Gestational Exposure to Anticonvulsant Valproic Acid (VPA) Stimulates Forebrain Neurogenesis and Leads to Postnatal Brain Enlargement

Background: Exposure of the developing embryo to VPA is known to induce developmental defects in central nervous system, including signs of autism spectrum disorder (ASD). Mechanistic studies in developmental models have identified many signaling pathways including AP-1 transcription factor, P-ERK, GSK3b, ion channels, HDAC and epigenetic changes. Although recent studies of chronic drug exposure in adult brain models of depression and neurogenesis suggest VPA stimulates mitogenesis via P-ERK and HDAC, earlier studies in neural crest, neuroblastoma and glial cell lines indicate VPA inhibits proliferation and promotes cell differentiation. We now define effects of VPA on embryonic rat cerebral cortical precursors both in culture and in developing animals.

Objectives: To define the developmental effects of VPA on forebrain neurogenesis through in vitro and in vivo experiments.

Methods: In vitro, embryonic day 14.5(E14.5) cerebral cortex precursors were cultured without or with VPA. In vivo, pregnant moms at E16.5 received 2 doses of VPA/day at 300mg/kg body weight for a total of 1~5 doses. DNA synthesis was analyzed using incorporation of cell cycle markers [³H]-thymidine and BrdU; VPA effects on neurite outgrowth were analyzed using phase microscopy of live cells. Second messenger and cell cycle mechanisms were assayed using Western blotting. The differentiated cell populations of the brain were assessed at postnatal day 21 (P21) through unbiased stereology and immunohistochemistry for cell type-specific markers. Brains were weighed at P21, with 35 rats for the control and 33 rats for VPA exposed groups.

Results: VPA had similar effects on DNA synthesis of cortical precursors both in culture and in developing brain, increasing [³H]-thymidine incorporation by 25% and 23%, and BrdU labeling index by 20% and 53.7%, respectively, compared to control (N=10-15/group; p<0.01). These observations indicate that VPA increased the entry of cells into mitotic S phase. In addition, VPA increased the percent of cells expressing precursor marker nestin by 68.8% while decreasing process-bearing cells by ~50%, suggesting VPA inhibited the transition from proliferation to differentiation. At the level of mechanism, VPA stimulated cell cycle regulators, increasing levels of both cyclin-D3 and E proteins as early as 1h after exposure, suggesting that VPA rapidly alters cell cycle mechanisms to enhance neurogenesis. Further, to define mediating pathways we assessed second messengers and found VPA increased levels of acetylated histone H3, but not levels of activated ERK, AKT, GSK3 or PKC. Finally, the stimulatory effects of VPA in vitro were directly relevant to brain development in vivo: VPA exposure during gestation increased brain weight measured at P21 by 4% and total cell number by 13.8%. The increase in total cell number consisted primarily of neurons, as indicated by pan neuronal marker, NeuN, and neuronal layer-specific markers, Cux1 and Tbr1, which were increased by 14.3%, 26.4%, and 15.4%, respectively.

Conclusions: Embryonic exposure to VPA maintains forebrain precursors in the cell cycle and reduces neuronal differentiation. VPA likely acts via inhibiting histone de-acetylation and increasing cyclin-D3 and E to promote forebrain neurogenesis. These developmental effects of VPA may be relevant to brain enlargement observed in some cases of ASD.