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. 2019;20(3):253-263.
doi: 10.1631/jzus.B1800340.

Valproic acid withdrawal ameliorates impairments of hippocampal-spatial working memory and neurogenesis

Wanassanun Pannangrong  1 , Apiwat Sirichoat  1 , Trai Wongsiri  2 , Peter Wigmore  3 , Jariya Umka Welbat  1   4
Affiliations

Valproic acid withdrawal ameliorates impairments of hippocampal-spatial working memory and neurogenesis

Wanassanun Pannangrong et al. J Zhejiang Univ Sci B. 2019.

Abstract

Valproic acid (VPA), an agent that is used to treat epileptic seizures, can cause spatial memory impairment in adults and children. This effect is thought to be due to the ability of VPA to inhibit neurogenesis in the hippocampus, which is required for learning. We have previously used an animal model to show that VPA significantly impairs hippocampal-spatial working memory and inhibits neuronal generation in the sub-granular zone of the dentate gyrus. As there are patient reports of improvements in memory after discontinuing VPA treatment, the present study investigated the recovery of both spatial memory and hippocampal neurogenesis at two time points after withdrawal of VPA. Male Wistar rats were given intraperitoneal injections of 0.9% normal saline or VPA (300 mg/kg) twice a day for 10 d. At 1, 30, or 45 d after the drug treatment, the novel object location (NOL) test was used to examine spatial memory; hippocampal cell division was counted using Ki67 immunohistochemistry, and levels of brain-derived neurotrophic factor (BDNF) and Notch1 were measured using western immunoblotting. Spatial working memory was impaired 1 and 30 d after the final administration, but was restored to control levels by 45 d. Cell proliferation had increased to control levels at 30 and 45 d. Both markers of neurogenesis (BDNF and Notch1 levels) had returned to control levels at 45 d. These results demonstrate that memory recovery occurs over a period of six weeks after discontinuing VPA treatment and is preceded by a return of hippocampal neurogenesis to control levels.

Keywords: Hippocampus; Neurogenesis; Spatial memory; Valproic acid.

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Conflict of interest statement

Compliance with ethics guidelines: Wanassanun PANNANGRONG, Apiwat SIRICHOAT, Trai WONGSIRI, Peter WIGMORE, and Jariya Umka WELBAT declare that they have no conflict of interest.

All institutional and national guidelines for the care and use of laboratory animals were followed. The animal investigation was accepted by the Animal Ethics Committee of Khon Kaen University, Thailand, based on the Ethics of the Animal Experiment of National Research Council of Thailand under permit number AEKKU 36/2557.

Figures

Fig. 1
Fig. 1
Timeline showing protocol of saline/VPA administration and experimental paradigm Saline/VPA was administrated twice daily for 10 d (a). The animals were allowed to recover for 30 (b) or 45 (c) d after saline/VPA injection. Their behavior was tested using the NOL test
Fig. 2
Fig. 2
Exploration time of the novel object location task after treatment (a) In the familiarization trial, objects A and B are the objects placed in separate locations in the arena. Each animal was allowed to explore the objects for 3 min. No significant difference was found in the exploration time of either object for any group (P>0.05). (b) There was a significant difference comparing the replica objects between familiar and novel locations in the control 10 d, control withdrawal 30 d, control withdrawal 45 d, and VPA withdrawal 45 d groups (* P<0.05) in the choice trial. In contrast, the rats receiving VPA and which were tested shortly after treatment and 30 d after treatment, did not show a significant difference (P>0.05). (c) For the preference index (PI) of control and VPA-treated rats performing the novel object location test shortly after VPA treatment and 30 and 45 d after treatment, a significant difference was found in comparison of all experimental groups (P=0.0185). The PI of rats showed a significant difference compared to 50% chance (dashed line) in the control 10 d, control withdrawal 30 d, control withdrawal 45 d, and VPA withdrawal 45 d groups (* P<0.05); however, there were no significant differences in the VPA 10 d or VPA withdrawal 30 d group (P>0.05). (d) Total exploration time of control, VPA 10 d, VPA withdrawal 30 d, and VPA withdrawal 45 d groups did not show a significant difference among groups (P>0.05). Data are expressed as mean±SEM (n=10)
Fig. 3
Fig. 3
Cell proliferation in the sub-granular zone (SGZ) of the dentate gyrus using Ki67-positive staining (a–f) In the SGZ, the nuclei of Ki67-positive cells were stained green and cells of the dentate gyrus were stained red. (g) Total numbers of Ki67-positive cells (mean±SEM, n=10) in the dentate gyrus were determined from cell counts. The number of Ki67-positive cells in the SGZ of rats receiving VPA was not significantly different from that of rats receiving saline (P>0.05) in both the VPA withdrawal 30 and 45 d groups. However, rats administered with VPA for 10 d showed a significantly lower number of Ki67-positive cells in comparison with the control 10 d group (* P<0.05) (Note: for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article)
Fig. 4
Fig. 4
Effects of VPA withdrawal on BDNF and Notch1 expression in the hippocampus (a) GAPDH was used as a loading control. Western blotting was used to measure Notch1 (120 kDa) and BDNF (15 kDa) protein expression. (b) Notch1 expression showed a significant difference among groups (P<0.0001). There was no significant difference between the control and VPA-treated rats at 45 d after treatment (P>0.05). However, Notch1 levels in both time scales after treatment showed significantly lower expression than controls (* P<0.05). (c) BDNF protein expression showed significant differences between groups (P<0.0001). Levels of BDNF in both the VPA-treated 10 d and VPA withdrawal 30 d groups revealed a significant decrease after treatment compared to controls (* P<0.05), but not in the VPA withdrawal 45 d group (P>0.05). Data are expressed as mean±SEM (n=10)
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