Memantine stimulated the formation of inhibitory synapses in <i>Fmr1</i>-KO CGCs.

<p>Representative photomicrographs of labeling of VGAT (arrow) in WT CGCs, KO CGCs and KO CGCs treated with memantine. Green, VGAT; Blue, Hoechst, scale bars, 25 µm. The histogram shows quantification of puncta size using image analysis and ratio of excitatory synapses to inhibitory synapse. WT, n = 23; <i>Fmr1</i>-KO, n = 25; <i>Fmr1</i>-KO+Memantine, n = 25. ^*p<0.05, ^**p<0.01.</p

Memantine fully restored the intensity of excitatory synapses to a normal range in <i>Fmr1</i>-KO CGCs.

<p>Representative photomicrographs of labeling of VGLUT1 (arrow) in WT CGCs, <i>Fmr1</i>-KO CGCs and <i>Fmr1</i>-KO CGCs treated with memantine. Red, VGLUT1; Blue, Hoechst, scale bars, 25 µm. The histogram shows quantification of puncta size using image analysis. WT, n = 23; <i>Fmr1</i>-KO, n = 25; <i>Fmr1</i>-KO+Memantine, n = 26. ^*p<0.05.</p

Memantine stimulated the formation of total synapses in <i>Fmr1</i>-KO CGCs.

<p>Representative photomicrographs of labeling of Syp (arrow) in WT CGCs, <i>Fmr1</i>-KO CGCs and <i>Fmr1</i>-KO CGCs treated with memantine. Green, Syp; Blue, Hoechst, scale bars, 25 µm. The histogram shows quantification of puncta size using image analysis. WT, n = 23; <i>Fmr1</i>-KO, n = 25; <i>Fmr1</i>-KO+Memantine, n = 26. ^**p<0.01.</p

Schematic representation of memantine and NMDA receptor.

<p>Schematic representation of memantine and NMDA receptor.</p

Memantine had little effect on CGCs migration.

<p>A. Cultured CGCs. Scale bar, 100 µm. B. CGCs stained with Calcein AM dye in modified Boyden chamber migration assay. Scale bar, 100 µm. C. Comparison of CGCs migration. The values are mean ±SE from three independent experiments with five replicates in each experiment. AFU: arbitrary fluorescence units.</p

Memantine increased the adhesion property in <i>Fmr1</i>-KO CGCs.

<p>Comparison of CGCs adhesion. The values are mean ±SE from three independent experiments with five replicates in each experiment, ^*p<0.05, ^**p<0.01. OD: optical density.</p

Neural genes altered ≥2 fold in the cerebral hemisphere of female pups from mothers having FA supplementation during gestation at 4 mg/kg in comparison to mothers at 0.4 mg/kg diet.

<p>Neural genes altered ≥2 fold in the cerebral hemisphere of female pups from mothers having FA supplementation during gestation at 4 mg/kg in comparison to mothers at 0.4 mg/kg diet.</p

High maternal FA alters the expression of Slc17a7p and Vgll2p in the cerebral hemispheres of female newborn pups.

<p>Total cell lysates from the cerebral hemispheres of female (F) newborn pups were prepared from both 0.4 mg/kg and 4 mg/kg FA diet groups and proteins were resolved on 10% Tris/HEPES/SDS-polyacrylamide gels. The blot was probed with Slc17a7p antibody (<b>A</b>, <b>B</b>) or with Vgll2p antibody (<b>C</b>, <b>D</b>). To confirm equal protein loading, the membranes were re-probed with GAPDH/β-tubulin antibodies. The left panel represents one representative blot and the right panel shows densitometric evaluation of two independent experiments. Densitometric evaluation of the bands were calculated by using the Image J software and normalized to the densities of GAPDH//β-tubulin. The values corresponding to 0.4 mg/kg FA diet were arbitrarily set to 100%, and the 4 mg/kg values are presented relative to this. The densitometric values represent the mean, and the error bar represents the inter-variability among independent experiments.</p

A: Quantitative RT-PCR confirmation of microarray data.

<p>Relative expression of the <i>Cpn2</i>, <i>Zfp353</i>, <i>Htr4</i>, <i>Nkx6-3</i> and <i>Xist</i> transcripts in the cerebral hemispheres of male newborn pups, from mothers supplemented with FA at 4 mg/kg of diet. The results were normalized to <i>Hprt</i> transcript expression, and expressed as relative values in comparison to corresponding transcripts from newborn pups with FA at 0.4 mg/kg diet. Results represent means ± S.D; asterisks denote statistically significant change (***<i>P</i><0.001, **<i>P</i><0.01 and *<i>P</i><0.05). <b>B</b>): <b>Quantitative RT PCR confirmation of microarray data</b>. Relative expression of the <i>Vgll2</i>, <i>Leprel1</i>, <i>Nfix</i> and <i>Slc17a7</i> transcripts from the cerebral hemispheres of female newborn pups, from mothers supplemented with FA at 4 mg/kg of diet. The results were normalized to <i>Gapdh</i> transcript expression, and were expressed as relative values in comparison to corresponding transcripts from newborn pups with FA at 0.4 mg/kg diet. Results represent means ± S.D; asterisks denote statistically significant change (***<i>P</i><0.001, **<i>P</i><0.01 and *<i>P</i><0.05).</p

The schematic diagram of the experimental design for this study.

<p>The schematic diagram of the experimental design for this study.</p