Nrg3 promotes the formation of excitatory synapses onto ErbB4+ interneurons in vitro.Figure 3 A) Schematic display of the culture model using hippocampal neurons that were infected by a lentivirus co-expressing a synaptophysin-CFP fusion protein (SypCFP) together with either Nrg3 or Nrg3∆EGF at a low titer so that 10-20% of the hippocampal neurons were transduced with the lentivirus (depicted as a light blue colored neuron with a red nucleus). Presynaptic boutons formed by non-transduced (depicted as a light grey neuron with a white nucleus) and transduced neurons can be distinguished by the absence or presence of CFP labeling (grey and blue boutons, respectively)  B) Hippocampal Nrg3-/- neurons after transduction with lentivirus co-expressing SypCFP/Nrg3 were analyzed by immunocytochemistry. (B) Antibodies against CFP, ErbB4, Nrg3 and vGlut1/2; (B), (B') and (B'') show the same image and display SypCFP/ErbB4 and SypCFP/Nrg3 and SypCFP/vGlut1/2 signals, respectively. The images were assigned false colors for better visualization of the signals. SypCFP, Nrg3 and ErbB4 co-localize in excitatory vGlut1/2+ synapses C, D) Correlation analyses of the levels of ErbB4 and Nrg staining in vGlut1/2+ synapses; every dot corresponds to one synapse. Cultured Nrg3-/- neurons were transduced with either SypCFP/Nrg3 (black in C,D lentiviruses C, D) Correlation analyses of the levels o ErbB4 and SypCFP (F) staining in vGlut1/2+ synapses; every dot corresponds to one synapse. Cultured Nrg3-/- neurons were transduced wit SypCFP/Nrg3∆EGF (red in D) lentiviruses  E, F) Hippocampal neurons were transduced with (E) SypCFP/Nrg3 or (F) SypCFP/Nrg3∆EGF viruses and immunostained for vGlut1/2, ErbB4 and SypCFP. Both viruses express similar amounts of protein (Fig. EV2). In cultures transduced with the SypCFP/Nrg3 virus, synaptic enrichment of ErbB4 and SypCFP was very pronounced, but not in cultures transduced with the SypCFP/Nrg3∆EGF virus. Note that (E-E''') and (F-F''') display the same images but color channels were separated for better visualization  G) ErbB4 enrichment in synapses formed in hippocampal cultures after infection with either the SypCFP/Nrg3 or the SypCFP/Nrg3∆EGF virus. We compared neuronal cultures of different genotypes (Nrg3-/- and wildtype cultures indicated by N3- and wt, respectively). ErbB4 enrichment was calculated as the level of ErbB4 in synapses formed by infected neurons (vGlut1/2+/SypCFP+ boutons) divided by the level of ErbB4 in synapses formed by uninfected neurons (vGlut1/2+/SypCFP- boutons). The dashed line indicates a ratio of 1 at which SypCFP positive and negative boutons would behave identical H) Quantification of the preference for neurons expressing SypCFP/Nrg3 or SypCFP/Nrg3∆EGF to form synapses on ErbB4+ neurons. The synapse preference was calculated as the proportion of vGlut1/2 boutons containing SypCFP that were present on ErbB4-positive neurons divided by the proportion of such boutons present on ErbB4-negative neurons. I) Quantification of SypCFP enrichment in excitatory synapses on ErbB4+ neurons. Enrichment was determined as the ratio between SypCFP levels in SypCFP+/vGlut1/2 boutons on ErbB4-positive and -negative neurons  J, K) Hippocampal neurons from Nrg3-/- (J) and Nrg3-/-;ErbB4-/- (K) mice were transduced with the SypCFP/Nrg3 expressing virus and immunostained for Nrg3, ErbB4/parvalbumin and SypCFP. Nrg3 and SypCFP were strongly enriched in synapses on dendrites of PV neurons in Nrg3-/- (J) but not Nrg3-/-;ErbB4-/- cultures (K)  I) Quantification of SypCFP enrichment in excitatory synapses on ErbB4+ neurons. Enrichment was determined as the ratio between SypCFP levels in SypCFP+/vGlut1/2 bouton L-N) Quantification of synapse preference (L), enrichment of SypCFP (M) and of Nrg3 (N) in Nrg3-/- (N3-) and Nrg3-/-;ErbB4-/- (N3-E4-) hippocampal cultures transduced with the SypCFP/Nrg3 lentivirus. Synapse preference (L) was calculated as the proportion of vGlut1/2 boutons containing SypCFP present on parvalbumin-positive neurons divided by the proportion of such boutons present on parvalbumin-negative neurons. SypCFP enrichment (M) was determined as the ratio of SypCFP levels in SypCFP/vGlut1/2+ boutons present on parvalbumin-positive and -negative neurons. Nrg3 enrichment (N) was determined as the ratio of Nrg3 levels in SypCFP+/vGlut1/2+ boutons present on parvalbumin-positive and -negative neurons