Abstract
NEUROBIOLOGY & PHYSIOLOGY Animal Cells and Systems, 2014 Vol. 18, No. 2, 101–108, http://dx.doi.org/10.1080/19768354.2014.905491 Yunkyoung Lee, Bongkyu Lee and Chang-Joong Lee* Department of Biological Sciences, Institute of Molecular and Cellular Biology, Inha University, Incheon 402-751, South Korea (Received 13 March 2014; received in revised form 13 March 2014; accepted 13 March 2014) Valproic acid (VPA) is used as an antiepileptic drug or mood stabilizer. Recent studies have shown that exposure to VPA during embryonic development alters neural progenitor cell proliferation. The main goal of this investigation was to elucidate the effects of VPA on cell proliferation in the cerebellum of zebrafish larvae using immunohistochemistry. 5-bromo-2’-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA)-labeled cells were mostly found in the ventral cerebellum proliferation zone and external granular layer of the cerebellum from 5 days postfertilization zebrafish immediately after treatment with BrdU for 12 h. Subsequently, BrdU-labeled cells were mostly found in the medial zone of the cerebellar plate 48 h after terminating BrdU treatment. Pretreatment with 2-mM VPA for 3 h prior to BrdU exposure reduced the number of BrdU-labeled cells both immediately and 48 h after terminating BrdU exposure. Posttreatment with VPA following BrdU exposure reduced the migration of BrdU-labeled cells, represented by a decreased number of BrdU- positive cells in the medial zone of the cerebellar plate. These results suggest that VPA may delay brain development by hindering cell proliferation and migration during the early developmental period. Keywords: zebrafish; valproic acid; cerebellum; development 1. Introduction on histone deacetylase activity have been found to reduce cell proliferation in the rat hippocampal dentate gyrus, Valproic acid (VPA) is a well-known drug that is useful for which may be due to decreased levels of neurotrophic managing various neurological diseases. This compound is factors such as brain-derived neurotrophic factor (BDNF) one of the major drugs administered to suppress seizures in in the neurosphere from the subventricular zone (SVZ) of humans and a variety of animal models (Gobbi & Janiri adult mice (Umka et al. 2010; Zhou et al. 2011). In contrast, 2006; Jessberger et al. 2007). VPA is also used to treat it was recently reported that VPA exposure to pregnant bipolar disorders, social phobia, neuropathic pain (Winterer mice transiently increases BDNF mRNA and protein levels & Hermann 2000; Hosák & Libiger 2002; Smith et al. in the fetal mouse brain, which may cause aberrant brain 2010), and neurodegenerative disorders such as Hunting- development (Almeida et al. 2014). Similarly, VPA ton’s disease and Alzheimer’s disease (Ren et al. 2004; increases the proliferation of hippocampal neuronal pro- Qing et al. 2008; Zádori et al. 2009). However, VPA genitors in adult mice and prenatal rats (Hao et al. 2004;Go exposure during the prenatal and postnatal periods is also et al. 2012). known to have severe negative effects on the brain and Reduced cell proliferation of neuronal progenitors behavior at later adult stages. Adult rats that were prenatally during the early developmental period is likely to influence exposed to VPA were found to have behavioral impair- a variety of behavioral and cognitive functions in adults. ments such as decreased rearing and hole-poking along For example, severe proliferation defects are prominent in with increased locomotor activity in novel environments various regions of the developing brain including the (Sandhya et al. 2012). VPA reduce slow threshold (T type) cerebellum, dentate gyrus, and neocortex in mouse models calcium channel current and voltage-gated Na channel of Down syndrome (DS; Haydar et al. 2000; Lorenzi & activity while γ-aminobutyric acid (GABA) synthesis in the Reeves 2006; Roper et al. 2006; Chakrabarti et al. 2007; substantia nigra is increased (Kelly et al. 1990; Löscher Contestabile et al. 2007) as well as the hippocampal region 1999; Ziyatdinova et al. 2011). Furthermore, VPA has anti- excitotoxic effects on N-methyl-D-aspartate-induced tran- of human fetuses with DS (Contestabile et al. 2007; Guidi sient depolarization (Zeise et al. 1991) and decreases et al. 2008). Similarly, impaired proliferation of neural progenitors results in a reduction of cortical thickness that glutamate-mediated excitotoxic damage in a porcine model of traumatic brain injury (Hwabejire et al. 2013). eventually leads to microcephaly and developmental delay (Samuels et al. 2008). Neurogenesis defects such as The effects of VPA on cell proliferation in the brain remain unclear. Recent studies have shown that VPA reductions of neural progenitors and decreased neuronal functions as a histone deacetylase inhibitor (Venkataramani differentiation have also been observed in the SVZ of a et al. 2010; Chen et al. 2013). The inhibitory effects of VPA mouse DS model (Hewitt et al. 2010). *Corresponding author. Email: changlee@inha.ac.kr © 2014 Korean Society for Integrative Biology 102 Y. Lee et al. Cell proliferation and neurogenesis occur in various Leica, Wetzlar, Germany) in the transverse plane at a regions throughout the brain of zebrafish larvae that are thickness of 7 µm, mounted onto coated slides (Matsu- susceptible to chemical or physical insults. For example, nami Glass Ind., Ltd., Osaka, Japan), and stored at 20°C stab injury increases the proliferation of neuronal pre- until further use. cursor cells in the ventral zone of the injured telencepha- lon of adult zebrafish (Kishimoto et al. 2012). Therefore, 2.2.2. BrdU/proliferating cell nuclear antigen or HuC the zebrafish is a suitable model for studying alterations of double-immunohistochemistry cell proliferation following chemical-induced insults. In To determine the progenitor cell phenotype, double- particular, strong proliferative activity has been observed labeling was conducted to detect the colocalization of in the cerebellum of zebrafish larva, which is present in BrdU with proliferating cell nuclear antigens (PCNAs) or the anterior hindbrain during the larval period (Kaslin HuC. We used three to six zebrafish larvae from each et al. 2013). Three major proliferation zones of the experimental group and two sections (each 7-µm thick) of zebrafish cerebellum are the ventral cerebellar prolifera- cerebellum per larva. Sections of the brain were rinsed in tion zone at the bottom of cerebellar plate, the medial 0.1% phosphate buffer saline with Triton X-100 (PBS-T) cerebellar proliferation zone in the midline of the cerebel- and blocked with 10% normal goat serum (Jackson lar hemispheres, and the superficial proliferation zone at Immuno Research, West Grove, PA, USA) in 0.1% PBS- the top of the cerebellar plate (Hashimoto & Hibi 2012). T. After washing, the sections were incubated overnight at In the present study, we evaluated effects of VPA on cell 4°C in a solution containing antibodies specific for rat proliferation and migration in the cerebellum of zebrafish BrdU (monoclonal, 1:1000; Abcam, Cambridge, UK), larvae using immunohistochemical technique. PCNA (monoclonal, 1:300; DAKO, Glostrup, Denmark), or human HuC (1:100; Molecular Probes, Eugene, OR, 2. Materials and methods USA). The sections were rinsed in 0.1% PBS-T, and then 2.1. Animals incubated for 1 h at room temperature in a mixture of Adult zebrafish (around 2.5 cm in length) were purchased secondary antibodies including goat anti-rat IgG conju- from a local fish shop (Seoul, South Korea) and main- gated to Alexa Flour 594 (1:1000; Invitrogen, Carlsbad, tained at 28.0 ± 1.0°C with a 14-h light/10-h dark cycle in CA, USA) to detect BrdU and goat anti-mouse IgG aquariums. Tap water that was supplied to the aquariums conjugated to Alexa Flour 488 (1:1000; Invitrogen) to passed through a multistage filtration system equipped visualize PCNA or HuC. with a sediment filter, a post-carbon filter, and a fluores- For the quantitative analyses, cells stained for BrdU, cent UV light sterilizing filter (Zebrafish Auto System, PCNA, or HuC in the ventral cerebellum proliferation Genomic Design, Daejeon, South Korea). Water in the (VCP) zone, medial cerebellum proliferation (MCP) zone, containers was aerated and the pH was maintained and external granular layer (EGL) of the cerebellar area between 6.5 and 7.5. The zebrafish were fed twice a day were counted under 63× magnification with an oil with flake food (TetraBits, Luzerne, Singapore). To obtain immersion lens using a Zeiss AXIO Imager A1 micro- zebrafish embryos, females and males were placed in scope (Carl Zeiss, Jena, Germany). The stained cells in breeding tanks at a 2:3 ratio and the fertilized eggs were two consecutive sections were counted and used to maintained at 28.5°C. calculate the total number of stained cells in each larva. The sections were viewed under a 40× objective lens with 2.2. Immunostaining a 1.0 airy unit pinhole for each channel using a Zeiss LSM 510 META (Carl Zeiss) equipped with Argon/2 (488 nm) 2.2.1. Drug treatment and tissue preparation and HeNe (594 nm) lasers for excitation. Images were In order to assess the effects of VPA on neurogenesis, obtained with an averaging scan number of 16 in the XY- zebrafish larvae (5 days postfertilization [dpf]) were plane and a frame size of 512 × 512 pixels for 12-bit placed for 3 h in aquarium water containing 0.2 or sampling. The images were then analyzed with ZEN Light 2 mM VPA (Sigma-Aldrich, St. Louis, MO, USA). For Edition software (Carl Zeiss). the control experiment, age-matched larvae were placed in normal aquarium water for the corresponding period. After terminating the VPA treatment, the larvae were 2.3. Statistical analysis maintained for 12 h in aquarium water containing 10 Data are expressed as the mean ± standard error of the mM5-bromo-2′-deoxyuridine (BrdU; Sigma-Aldrich, St. mean (SEM). The number of the stained cells was Louis, MO, USA). All larvae were fixed with 4% analyzed with an analysis of variance (ANOVA) followed paraformaldehyde in 0.01 M phosphate buffer (pH 7.4), by post hoc Tukey’s tests. p-Values < 0.05 were con- and cryoprotected overnight in 30% sucrose at 4°C. The larvae were then sectioned with a cryostat (CM1800; sidered to be statistically significant. Animal Cells and Systems 103 DAPI BrdU PCNA Merge A A DAPI BrdU PCNA Merge MCP EGL 0 h 48 h CeP Control Control VCP 2 mM 2 mM VPA VPA B + BrdU -cells BrdU -cells PCNA -cells PCNA -cells Control 0.2 mM VPA 2 mM VPA Control 0.2 mM VPA 2 mM VPA C 120 Control 0.2 mM VPA 2 mM VPA 0 Control 0.2 mM VPA 2 mM VPA Figure 1. Changes in the numbers of BrdU- and PCNA-positive Figure 2. Changes in the number of BrdU- and PCNA-positive cells in the cerebellum of zebrafish larvae induced by VPA cells in the cerebellum of zebrafish larvae induced by VPA 48 h immediately after terminating BrdU treatment. after terminating BrdU exposure. (A) Confocal images showing Note: (A) Confocal images show the immunostaining patterns the immunostaining patterns corresponding to DAPI (blue), corresponding to 4′,6-diamidino-2-phenylindole (DAPI, blue), BrdU (red), and PCNA (green), (B) Quantification of BrdU- BrdU (red), and PCNA (green), (B) Quantification of BrdU- and and PCNA-positive cells in the cerebellum, and (C) Bars PCNA-positive cells in the cerebellum, and (C) Bars represent represent the percentage of PCNA-positive cells among the the percentage of PCNA-positive cells among the BrdU-positive BrdU-positive cells. Data are expressed as the mean ± SEM and cells. Data are expressed as the mean ± SEM and were analyzed were analyzed with post hoc Tukey’s multiple comparison tests. with post hoc Tukey’s multiple comparison tests. *p < 0.05 *p < 0.05 compared to the control. Scale bar = 50 µm. compared to the control. Scale bar = 50 µm. VPA was reduced significantly to 53.1% of the control (61.8 ± 3.0, p < 0.05, n ≥ 3; Figure 1B). 3. Results PCNA-positive cells were also observed in the EGL, 3.1. VPA reduces the number of BrdU- and PCNA- MCP, and VCP of the cerebellum (Figure 1A). The number positive cells of PCNA-positive cells in the cerebellum of larvae treated To determine whether VPA affects cell proliferation in the with 2-mM VPA was significantly reduced to 52.3% of the cerebellum of zebrafish larvae at 5 dpf, the fish was control (84.8 ± 4.2, p < 0.05, n ≥ 3; Figure 1B). The sacrificed immediately after 12 h of treatment with BrdU majority of BrdU-positive cells were positive for PCNA; following 3 h of exposure to 0.2 or 2-mM VPA. In the over 90% of the BrdU-positive cells were also positive for cerebellum of the zebrafish larvae (Figure 1A), cells PCNA in the control. The percentage of PCNA-positive stained for BrdU were found predominantly in the EGL, cells among BrdU-positive cells was not decreased in the 2- MCP zone, and VCP zone, consistent with findings from a mM VPA-treated zebrafish compared to the control with previous study (Mueller & Wullimann 2002). The number above 90% of the BrdU-positive cells positive for PCNA in of BrdU-positive cells in zebrafish treated with 2-mM the zebrafish exposed to 2 mM VPA (Figure 1C). The number of + + + + PCNA amog BrdU -cells (%) BrdU and PCNA -cells (n) The number of + + + + PCNA amog BrdU -cells (%) BrdU and PCNA -cells (n) 104 Y. Lee et al. At 48 h after terminating BrdU treatment, the number of fish at 48 h, significantly higher than the percentage BrdU-positive cells in the cerebellum was not different measured immediately after terminating the BrdU treat- compared to that measured immediately after terminating ment (Figure 4C). Only 7–10% of the BrdU-positive cells BrdU treatment in the control (Figures 1B and 2B). were also positive for HuC in the VCP of the control and Interestingly, BrdU-positive cells were mostly found in the VPA-treated larvae. In the VCP and medial zone of the medial zone of CeP and marginally in the proliferating CeP of 2-mM VPA-treated larvae. The number of BrdU- zones of the MCP and VCP 48 h after BrdU treatment had positive cells in the VCP and medial zone of the CeP of ended (Figure 2A). PCNA-positive cells were still present in 2-mM VPA-treated larvae was significantly decreased to the MCP and VCP as was observed immediately after BrdU 26.3% of 43.1% of the control, respectively (Figure 4B treatment had ceased (Figures 1A and 2A). Therefore, only and 4C). 20.9% of the BrdU-positive cells were also PCNA-positive 48 h after terminating BrdU treatment in the control 3.3. Posttreatment with 2-mM VPA after BrdU (Figure 2C). In the larvae exposed to 2-mM VPA, the exposure reduces the migration of BrdU-positive cells in number of BrdU-positive cells was significantly reduced to the CeP 77.1% of the control (control, 73.8 ± 1.8; n =4, p < 0.05). The zebrafish larvae were treated with BrdU for 12 h and However, the number of PCNA-positive cells (Figure 2B) subsequently exposed to 2-mM VPA for 3 h. The larvae was similar to that found in the control fish (control, 86.4 ± were sacrificed 45 h after terminating the VPA treatment 2.1; 2 mM VPA, 79.8 ± 1.4; n = 3). It is also noted that above (48 h after terminating the BrdU exposure). BrdU-positive 50% of the BrdU-positive cells were also PCNA-positive 48 cells were mostly found in the medial zone of the CeP in h after terminating BrdU treatment in 2-mM VPA-treated the control. In contrast, these cells were predominantly larvae (Figure 2C), suggesting that VPA suppressed migra- observed in the VCP of the 2-mM VPA-treated larvae tion of BrdU-positive cells to the medial zone of CeP. (Figure 5A). The number of BrdU-positive cells in the VCP was reduced (Figure 5B) but to a much lesser extent 3.2. The number of BrdU- and HuC-double-labeled compared to the reduction in the medial zone of the cells increases 48 h after terminating BrdU treatment CeP of the 2-mM VPA-treated larvae (control, 20.9 ± 1.2; 2 mM VPA, 13.1 ± 0.5; n = 3). The number of BrdU- In order to determine the phenotype of the BrdU-positive positive cells in the medial zone of the CeP of the 2-mM cells, the brain sections were double-labeled for BrdU and VPA-treated larvae (Figure 5C) was significantly decreased Hu, a neuronal marker, in the cerebellum of 5-dpf to 15% of the control (control, 124.1 ± 04; n =3, p <0.05). zebrafish larvae. While BrdU-positive cells were present Over 90% of the BrdU-positive cells were also positive to in the MCP and VCP, HuC-positive cells appeared in the HuC in the CeP of the control fish as previously observed medial zone of the CeP and medullar oblongata (Figure 3A). (Figure 4). Only 5–10% of the BrdU-positive cells were Immediately after 12 h of BrdU treatment, approximately also positive for HuC in the VCP of both the control and 5% of the BrdU-labeled cells in the cerebellum of the 5-dpf the VPA-treated larvae. larvae were positive for HuC in the VCP of the control and 2-mM VPA-treated groups (Figure 3B). The numbers of cells positive for both BrdU and HuC in the control and 2- 4. Discussion mM VPA-treated larvae were not significantly different. The number of BrdU-positive cells in the VCP of larvae treated Results of this study demonstrated that the newborn cells with 2-mM VPA was significantly reduced to the half of the were mostly found in the proliferating zones of the VCP, control (48.3 ± 0.7, n =3, p <0.05; Figure 3B) as previously MCP, and EGL of the cerebellum in the zebrafish larvae. observed (Figure 1B). In the medial zone of the CeP, the Most newborn cells migrated to the medial zone of the numbers of BrdU-positive cells were only 3–5 in the control CeP within 48 h after BrdU treatment had ended and and VPA-treated larvae although most BrdU-labeled cells underwent differentiation into HuC-positive neurons in the were also positive for HuC (Figure 3C). medial zone of the CeP. After 3 h of treatment with 2-mM VPA, migration of the newborn cells was decreased along In both the control and the VPA-treated larvae with cell proliferation in the zebrafish larvae cerebellum. (Figure 4A and 4B), the number of BrdU-positive cells was higher in medial zone of the CeP but lower in the Since VPA reduced not only the number of BrdU- VCP at 48 h than immediately after the termination of positive cells but also the number of PCNA-positive cells, BrdU treatment. Cells positive for both BrdU and HuC it is possible that 2-mM VPA promoted the death of were mostly found in the medial zone of the CeP, progenitor cells. However, a previous report has shown suggesting that BrdU-positive cells migrated to the CeP that apoptosis is not induced by 2-mM VPA in the during the 48-h period and differentiated into HuC- telencephalic area of the zebrafish larva brain (Lee et al. positive cells (Figure 4C). Over 90% of the BrdU-positive 2013). Cell proliferation appeared to recover during cells were also positive for HuC in the CeP of the control the 60-h period following termination of VPA treatment. Animal Cells and Systems 105 DAPI BrdU HuC Merge 0 h Control 2 mM VPA VCP CeP B C 80 + + 80 + + HuC /BrdU -cells HuC /BrdU -cells - + - + HuC /BrdU -cells HuC /BrdU -cells 60 60 40 40 20 20 0 0 Control 0.2 mM 2 mM Control 0.2 mM 2 mM VPA VPA VPA VPA Figure 3. Changes in the numbers of BrdU- and HuC-positive cells in the cerebellum of zebrafish larvae induced by VPA immediately after BrdU treatment had ended. (A) Confocal images showing the immunostaining patterns corresponding to DAPI (blue), BrdU (red), and HuC (green). (B and C) Quantification of BrdU- and HuC-positive cells in the VCP (B) and medial zone of the CeP (C). Bars represent the numbers of HuC-positive and HuC-negative cells among the BrdU-positive cells. Data are expressed as the mean ± SEM and were analyzed with post hoc Tukey’s multiple comparison tests. p < 0.05 compared to the control. Scale bar = 50 µm. DAPI BrdU HuC Merge 48 h Control 2 mM VPA B VCP C CeP 160 + + + + HuC /BrdU -cells HuC /BrdU -cells - + - + HuC /BrdU -cells HuC /BrdU -cells 120 120 80 80 40 40 0 0 Control 0.2 mM 2 mM Control 0.2 mM 2 mM VPA VPA VPA VPA Figure 4. Change in the numbers of HuC-positive cells in the cerebellum of zebrafish larvae induced by VPA48 h after BrdU treatment was terminated. (A) Confocal images showing the immunostaining patterns for DAPI (blue), BrdU (red), and HuC (green). (B and C) Graphs showing the quantification of BrdU- and HuC-positive cells in the VCP (B) and medial zone of the CeP (C). Bars represent the numbers of HuC-positive and -negative cells among the BrdU-positive cells. Data are expressed as the mean ± SEM and were analyzed with post hoc Tukey’s multiple comparison tests. p < 0.05 compared to the control. Scale bar = 50 µm. + + + + The number of HuC /BrdU - The number of HuC /BrdU - - + - + and HuC /BrdU -cells (n) and HuC /BrdU -cells (n) + + + + The number of HuC /BrdU - The number of /BrdU - HuC - + - + and HuC /BrdU -cells (n) and HuC /BrdU -cells (n) 106 Y. Lee et al. A DAPI BrdU HuC Merge 48 h Control 2 mM VPA B C VCP CeP 160 + + 160 + + HuC /BrdU -cells HuC /BrdU -cells - + - + HuC /BrdU -cells HuC /BrdU -cells 120 120 40 40 0 0 Control 0.2 mM 2 mM Control 0.2 mM 2 mM VPA VPA VPA VPA Figure 5. Changes in the numbers of BrdU- and HuC-positive cells in the cerebellum of zebrafish larvae after treatment with VPA following BrdU exposure. (A) Confocal images showing the immunostaining patterns for DAPI (blue), BrdU (red), and HuC (green). (B and C) Graphs presenting the quantification of BrdU- and HuC-positive cells in the VCP (B) and medial zone of the CeP (C). Bars represent the numbers of HuC-positive and -negative cells among the BrdU-positive cells. Data are expressed as the mean ± SEM and were analyzed with post hoc Tukey’s multiple comparison tests. *p < 0.05 compared to the control. Scale bar = 50 µm. The number of BrdU-positive cells was 53.1 or 77.1% of immediately after terminating BrdU treatment than 48 h. the control at 12 and 60 h after termination of VPA This could be due to changes in the number of cells treatment, respectively. Additionally, the numbers of positive for neither PCNA nor BrdU at 48 h. In fact, the PCNA-positive cells in the control and 2-mM VPA-treated numbers of PCNA- and BrdU-positive cells were not larvae were not significantly different 60 h after VPA different immediately and 48 h after terminating BrdU treatment (Figures 1 and 2). These results are consistent treatment (Figures 1 and 2). Therefore, the decreased with data from a previous report showing that the numbers percentage of PCNA-positive cells among BrdU-positive of PCNA- and BrdU-positive cells in the telencephalic cells at 48 h is most likely attributed to the migration of area of 2-mM VPA-treated larvae returned to levels BrdU-positive cells to the medial zones of the CeP. similar to those observed in the control animals at 4 and Consistent with a previous study (Wullimann et al. 10 days after VPA treatment, respectively (Lee et al. 2011), three distinct proliferative zones of the MCP, VCP, 2013), suggesting that the recovery of BrdU-positive cell and EGL were also identified in the present investigation. numbers is delayed. According to this study, the impair- The presence of BrdU-positive cells in the EGL becomes ment and recovery of cell proliferation may be related to apparent with BrdU treatment over time. After 10 h of β-catenin, lef1, and GSK3β expression. Both β-catenin BrdU exposure, BrdU-positive cells are sparsely distrib- and lef1 mRNA levels are increased while GSK3β uted in the VCP close to the ventricles and are rarely expression is reduced 10 days after VPA treatment (Lee found in the EGL (Mueller & Wullimann 2002). However, et al. 2013). In contrast, β-catenin and lef1 mRNA both the VCP and the EGL are increasingly filled with expression is decreased 12 h after VPA treatment but BrdU-positive cells after 12 h of BrdU treatment as shown GSK3β expression is increased (Lee et al. 2013). in the present study (Figure 1). In the control fish, the percentage of PCNA-positive Spatiotemporal distribution of PCNA-, BrdU-, and cells among BrdU-positive cells was much higher HuC-positive cells was also distinctly different over time + + The number of HuC /BrdU - - + and HuC /BrdU -cells (n) + + The number of HuC /BrdU - - + and HuC /BrdU -cells (n) Animal Cells and Systems 107 Gobbi G, Janiri L. 2006. Sodium- and magnesium-valproate after termination of BrdU exposure. PCNA-positive cells in vivo modulate glutamatergic and GABAergic synapses in that are potentially mitotic were detected only in the the medial prefrontal cortex. Psychopharmacology (Berl). proliferation zones of the VCP, EGL, and MCP both 185:255–262. immediately and 48 h after terminating the BrdU treatment. Guidi S, Bonasoni P, Ceccarelli C, Santini D, Gualtieri F, Ciani On the other hand, HuC-positive cells, which are presum- E, Bartesaghi R. 2008. Neurogenesis impairment and increased cell death reduce total neuron number in the ably neuronal cells, were located between the VCP and the hippocampal region of fetuses with down syndrome. Brain EGL both immediately and 48 h after ending the BrdU Pathol. 18:180–197. exposure. Since the PCNA-positive cells localize adjacent Hao Y, Creson T, Zhang L, Li P, Du F, Yuan P, Gould TD, Manji to the ventricles and in a subpial layer of the cerebellum, the HK, Chen G. 2004. 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Journal
Animal Cells and Systems
– Taylor & Francis
Published: Mar 4, 2014
Keywords: zebrafish; valproic acid; cerebellum; development
