Valproic Acid Decreases Cell Proliferation and Color Preference in the Zebrafish Larvae

Valproic acid (VPA) is widely used as an antiepileptic drug or mood stabilizer. In this study, we evaluated the effects of treatment with 2 mM VPA for 3 h on cell proliferation in the telencephalic area of zebrafish larvae using bromodeoxyuridine (BRDU) to label dividing cells. It was demonstrated that 2 mM VPA exposure for 3 h at 2 and 3 days post-fertilization (dpf) larvae decreased cell proliferation in the telencephalic area of 5 dpf larvae. The reduced cell proliferation was not restored at 10 dpf larvae. The quantitative real-time PCR (qRT-PCR) data indicated that mRNA expression levels of WNT signaling pathway-related factors such as β-catenin, LEF1, and gsk3β were altered in the zebrafish larvae treated with 2 mM VPA at 2 and 3 pdf. It was also demonstrated that 2 mM VPA exposure affected color preference of the zebrafish larvae, reducing blue color preference at 5 dpf larvae. The altered color preference was restored at 10 dpf larvae. These results suggest that VPA exposure may cause molecular, cellular, and behavioral alterations in early developmental stage of the zebrafish.


Introduction
Exposure to valproic acid (VPA) during the prenatal and postnatal period is known to have some deleterious effects on the brain development and behavior.Accumulating studies have shown that extensive reduction of neurogenesis in the developing brain may be involved in a variety of neurological functions in adult mice and humans [5] [6].For example, severe cell proliferation defects are present in the developing neocortex, dentate gyrus, and cerebellum of mouse of Down Syndrome (DS) models [7], and the hippocampal and cerebellar regions of human fetuses with DS [8].In the brain of zebrafish larvae cell proliferation and neurogenesis actively occur in diverse areas and are affected by external factors such as treatment with pentylenetetrazol, a GABA receptor blocker [9].
Therefore, the zebrafish is an appropriate model for researching behavioral consequences in adulthood that may be caused by alterations of cell proliferation in the brain during early development stages.
WNT signaling factors such as β-catenin and LEF1 are involved in regulation of cell proliferation in the mammalian brain [10].A nuclear mediator of wnt signaling, LEF1 accelerates cell proliferation in the developing forebrain of the mouse [11].These findings indicate the association between alterations in the WNT signaling gene expression and changes in cell proliferation following VPA exposure.In this study, the effects of VPA on the brain development and cell proliferation were studied in the telencephalic area of the zebrafish larvae.Furthermore, modification of color preference was also examined in the VPA-treated larvae.

Animals
Adult zebrafish (around 2.5 cm in length) were purchased from a local fish shop (Incheon, South Korea) and maintained at 28.5˚C ± 1.0˚C with a 14-h light/10-h dark cycle in aquariums as described previously [12].

Immunostaining
VPA (Sigma-Aldrich) was dissolved in distilled water at a concentration of 1 M, and then diluted to final concentrations of 2 mM in containers of aquarium water.Zebrafish larvae (2 and 3 days postfertilization [dpf]) were placed for 3 h in aquarium water containing 2 mM VPA.For the control experiment, age-matched larvae were placed in normal aquarium water during the corresponding period.
All larvae were fixed with 4% PFA in 0.01 M PBS immediately after termination of the BRDU treatment, and cryoprotected overnight in 30% sucrose at 4˚C.
The larvae were then sectioned with a cryostat (CM1800; Leica, Wetzlar, Germany) in the coronal plane at a thickness of 10 μm.The sections were mounted onto coated slides (Matsunami Glass Ind., Ltd., Osaka, Japan), and stored at −20˚C until further use.

Color Preference Tests
For measurements of color preference in the zebrafish larvae, a color maze kit (8.2 cm long × 12.4 cm wide × 2.1 cm high) was subdivided into eight compartments, each compartment with divided into two colors (blue and yellow) by color sleeves.The color preference of the zebrafish larvae was monitored using a digital camera; fish's position (blue or yellow sleeve) was monitored every minute for 1 h.The data were expressed as the means ± S.E.M and subjected to ANOVA, followed by post hoc Tukey's tests.
For the experiment, ten zebrafish larvae (5 and 10 dpf) were placed in a compartment of color maze filled with normal aquarium water (4 ml) on an illumination box.

Whole-Mount Skeletal Staining
Cartilage of the zebrafish larvae was stained as described previously [13].The larvae were fixed at 10 dpf with 4% PFA in 0.01 M PBS (pH 7.4) for 4 h at room temperature, washed in 0.1% PBS-T, and stained overnight with 0.1% Alcian blue 8GX (Sigma-Aldrich) in 80% ethanol/20% acetic acid.For clarification, the larvae were treated with 1% KOH/3% H 2 O 2 for 6 h, followed by digestion for 15 min with 0.02% trypsin (Sigma-Aldrich) dissolved in PBS.The larvae were then rehydrated in 70%, 50%, and 30% EtOH/0.01M PBS, cleared in 30% glycerol/0.01M PBS, and stored in 100% glycerol.Larvae stained with Alcian blue were dissected and flat-mounted.The cartilage was examined with a Leica MZ75 microscope at 50X magnification and images were captured by a Nikon D200 camera (Nikon, Tokyo, Japan).
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Quantitative Real-Time PCR (qRT-PCR)
The heads of 50 larvae were flash-frozen on dry ice at 12 h, 2 d, 3 d, 7 d, and 8 d of recovery in normal water after 2 mM VPA treatment.Zebrafish larvae heads were lysed in TRIzol reagent (Invitrogen), and total RNA was isolated following the manufacturer's protocols.RNA was then treated with DNase I (Promega) to eliminate genomic DNA contamination and further purified using the RNeasy mini kit (Qiagen) following the manufacturer's protocols.

The Number of BRDU-and PCNA-Positive Cells Was Reduced by 2 mM VPA
The number of BRDU-positive cells in the telencephalic area of zebrafish treated ).The number of PCNA-positive cells was also significantly reduced to 34.0% and 32.6% of that found in the control (p < 0.01, n = 8) (Figure 1(c)).To find out whether the suppressive effect of VPA on cell proliferation is lasting, BRDU-or PNCA-positive cells in the telencephalic area of larvae that had been treated with 2 mM VPA for 3 h at 2 and 3 dpf were measured at 5 and 10 dpf.
The numbers of BRDU-or PCNA-positive cells measured 12 h following BRDU treatment were not significantly different at 2, 3, 5 and 10 dpf in the control.
However, the numbers of BRDU-or PCNA-positive cells were significantly lower in the 2 mM VPA-treated larvae of 2, 3, 5, and 10 dpf compared to the age-matched controls (Figure 1).

Cranial Malformation Was Not Observed in the VPA-Treated Larvae
Evaluation of the cartilage structures showed that the size and shape of the vis-

Color Preference Was Transiently Altered by 2mM VPA
In the color preference test at 5 dpf, blue color preference for the control were measured 9.01 ± 0.06 and yellow color preference were 0.98 ± 0.06 (p < 0.01, n = 7) (Figure 4(a)).In contrast, blue color preference in the larval treated with VPA at 2 and 3 dpf significantly decreased to 5.17 ± 0.24 and 6.11 ± 0.21 and yellow color preference significantly increased to 4.82 ± 0.24 and 3.88 ± 0.21 for the 5 dpf larvae, respectively (p < 0.01, n = 7) (Figure 4(a)).However, altered color preference was recovered at 10 dpf.Blue color preference in the control was measured to 7.11 ± 0.03 and yellow color preference was 2.92 ± 0.02 at 10 dpf (n = 8) (Figure 4(b)).Similarly, blue color preference in the larval treated with VPA at 2 and 3 dpf were 6.56 ± 0.05 and 6.81 ± 0.15 and yellow color preference were to 3.43 ± 0.05 and 3.18 ± 0.15, respectively (p < 0.01, n = 8) (Figure 4(b)).Locomotion activity increased as zebrafish larvae grew with no significant difference between groups (data not shown).

Discussion
This study showed that 2 mM VPA reduced cell proliferation in the telencephalic  previous research has shown that VPA increases hsp70 protein levels in the rat cortical neurons [15].Similarly, hsp70 mRNA levels were increased in the zebrafish brain treated with 2 mM VPA for 3 h in this study.
It has been reported that young epilepsy patients suffered hyperammonemic encephalopathy following treatment with VPA for 1 ~ 2 weeks [16].The serum valproic acid level ranged 50 ~ 269.9 mg/L, which roughly corresponds to 0.5 ~ 2.5 mM [16] [17].Our previous study has also shown that treatment with 2 mM VPA induced learning impairments in the adult zebrafish [18].In this study, therefore, 2 mM VPA was applied for 3 h in aquarium water in order to evaluate effects of 2 mM VPA on cell proliferation and behavior of zebrafish larvae.
The decreased cell proliferation in the larvae of 5 and 10 dpf treated with 2 mM VPA for 3 h at 2 and 3 dpf is consistent with a decreased cell proliferation in the larvae treated with the same dose of VPA for 3 h at 5 dpf [11].An interesting difference is that the decreased cell proliferation was gradually recovered within 10 days following VPA treatment at 5 dpf, while it was not recovered following VPA treatment at 2 and 3 dpf.This discrepancy in the recovery of cell proliferation depending on the treatment time may be attributed to different expression patterns β-catenin and LEF1 mRNA in the two groups.Expression patterns of β-catenin and LEF1 mRNA were altered following VPA treatment at 2 and 3 dpf larvae in this study.When 2 mM VPA was treated at the 2 and 3 dpf larvae, β-catenin and LEF1 expression was lower in the VPA-treated larvae than the control at 2 ~ 8 d following VPA treatment.In contrary, gsk3β mRNA expression was higher in the VPA-treated larvae.Conversely, when the same does of VPA was treated at the 5 dpf larvae, β-catenin and LEF1 expression was higher in the VPA-treated larvae than the control 2 ~ 10 d following VPA treatment B. Lee et al. [11].On the contrary, gsk3β expression was lower in the VPA-treated larvae.
The reduced cell proliferation in the brain didn't cause either the cranial malformation or abnormal locomotive activity in this study.Locomotive activity increased as the larvae grew, but it was not significantly different between the control and VPA-treated larvae.However, color preference was altered in the 5 dpf larvae treated with 2 mM VPA at 2 and 3 dpf.VPA-treated larvae showed a decreased blue color preference compared to the control.Interestingly, the color sense disorder has been reported from adolescent patient who underwent seizure treatment by carbamazepine (CBZ) and VPA [19].

Conclusion
VPA is known to detrimentally affect the embryonic and fetal development when a pregnant woman is exposed to it in pregnancy.Taken our studies together, 2 mM VPA had molecular and cellular effects on the brain development of zebrafish larvae.Especially, VPA treatment at 2 and 3 dpf reduced cell proliferation in the telencephalic area of zebrafish larvae of 5 and 10 dpf.In addition, it was also demonstrated that VPA exposure might cause visual deficits.Furthermore, VPA altered expression patterns of LEF1, β-catenin, and gsk3β RNA, which might underlie the reduced cell proliferation.Thus, our study suggests the possible cellular and molecular factors underlying detrimental effects of VPA on the brain development.
with 2 mM VPA at 2 and 3 dpf was reduced significantly to 43.2% and 43.5% of the control at 12 h after BRDU injection (p < 0.01, n = 8) (Figure 1(a) and Figure 1(b)

Figure 1 .
Figure 1.Reduced number of BRDU-and PCNA-positive cells in the zebrafish larvae treated with 2 mM VPA: (a) A representative hematoxylin and eosin stained section of the telencephalic area of zebrafish larvae (5 dpf) is shown at the top.Fluorescence microscope images show the immunostaining patterns corresponding to BRDU (red) and PCNA (green); (b) Quantification of BRDU-positive cells in the pallium (P) and dorsal subpallium (Sd); (c) Quantification of PCNA-positive cells in the pallium (P) and dorsal subpallium (Sd).Data were expressed as the means ± S.E.M and were analyzed via post hoc Tukey's multiple comparison tests.* P < 0.01 (VPA-treated at 2 dpf); # P < 0.01 (VPA-treated at 3 dpf) compared to the control; Scale bar = 0.5 mm.

Figure 3 .
Figure 3. Effects of 2 mM VPA on craniofacial development in the zebrafish larvae: (a) Image of Alcian blue staining of the control and VPA-treated larvae of 10 dpf.Meckel's and palatoquadrate cartilage (M/PQ), ceratohyals (Ch) cartilage were clearly identified in the neurocranium (left); ethmoid plate and trabeculae (Et/Tr), ethmoid plate (Et), and fenestra (Fen) in viscerocranium (right) of the control and VPA-treated larvae; (b) Quantitative analysis of the M/PQ length, Ch length, Et/Tr length, Et width, and Fen width.Scale bar = 200 μm, n = 5.

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Figure 4 .
Figure 4. Altered color preference by 2 mM VPA: Color preference test measured at 5 dpf.Yellow color preference was increased in the zebrafish larvae treated with 2 mM VPA at 2 and 3 dpf compared to the control; (b) Color preference test measured at 10 dpf.Color preference was recovered in the zebrafish larvae treated with 2 mM VPA.The data were expressed as the means ± S.E.M and were analyzed via post hoc Tukey's multiple comparison tests.* P < 0.01 compared to the control.