Development of synapses in postnatal day 0 (P0) hippocampal cultures. (A) Representative images of immunofluorescence staining of Microtubule associated protein 2 (MAP2; dark blue) and Vesicular glutamate transporter 1 (VGLUT1; green) at 8 days in vitro (8 DIV) (left) and 14 DIV (right). Scale bar = 2 μm. (B) Quantification of presynaptic inputs per area of dendrite at 8 DIV and 14 DIV (± standard error of the mean). To ensure equivalent staining, cells were fixed at respective days and then stained with a single reaction. The number of inputs at 14 DIV is normalized to the number of inputs at 8 DIV. n = 47 fields of view. *p < 0.001, Students t-test. (C-F) Colocalization of VGLUT1 puncta (C, F, red) with MAP2 (E, F, blue) and Membrane associated guanylate kinase (MAGUKs) (D, F, green). Scale bar = 2 μm.