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.

3). The levels of hsp70 mRNA decreased by 80.6% and 53.7% of the 2 dpf larvae treated with VPA in the 5 and 10 dpf larvae, respectively (p < 0.01, n = 3) (Figure 2(a)). Similarly, the levels of hsp70 mRNA decreased by 47.8% and 37.5% of the 3 dpf larvae treated with VPA in the 5 and 10 dpf larvae, respectively (p < 0.01, n = 3) (Figure 2(b)). β-catenin mRNA expression was not significantly different from the control 12 h after VPA treatment, but gradually decreased by 31.6% and 53.1% of the control in the larvae treated with VPA at 2 dpf, and by 45.7% and 56.2% of the control in the larvae treated with VPA at 3 dpf when measured at 5 and 10 dpf, respectively (p < 0.01) (Figure 2(c) and Figure 2(d)). LEF1 mRNA levels increased by 18.4% and 35.6% of the control 12 h after VPA treatment at 2 and 3 dpf. LEF1 mRNA levels gradually decreased by 40.6% and 49.7% of the control in the larvae treated with VPA at 2 dpf, and by 20.4% and 60.8% of the control in the larvae treated with VPA at 3 dpf when measured at 5 and 10 dpf, respectively (p < 0.01) (Figure 2(e) and Figure 2(f)). The expression levels of gsk3β were not significantly different from the control 12h after the VPA treatment at 2 dpf, but gsk3β mRNA expression increased by 16.4% in the VPA-treated zebrafish of 10 dpf relative to the control (p < 0.01, n = 3) (Figure 2(g)). In the VPA-treated larvae at 3 dpf gsk3β mRNA expression significantly increased by 19.5% of the control when measured at 5 dpf, respectively (p < 0.01, n = 3) (Figure 2(h)).

3.3. Cranial Malformation Was Not Observed in the VPA-Treated Larvae

Evaluation of the cartilage structures showed that the size and shape of the vis-

Figure 2. mRNA expression of hsp70, β-catenin, LEF1, and gsk3β in the zebrafish larvae treated 2 mM VPA at 2 and 3 dpf: ((a), (c), (e), (g)) Expression levels of hsp70, β-catenin, LEF1, and gsk3β mRNA zebrafish larvae treated with 2 mM VPA at 2 dpf compared to the control; ((b), (d), (f), (h)) Expression levels of hsp70, β-catenin, LEF1, and gsk3β mRNA zebrafish larvae treated with 2 mM VPA at 3 dpf compared to the control. The data were expressed as the means ± S.E.M and were analyzed with Student’s t-tests along with post hoc Tukey’s multiple comparison tests. *P < 0.01 versus the control at each time point (Student’s t-tests); #P < 0.01 compared to the 2 or 3d group (post hoc Tukey’s tests).

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.

cerocranium as well as neurocranium of the VPA-treated larvae were unaffected when measured at 10 dpf (Figure 3(a)). The Meckel’s and palatoquadrate cartilage (M/PQ), ceratohyals (Ch), ethmoid plate and trabeculae (Et/Tr), ethmoid plate (Et), and fenestra (Fen) were 520.3 ± 2.8, 234.4 ± 3.4, 462.8 ± 4.4, 248.4 ± 3.1, and 148.0 ± 4.8 µm, respectively, in the control (n = 5); 485.7 ± 6.4, 232.5 ± 2.7, 440.1 ± 5.9, 218.7 ± 3.6, and 135.1 ± 1.5 µm in the larvae treated with VPA at 3 dpf (n = 5); 490.6 ± 0.6, 225.2 ± 3.1, 431.2 ± 1.8, 224.0 ± 5.2, and 138.8 ± 1.5 µm in the larval treated with VPA at 2 dpf (n = 5). No significant differences in these elements were observed the three groups (Figure 3(b)).

3.4. 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).

4. Discussion

This study showed that 2 mM VPA reduced cell proliferation in the telencephalic

Figure 4. Altered color preference by 2 mM VPA: (a) 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.

area of zebrafish larvae. Expression levels of LEF1 and β-catenin mRNA, nuclear mediators of wnt signaling, were also decreased following treatment with 2 mM VPA. LEF1 and β-catenin have been shown to increase cell proliferation in the developing brain of the mouse and neural stem cell [10] [11] [14] . In addition, a 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 [11] . On the contrary, gsk3β mRNA 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] .

5. 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.

Acknowledgements

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-0002753).

Cite this paper
Lee, B. , Lee, S. , Choi, M. and Lee, C. (2017) Valproic Acid Decreases Cell Proliferation and Color Preference in the Zebrafish Larvae. Journal of Biosciences and Medicines, 5, 56-66. doi: 10.4236/jbm.2017.512007.
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