DG-CA3 synapses are reduced in NeuroD2 null neurons in vitro. Hippocampal neurons from P0 WT and NeuroD2 null mice were cultured at low density on a glial monolayer and fixed for immunostaining at 16 days in vitro for the following markers: (A, E) PSD-95; (B, F) synaptoporin (SPO); (C, G) Vglut1; (D, H) merged images of staining for synaptically localized proteins with PSD-95 in red, SPO in green and Vglut1 in blue. (I) Quantification of DG synapse density by triple co-localization of synaptic markers on large primary dendrites (WT, n = 25; knockout (KO), n = 21). Cultures were also immunostained with the DG- and CA1-specific nuclear marker CTIP2 to investigate the localization of synaptic terminals onto putative hippocampal CA3 neurons in vitro. (J, M, N, Q) CTIP2-negative neurons, putative CA3 neurons, lacked staining in their nucleus; the asterisk indicates lack of nuclear staining for CTIP2. (R, U, V, Y) CTIP2-positive neurons, non-CA3 neurons, had staining that included the nucleus. (K-M, O-Q, S-U, W-Y) SPO and Vglut1 immunohistochemistry were used to localize MF terminals onto the proximal dendrites of these neuron types as indicated. (Z) Quantification of DG terminal density onto CTIP2-negative, putative CA3 neurons (WT, n = 14; KO, n = 16), and CTIP2-positive non-CA3 neurons (WT, n = 9; KO, n = 8). ***P < 0.01, t-test; n.s., not significant. Scale bars: 10 μm. Error bars represent ± standard error of the mean. (Note that the secondary antibody used to detect CTIP2 cross-reacts with an additional primary antibody against CamKII used as a dendritic marker, leading to dendritic signal in the red channel that did not interfere with assessing nuclear CTIP2 signal).