Valproic acid increases histone 3 acetylation and GSK3 phosphorylation in the hippocampus and enhances fear memory in mice

• Main Authors: Shang-Meng Yang, 楊尚孟
• Other Authors: Yn-Ho Huang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/68406737901093980179

碩士 === 國立陽明大學 === 解剖學及細胞生物學研究所 === 101 === Background: Valproic acid (VPA) has long been used as a mood-stabilizing drug in the treatment of bipolar disorder, however, the mechanism of how Valproic acid stabilizes mood is yet unknown. We speculate that VPA modifies emotions through inhibiting histone acetylation and GSK3 phosphorylation because of the following reasons: (1) Increasing evidences implicate that chromatin remodeling affects emotional behaviors in mice; (2) VPA inhibits histone deacetylases (HDAC) which remove the acetyl groups from the lysine residues leading to the formation of a condensed and transcriptionally silenced chromatin; (3) VPA has almost the same effects on bipolar disorder as Lithium which affects the Wnt signaling pathway and inhibits GSK3. Materials and methods: 8-10 week-old male C57BL/6J Narl mice were used for experiments. The dose effect of VPA on hippocampal histone H3 acetylation was studied by administering different doses of VPA and saline to mice which were sacrificed 45 min later. Hippocampal proteins were extracted with the sulfuric acid histone extraction method and the RIPA protein extraction method for histone H3 and GSK3 western blotting, respectively. In the time course study, 400 mg/kg of VPA was administered to 4 groups of mice which were sacrificed 45min, 3hr, 6hr, 12hr later. According to the sustainability of VPA’s acetylating effect in the hippocampus, 400 mg/kg of VPA was administered to two groups of fear-conditioned mice. One was administered every 6 hours and the other only right after the fear-conditioning. In the fear-conditioning test, mice were given electrical shocks (0.6mA, 4sec x 3) which were preceded by 20 seconds of a cue sound. Memory of fear was evaluated by the percentage of freezing behavior 24 hours after the conditioning training. In the multiple T-maze test, mice were trained to find the 12μl-sweet condensed milk in the goal arm 15 times per day. The one-hit rate and mean number of misses were plotted day by day. All the experimental data were analyzed using SPSS statistical software analysis. Results: The ratio of acetylated histone H3 in mice hippocampus showed a trend of increasing with administered dose of VPA. The ratios of acetylated histone H3 are significantly higher with the 200 mg/kg and 400 mg/kg groups than the saline group. In the time course study, acetylated histone H3 was significantly higher in the 45-min, 3-hrs and 6-hrs groups than the saline and 12-hrs groups, which also showed an attenuating trend with time. Phosphorylated GSK3β-Ser9, GSK3α-Ser21 in the hippocampus were significantly increased 6 hours after the mice were injected with 400 mg/kg of VPA. In the freezing test for emotional memory evaluation, the group given VPA every 6 hours showed a higher freezing response than the only once -treated group which displayed similar response to the control group. VPA-treated mice did not better spatial memory or learning in the multiple T-maze test. Conclusion: VPA’s acetylating effect on histone H3 in the hippocampus is dose and time-dependent. Adequate dose and time in administering VPA increases H3 acetylation, GSK3 phosphorylation and emotional memory. Whether VPA’s mood-stabilizing effects comes from enhancing emotional memory needs further exploration.