Figure 7: Effect of VPA treatment is occluded by blockade of NMDAR activation in in vitro VPA model.

(a) Scheme of procedure of experiments using the NMDAR antagonist AP5. During the first 6 days of culture, neocortical neurons were exposed to 2 mM VPA or vehicle. Subsequently, AP5 (200 μM) was added to from day-in-vitro (DIV) 9–15. (b,c) Relative synaptic protein expression levels for neuronal preparations treated as described in panel A. The alteration in GAD65, GAD67 and vGluT1 by VPA is occluded by AP5 treatment (VPA; n = 5–6, AP5; n = 7–13, VPA + AP5; n = 7–9). (d) Representative images of endogenous GAD65, vGAT and GluA2/3 protein immunocytochemistry in VPA-, AP5-, or VPA plus AP5-treated cultures. Cultured cortical neurons are triple-stained with anti-GAD65 (red), vGAT (green), and GluA2/3 (blue) antibodies. Scale bar = 20 μm. Blots were cropped to display the relevant molecular weight range. Uncropped blots are presented in Fig. S4f. (e,f) Co-application of VPA and AP5 does not have an additive effect on reducing the number of GAD65 puncta. The number of GAD65 perisomatic puncta per cell (e) was reduced in VPA, AP5 or VPA + AP5 treated neurons, whereas intensity of GAD65 puncta (f) was not altered. 15 or 17 images per condition were analyzed from 3 independent experiments.