FIGURE 2

Lithium and valproic acid (VPA) induce activation of brain-derived neurotrophic factor promoter IV. (a) The scheme delineates the promoter IV region and each number indicates bp position from the transcriptional initiation site of the exon. The enlarged promoter region between -20 and -80 bp contains three calcium-responsive elements (CaREs). (b) Firefly luciferase reporter vectors, pGL3 basic, were constructed to certain specific lengths of the promoter, namely, -2000, -710, -170 or -80 bp. 'Basic' refers to pGL3 basic without promoter. Cortical neurons at 7 days in vitro (DIV) were treated with 1 mM LiCl or 0.5 mM VPA for 3 days. At 8-DIV, neurons were transfected with each reporter vector (pGL3-2000 luc, -710 luc, -170 luc, -80 luc or -Basic) and pRL-TK using Lipofectamine 2000. Cell lysates were prepared at 10-DIV and assayed for luciferase activity. The values were normalized to those of pGL3 basic. (c) Cortical neurons were treated with indicated concentrations of LiCl, VPA, or their vehicle (normal saline), and transfected with pGL3-710 luc. The values were normalized to those of vehicle-treated cells. Quantified data are expressed as meanss.e.m. from four experiments. ^* P<0.05; ^** P<0.01, ^*** P<0.001 compared with respective vehicle control. (d) Cortical neurons at 8-DIV were treated with LiCl at indicated concentrations for 48 h. Cell lysates were then prepared for western blotting of p-GSK-3 (Ser21/9) and -actin. (e) Cortical neurons at 8-DIV were treated with indicated concentrations of VPA for 48 h prior to western blotting analysis of acetylated histone H3 (AcH3), HDAC1 and -actin.