Cell cycle kinetics and immunohistochemical characterization of dissociated fetal neocortical cultures: evidence that differentiated neurons have mitotic capacity

Developmental Brain Research 122 (2000) 67–80 www.elsevier.com / locate / bres

Research report

Cell cycle kinetics and immunohistochemical characterization of dissociated fetal neocortical cultures: evidence that differentiated neurons have mitotic capacity Julie S. Jacobs a , Michael W. Miller a,b,c,d , * a Neuroscience Program, University of Iowa, Iowa City, IA 52242 -1087, USA Research Service, Veterans Affairs Medical Center, Iowa City, IA 52246 -2208, USA c Department of Psychiatry, University of Iowa College of Medicine, Iowa City, IA 52242 -1000, USA Department of Pharmacology, University of Iowa College of Medicine, Iowa City, IA 52242 -1109, USA b

d

Accepted 16 May 2000

Abstract Neurons in the neocortex (regardless of their developmental state) are considered to be post-mitotic and incapable of dividing. We used dissociated primary cultures derived from the neocortices of 16-day-old fetuses to test the counter-hypothesis, that is, differentiating neocortical neurons can divide. The cultured cells experienced considerable cell death, yet the number of viable cells remained relatively constant over the first 5 days in vitro. The implication was that the cultures contained proliferating cells. This was confirmed with a [ 3 H]thymidine ([ 3 H]dT) incorporation study and cumulative bromodeoxyuridine labeling. In fact, over 1 / 4 of the cells were cycling and the length of the cell cycle was 20.0 h; kinetics which mirror those of the developing cortex in vivo. This population of proliferating cells was eliminated by 48 h treatment with fluorodeoxyuridine. Immunohistochemical procedures determined that most cultured cells ($90%) expressed proteins associated with differentiating or mature neurons, e.g., neurofilament (NF) 200 and isoforms of microtubule-associated protein (MAP) 2. Markers for immature neurons (e.g., nestin) were expressed by 10% of the cells. In contrast, markers for glia and their precursors were expressed by #2% of the population. Double-labeling with [ 3 H]dT and a neural-specific antibody showed that cells expressing an antigen for immature neurons constituted most of the proliferating cells, however, a considerable number of [ 3 H]dT-labeled cells expressed markers for differentiating neurons (e.g., NF200 and MAP2). Thus, differentiating neocortical neurons can be mitotically active and it appears that differentiating neurons are derived from both the ventricular and subventricular proliferative zones.  2000 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Genesis of neurons and glia Keywords: Cell cycle kinetics; Cerebral cortex; Microtubule associated protein; Nestin; Neurofilament; Subventricular zone; Ventricular zone

Abbreviations: bFGF, basic fibroblast growth factor; BrdU, 5-bromo29-deoxyuridine; BSA, bovine serum albumin; CNPase, 29-39-cyclic nucleotide 39-phosphodiesterase; cpm, counts per minute; FCS, fetal calf serum; FdU, 5-fluoro-29-deoxyuridine; G, gestational day; GF, growth fraction; GFAP, glial fibrillary acidic protein; [ 3 H]dT, tritiated thymidine; LI, labeling index; MAP, microtubule-associated protein; MBP, myelin basic protein; NF, neurofilament; NSE, neuron specific enolase; PBS, phosphate buffered saline; SZ, subventricular zone; SZa, anterior subventricular zone; T c , total length of the cell cycle; TCA, trichloroacetic acid; T s , length of the S-phase; TUNEL, terminal dUTP nick end labeling; VZ, ventricular zone *Corresponding author. Tel.: 11-319-353-4885; fax: 11-319-3533003. E-mail address: [email protected] (M.W. Miller)

1. Introduction It is generally believed that neurons throughout the CNS are post-mitotic [26]. This includes the neurons of the neocortex. The proliferation of the precursors of neocortical neurons occurs only prenatally. Neuronal generation in the rat neocortex begins on gestational day (G) 12 and ends on G21 [42]. During this period, cell proliferation occurs in three locations. Most activity occurs in two zones (the ventricular and subventricular zones) surrounding the ventricles [5,6,15,25,42,44,45,60,61,67]. A small amount

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of glial proliferation takes place in the parenchyma of the cortical plate and intermediate zone [1,7,19]. Over the neuronogenetic period, increasing numbers of cells exit the cell cycle [47,49,66]. This process is considered to be irreversible. Soon after exiting from their final cell cycle, a daughter cell begins to elaborate neuronspecific proteins [17,18,23,27,32,41,46]. Such proteins include markers for undifferentiated cells (e.g., nestin) and for mature neurons (e.g., neurofilament (NF) 200 and isoforms of microtubule-associated protein (MAP) 2). Not all CNS structures have a restricted period of neuronogenesis. In select sites, neuronal precursors can proliferate well into adulthood. These sites include the olfactory bulb and the dentate gyrus [2,20,24,30,35,36,40,48]. In these areas, neurons are derived from a long-persisting population of neuronal precursors. Cell culture models show that some proliferating cells derived from the olfactory and hippocampal proliferative zones express differentiating and / or mature neuronal markers [8,35,36,39]. In vitro, postmitotic neurons are commonly derived from dissociated fetal neocortices [14,28]. Given our knowledge of neocortical development in vivo, cultures obtained from the cortices of two-week-old rat fetuses might be expected to be quite heterogenous. After all, cortex at this age contains proliferating, migrating, and differentiating cells [7,26,42]. Nevertheless, investigators generally find that such cultures are largely composed of post-mitotic neurons [28]. We used such ‘purified’ cultures of cortical neurons to test the hypothesis that differentiated (or differentiating) neocortical neurons are capable of dividing.

2. Materials and methods

The cortices from the fetuses in a single litter were pooled and the cells were dissociated using a standard procedure [16,62]. Briefly, the tissue was minced with a razor blade and treated with 0.25 mg / ml trypsin at 378C for 10 min. The trypsin activity was terminated (a) by the addition of 0.050 mg / ml trypsin inhibitor and (b) by centrifuging the tissue and treating the pellet with 0.25 mg / ml trypsin inhibitor. The tissue was resuspended and triturated to dissociate the cells. Debris was removed by centrifuging the sample through a bovine serum albumin (BSA) gradient, and the cells were diluted with a neuronal medium composed of 25.0 mM KCl, 1.0 mM glutamine, 33.0 mM glucose, and 180 mM gentamicin in minimal essential medium (GibcoBRL, Long Island, NY). This medium was supplemented with 10.0% fetal calf serum (FCS). Dissociated neurons were plated at a density of 3.4310 5 cells per cm 2 on poly-d-lysine-coated culture dishes. The cultures were incubated at 378C in an atmosphere of 6.0% CO 2 and 94.0% air. Five hours after being plated, the culture medium was removed and replaced with neuronal medium supplemented with a reduced amount of serum (1.0% FCS). A medium containing reduced serum content was used in the present study in order to balance the restrained growth occurring when cells are raised in a serum-free medium with the high amount of growth and differentiation induced by the addition of exogenous factors. The time of the initial medium change was designated experimental Day 0. In some experiments, the cultures were treated with 5fluoro-29-deoxyuridine (FdU; 15–140 mM; [F-0503, Sigma, St. Louis, MO) to inhibit proliferation. The cultures were placed on racks in sealed containers. Two hundred ml of water was added to the containers, and 60 cc CO 2 was injected into the sealed container. On each culture day, the containers were opened, the bath water was changed, and the CO 2 was reintroduced.

2.1. Culturing technique 2.2. Measures of cell proliferation Timed-pregnant Sprague–Dawley dams were obtained from Harlan–Sprague–Dawley (Indianapolis IN). The day a sperm-positive vaginal plug was first found was designated as G1. On G16, the dams were anesthetized with a cocktail of ketamine (60 mg / kg) and xylazine (7.5 mg / kg) and the fetuses were delivered by Cesarean section. The fetal crania were incised to expose the brain. The meninges were removed. A portion of the neocortex was dissected. This included the segment lateral to cingulate cortex, posterior to the olfactory bulb and rostral cortex, and dorsal to the lateral ventricle and rhinal sulcus. This dissection procedure eliminated the hippocampal, septal, and basal ganglia neuroepithelia, as well as the contribution of cells in the anterior subventricular zone that is contiguous to the olfactory bulbs [36,40]. Samples were placed in H-EBSS (13.8 mM NaCl, 5.0 mM KCl, 25.0 mM HEPES, 4.2 mM NaHCO 3 , 1.0 mM NaH 2 PO 4 ?H 2 O, and 0.010% Phenol red).

2.2.1. Cell counts An index of cell growth was obtained by tracing the change in the number of cells in culture over time. Dissociated cells were plated in 24-well culture dishes. Cell counts were obtained at 0, 24, 48 h, etc. (on Day 0, Day 1, Day 2, etc., respectively) after the initial change of the medium. Medium was removed from each well and 150 ml of 0.25% trypsin / 0.10% EDTA was added to each culture dish to aid in the detachment of the cells from the plate. After a 2-min incubation at 378C, 150 ml of culture medium supplemented with 10% FCS was added to inactivate the trypsin activity. The cells were further dissociated by trituration. Trypan blue in 0.10 M phosphate buffered saline (PBS) was added to the cells to a final concentration of 0.13%. The cells were incubated for 1 min and then counted with a Neubauer hemocytometer. The total number of cells, the number of cells that

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excluded trypan blue, and the number of trypan bluestained somata per well was determined. Four counts were made on each dish and an average value per time point was obtained. Each mean (6the standard error of the mean) was based on the data from three dishes.

2.2.2. Thymidine incorporation assay Cell proliferation requires that cells duplicate their DNA. Thus, an index of cell proliferation was based on the amount of tritiated thymidine ([ 3 H]dT) incorporated into the cells. Cells were plated in 60 mm Petri dishes. Twentyfour or 48 h after the initial change of medium (on Day 1 or Day 2, respectively), [ 3 H]dT (specific activity 1.0 mCi / ml; NEN, Boston MA) was added (5.0 mCi / ml) to the culture medium. Cells were incubated in the radioactive medium for 1 h. The medium was then aspirated and the cells were washed with 2.0 ml of ice cold PBS to remove unincorporated [ 3 H]dT. Cells were then detached from the plate by adding 500 ml of a solution of 0.25% trypsin and 0.10% EDTA to each dish, incubating the cells at 378C for 3 min, and resuspending the cells in 2.0 ml of ice-cold PBS. The detached cells were transferred to test tubes and representative aliquots were removed from each sample for cell counts. DNA was extracted from the remaining cell suspension. The suspension was treated with 2.0 ml ice-cold 10% trichloroacetic acid (TCA) and 40 ml of 10 mg BSA per ml PBS. After a brief mix and a 20-min incubation on ice, the lysed cells were filtered through a Whatman GF /A fiberglass filter (Fisher, Pittsburgh PA). The filter was rinsed twice with 5.0% ice cold TCA, and dried with 95% ethanol. Each filter was placed in a scintillation vial containing 10 ml of counting cocktail (Econo-Safe; Research Products Int. Corp., Mount Prospect, IL). The radioactivity in each vial was counted over 1.0 min with a Packard Tri-Carb 4530 scintillation counter (Packard Instrument Co., Downer’s Grove, IL). Two samples, obtained from three litters per time point, were independently processed. Three negative controls were performed. Some cells were not exposed to [ 3 H]dT. Some petri dishes devoid of cells were pulsed with [ 3 H]dT. Other vials contained filters that were washed with extraction buffer only. The results of these controls were consistent; only background amounts of activity were detected. 2.2.3. Cumulative labeling method The cell cycle kinetics and the proportion of cells that were actively cycling (the growth fraction; GF) were determined using a cumulative labeling technique [34,54]. Cells were plated on chamber slides. On Day 1 (i.e., after the cells had a day to acclimatize to the medium with a reduced serum content), 10 mM 5-bromo-29-deoxyuridine (BrdU; Sigma) was added to the cultures. Cells were harvested 0.50, 1.0, 2.0, 4.0, 12.0, 24, 48, and 72 h after the BrdU was added. The medium was removed, the

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cultures were washed thrice with PBS, and the cells were fixed with 70% ethanol in 50 mM glycine buffer (pH 2.0) for 30 min at 2208C. The slides were again washed in PBS. BrdU detection relied a standard immunolabeling procedure [34,51]. The cells were incubated with an antiBrdU antibody (diluted 1:10 in 66.0 mM Tris, 0.66 mM MgCl 2 , and 1.0 mM 2-mercaptoethanol) for 30 min at 378C. This antibody (mouse monoclonal antibody BMC 9318; Boehringer–Mannheim, Indianapolis, IN) was specific for BrdU and exhibited no cross-reactivity with FdU, endogenous thymidine, or uridine. The primary antibody solution was removed, the slides were washed thoroughly with PBS, and an anti-mouse IgG antibody conjugated to alkaline phosphatase (Vector, Burlingame, CA; diluted 1:10 in PBS) was added (30 min at 378C). After rinsing with PBS, the slides were incubated with alkaline phosphatase substrate (BCIP/ NBT; Vector) in 100 mM Tris–HCl, 100 mM NaCl, and 50 mM MgCl 2 for 20 min at room temperature. The slides were rinsed with distilled water, dehydrated through gradient alcohols, cleared with xylenes, and coverslipped. The number of BrdU-positive cells and the total number of cells in a box 150 mm by 150 mm were counted. A labeling index (LI; the number of labeled cells divided by the total number of cells) was calculated at each time point. Four slides were individually prepared for each time point and three counts were made on randomly chosen fields from each slide. Control slides were prepared in which the BrdU, primary antibody, or secondary antibody was omitted. In all cases, the results of the control studies were negative.

2.3. Protein immunohistochemistry 2.3.1. Preparation Cultures were grown on chamber slides, and harvested 24, 48, 72, and 96 h after the initial medium change (Day 1, 2, 3, and 4, respectively). The cells were fixed for 30 min with 4.0% paraformaldehyde in PBS (pH 7.4) at room temperature. The fixative was removed, the slides were rinsed in PBS, and cells were permeabilized with 1.0% Triton X-100 in PBS for 30 min. Endogenous peroxidase activity was quenched with a 5 min wash in 2.0% hydrogen peroxide in PBS. The slides were briefly rinsed, and non-specific activity was minimized by washing the cells with a blocking buffer of 10% dry milk in PBS for 1 h. 2.3.2. Primary antibodies Following the blocking steps, the cells were incubated with a primary antibody (diluted in 2.5% dry milk in PBS) overnight at 48C. A variety of antibodies was used to characterize the phenotype of the cultured cells. The markers were for neuron- or glia-specific lineages or for maturing cells of both lineages. Neuronal markers included

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antibodies against NF200 ([N-4142 and [N5389, Sigma), NF160 ([N5264, Sigma), NF68 ([N5139, Sigma), MAP2abc and MAP2ab ([M4403 and M1406, Sigma), and neuron specific enolase (NSE; [MAB314, Chemicon Intl., Temecula, CA). Glial markers included 29–39-cyclic nucleotide 39-phosphodiesterase (CNPase; [C-5922, Sigma), myelin basic protein (MBP; [1118-099, Boehringer–Mannheim), and glial fibrillary acidic protein (GFAP; [G-3893, Sigma). To identify cells in a developmental lineage, anti-A2B5 ([1300-016; Boehringer– Mannheim), anti-O4 ([1518-925, Boehringer–Mannheim), 8C10 (generously provided by Dennis Landis, Case Western Reserve Sch. of Med., Cleveland, OH), and anti-nestin (courtesy of Ron McKay, NINDS, Bethesda MD) antibodies were used. Anti-fibronectin ([F-3648, Sigma) was also used to label fibroblasts and / or endothelial cells. Three of the labels were rabbit polyclonal antibodies; those directed against NF200 ([4142), nestin, and fibronectin. The others were monoclonal antibodies. The working dilutions of the antibodies were: NF200 ([4142 and [5389), 1:100 and 1:40, respectively; NF160, 1:40; NF68, 1:100; MAP2abc, 1:100; MAP2ab, 1:200; NSE, 1:10; CNPase, 1:200; MBP, 1:100; GFAP, 1:100; A2B5, 1:100; O4, 10 mg / ml; nestin, 1:100; and fibronectin, 1:200.

2.3.3. Visualization of the immunolabeled cells The cells were washed with blocking buffer and then with PBS. A peroxidase-conjugated secondary antibody (anti-mouse IgG [NA-931 or anti-rabbit IgG [NA-934, Amersham, Arlington Heights, IL) was then added to the chambers at a dilution of 1:500 in 2.5% dry milk in PBS. After a 1-h incubation at room temperature, the slides were rinsed in blocking buffer and PBS. The distribution of bound secondary antibody was determined by reacting the peroxidase with hydrogen peroxide in the presence of the chromogen diaminobenzidine (DAB; Vector) for 5 min. Subsequently, the slides were rinsed with water, dehydrated through graded alcohols, cleared with xylenes, and coverslipped. Two slides were prepared per time point, and three repetitions of the experiment were performed. Control reactions included omission of the primary or secondary antibody steps, and use of an inappropriate secondary antibody. 2.3.4. Quantitative analysis Labeling with the various histochemical methods was evaluated qualitatively. Labeled and unlabeled cells were distinguished using both light and phase contrast optics, respectively. For a cell to be identified as specifically labeled, the soma and proximal processes had to show moderate to intense immunoreactivity. Cell counts were performed with the aid of the Bioquant Image Analysis System (R&M Biometrics, Nashville, TN). The numbers of cells in a box (1503150 mm) were counted. A minimum of four counts was made on each

slide, and a mean number of cells was determined. LIs were calculated for each label, as described for the BrdU study.

2.4. Combined immunohistochemistry and [ 3 H] dT autoradiography A double-labeling procedure was used to trace the generation of cells expressing a particular protein. On Day 1, [ 3 H]dT (5.0 mCi / ml) was added to the medium. After a 1-h incubation at 378C, the radioactive medium was replaced with a medium conditioned from cultures that were matched for age and plating density. The cells were incubated at 378C until harvest 1.0, 24, 48, or 72 h later. The cultures were fixed with a solution of 4.0% paraformaldehyde in PBS and labeled immunohistochemically as described above. Under a photographic safe light, the slides were rinsed in water, coated with Nuclear Track Emulsion (NTB-2; Kodak, Rochester, NY), and placed in light-tight boxes. The emulsion was exposed for 7 days at 2208C before being processed in D-19 Developer (Kodak) at 138C for 3 min. Following a brief water rinse, the emulsion was cleared and stabilized with a 2-min wash with Rapid Fixer (Kodak) at 138C. After a series of rinses in cold water, the excess emulsion was scraped from the back of the slides, the cells were dehydrated, and the slides were coverslipped. Two slides were prepared for each immunolabel, at each time point. Control slides covered with cells that were not treated with [ 3 H]dT and / or did not undergo an immunoreaction were processed with each autoradiography batch. One slide from each time point was stained with cresyl violet after the emulsion was developed. The emulsion itself was pretested for light sensitivity. A two-stage quantitative analysis was performed. The LI of radiolabeled cells in cresyl violet-stained sections was determined using the method described above for analyzing the immunolabeled sections. The number of doublelabeled cells per 100 [ 3 H]dT-positive cells was then determined for each antibody. Three counts were performed per slide at each time point.

2.5. Statistical methods One-way analyses of variance were used to determine statistical differences among the data. In situations where significant differences were detected, post-hoc t-tests (Bonferroni’s method) were performed.

3. Results

3.1. Analyses of cell proliferation and cell cycle kinetics Cells in the primary cultures were derived from the cerebral wall (including the neocortex and the associated

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neuroepithelium) that was anterolateral to the cingulate cortex and posterior to the olfactory bulb. An initial plating density of 3.4310 5 cells / cm 2 yielded cultures of welldispersed cells. Most cells adhered to the vessel. The non-adherent cells were removed at the medium change, and by Day 0 cell numbers had fallen by 240% (Fig. 1). By Day 1, most cultured cells exhibited three or more processes extending from their cell bodies (Fig. 2). Between Days 1 and 5 the number of cells per culture well was stable (|6.7310 5 cells / well), i.e., there was no significant change in cell number over this period (Fig. 1). On the other hand, non-adherent cells were observed floating in the medium and trypan blue-stained somata were common. Accurate quantification of the number of dead / dying cells was not possible, because the floating cells were aspirated with the medium prior to the assay. By Day 7, the number of cells in the cultures had dropped significantly (F6,256 55.74; P,0.001). The consistency in cell density between Days 1 and 5 implied either that the population was stable or that the amount of cell loss and cell acquisition was balanced. Since cell death was common, it appeared that the latter was true. This contention was supported by data showing that the addition of the anti-mitotic agent FdU (60 mM) caused a steady, significant (F6,225 597.4; P,0.001) decrease in cell numbers (Fig. 1). The occurrence of cell proliferation was further verified using two direct assays: [ 3 H]dT incorporation and BrdU cumulative labeling. A [ 3 H]dT incorporation study was performed with cultures exposed to the radiolabel for only 1 h. Significant (F4,14 5141; P,0.001) amounts of radiolabeled thymidine were taken up by the cultured cells during the short exposure (Fig. 3). FdU significantly (F6,35 542.7; P, 0.001) decreased this incorporation. Not only were the

Fig. 1. Trypan blue exclusion assay for counts of viable cells. On Day 0, cultures were treated with medium containing 1.0% fetal calf serum with or without 60.0 mM fluorodeoxyuridine (FdU). Without the anti-mitotic, viable cell numbers dropped over the first 24 h, then remained stable for 4 days. In the presence of FdU, there was a steady decrease in viable cell number throughout the culturing period. Symbols and bars denote the means and standard errors of the means for three trials.

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effects of FdU concentration-dependent, the duration of the FdU exposure significantly (F11,52 544.4; P,0.001) affected the amount of [ 3 H]dT incorporation. For cells treated with FdU for 24 h, 90 mM FdU was required to attain maximal effect. Note that treatment with FdU for 24 h did not totally eliminate [ 3 H]dT incorporation. This contrasts with the cultures that were treated with FdU for 48 h. For these cells, only 15 mM FdU was required to achieve maximal inhibition; that is, no statistical difference (P, 0.05) was found among the FdU concentrations tested at this time point. The proportion of the cultured cells that were actively cycling and the cell cycle kinetics were determined with a cumulative BrdU labeling study (Fig. 4). The BrdU LIs at various times were best fit to a one-population model [54] (Fig. 5). There were two phases in the change of LI over time. In the first phase, the LI increased steadily; data fit a linear regression line for which R was 0.988 (P,0.05). The LI reached a maximum after 14.2 h. Subsequently, the LI was stable. From these data, the total length of the cell cycle (T c ), the length of the S-phase (T s ), and the GF were calculated [34,54]. Accordingly, the T c and T s were 20.061.5 h and 5.861.3 h, respectively, and the GF was 28.461.0%.

3.2. Immunohistochemical identity of the cultured cells The identity of the cultured cells was explored immunohistochemically (Fig. 2). The cultures were heterogeneous, in that cells expressed many different proteins. On Day 2, the vast majority of cells (.80%) expressed markers typical of maturing neurons (e.g., NF and MAP2 isoforms; Table 1). In contrast, nestin, a protein elaborated by immature cells [69], was expressed by 1 in 12 somata. Cells expressing glial-specific proteins were only rarely detected (#2% of the cells). Likewise, fibroblast contamination (as determined by fibronectin staining) was ,1% (data not shown). To assess temporal changes in protein expression, the proportion of cells expressing a particular marker was examined at various times after the cells were placed in the medium with reduced serum content (Day 0). The expression of NF200 (F3,28 515.0, P,0.001) and MAP2abc (F3,20 516.8, P,0.001) increased significantly over the culturing period (Fig. 6). The number of MAP2ab-positive cells did not change significantly. By deduction, the number of MAP2c expressing cells was calculated as the difference between the numbers of MAP2abc and MAP2ab immunoreactive cells. Accordingly, expression of MAP2c also rose substantially over the 4 days. The number of cells expressing nestin and GFAP did not vary significantly. FdU treatment altered the temporal pattern of expression for several cell-specific markers (Fig. 6). By Day 3, the number of cells expressing nestin was significantly decreased in FdU-treated cultures (t52.74; P,0.05). FdU treatment significantly increased the number of cells

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Fig. 2. Immunolabeling and [ 3 H]dT autoradiography on Day 2. Plates on the left side of the figure were labeled with markers for mature neurons (neurofilament (NF) 200), developing cells (nestin), or mature glia (glial fibrillary acidic protein; GFAP). Labeled cells (solid arrows) were identified by specific immunoreactivity in their cells bodies that could be traced into their processes. This contrasts with the weak or lack of labeling in the so-called unlabeled cells (open arrows). At the right, the results of a combined immunochemistry and tritiated thymidine ([ 3 H]dT) labeling experiment are demonstrated. In these micrographs taken through the plane of the autoradiographic grains, cells which expressed only the [ 3 H]dT (seen as grains over the nuclei) are indicated by open arrows. Cells labeled both immunohistochemically and autoradiographically for [ 3 H]dT are denoted with solid arrows. Both ‘mature’ (NF200) neurons and immature (nestin) cells are double-labeled. These examples were taken from cultures fixed 24 h after a 1 h pulse with [ 3 H]dT. Scale bars are 50 mM.

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Fig. 3. [ 3 H]dT incorporation assay of proliferation. A range of FdU concentrations was used to determine the threshold of the anti-mitotic effect. Average counts per minute (cpm)6standard errors are shown. At 24 h, none of the FdU concentrations eliminated [ 3 H]dT incorporation. By 48 h, all the FdU groups exhibited only background level counts. The means and standard errors of the means are represented by symbols (solid and open circles for cells treated with FdU for 24 and 48 h, respectively) and T-bars. Each mean is based on three independent trials.

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Fig. 5. Cell cycle kinetics. The BrdU cumulative labeling data were analyzed with a one population model of cell cycle kinetics. The growth fraction (GF), or cycling population, constituted over one fourth of the cells. By combining this data with a linear regression over the period of increasing BrdU incorporation, the length of the cell cycle (T c ) the length of the S-phase (T s ) were also determined. Error bars indicate standard errors of four replicates.

Fig. 4. Cumulative BrdU labeling in 1.0% serum-containing medium (without FdU). Cells were labeled immunohistochemically for BrdU incorporation at 2 and 24 h after addition of BrdU to the culture medium. A larger proportion of the cells (indicated by solid arrows) were labeled at the later time point. Unlabeled cells are denoted by open arrows, for comparison. Scale bars are 50 mM.

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Table 1 Proportions of cells expressing a specific antigen a Neuronal antigens MAP2abc MAP2ab NF68 NF160 NF200 (mono.) NF200 (poly.) NSE

Glial antigens 82.863.4% 16.063.1% 5.061.9% 93.861.1% 80.265.4% 90.260.8% 55.765.0%

CNPase GFAP MBP O4

Immature cell antigens 0.160.1% 1.360.3% 0.960.4% 0.060.0%

Nestin 8C-10 A2B5

8.660.3% 0.060.0% 2.360.5%

a This table describes the labeling indices (6standard errors) for various immunochemical markers 48 h after the neuronal cultures were plated. Labeling indices are calculated as a percent of the total number of cells, using medium without FdU. The vast majority of cells expressed neuron-specific markers. Only a small minority of the cells (#2%) were in a glial lineage.

Fig. 6. Changes in immunolabeling patterns over time in culture. Labeling indices for each antibody varied both over time and with anti-mitotic treatment. Bars denote standard errors. The indices for microtubule-associated protein (MAP) 2c were derived from the difference between the labeling frequencies for MAP2abc and MAP2ab. The means (6the standard errors of the means) are based on three samples. In some studies, cells were treated with 60 mM of fluorodeoxyuridine (FdU).

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expressing NF200 (t53.07; P,0.01) on Day 3. The implication from these data was that the relative number of proliferating cells decreased as more cells moved into the subpopulations of differentiating neurons.

3.3. Expression of neuronal antigens by proliferating cells The identity of the proliferating cells was directly determined using [ 3 H]dT autoradiography. Overall, one in every eight to twelve cells was labeled by a pulse administration of [ 3 H]dT (Table 2). This LI did not change significantly over the 4 days in culture. Interestingly, the present data on the LI after 1 h of [ 3 H]dT exposure (LI 1 h ) concur with the data from the BrdU cumulative labeling study. Furthermore, assuming that the LI 1 h is similar to the theoretical LI at time 0 h [54], the LI 1 h in conjunction with the data on T c and T s , can be used to generate an estimate of the GF using the formula, GF5(LI 1 h )4T s /T c . T s /T c is proportional to the number of cells that theoretically incorporate [ 3 H]dT immediately on administration of the radiolabel. Accordingly, the GF for the cultured cells was 31.4%. This is remarkably similar to the data obtained empirically in the present BrdU cumulative labeling study. The frequency of cells expressing a particular antigen was determined with a combined autoradiographic-immunohistological method (Fig. 2). Double-labeled cells were identified by silver grains over their nuclei and immunoreaction product in their perikarya. The most common antigen expressed by the [ 3 H]dT-labeled cells was nestin (Table 2). It accounted for 2 / 3 of the radiolabeled cells. Neuronal markers (MAP2abc, MAP2ab, and NF200) were also expressed by a small proportion of the [ 3 H]dT-labeled cells. Of these, only the frequency of MAP2abc double-labeled cells exhibited a significant change in expression over time; its LI fell significantly (P,0.05) by 72 h.

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4. Discussion

4.1. Primary neocortical cultures contain a substantial proportion of proliferating cells Cells isolated from the developing cerebral cortex can proliferate in dissociated culture, however, the timing of the cell harvest is often chosen to minimize the contribution of cycling cells. The generation of neurons in rat cortex in vivo occurs between G12 and G21 [7,9,42,43]. Cultures derived from 13- to 14-day-old fetuses contain many proliferating cells [14], however, it is generally accepted that cells derived from older (e.g., 16-day-old) fetuses yield cultures with minimal numbers of actively cycling cells [28]. In contrast to this understanding, the present study shows that cultures of cortical samples harvested on G16 contain a considerable proportion of actively proliferating cells. The contribution of these cells, and their effect on data interpretation, may be underestimated in the literature. Two other factors, the plating density and the medium composition, affect the amount of cell proliferation in a dissociated culture system. In general, high plating densities can be expected to reduce proliferative activity due to contact inhibition, however, there may be instances in which high plating densities can promote neurogenesis [21]. In the current study, a plating density was chosen that would neither promote cell death because it was too low nor hinder cell proliferation because it was too high. The use of medium supplemented with FCS may provide growth factors which promote cell proliferation. For example, the proliferation rate of neuroblastoma cells (a model of proliferating neuronal precursors) is directly dependent upon the amount of serum supplementation [34]. Similarly, the proliferation of CNS stem cells grown in neurosphere cultures is positively affected by epidermal growth factor [58] and basic fibroblast growth factor (bFGF) [70]. In the present study, the cells were raised in a

Table 2 Identity of proliferating cells a Time after [ 3 H]dT pulse 1h 3

Total [ H]dT-labeled cells (%) Double-labeled cells (%) MAP2abc MAP2ab MAP2c (derived) Nestin NF200 a

24 h

48 h

72 h

7.960.4

11.660.8

9.960.9

12.161.2

15.361.5 1.760.9 13.7 62.362.3 4.361.5

17.362.9 3.061.5 14.3 68.065.5 8.362.0

10.561.5 4.361.2 6.1 59.063.4 3.761.6

9.261.3 4.861.0 4.2 57.566.0 6.261.5

In the top line of the table, the percentage of [ 3 H]dT-labeled cells are shown for each time point after the [ 3 H]dT pulse. The fraction of [ 3 H]dT-labeled cells expressing each immunolabel is shown underneath. The numbers of MAP2c expressing cells was derived by subtracting the frequency of MAP2ab-labeled cells from the frequency of MAP2abc-positive cells. Nestin-labeled cells had frequently incorporated [ 3 H]dT, as determined by grains of exposed emulsion over the nuclei, produced via decay of the tritium. Interestingly, however, cells labeled with several markers for ‘mature’ neurons (e.g., NF200) were also double-labeled with [ 3 H]dT.

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low serum (1.0% FCS) containing medium. This medium contains barely detectable amounts of growth factors. Radioimmunoassays show that this medium contains ,50 pg / ml of bFGF and nerve growth factor, below that needed to promote cell proliferation (unpublished results). Nevertheless, we cannot eliminate the possibility that components of FCS contains may be sufficient to foster cell proliferation.

The data on the growth fraction and the cell cycle kinetics in vitro are similar to the activity of proliferating cerebral cells in vivo. In cortical cultures, 28% of the cells are cycling. The size of this cycling population is similar to that in neocortex in vivo. The growth fraction in the fetal neocortex on G18 (analogous to Day 2 in our in vitro preparations) is 26% [50]. The T c and the T s for cultured cortical cells on Day 2 are 20 and 6 h, respectively. These data are virtually identical to those from an anatomical study using the percent labeled mitoses procedure: on G18 the T c is 19 h and the T s is 6–8 h [71]. Further corroboration comes from a flow cytometric study of whole cortical samples: on G18, the T c and the T s are 16.1 and 8.8 h, respectively [50]. Thus, the proliferative activity of the cells in vitro parallels that in vivo.

mature. MAP2c is produced first, followed by the expression of MAP2b. The expression of these isoforms may overlap for a short time. The last MAP2 isoform to be produced is MAP2a. Its expression is concurrent with the termination of MAP2c expression [27,59]. The pattern of immunoexpression in vitro reflects the developmental sequence in vivo. For example, the frequency of nestin-immunolabeled cells and the number of nestin-positive cells that incorporated [ 3 H]dT remains relatively stable over time. This implies that half of the daughters of the nestin-labeled cells remain in the cycling population. In contrast, the numbers of cells expressing NF200 and MAP2 increase over time, suggesting that the cells are differentiating in vitro. This differentiation seems to follow the pattern described for neurons in vivo [27,32,33,53,59]. That is, the cultured cells express a marker for immature cells for a limited time, followed by the expression of intermediate filaments associated with neuronal maturation. From a procedural point-of-view, the present data show that it is imperative to specify the ages of the cultures when immunolabeling data are obtained. Two commonly used markers for ‘mature’ neurons, MAP2 and NF200, have a lag time of approximately 2 days before reaching stable expression levels in vitro. Therefore, immunolabeling studies performed at only one time point do not account for dynamic changes in protein expression.

4.3. Cultured cells include differentiating neurons at several developmental stages

4.4. Differentiating neocortical neurons are mitotically active

Immunolabeling data show that most cultured cortical cells are dedicated to a neuronal lineage. As many as 90% of the cells express an antigen(s) found in differentiating / mature neurons (e.g., MAP2 or NF200). Furthermore, nestin is expressed by about one in ten cells. Although nestin-positive cells can differentiate into either neurons or glia [18,33,69], we can infer from the present data that the nestin-positive cells are directed into a neuronal lineage. After all, there is no change in the proportion of cells expressing a marker for mature or immature glial labels, and the frequency of labeling with glial markers is low. It is unclear whether cultured cells dually-express nestin and a marker for more mature neurons, however, data from in vivo studies show that nestin and NF200 are not coexpressed. Nestin is expressed in proliferating neuroectodermal cells [33]. After a cell exits from the cycling population and begins to differentiate, the neuron-specific neurofilaments are expressed [32,53]. NF68 and NF160 are expressed first. They may even be expressed as cells pass through their terminal mitotic division [12]. NF200 is expressed last. It is found at low levels during neurite formation and at higher levels after the onset of synaptogenesis [11]. The mature neurofilament is a heteropolymer of all three neurofilament species [53]. Like the neurofilaments, MAP2 expression also changes as neurons

Many investigators believe that neocortical cells that have already differentiated into neurons are incapable of proliferating. The present study contradicts this belief and shows that neocortical neurons can divide. In this regard, neocortex may be similar to the select areas, olfactory bulb and hippocampus, where the proliferation of neurons is well acknowledged. Virtually all of the [ 3 H]dT-labeled cells express a neuronal antigen(s). Interestingly, however, only 2 / 3 of the [ 3 H]dT-labeled cells express the neural stem cell marker nestin. Another 15–20% of cells that have incorporated [ 3 H]dT express mature neuronal labels. These data are supported by the mathematical inference that as many as 90% of the cultured cells express MAP2 and / or NF200, yet 28% of the total population is labeled with BrdU. Conceivably, the BrdU (or [ 3 H]dT) labeling could result from incorporation by dying cells [68]. Various data argue against this. (1) Treatment with the anti-mitotic agent FdU for 48 h virtually eliminates [ 3 H]dT incorporation. (2) In cultures that are not treated with FdU, there is a considerable amount of DNA nick end labeling (TUNEL staining), hence death (Jacobs, unpublished data). Since the number of cells in the cultures is stable, despite this death, we can only conclude that two processes (proliferation and death) occur concurrently. (3) The cumulative labeling study is

4.2. Neocortical cells in vitro display cell cycle kinetics parallel to that of the developing cortex in vivo

J.S. Jacobs, M.W. Miller / Developmental Brain Research 122 (2000) 67 – 80

based on the premise that cycling cells pick up the BrdU. This contributes to the linear increase in the percentage of labeled cells. At the same time, the death of cells in cortical cultures continues throughout the first week postplating, albeit in a non-linear fashion (Jacobs, unpublished results). Should dying cells also incorporate BrdU, then we would expect two changes in the cumulative labeling: the percentage of cells labeled should be much higher and the increase in labeling would not be linear. Empirical data are consistent with the notion that BrdU is taken up only by cycling cells. The [ 3 H]dT labeling of NF200- and MAP2-expressing cells is an intriguing result. Co-labeled cells in the cultures having long post-pulse periods may represent cells that were nestin-positive during the pulse, but with maturity, they have become NF200 or MAP2-positive. This scenario does not explain the preparations in which the cultures were fixed immediately after the [ 3 H]dT pulse, i.e., at the 1.0 h time point. Over 15% of the cells expressing [ 3 H]dT at this time co-express NF200 or MAP2. Therefore, these labels of differentiating neurons must be expressed in some cycling cells as they pass through the synthesis phase of the cell cycle. Previous results have also shown that differentiating neurons can divide. These studies (a) use markers for early events in neuronal differentiation and / or (b) examine areas where the proliferation of neuronal precursors in the adult is well acknowledged. The anterior subventricular zone (SZa) has TuJ1-positive cells that can be mitotically active [36]. TuJ1 is a neuron-specific tubulin that is expressed early in neuronal determination / differentiation, before the young neuron expresses MAP2ab or NF160 [13,22,27,38,39,56]. Places where dividing neuronal precursors have been identified in the adult CNS are the proliferative zone for the olfactory bulb (SZa) and the hippocampus [2,20,24,30,35,39,40,48,67]. Dissociated cultures derived from the SZa [36] and the hippocampus [8] contain cells that express neuronal markers (TuJ1 and NF200, respectively) and incorporate a DNA precursor. The present data extend this principle by showing that neocortical neurons also have the ability to divide. The expression of mature markers by mitotically active cells can be dismissed as a consequence of the in vitro conditions. At the very least, the data show that expression of a neuron-specific marker(s) can not be used as an index of the proliferative capacity of a cultured cell. That stated, the data do raise the interesting possibility that differentiating or differentiated neocortical neurons retain the ability to divide. This conclusion runs counter to the concept that cerebral cortical neurons exiting their ‘terminal’ mitoses (i.e. prior to expression of differentiated markers and / or mature morphology) become permanently post-mitotic. Nevertheless, there is evidence that neurons in rat visual cortex can incorporate [ 3 H]dT [29,30]. It is important to note that these cells are neither stem cells nor neuronal precursors, such as those described in recent in vitro [37,64] and in vivo studies [6,15,67]. The labeled cells are

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differentiated neurons, as defined by their somatodendritic morphology and by the density of synapses in which they participate [29,30].

4.5. Derivation of cycling cells from the ventricular zone and subventricular zone The question arises whether the cycling cells in culture are contributed by the ventricular zone (VZ) or subventricular zone (SZ). This issue is controversial since various investigators have shown that neurons are derived either from the VZ [42,45] or exclusively from the SZ [15,36,67]. Only the rare cells in non-proliferative zones (the marginal zone, the cortical plate, the subplate and the intermediate zone) are pulse-labeled with [ 3 H]dT [1]. Most [ 3 H]dTincorporating cells in a 16-day-old fetus are in the VZ and SZ which are both prominent at this time [44]. It is impossible to distinguish the ontogeny of the proliferating cells directly since there are no labels currently available that discriminate cells in one proliferative zone from another. That said, the dissection eliminated the SZa which can generate neurons in the olfactory bulb of the adult rat [2,24,35,36,40]. By comparing the data from in vivo studies with those from the present in vitro study, we can conclude that neurons in our cultures are likely derived from both proliferative zones. Based on previous data [44], 47% of the total population of cells in the cerebral wall in vivo are in the VZ, whereas only 10% reside in the SZ. The GFs of the VZ and SZ are 80% and 12%, respectively. These data on the growth fractions are for 21-day-old rat fetuses, however, it is likely that these values are similar to those for a 17-day-old fetus because the GF in the mouse is stable over the latter part of cortical neuronogenesis [65]. Thus, cycling VZ cells account for 38% (47380%) of all cells in the cerebral wall, and cycling SZ cells account for 1.2% of all cortical cells. In the dissociated cortical cell cultures, as many as 8.3% of the proliferating cells are labeled with NF200. The GF in vitro is 28.4%. Hence, 2.4% (8.3328.4%) of all the cultured cortical cells are cycling NF200-positive neurons. This value is twice the number of cycling SZ cells in the cerebral wall. It appears, therefore, that the number of cycling SZ cells cannot wholly account for the totality of cycling NF200-positive neurons. Mitotically active differentiated neurons are therefore derived from both the VZ and SZ. The dual contribution of the VZ and SZ to the population of cortical neurons is supported by data from in vivo studies in which ethanol differentially alters the timing of neuronal generation and cell proliferation in the VZ and SZ [44,45,52].

4.6. FdU toxicity and the recruitment of proliferating cells FdU treatment alters the expression of several immunolabels. MAP2ab and nestin labeling indices are signifi-

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cantly lower in FdU-containing medium, whereas NF200 expression is significantly higher on most days. Viable cell numbers are dramatically reduced by FdU treatment. These results raise the possibility that FdU is toxic to at least a subset of the cultured cells. Given the higher labeling indices for NF200 and MAP2 in FdU-treated cultures, as well as the decrease in nestin labeling, it is likely that FdU is toxic to the proliferative cells. After 24-h FdU exposure (Day1), the number of cells in the untreated cultures is reduced by half. Similar declines would occur if FdU is lethal to the entire cycling population. Not only would there be approximately 28% loss of cells from direct toxicity (the growth fraction), but the basal rate of death in culture would not be compensated by proliferation. Cell proliferation is totally eliminated by 48-h treatment with FdU (at concentrations as low as 15 mM), whereas a 24-h exposure only partially eliminates [ 3 H]dT incorporation. Given a cell cycle length of 20 h, these data suggest that FdU treatment must be available as cells pass through two cell cycles to eliminate proliferative activity. This concurs with other published data that treatment with an anti-mitotic agent must continue for at least 1 1 / 2 times the length of the cell cycle to stop cell proliferation [57]. Why must the exposure be longer than a single cell cycle? Some mitotically capable cells may require multiple insults to eliminate their proliferative capability. Alternatively, FdU may selectively eliminate all of the cells passing through the S phase of the cell cycle. As a result of this insult, non-proliferating cells (perhaps residing in the proliferative zone in vivo) are newly recruited into the cycling population. A longer exposure to FdU (e.g., through two cell cycles) is required to eliminate these new recruits. This further implies either that some cells persist in a quiescent, undifferentiated state, or that partially or fully differentiated cells can be recruited. Data from studies on the effects of X-irradiation support these conjectures. For example, a short exposure in vivo is not sufficient to eliminate the proliferating cells in the external granule cell layer of the cerebellum [3,4]. A longer exposure is required to cause lasting damage. In fact, following sub-lethal X-irradiation, a plastic response occurs such that lost proliferative cells are replaced by succeeding waves of cells recruited into the cycling population.

4.7. Implications Cortical cultures contain cells that are capable of dividing. These cultures are heterogeneous: whereas most cells are not dividing, a considerable number are proliferating. The existence of the proliferating neurons in the cortical cultures suggests that neurons routinely attempt to re-enter the cell cycle, but such activity is blocked by a molecular ‘gate-keeper’, e.g., cyclin D1 or Rb, that prohibits such activity [10,31,63]. Conversely, there may be a reservoir of cells in the fetal and adult cortices that are

determined to be neurons, but never lose the ability to enter the cell cycle [29,30,55]. We posit that all cortical populations contain cells that are determined to be neurons and retain the ability to proliferate. The recruitment of such cells may require an external signal, e.g., alterations in the expression of a factor normally provided by proliferating cells, or the release of a novel or re-expressed agent by dying neurons. Regardless, identification and isolation of proliferatively capable neurons, and an understanding of the signaling pathways that regulate this proliferative activity, is of great import for the treatment of neurondepleting diseases.

Acknowledgements This research was funded by the Department of Veterans Affairs and the National Institutes of Health (AA 05525, AA 06916, AA 07568, AA 09611, and DE 07734).

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