Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons (2018)
Figure 4

Figure 4 - ErbB4 but not ErbB4 kinase activity mediates Nrg3 functions in synaptogenesis.

A) His6-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.

B) Quantification 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, D) Neurons from Nrg3-/-;ErbB4-/-;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, (C',D') show the SypCFP signal.

E-G) Quantification of Synapse preference towards 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.

I) Co-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.

J) Schematic 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 Nrg3∆EGF (N3 and N3∆, 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-M) Sample traces of paired recordings (K), quantification of the amplitude of the evoked excitatory postsynaptic currents (eEPSC) (L) and quantification of paired pulse ratios (M).

Data information: Scale bars: 5µm (D') and 25µm (H'', I'''). (E-G) Data from n=27 (LV-Nrg3, ErbB4wt), n=27 (LV-Nrg3, ErbB4kd), n=29 (LV-Nrg3∆EGF, not shown) samples from three independent experiments were analyzed using 1-way-ANOVA with Tukey's multiple comparisons test to assess statistical significance (ns= not significant). (L,M) Data from n=8 (LV-Nrg3), n=9 (LV-Nrg3∆EGF) from more than 3 independent experiments were analyzed using (L) Mann-Whitney U test (unpaired, 2-tailed) and (M) t-test (unpaired, 2-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.