Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons
2018; Springer Nature; Volume: 37; Issue: 17 Linguagem: Inglês
10.15252/embj.201798858
ISSN1460-2075
AutoresThomas Müller, Stephanie Braud, René Jüttner, Birgit C. Voigt, Katharina Paulick, Maria E. Sheean, Constantin Klisch, Dilansu Gueneykaya, Fritz G. Rathjen, Jörg Geiger, James F.A. Poulet, Carmen Birchmeier,
Tópico(s)Neurogenesis and neuroplasticity mechanisms
ResumoArticle26 July 2018Open Access Source DataTransparent process Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons Thomas Müller Thomas Müller orcid.org/0000-0001-8096-1853 Developmental Biology/Signal Transduction Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Stephanie Braud Stephanie Braud Institute of Neurophysiology, Charité – Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author René Jüttner René Jüttner orcid.org/0000-0003-3565-0698 Developmental Neurobiology Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Birgit C Voigt Birgit C Voigt orcid.org/0000-0002-2520-0178 Neural Circuits and Behaviour Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Katharina Paulick Katharina Paulick orcid.org/0000-0002-8079-5944 Developmental Biology/Signal Transduction Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Maria E Sheean Maria E Sheean orcid.org/0000-0003-3055-0495 Developmental Biology/Signal Transduction Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Constantin Klisch Constantin Klisch Institute of Neurophysiology, Charité – Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Dilansu Gueneykaya Dilansu Gueneykaya Cellular Neuroscience Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Fritz G Rathjen Fritz G Rathjen orcid.org/0000-0001-5351-613X Developmental Neurobiology Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Jörg RP Geiger Jörg RP Geiger Institute of Neurophysiology, Charité – Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author James FA Poulet James FA Poulet orcid.org/0000-0003-0296-5415 Neural Circuits and Behaviour Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Neuroscience Research Center and Cluster of Excellence NeuroCure, Charité-Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Carmen Birchmeier Corresponding Author Carmen Birchmeier [email protected] orcid.org/0000-0002-2041-8872 Developmental Biology/Signal Transduction Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Thomas Müller Thomas Müller orcid.org/0000-0001-8096-1853 Developmental Biology/Signal Transduction Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Stephanie Braud Stephanie Braud Institute of Neurophysiology, Charité – Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author René Jüttner René Jüttner orcid.org/0000-0003-3565-0698 Developmental Neurobiology Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Birgit C Voigt Birgit C Voigt orcid.org/0000-0002-2520-0178 Neural Circuits and Behaviour Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Katharina Paulick Katharina Paulick orcid.org/0000-0002-8079-5944 Developmental Biology/Signal Transduction Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Maria E Sheean Maria E Sheean orcid.org/0000-0003-3055-0495 Developmental Biology/Signal Transduction Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Constantin Klisch Constantin Klisch Institute of Neurophysiology, Charité – Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Dilansu Gueneykaya Dilansu Gueneykaya Cellular Neuroscience Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Fritz G Rathjen Fritz G Rathjen orcid.org/0000-0001-5351-613X Developmental Neurobiology Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Jörg RP Geiger Jörg RP Geiger Institute of Neurophysiology, Charité – Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author James FA Poulet James FA Poulet orcid.org/0000-0003-0296-5415 Neural Circuits and Behaviour Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Neuroscience Research Center and Cluster of Excellence NeuroCure, Charité-Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Carmen Birchmeier Corresponding Author Carmen Birchmeier [email protected] orcid.org/0000-0002-2041-8872 Developmental Biology/Signal Transduction Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany Search for more papers by this author Author Information Thomas Müller1, Stephanie Braud2, René Jüttner3, Birgit C Voigt4, Katharina Paulick1, Maria E Sheean1, Constantin Klisch2, Dilansu Gueneykaya5, Fritz G Rathjen3, Jörg RP Geiger2, James FA Poulet4,6 and Carmen Birchmeier *,1 1Developmental Biology/Signal Transduction Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany 2Institute of Neurophysiology, Charité – Universitätsmedizin Berlin, Berlin, Germany 3Developmental Neurobiology Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany 4Neural Circuits and Behaviour Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany 5Cellular Neuroscience Group, Max-Delbrueck-Centrum in the Helmholtz Association, Berlin, Germany 6Neuroscience Research Center and Cluster of Excellence NeuroCure, Charité-Universitätsmedizin Berlin, Berlin, Germany *Corresponding author. Tel: +49 3094 063313; E-mail: [email protected] The EMBO Journal (2018)37:e98858https://doi.org/10.15252/embj.201798858 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low-affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin-positive (PV) interneurons. Nrg3 and ErbB4 are located pre- and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo. Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short-term plasticity, and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4+ interneurons and affects short-term plasticity. Nrg3 mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non-neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4+ interneurons. Synopsis Nrg3 is a low affinity ligand of the ErbB4 tyrosine kinase receptor that has poor signaling activity. Nrg3/ErbB4 interactions provide adhesive cues that promote formation and function of excitatory synapses onto ErbB4+ inhibitory neurons. Nrg3 promotes formation and maturation of excitatory synapses onto ErbB4+ interneurons in the hippocampus. Nrg3 presented by non-neuronal cells induces postsynaptic membrane specialization in ErbB4+ interneurons. Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4+ interneurons. Nrg3 is an important ErbB4 interaction partner for synaptogenesis and synapse function. Introduction Genetic and environmental factors contribute to the manifestation of neuropsychiatric disorders like schizophrenia. Taking into account the enormous complexity of the human brain, it is difficult to define causative mechanisms. One route toward a better understanding is provided by human genetics, which correlates the occurrence of neuropsychiatric disease with mutations or sequence variants in particular genes (Escudero & Johnstone, 2014; Fromer et al, 2014; Hall et al, 2015). The functional analysis of such genes in model organisms provides an entry point to study the mechanisms and pathophysiology of neuropsychiatric diseases such as schizophrenia. Neuregulins are a family of growth factors containing an EGF-like domain that bind and activate tyrosine kinase receptors of the ErbB family. Four different Neuregulin genes (Nrg1, Nrg2, Nrg3, and Nrg4) exist. Neuregulins bind to the ErbB4 receptor with variable affinities and activate its tyrosine kinase with different efficacies (Yarden, 2001). NRG1, NRG3, and ERBB4 mutations and gene variants have been implicated in several neuropsychiatric diseases in humans, but are most frequently associated with schizophrenia in several ethnic groups (Stefansson et al, 2002; Chen et al, 2009; Kao et al, 2010; Greenwood et al, 2011; Morar et al, 2011; Hatzimanolis et al, 2013). ErbB4 is expressed in various neuronal types in the brain where it controls physiology and behavior (Li et al, 2007; Fazzari et al, 2010; Gu et al, 2016; Sun et al, 2016; Geng et al, 2017). In the neocortex and hippocampus, ErbB4 expression is restricted to GABAergic interneurons. ErbB4 expression is particularly high in hippocampal PV inhibitory interneurons, where it is enriched at postsynaptic sites (Vullhorst et al, 2009; Fazzari et al, 2010; Neddens et al, 2011; Mitchell et al, 2013). The mechanisms responsible for this postsynaptic clustering of ErbB4 remain open. Prior work has shown that mutating ErbB4 reduced the number of excitatory synapses on PV inhibitory neurons in the hippocampus, altered synapse function, and resulted in hyperactivity of the cortical circuit (Chen et al, 2010; Fazzari et al, 2010; Del Pino et al, 2013; Yang et al, 2013). In addition, ErbB4 has been assigned both a kinase-dependent and kinase-independent role in inhibitory synapse function (Krivosheya et al, 2008; Mitchell et al, 2013). Various responses of ErbB4+ interneurons to recombinant or transgenic overexpression of Nrg1 have been reported both in vitro and in vivo (Abe et al, 2011; Yin et al, 2013; Agarwal et al, 2014; Sun et al, 2016), and cortical ablation of Nrg1 can result in behavioral changes and unbalanced excitatory–inhibitory neurotransmission (Agarwal et al, 2014). In comparison with Nrg1, little is known about another member of the Neuregulin family, Nrg3, probably because of its low affinity for ErbB4 and its poor signaling activity (Jones et al, 1999; Hobbs et al, 2002). This changed with the discovery that Nrg3 sequence polymorphisms are associated with schizophrenia and other psychiatric disorders (Wang et al, 2008; Chen et al, 2009; Xu et al, 2009; Kao et al, 2010; Morar et al, 2011; Meier et al, 2013). Ablation of Nrg3 and its overexpression in the prefrontal cortex of mice were associated with decreased and increased impulsivity, respectively, while transient overexposure of Nrg3 had lifelong consequences on behavior (Loos et al, 2014; Paterson & Law, 2014). It is interesting to note that Nrg3 is a transmembrane molecule and that transfected Nrg3 was recently observed to be located presynaptically in boutons on cultured inhibitory neurons where ErbB4 is present postsynaptically (Vullhorst et al, 2017). However, the cellular and molecular functions of Nrg3 at the synapse are unknown. Here, we demonstrate that Nrg3 is a functionally important interaction partner of ErbB4 using biochemical, cell biological, electrophysiological, and genetic analyses. We focused our analyses on excitatory synapses on PV inhibitory neurons in the hippocampus because we observed pronounced Nrg3 and ErbB4 co-clustering at such synapses in vivo. Our in vivo analysis demonstrates that the loss of Nrg3 weakens the excitatory input to PV neurons, changes the paired pulse ratio at this synapse type, and alters hippocampal network activity. The extensive similarities between the Nrg3 phenotype and the phenotype previously reported for mice that lack ErbB4 in the entire brain or specifically in interneurons indicate that a dominant function of Nrg3 is exerted in excitatory synapses on ErbB4+ interneurons. Our mechanistic analyses performed in cultured neurons show that presynaptic Nrg3 stimulates the formation and/or stabilization of excitatory synapses onto ErbB4+ neurons. Furthermore, Nrg3 enhances the recruitment of synaptophysin, indicating that it participates in synapse maturation, and increases the efficacy of excitatory transmission. The effect of Nrg3 on the number and maturation of excitatory synapses in cultured inhibitory neurons depends on ErbB4, but not on ErbB4 tyrosine kinase activity. In conclusion, Nrg3 enhances synaptogenesis onto inhibitory neurons, and we suggest that it provides adhesive cues that facilitate the selection of ErbB4+ interneurons as synaptic partners of cortical excitatory neurons. Results Nrg3 is enriched in excitatory synapses on inhibitory neurons in vivo The Nrg3 gene is expressed broadly and abundantly throughout the nervous system (Zhang et al, 1997). In the hippocampus and the cortex, Nrg3 transcripts can be detected in both excitatory and inhibitory neurons (Fig 1A–A″). Nrg3 mRNA levels in the cortex and hippocampus are higher than those of either Nrg1 or Nrg2 (Fig 1B). Nrg3 encodes a transmembrane protein whose domain structure was previously determined (Vullhorst et al, 2017; see Fig 1C for a scheme of the Nrg3 structure). Antibodies directed against Nrg3 detected a protein with a molecular weight of around 95kD in brain extracts of control mice. Subcellular fractionation demonstrated that Nrg3 was enriched in fractions containing synaptic membranes, including synaptosomes (P2′), synaptosomal membranes (P3), and synaptic plasma membranes (SPM) but not in synaptic vesicle or soluble fractions (S2′, S3′) (Fig 1D). Figure 1. Expression and localization of Nrg3 in vivo A. Double fluorescence in situ hybridization using Nrg3 (green)- and Gad1 (red)-specific probes; DAPI was used as a counterstain. Nrg3 is present in Gad1-positive (filled arrowheads) and Gad1-negative neurons. (A′) and (A″) show higher magnifications of boxed areas indicated in (A). Nrg3-negative neurons are indicated by open arrowheads; note that many but not all neurons express Nrg3. B. Comparison of mRNA levels of Nrg3, Nrg1β type III (Nrg1β−III), Nrg2β, Nrg1β type II (Nrg1β−II), Nrg2α, and Nrg1β type I (Nrg1β−I) in the cortex (Cx), hippocampus (HC), and striatum (Str) of juvenile mice (P30–60) by quantitative RT–PCR. Data represent means ± SD of four biological replicates. Two-way ANOVA with Bonferroni's multiple comparisons test was performed to assess statistical significance (****P < 0.0001, ***P < 0.001, ns = not significant). C. Schematic display of the structure of Nrg3 before (left) and after cleavage by Bace1 (right). The following domains of Nrg3 are indicated: black, N-terminal intracellular domain and transmembrane domain; blue, extracellular domain; red EGF domain; black, stalk region with Bace1 cleavage site indicated by an arrow and C-terminal transmembrane domain; and green, intracellular domain. D. Detection of Nrg3 by Western blot analysis in fractions from brain lysates: S1, crude lysate; S2, cytosol and light membranes; S2′, cytosol; LM, light membranes; P2′, crude synaptosomes; S3′, synaptosomal cytoplasm; SV, synaptic vesicles; P3, synaptosomal membranes; and SPM, synaptic plasma membranes. E, F. Immunohistochemical analysis in the hippocampus of wildtype (E, E′, E″) and ErbB4 mutant mice (F) at 2 months of age using Nrg3-, ErbB4-, and parvalbumin (Parv)-specific antibodies. The white box in (E) is displayed magnified in (E′, E″). In control mice, Nrg3 is enriched on dendrites of ErbB4+ PV interneurons, compared to the surrounding neuropil (E′, E″). In ErbB4 mutants, the Nrg3 enrichment on PV interneurons is not apparent (F). G. Nrg3, ErbB4, and GluA4 were identified by immunohistology and co-localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1. Note that (G) displays Nrg3/ErbB4, (G′) Nrg3/GluA4, and (G″) ErbB4/GluA4 signals of the same triple-stained image; false colors were assigned for better signal visualization. H. Quantification of GluA4 puncta present on ErbB4+ dendrites in the CA1 stratum radiatum of adult wildtype (wt) and Nrg3−/− mice (age P90–120). Data are presented as box plots with Tukey's whiskers and outliers; means are indicated by a plus symbol. n = 65 (wt) and n = 62 (Nrg3−/−) from five animals each. Unpaired t-test (two-tailed) with Welch's correction was performed to assess statistical significance (****P < 0.0001). Data information: Scale bars: 500 μm (A, E), 50 μm (F), and 5 μm (G″). Source data are available online for this figure. Source Data for Figure 1 [embj201798858-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint We used immunohistochemistry to analyze the distribution of Nrg3 in the brains of 2-month-old mice. An antibody against Nrg3 detected the protein in the neuropil where it was particularly abundant in the molecular layer of the dentate gyrus and the mossy fiber tract. In addition, puncta with higher levels of Nrg3 were detected. Nrg3 puncta co-localized with ErbB4 in the dendrites of ErbB4+ PV neurons in the hippocampus (Figs 1E–E″ and EV1; see Fig EV1 for antibody specificity). In the cortex, Nrg3 clusters were observed on PV ErbB4+ interneurons and on a subset of ErbB4+ neurons that did not co-express PV (Fig EV1). In ErbB4 mutant mice, Nrg3 association with the dendrites of PV interneurons was no longer observed (Fig 1F). This indicates that Nrg3 binds to the dendritic surface of PV interneurons in an ErbB4-dependent manner. Click here to expand this figure. Figure EV1. Specificity of anti-Nrg3 and anti-ErbB4 antibodies, Nrg3/ErbB4 co-clustering on PV-negative neurons, and ErbB4 interneuron numbers in the hippocampus of Nrg3 mutant mice A, B. Hippocampus sections from wildtype (A, B) and Nrg3−/− mice (A′, B′), age P35, were immunostained for Nrg3 (A, A′) and ErbB4 (B, B′). Nrg3 is widely distributed throughout the neuropil with higher levels being associated with ErbB4+ interneurons (A). Nrg3 immunostaining is abolished in Nrg3 mutant tissue (A′), ErbB4 levels are unchanged (B, B′). C. Western blot analysis of lysates from cortical tissue (brain) and cultured cortical neurons (culture). The anti-Nrg3 antibody detects a main band of about 95 kD (arrow) in lysates from wildtype (+/+) but not from Nrg3 mutant mice (−/−). D. Hippocampus sections from wildtype (D) and heart-rescued ErbB4 mutant mice (D′), 2 months of age, were immunostained for ErbB4. ErbB4 immunostaining is detected in wildtype (D) but not ErbB4 mutant hippocampus (D′). E. Western blot analysis of lysates from cortical tissue. The anti-ErbB4 antibody detects a main band of 180 kD (black arrow) and a minor band of about 70 kD (gray arrow) in lysates from wildtype (+/+) but not from ErbB4 mutant mice (−/−). F. Hippocampus section was immunostained against pro-CCK, ErbB4, and Nrg3; shown is the stratum pyramidale of CA1. CCK+ interneurons express variable amounts of ErbB4 as indicated by white (high expression), gray (middle), and open arrowheads (no detectable ErbB4). Association of Nrg3 with soma or dendrites of CCK+ interneurons is not detectable. G. Section of the prefrontal cortex was immunostained against Nrg3, ErbB4, and parvalbumin (Parv). Nrg3 is co-localized with ErbB4 on soma and dendrites of PV-positive (filled arrow and arrowheads) and PV-negative neurons (open arrow and arrowheads). H. Densities of ErbB4+ interneurons in different layers of the hippocampal CA1 region are identical in adult wildtype and Nrg3 mutant mice at postnatal days 90–120; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum-moleculare. Data represent mean ± SD of six biological replicates for each genotype. Two-way ANOVA with Bonferroni's multiple comparisons test was performed to assess statistical significance (ns = not significant). Scale bars: 500 μm (D′), 40 μm (F″), 50 μm (G‴). Source data are available online for this figure. Download figure Download PowerPoint Nrg3/ErbB4 co-localization in puncta was particularly obvious on dendrites of PV interneurons in the stratum radiatum of the CA1 hippocampus, where 95.8 ± 3.4% of Nrg3 puncta contacted clustered ErbB4, and vice versa, 93.1 ± 6.4% of ErbB4 clusters contacted Nrg3 puncta (573 puncta analyzed in 12 dendritic segments from 6 neurons). These Nrg3/ErbB4 puncta also co-localized with the AMPA receptor subunit GluA4 (Fig 1G–G″), a marker for excitatory synapses on fast-spiking interneurons (Geiger et al, 1995). We then examined excitatory synapse numbers on interneurons in the hippocampal CA1 region in control and Nrg3−/− mice. Quantification of GluA4+ puncta on the dendrites of PV interneurons in the stratum radiatum of the hippocampal CA1 region demonstrated a significant reduction in Nrg3−/− mice (Fig 1H). However, the number and overall distribution of ErbB4+ interneurons in the hippocampus was apparently unchanged (Fig EV1). The role of ErbB4 in the formation and function of excitatory synapses on PV interneurons has been well documented (Fazzari et al, 2010). Since we observed a similar reduction in the number of excitatory synapses on PV interneurons in Nrg3−/− brains in vivo as the one reported for ErbB4 mutants, we concentrated our further investigations on the cell biological mechanisms of Nrg3 function in this synapse type. Presynaptic Nrg3 promotes ErbB4 clustering and synapse formation We used cultures of hippocampal neurons to further investigate the mechanisms of Nrg3 synaptic function. Nrg3 distribution was analyzed in neurons cultured for 21–23 days (Fig 2A and A′). We observed clusters of Nrg3/ErbB4 on the dendrites of both PV-positive and PV-negative neurons in culture (Fig EV2). Most synaptic Nrg3 clusters (81.6 ± 1.4%, mean ± SEM) co-localized with ErbB4 and, similarly, most (80.8 ± 1.6%) ErbB4 puncta co-localized with Nrg3 (42 dendrites from 30 neurons were quantified in three independent experiments). Next, we analyzed the presence of endogenous Nrg3 in excitatory and inhibitory synapses identified by antibodies against the vesicular glutamate transporters vGlut1/2 and the vesicular GABA transporter vGAT, respectively. Among the vGlut1/2+ and vGAT+ synapses, about 54.1 ± 3.2% and 21.5 ± 1.4% contained Nrg3, respectively (Fig 2B). Nrg3 levels in vGAT+ synapses were lower than the levels observed in vGlut1/2+ synapses (Fig 2C). The comparatively low number of inhibitory synapses and the small ratio of those containing Nrg3 provide an explanation for our difficulties detecting Nrg3 in inhibitory synapses in vivo. To assess whether Nrg3 affects synaptogenesis in cultured neurons, we counted the number of excitatory vGlut1/2+ synapses on the dendrites of PV interneurons (Fig 2D). Similarly to the in vivo situation, we observed a significant reduction in the number of excitatory synapses in Nrg3−/− compared to control cultures. Figure 2. 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. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Nrg3/ErbB4 co-clustering in cultured PV-positive and PV-negative interneurons A, B. Neurons, cultured for 23 days, were co-immunostained with antibodies against Nrg3, ErbB4, parvalbumin (Parv), and vGlut1/2. Nrg3 and ErbB4 co-clustered on the dendrites of PV-positive (A) and PV-negative neurons (B). Note that (A–A″) and (B–B″) display false colors of the same images. Scale bar: 10 μm (B″). Download figure Download PowerPoint We next tested whether recruitment of Nrg3 to synaptic sites in culture also depends on ErbB4. For this, we used cultured neurons from ErbB4−/− mice that carried one Gad1/Gad67-GFP allele (Tamamaki et al, 2003). The Gad1/Gad67-GFP allele is expressed by inhibitory neurons, which allowed their identification by GFP. We also mixed neurons from wildtype mice into these cultures. Interneurons from wildtype animals were identified by staining for ErbB4, while interneurons from ErbB4−/− animals were identified by GFP. In these mixed cultures, Nrg3 puncta were present on ErbB4+ but not on GFP-positive ErbB4−/− interneurons (Fig 2E–E″). Thus, in vitro and in vivo, Nrg3 recruitment to synapses on inhibitory neurons depends on ErbB4. Nrg3 is also expressed by GABAergic interneurons (Fig 1A). This raises the question of whether clustered Nrg3 detected on dendrites is derived from the postsynaptic cell. To address this, we again used mixed neuronal cultures, i.e., cultures containing neurons from Nrg3−/− mice that carried one Gad1/Gad67-GFP allele mixed with neurons from wildtype mice. Immunohistochemical analysis demonstrated indistinguishable punctate patterns of Nrg3 on GFP-positive Nrg3−/− and GFP-negative wildtype neurons (Fig 2F–F″), thus demonstrating that Nrg3 is produced presynaptically and provided in trans. We subsequently used lentivirus-infected cultures of hippocampal neurons, the lentivirus co-expressing Nrg3 and synaptophysin fused to cyan fluorescent protein (SypCFP) (Fig EV3). Nrg3 expression was driven by the synapsin (Syn1) promoter. In vGlut1/2+ synapses, the virally produced Nrg3 was present at a 2.2-fold higher level than the endogenous Nrg3 protein as determined by immunofluorescence (Fig EV3). We used the Nrg3/SypCFP lentivirus at a titer that infected 10–20% of the cultured neurons. In these cultures, infected and non-infected neurons formed synapses, and these two types of synapses were distinguished by the presence or absence of SypCFP, respectively (see scheme in Fig 3A). Most SypCFP+ puncta were stained with antibodies against vGlut1/2 and thus represented excitatory synapses (mean ± SD: 79.2 ± 11.5% and 60.3 ± 14.3% of the SypCFP+ puncta on ErbB4-positive and ErbB4-negative neurons, respectively; n = 27 neurons from four independent experiments; see also Fig 3B–B″). The SypCFP+ puncta apposed clustered glutamate receptors detected by an anti-GluA antibody (Fig EV3). A low number of SypCFP+ boutons contained the vesicular GABA transporter vGAT and therefore correspond to inhibitory synapses (4.6 ± 5.1% and 8.8 ± 9.4% of SypCFP+ puncta on ErbB4+ and ErbB4− neurons were vGAT+, respectively; n = 28 neurons from four independent experiments). These transduced cultures provided an experimental strategy that allowed us to compare and quantify excitatory synapses generated by neurons that presented/did not present Nrg3 in a single culture. Click here to expand this figure. Figure EV3. Lentiviral expression of Nrg3 and Nrg3ΔEGF A, B. Schematic 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 synaptophys
Referência(s)