Figure 7.
NRG3 accumulates on axonal terminals by trans-synaptically interacting with postsynaptic ErbB4 receptors. (A) Representative immunofluorescence histochemistry of hippocampal area CA1 from a P30 rat brain section co-labeled for NRG3 and ErbB4, showing extensive overlap of punctate NRG3 and ErbB4 signals on soma and dendrites of presumptive stratum pyramidale GABAergic interneurons (arrowheads). (B) Representative Airyscan immunofluorescence micrograph of a cultured DIV24 hippocampal GABAergic interneuron that reveals extensive co-localization of punctate NRG3 signals with ErbB4 and the glutamatergic presynaptic marker protein vGluT1 (arrowheads). Boxed area on the left is magnified on the right and separated by channels. (C) Successful knockdown of ErbB4 protein in hippocampal cultures transduced with an AAV expressing shRNA 592. Cultures transduced with NTC shRNA are included as negative controls. GFP, co-expressed from the shRNA construct, serves as a transduction control. (D) Representative micrographs of GABAergic interneurons transduced with NTC (top) or ErbB4_592 shRNA (ErbB4-KD, bottom), illustrating dramatic reduction of ErbB4 immunoreactivity. (E) Representative micrographs illustrating strongly attenuated NRG3 signals in a GABAergic interneuron (labeled for NRG2) transduced with ErbB4_592 (bottom), as compared to a neuron transduced with NTC shRNA (top). (F) Summary analysis of results shown in E. Data are plotted as integrated pixel densities and represent the mean ± SEM of 22–24 neurons from three independent experiments. (G) Representative micrographs illustrating reduced presynaptic NRG3 signals on ErbB4+ GABAergic interneurons in cultures transduced with an AAV for DN Rab4, as compared to cultures transduced with WT Rab4. (H) Summary analysis of results shown in G. Data are plotted as integrated pixel densities and represent the mean ± SEM of 36 neurons from three experiments. ****, P < 0.0001; ns, P > 0.05 (F: Kruskal-Wallis test with Dunn’s multiple comparisons test; H: Mann-Whitney test). Scale bars: A, 50 µm; B, 10 µm; D, E, and G, 20 µm. Source data are available for this figure: SourceData F7.

NRG3 accumulates on axonal terminals by trans-synaptically interacting with postsynaptic ErbB4 receptors. (A) Representative immunofluorescence histochemistry of hippocampal area CA1 from a P30 rat brain section co-labeled for NRG3 and ErbB4, showing extensive overlap of punctate NRG3 and ErbB4 signals on soma and dendrites of presumptive stratum pyramidale GABAergic interneurons (arrowheads). (B) Representative Airyscan immunofluorescence micrograph of a cultured DIV24 hippocampal GABAergic interneuron that reveals extensive co-localization of punctate NRG3 signals with ErbB4 and the glutamatergic presynaptic marker protein vGluT1 (arrowheads). Boxed area on the left is magnified on the right and separated by channels. (C) Successful knockdown of ErbB4 protein in hippocampal cultures transduced with an AAV expressing shRNA 592. Cultures transduced with NTC shRNA are included as negative controls. GFP, co-expressed from the shRNA construct, serves as a transduction control. (D) Representative micrographs of GABAergic interneurons transduced with NTC (top) or ErbB4_592 shRNA (ErbB4-KD, bottom), illustrating dramatic reduction of ErbB4 immunoreactivity. (E) Representative micrographs illustrating strongly attenuated NRG3 signals in a GABAergic interneuron (labeled for NRG2) transduced with ErbB4_592 (bottom), as compared to a neuron transduced with NTC shRNA (top). (F) Summary analysis of results shown in E. Data are plotted as integrated pixel densities and represent the mean ± SEM of 22–24 neurons from three independent experiments. (G) Representative micrographs illustrating reduced presynaptic NRG3 signals on ErbB4+ GABAergic interneurons in cultures transduced with an AAV for DN Rab4, as compared to cultures transduced with WT Rab4. (H) Summary analysis of results shown in G. Data are plotted as integrated pixel densities and represent the mean ± SEM of 36 neurons from three experiments. ****, P < 0.0001; ns, P > 0.05 (F: Kruskal-Wallis test with Dunn’s multiple comparisons test; H: Mann-Whitney test). Scale bars: A, 50 µm; B, 10 µm; D, E, and G, 20 µm. Source data are available for this figure: SourceData F7.

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