clozapine-n-oxide br Results br Discussion The cognitive def

Results
Discussion The cognitive deficits associated with prenatal VPA exposure might not due solely to the reduced neurogenesis with the abnormal neuronal morphology in the hippocampus, and there is a possibility that the low freezing responses in fear associative tests were contributed by the deficiency in amygdala, nociception, and/or motoric functions. Nevertheless, our data suggested that the reduced neurogenesis associated with the abnormal neuronal morphology in the hippocampus were very likely to be correlated with the observed cognitive deficits for several reasons. First, voluntary running is well known for its effect on enhancing both adult neurogenesis in the DG of the hippocampus and hippocampus-dependent learning and memory (Zhao et al., 2008), and this voluntary running could recover the cognitive deficit, if not all, in VPA-treated mice with reduced neurogenesis in the DG. Second, to the best of our knowledge, there are no reports to date that show a direct contribution of voluntary running to the enhancement of amygdala function that subsequently leads to an improvement in the cued fear response. Third, based on experiments that we have conducted, we could not find any significant differences in amygdala size and in the expression levels of cortical layer-specific genes of MC- and VPA-treated mice, with or without voluntary running. Fourth, total traveled distance in the open field, elevated plus and the Y-maze tests, and the number of light/dark transitions were not significantly different between MC- and VPA-treated mice, although in our earlier experiment some of these parameters showed modest differences. Moreover, in fear associative test, VPA-treated mice move similarly to MC-treated mice before the start of the tone (pre-tone), which indicate that motor deficiency is unlikely to be the main cause of low freezing responses in VPA-treated mice. Fifth, MC- and VPA-treated mice have similar basal nociceptive response and startle response to electric footshock during the conditioning for fear-associative test, thus it seems unlikely that VPA-treated mice have abnormal nociception and cannot sense the foot shock. Taking these facts into consideration, we therefore suggested that the reduced neurogenesis associated with the abnormal neuronal morphology in the hippocampus were very likely to be a critical cause of the observed cognitive deficits. However, we still cannot completely exclude the possibility that changes in other clozapine-n-oxide areas may also contribute to the deficits, warranting further future investigation. We and others have shown previously that VPA treatment induces neuronal differentiation but suppresses glial differentiation of cultured multipotent NPCs (Hsieh et al., 2004; Balasubramaniyan et al., 2006; Murabe et al., 2007; Abematsu et al., 2010; Juliandi et al., 2012). We have now demonstrated that VPA also increases histone acetylation in the embryonic forebrain and induces neuronal differentiation of embryonic NPCs. Previous study have shown that VPA promotes neuronal differentiation by increasing histone H4 acetylation at proneural gene promoters (Yu et al., 2009). However, several studies have suggested that the activation of GSK-3β/β-catenin and/or ERK pathway is the main cause for the increase neurogenesis of NPCs by VPA (Yuan et al., 2001; Jung et al., 2008; Hao et al., 2004; Go et al., 2012). It has been suggested that VPA might have various cellular effects that will depend on the context of VPA usage and/or cell type and experimental design used in the study, which warrant further research to reveal the connection between these effects (Kostrouchová et al., 2007; Rosenberg, 2007). We suggest that gene expression change caused by VPA is attributable mainly to its HDAC-inhibiting activity. To date, more than a dozen HDACs have been characterized and they are classified into at least three major groups. In particular, HDAC1 and HDAC2, belonging to the class I group, have been reported to regulate NPC differentiation (Sun et al., 2011). NPCs express high levels of HDAC1 and some of them also express low levels of HDAC2 (MacDonald and Roskams, 2008). Interestingly, as NPCs are committed to the neuronal lineage, expression of HDAC2 is upregulated while that of HDAC1 is downregulated and becomes undetectable in most post-mitotic neurons (MacDonald et al., 2005; MacDonald and Roskams, 2008); on the other hand, HDAC1 expression is sustained in the majority of cells in glial lineages (astrocytes and oligodendrocytes), in which HDAC2 is not detected (Shen et al., 2005; MacDonald and Roskams, 2008). Moreover, HDAC2, but not HDAC1, was found to inhibit astrocytic differentiation (Humphrey et al., 2008). Therefore, although VPA is capable of inhibiting both HDAC1 and HDAC2 (Kazantsev and Thompson, 2008), it is tempting to speculate that the main target of VPA in HDAC inhibition-mediated neuronal differentiation of NPCs is HDAC1. It will be of interest to explore this possibility in a future study.