Fig 2: Lentiviral expression of Nrg3 and Nrg3ΔEGF A, BSchematic display of the expression cassettes. The synapsin I (Syn1) promoter is used for neuronal expression. Coding sequences for red fluorescence protein with a nuclear localization sequence (nRFP) and a fusion protein of synaptophysin and the cyan fluorescent protein (SypCFP) are followed by coding sequences for Nrg3 (A) or Nrg3ΔEGF (B). nRFP, SypCFP, and Nrg3/Nrg3ΔEGF coding sequences are separated by 2A sequences.C, DComparison of Nrg3 (C) and SypCFP (D) expression levels in Nrg3 −/− neurons that were transduced with the Nrg3/SypCFP (middle lanes) and Nrg3ΔEGF/SypCFP lentiviruses (right lanes) at high titer, respectively, by Western blot analysis using antibodies against Nrg3 (C) and GFP (D). The lanes indicated by (−) show lysates from non‐transduced neurons as negative control. Both lentiviruses express similar protein levels of Nrg3/Nrg3ΔEGF and SypCFP. Note that the main Nrg3 band in lysates from neurons transduced with the Nrg3ΔEGF/SypCFP lentivirus is smaller due to the deletion.EImmunocytochemical comparison of endogenous and lentivirally expressed Nrg3 levels in vGlut1/2+ excitatory synapses on ErbB4+ neurons in cultures of wildtype (wt, left) or Nrg3 −/− neurons transduced with the SypCFP/Nrg3 lentivirus (right). Fluorescence levels were normalized to the mean fluorescence level in wildtype neuron cultures. The Nrg3 lentivirus produced 2.17 ± 0.14‐fold higher Nrg3 levels than wt neurons, and n = 13 and 11 neurons were analyzed in cultures from wildtype and Nrg3 mutants, respectively. Data are presented as box plots with Tukey's whiskers and outliers. Plus symbols denote the mean.FHippocampal Nrg3 −/− neurons after transduction with lentivirus expressing SypCFP/Nrg3 were analyzed by immunocytochemistry using antibodies against CFP, ErbB4, vGlut1/2, and GluA. Panels show the same image and display SypCFP/ErbB4, SypCFP/vGlut1/2, and SypCFP/GluA signals, respectively. The images were assigned false colors for better visualization of the signals. SypCFP, ErbB4, vGlut1/2, and GluA co‐localize in excitatory synapses. Scale bar: 5 μm. Source data are available online for this figure.

Fig 3: Nrg3 locates to synapses on ErbB4+ interneurons in vitro Hippocampal neurons cultured for 21 days were analyzed by immunocytochemistry using antibodies against Nrg3, ErbB4, and vGlut1, demonstrating co‐clustering of Nrg3 and ErbB4 in excitatory synapses. Note that (A) displays Nrg3/vGlut1 and (A′) Nrg3/ErbB4 signals of the same triple‐stained image.Quantification of the proportions of vGlut1/2+ and vGAT+ presynaptic boutons that are positive for Nrg3 (two‐tailed Wilcoxon test, ****P < 0.0001, n = 18 from two independent experiments).Quantification of Nrg3 immunofluorescence levels in vGlut1/2+ and vGAT+ presynaptic boutons that are positive for Nrg3 (A.U., arbitrary units, two‐tailed Wilcoxon test, ****P < 0.0001, n = 17 from two independent experiments).Quantification of vGlut1/2+ synapse numbers on secondary dendrites of PV interneurons in cultures from wildtype (wt) and Nrg3 −/− mice (21–24 days in culture). Data from n = 27 (wt) and n = 26 (Nrg3 −/−) neurons from three independent experiments were analyzed using two‐tailed Mann–Whitney U‐test, ****P < 0.0001.Immunocytochemical analysis of a mixed culture containing neurons of wildtype and ErbB4 −/−;Gad1/Gad67‐GFP animals triple‐stained against ErbB4, GFP, and Nrg3. To improve the visibility, ErbB4 and Nrg3 signals are also shown separately (E′, E″). ErbB4+ interneurons from wildtype animals are indicated by filled arrowheads, and interneurons from ErbB4 −/− mice were identified by GFP and are indicated by open arrowheads.Immunocytochemical analysis of a mixed culture containing neurons from wildtype and Nrg3 −/− ;Gad1/Gad67‐GFP animals triple‐stained against ErbB4, GFP, and Nrg3. To improve visibility, ErbB4 and Nrg3 signals are also shown separately (F′, F″). ErbB4+ interneurons from Nrg3 −/− animals are GFP‐positive and are indicated by open arrowheads; ErbB4+ interneurons from wildtype animals are GFP‐negative and are indicated by filled arrowheads.Data information: Scale bars: 10 μm (A′) and 50 μm (F″). Data in (B–D) are presented as box plots with Tukey's whiskers and outliers; means are indicated by plus symbols.

Fig 4: Nrg3 promotes the formation of excitatory synapses onto ErbB4+ interneurons in vitro ASchematic 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 Nrg3ΔEGF 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 gray neuron with a white nucleus) and transduced neurons can be distinguished by the absence or presence of CFP labeling (gray and blue boutons, respectively).BHippocampal 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, 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, DCorrelation analyses of the levels of ErbB4 and Nrg3 (C) or ErbB4 and SypCFP (D) staining in vGlut1/2+ synapses; every dot corresponds to one synapse. Cultured Nrg3 −/− neurons were transduced with either SypCFP/Nrg3 (black in C, D) or SypCFP/Nrg3ΔEGF (red in D) lentiviruses.E, FHippocampal Nrg3 −/− neurons were transduced with (E) SypCFP/Nrg3 or (F) SypCFP/Nrg3ΔEGF 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/Nrg3ΔEGF virus. Note that (E–E‴) and (F–F‴) display the same images, but color channels were separated for better visualization.GErbB4 enrichment in synapses formed in hippocampal cultures after infection with either the SypCFP/Nrg3 or the SypCFP/Nrg3ΔEGF 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 SypCFP‐negative boutons would behave identical.HQuantification of the preference for neurons expressing SypCFP/Nrg3 or SypCFP/Nrg3ΔEGF 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.IQuantification 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 ErbB4‐negative neurons.J, KHippocampal 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).L–NQuantification 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 parvalbumin‐negative neurons. Nrg3 enrichment (N) was determined as the ratio of Nrg3 levels in SypCFP+/vGlut1/2+ boutons present on parvalbumin‐positive and parvalbumin‐negative neurons.Data information: Scale bars: 5 μm (B″, F‴, K‴). (G–I) Data from n = 31 (LV‐Nrg3, N3 −), n = 29 (LV‐Nrg3, wt), and n = 29 (LV‐Nrg3ΔEGF) samples from more than three independent experiments were analyzed. (L–N) Data from n = 55 (LV‐Nrg3, N3 −), n = 53 (LV‐Nrg3, N3 − E4 −), and n = 45 (LV‐Nrg3ΔEGF, not shown in graphs) samples from more than three independent experiments were analyzed. Data in (G–I, L–N) are presented as box plots with Tukey's whiskers and outliers; means are indicated by plus symbols. One‐way ANOVA with Tukey's multiple comparisons test was performed to assess statistical significance (****P < 0.0001, ***P < 0.001, **P < 0.01).

Fig 5: ErbB4 but not ErbB4 kinase activity mediates Nrg3 functions in synaptogenesis AHis6‐tagged EGF domains of Nrg1β and Nrg3 were added to neuronal cultures at the indicated concentrations. Western blot analyses of ErbB4 immunoprecipitates using antibodies against phospho‐tyrosine (pY) and ErbB4, and of whole lysates using antibodies against pErk1/2 and pAkt.BQuantification of Nrg1 and Nrg3‐dependent changes in phosphorylation of ErbB4, Erk1/2 and Akt normalized to input, where 100% corresponds to the maximal change observed for His6‐Nrg1β. Data represent mean ± SD from three independent experiments.C, DNeurons from Nrg3 −/−;ErbB4 flox/− ;vGAT‐Cre mice were transduced with a lentivirus expressing either (C) ErbB4wt or (D) ErbB4kd (kinase‐dead ErbB4) in a Cre‐dependent manner, and with a second virus at a low titer expressing Nrg3/SypCFP. Immunocytochemical analysis of ErbB4 and SypCFP; (C, C′) and (D, D′) each show the same image, and (C′, D′) show the SypCFP signal.E–GQuantification of Synapse preference toward ErbB4+ neurons (E), ErbB4 enrichment (F), and SypCFP enrichment (G) in synapses on neurons expressing wildtype ErbB4 (wt) or ErbB4kd (kd).H Nrg3 −/− neuron cultures were transfected with a plasmid that strongly overexpresses Nrg3 and GFP and immunostained with antibodies against ErbB4, GFP, and GluA; (H) shows the triple‐stained image, while (H′) and (H″) only show ErbB4 and GluA signals, respectively.ICo‐cultures of Nrg3 −/− neurons with HEK293 cells expressing Nrg3. Cultures were stained with antibodies against Nrg3, ErbB4, and GluA. Note that Nrg3, ErbB4, and GluA are enriched at the contact sites between Nrg3‐expressing HEK293 cells and ErbB4+ neurons.JSchematic diagram of the strategy used to obtain paired recordings in cultures obtained from Nrg3 −/−;Gad1/Gad67GFP mice. Inhibitory neurons were identified by GFP expression. The cultures were transduced with lentiviruses expressing either Nrg3 or Nrg3ΔEGF (N3 and N3Δ, respectively) together with nuclear red fluorescence protein (nRFP). Transduced neurons, identified by nuclear RFP fluorescence, were stimulated, and evoked responses were recorded from interneurons that were GFP‐positive and nRFP‐negative at 12–15 days in culture.K–MSample traces of paired recordings (K), quantification of the amplitude of the evoked excitatory postsynaptic currents (eEPSCs) (L), and quantification of paired pulse ratios (M).Data information: Scale bars: 5 μm (D′) and 25 μm (H″, I‴). (E–G) Data from n = 27 (LV‐Nrg3, ErbB4wt), n = 27 (LV‐Nrg3, ErbB4kd), and n = 29 (LV‐Nrg3ΔEGF, not shown) samples from three independent experiments were analyzed using one‐way ANOVA with Tukey's multiple comparisons test to assess statistical significance (ns = not significant). (L, M) Data from n = 8 (LV‐Nrg3) and n = 9 (LV‐Nrg3ΔEGF) from more than three independent experiments were analyzed using (L) Mann–Whitney U‐test (unpaired, two‐tailed) and (M) t‐test (unpaired, two‐tailed) to assess statistical significance (**P < 0.01). Data in (E–G, L, M) are presented as box plots with Tukey's whiskers and outliers. The means are indicated by plus symbols.Source data are available online for this figure.