Fig 2: Ang II enhances synaptic connectivity of R312H primary neurons.A Representative images showing DIV14 primary cortical neurons of the indicated genotypes co-stained with Syn-1 (green), PSD-95 (red) to visualize functional synapses (arrows), and DAPI (blue), in control or incubated in the presence of 0.7 µM Ang II. B Magnifications are taken from the images on the left. Scale bars 50 µm for small magnification images and 10 µm for large magnification images. C Average number of dendritic spines for the cells of the indicated genotypes incubated in the absence/presence of 0.7 µM Ang II. For a single neuron, the number of spines was counted over a single dendrite for a continuous length of ~100 µm. N = 20 neurons/genotype from: WT: 4 pups; HETERO: 6 pups; HOMO: 6 pups. D Number of functional synapses in co-cultures of primary neurons of the indicated genotypes. Cells were co-stained with SYN-1, PSD-95 and DAPI and functional synapses were identified by SYN-1 and PSD-95 signals co-localizations. N = WT (cnt.-Ang): 8–9 cultures; HETERO: 13–14 cultures; HOMO: 8–7 cultures obtained from: 3 WT, 5 HETERO and 4 HOMO pups. Two technical replicates/culture. Data were analyzed with ImageJ/Fiji software and normalized to WT in control conditions. *P < 0.05; ***P < 0.001, and ****P < 0.0001 (one-way ANOVA, Tukey’s post hoc).