Fig. 4
Hippocampal neurons isolated from VPA animals formed fewer glutamatergic synapses after active synaptogenesis in vitro. Hippocampal neurons isolated from control and VPA animals were cultured in the absence of glia. a GABAergic synapses immunostained for pre-synaptic marker GAD-67 and overlapped with phase contrast images (DIV14). Glutamatergic synapses were immunostained (DIV14) for b vesicular glutamate transporter vGLUT1 (3 × magnification inset depicts vGLUT1 synaptic puncta), c post-synaptic marker PSD-95 (3 × magnification inset shows PSD-95 synaptic puncta) and d NMDA receptor subunit NR1 (3 × magnification inset shows NR1/MAP2 co-staining). e Puncta number quantification revealed no statistical changes in GAD-67 and a reduction in vGLUT1, PSD-95 and NR1 in neurons from VPA animals. f vGLUT1 and NR1 individual puncta exhibited smaller size in neurons from VPA animals. g Total puncta area was coincident with puncta number. h Decreased NR1 puncta number regardless dendritic tree reduction in neurons from VPA animals. i Experimental protocol for FM4-64 synaptic loading and unloading. j FM4-64 loading images revealed a reduced number of active pre-synaptic terminals in neurons from VPA animals. k Analysis of FM4-64 puncta parameters confirmed fewer functional synapses, smaller functional vesicular pools and a diminished total puncta area in neurons from VPA animals. l Representative images at different time points showed conserved unloading capacity of neurons from VPA animals. Arrows depict fluorescent puncta over time analyzed in unloading studies; t0 = 5 s, t1 = 25 s and t2 = 50 s. m Conserved unloading kinetics in neurons from VPA animals (Control: m: − 0.2169 s−1 and VPA: m: − 0.2498 s−1, no significant differences between slopes, Student’s t test). Results are expressed as mean values (± SD, e–h control n = 20–50 neurons, VPA n = 20–50 neurons from 2 independent cultures; k control n = 39 neurons, VPA n = 36 neurons from 2 independent cultures). **p < 0.01; ***p < 0.001 between groups by Student’s t test and ns: non-significant; #p < 0.05; ##p < 0.01; ###p < 0.001 between groups by Mann–Whitney U Test. Scale bars: 50 µm (a–d, j); 5 µm (l)