Fig. 3

Hippocampal neurons isolated from VPA animals mimicked, in vitro, the synaptic pattern found in vivo. Hippocampal neurons isolated from control and VPA animals were cultured in the absence of glia. a Synaptophysin (SYN) immunostaining of hippocampal neurons at DIV7, 10 and 14. Insets (×4 magnification) detail SYN immunostaining pattern. b Neurons from VPA animals showed conserved SYN puncta number at DIV7 but reduced at DIV10 and 14 compared to controls. c No deep changes were observed in synaptic size throughout differentiation. d Quantification of total SYN puncta area matched puncta number results. e No statistical differences in total SYN expression by western blot, ruling out changes in SYN expression. f Actin arborization was similar between groups throughout neuronal differentiation. g MAP-2 immunostaining and SYN/MAP-2 co-staining (×3 magnification insets) at DIV14. h MAP-2 immunoreactive area quantification revealed a preserved dendritic tree at DIV7 but a smaller one at DIV14 in neurons from VPA animals. i SYN puncta number decreased regardless of MAP-2 immunostaining reduction in neurons from VPA animals at DIV14. j, k Decreased PSA-NCAM immunoreactivity in hippocampal neurons from VPA animals (DIV14). l Increased NCAM expression in hippocampal neurons from VPA animals (DIV14). Results are expressed as mean values (± SD; b-d, h, k control n = 30–60 neurons, VPA n = 30–60 neurons from 3 independent cultures; e, l control n = 3 lysates, VPA n = 3 lysates from 3 independent cultures; f, i: control n = 30–40 neurons, VPA n = 30–40 neurons, except for f DIV10 n = 15 neurons, from 2 independent cultures). b, c, d, f and h ns: non-significant; ^#p < 0.05; ##p < 0.01 between groups by Kruskal Wallis; e, i, l ns: non-significant; **p < 0.01; ***p < 0.001 between groups by Student’s t test; k ^###p < 0.001 between groups by Mann–Whitney U Test. Scale bar: 50 µm