueghlani et al., 2003). Based on those results, we used only light microscopy to look for MBP+ elements in this study. We maintained the P0-BT3 slices 21 DIV. In these cultures, using the criteria described in Materials and methods for the quantification of the MBP+ elements, we observed that about 10% of the slices, pooled from different series of experiments, were in group III, the group that contains the slices with high number of MBP+ elements (Fig. 6B). However, the vast majority of the slices (85%) were in group I, with rare or no myelinated fibers (Fig. 6D). The occurrence of MBP+ elements in some of these cultures could indicate that the process of oligodendrocyte maturation was delayed and that myelination occurred at slow rate. Due to limitations of our approach (organotypic culture), we were unable to completely test the possibility of a long-term oligodendrocyte differentiation of

the BrdU-resistant NG2+ cells followed by a slow pace of myelination. In P0-BT3-30DIV, MBP+ elements were totally absent, but Purkinje cells, almost the only neurons with myelinated axons in cerebellar explants (the number of surviving deep nuclear neurons is very low), and consequently the unique population of neurons providing axons to myelinate, were in a process of degeneration. Purkinje cells exhibited disrupted axons, bearing numerous varicosities that certainly interfered with the myelination process (data not shown). In addition, when P0 slices were treated with BrdU during the first 6 DIV (P0-BT6-21DIV), myelin was absent from all examined slices. All these slices are in group I (Figs. 6C and 6D). They did not present a delayed myelination (Fig. 6C), at least within the limits of our experimental system. Despite certain limitations (notably no electron microscopy to confirm the presence of compact myelin in the P0BT6-21DIV cultures), these last results strongly indicate that the BrdU-resistant NG2+ cells have a restricted capacity to provide myelinating oligodendrocytes and delayed myelination, together with the occurrence of NG2+ cells in P0-BT321DIV slices. Finally, we investigated whether this limited myelination could be due to an indirect effect of the BrdU on the other cell populations present in the slices, provoking pathological alterations that could, for instance, cause the loss of oligodendrocyte differentiating factors. For that purpose,

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we developed an in vitro model to study migration and differentiation of control OPCs in BrdU-treated slices. The BrdU treatment does not affect the ability of the cerebellar slices to be myelinated To verify that the absence of myelin in the P0-BT314DIV is not due to BrdU toxicity on cell populations other than NG2+ cells, we tested the ability of neurons in these treated slices to be myelinated by OPCs originated from control slices (BrdU untreated). For that purpose, cerebellar explants taken from P0 mice were apposed to P0-BT3-7DIV (Fig. 7A). The co-cultures were maintained in vitro for 7 more days. In this case, numerous MBP+ elements, both oligodendrocytes and myelin sheaths, occurred in the P0BT3-14DIV slices (Fig. 7B), the latter being distributed in culture regions rich in Purkinje cell axons (Fig. 7C). Thus, the OPCs from the P0 slices seemed able to migrate deep into the BrdU-treated slices and differentiated into oligodendrocytes within 7 days. To rule out the possibility that P0 cerebellar explants could secrete some promoting factors accelerating the differentiation of the BrdU-resistant NG2+ cells, we carried out similar co-culture experiments but maintaining the two explants at a distance (Fig. 7D). In this case, no MBP+ elements were observed in the P0-BT314DIV slices (Fig. 7E). Thus, contact between explants is essential for the appearance of oligodendrocytes and myelin in the P0-BT3-14DIV slices. To prove that at least some NG2+ cells present in the P0-BT3-14DIV slices arise from the co-cultured P0-7DIV, we performed new co-culture experiments using as fresh P0 explants those taken from actin-GFP-transgenic cerebella (Fig. 7F). We observed NG2+/GFP+ cells within the P0-BT3-14DIV slices (Figs. 7G – 7I), showing that some NG2+ cells or their precursors originate from the actin-GFP-P0-7DIV slices and migrate into the BrdU-treated slices. Since OPCs lose their NG2 expression during maturation, we could not directly prove that these NG2+ cells differentiate into oligodendrocytes. To this purpose, using double labeling experiments we looked for the occurrence—among mature oligodendrocytes—of cells originated from the actin-GFP-P0 explants (GFP+), and myelinating axons in the P0-BT3-14DIV co-culture (MBP+). MBP+/GFP+ cells were observed (Figs. 7J –7L), supporting the fact that the BrdU-treated slices are able to attract NG2+ cells or their precursors, and to provide OPCs from the actin GFP slices with all required signals to pursue their differentiation program and to myelinate Purkinje cell axons present in the BrdU-treated slices. This study corroborates that a 3-day BrdU treatment completely eliminates the OPC population with young characteristics. Furthermore, the study revealed a BrdUresistant population of NG2+ cells. These cells may constitute either a truly independent cell population or a young OPC population that survives but is affected by the treatment. To eliminate the latter possibility, as reported above, we have verified that the BrdU treatment does not

impede subsequent cell division, since at least 50% of the NG2+ cells in treated slices divide during the 3 days following the treatment (Fig. 4). In addition, the treatment allows, at least in 10% of the cases, terminal differentiation into oligodendrocytes (MBP+; Fig. 6). These results strongly suggest that the resistant BrdU NG2+ cells constitute a different population than the young OPC NG2+ cells, since they exhibit different features: a slow proliferative rate and a low ability to differentiate into oligodendrocytes. Thus, we propose that from birth there are at least two populations of NG2+ cells in the cerebellum: the young OPCs and NG2+ cells with adult characteristics. One open question still remains: Why will only a very small fraction of these NG2+ cells with adult characteristics become oligodendrocytes despite the presence of axons to be myelinated in the slices? Here we provide some evidence that BrdU treatment does not indirectly affect the ability of the axon to be myelinated (Fig. 7). In this sense, our results parallel those obtained in vivo with cuprizone treatment, in which the long-term absence of myelination or remyelination appears to be the consequence of mature OPC depletion and not of the inability of the demyelinated axons to be remyelinated (Mason et al., 2004). Therefore, since most of the NG2+ cells resistant to the BrdU treatment are not able to myelinate axons, they cannot be considered to belong to mature OPCs. Do they constitute an NG2+ cell population non-responsive to the presence of demyelinated axons, as reported in vivo in the adult CNS? (Keirstead et al., 1998). In theory the answer to this question could be positive, because NG2+ cells or polydendrocytes in the adult CNS are considered today to form a heterogeneous population, containing at least two very different classes of cells: adult OPCs, with the ability to generate mature oligodendrocytes with myelinating potentiality; and a novel population of mature glial cells—named synantocytes—with still undefined function (Berry et al., 2002; Butt et al., 2002; Greenwood and Butt, 2003). The two different populations of NG2+ cells disclosed in this study suggest that the majority of the synantocytes derive from a separate NG2+ cell population present in the cerebellum from birth, whereas the majority of the adult OPCs originate from the maturation of the young OPCs. Another explanation is still possible, since we cannot exclude the possibility that in our culture conditions the adult NG2+ cells are preponderantly oriented towards the synantocyte phenotype. Indeed, unlike the in vivo cerebellum, in our in vitro study the number of NG2+ cells does not decrease in parallel with myelination. The density of NG2+ cells in control cerebellar slices at 7 DIV, 14 DIV and 21 DIV appears to remain constant (data not shown). The quantitative difference between the in vivo and in vitro conditions could result from the absence of climbing and mossy fibers (the other myelinated axons in the developing cerebellar cortex) in the explants, creating a numerical mismatch between OPCs and axons. However, this explanation does not appear to apply because it has been described that the number of OPCs depends on the number of axons (Fulcrand and Privat, 1977;

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Fig. 7. Migration of OPCs from P0 slice to P0-BT3-7DIV slices. Schematic representations of the co-culture system with contact (A) and without contact (D). A P0 slice is placed in culture and treated with BrdU during the first 3 days (P0-BT3). On the seventh day of culture, a fresh cerebellar slice taken from another P0 cerebellum is apposed to the P0-BT3 (A, co-culture with contact) or deposed at a certain distance (D, co-culture without contact). A thread is put on the fresh P0 explants. The co-cultures are maintained for 7 extra days in culture before fixation. Thus, at the end of the co-culture, we can identify the P0-7DIV culture by the presence of the thread. (B – C) Photomicrographs of P0-BT3-14DIV/P0-7DIV co-culture with contact, double immunostained with anti-MBP (B) and with anti-CaBP (C). The P0-7DIV donor culture is identified by the overlying thread (arrowheads in panels B and C). The line of contact between the slices is determined with CaBP staining (dashed line). The contact is made at the cortical surface (C). Note the presence of numerous MBP+ elements on the P0-BT3DIV14 slice. The arrows in panels B and C indicate the direction of migration. (E) Photomicrographs of P0-BT3-14DIV co-cultured with P0-7DIV (out of the field of the micrograph) without contact, stained with anti-MBP. The P0-BT3-14DIV does not contain MBP+ elements. Apposition between the P0-BT314DIV and P0-7DIV explants is therefore essential to detect MBP+ elements. (F) Photomicrograph of P0-BT3-14DIV/actin-GFP-P0-7DIV co-culture with contact, immunostained with anti-GFP (green fluorescent protein). Note the presence of numerous actin-GFP cells on the P0-BT3-14DIV slice (arrow), coming from the actin-GFP-P0-7DIV slice (*). (G – I) Confocal photomicrographs of P0-BT3-14DIV/actin-GFP-P0-7DIV co-culture with contact, double immunostained with anti-NG2 (red, G and I) and with anti-GFP (green, H and I) on the P0-BT3-14DIV. Note the presence of an actin-GFP+/NG2+ cell on the BT3 slices indicating the migration of an NG2+ cell or its precursor from the actin-GFP-P0-7DIV slice to the P0-BT3-14DIV slice. (J – L) Photomicrographs of P0-BT3-14DIV/actin-GFP-P0-7DIV co-culture with contact, double immunostained with anti-MBP (red, J and L) and with anti-GFP (green, K and L) on the P0-BT3-14DIV. Note the presence of two actin-GFP+/MBP+ cells (arrows) and of an MBP+ with a light expression of GFP (arrowheads) on the BT3 slices indicating the ability of migration and differentiation of oligodendrocyte precursor cells from the actin-GFP-P0-7DIV slice to the P0-BT3-14DIV slice. Scale bar is 300 Am in panels B, C and E; 360 Am in panel F; 20 Am in panels G – I; and 60 Am in panels J – L.

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David et al., 1984; Barres and Raff, 1993). We favor, therefore, two different hypotheses to explain the numerical constancy of NG2+ cells in aging cultures: (i) it could result from the presence of specific trophic factors in the culture medium, which would support the survival of OPCs, even in absence of axons; or (ii) our culture conditions increase the number of synantocytes, a new class of glial cells that are axon independent for their survival (Berry et al., 2002; Butt et al., 2002; Greenwood and Butt, 2003).

Conclusion From birth at least two different NG2+ cell subpopulations coexist: one composed of NG2+ cells with young OPC characteristics and another with mature characteristics (slow proliferation and differentiation rates). The majority of the NG2+ cells with mature characteristics are still able to divide but lack the capacity to generate oligodendrocytes, at least within 21 DIV. Further work will be necessary to determine what could be the function of this NG2+ cell population and if these cells are related to synantocytes. However, our results provide circumstantial evidence showing that newborn NG2+ cells are already heterogeneous, and that a fraction of adult NG2+ cells do not derive from OPCs with perinatal features. Acknowledgments We thank Marie-Ste´phane Aigrot and Boris Zalc for the preparation and the gift of the O4 antibody, Richard Schwarzman (Plateforme imagerie IFR83) for his help with the confocal microscopy and Ann Lohof for critical reading of the manuscript. This work was supported by the Paris VI University, the Centre National de la Recherche Scientifique (CNRS), the Institut National Scientifique pour la Recherche Me´dical (INSERM), the Fondation pour la Recherche Me´dicale, the Hunter’s Hope Foundation and ARC. References Barres, B.A., Raff, M.C., 1993. Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons. Nature 361, 258 – 260. Berry, M., Hubbard, P., Butt, A.M., 2002. Cytology and lineage of NG2positive glia. J. Neurocytol. 31, 457 – 467. Billings-Gagliardi, S., Adcock, L.H., Schwing, G.B., Wolf, M.K., 1980. Hypomyelinated mutant mice: II. Myelination in vitro. Brain Res. 200, 135 – 150. Bouslama-Oueghlani, L., Wehrle, R., Sotelo, C., Dusart, I., 2003. The developmental loss of the ability of Purkinje cells to regenerate their axons occurs in the absence of myelin: an in vitro model to prevent myelination. J. Neurosci. 23, 8318 – 8329. Butt, A.M., Kiff, J., Hubbard, P., Berry, M., 2002. Synantocytes: new functions for novel NG2 expressing glia. J. Neurocytol. 31, 551 – 565. Camand, E., Morel, M.P., Faissner, A., Sotelo, C., Dusart, I., 2004. Longterm changes in the molecular composition of the glial scar and

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