Article Figures & Data
Figures
- Figure 1.
Tests for expression of NRG1, NRG2, and ErbB4 in hippocampal newborn GCs and for shRNAmir-mediated depletion of NRG2. A1, Extraction of cytoplasm for single-cell RT-PCR. Left, Retrovirus-infected newborn GCs were identified by EGFP fluorescence at 5–6 dpi. Right, Cytoplasm was sucked into the whole-cell pipette with negative pressure. Arrowheads, EGFP-positive newborn GC; arrow, patch pipette. Scale bar, 10 μm. A2, Representative example for single-cell RT-PCR. Bands for NRG2β and β-actin (as a positive control) were detected at the predicted size of 282 and 414 bp, respectively. The leftmost lane is a size marker. B1, Immunostaining of endogenous NRG1 in the hippocampal slice culture (7 dpi) shows that NRG1 signal is weaker in dentate gyrus (DG) than in CA1 and CA3 regions (top). Bottom, Corresponding DIC image. Scale bar, 300 μm. B2, Double staining of ErbB4 transcripts (magenta) and DCX (green). Boxed regions of DG, CA3, and CA1 in top left panel are magnified images. ErbB4 signal was negative in the DCX+ cells (arrow) in the DG. Note that there are many ErbB4 fluorescent spots in DCX− ErbB+ cells (arrowhead). Hoechst33342 (blue) was used for nuclear counterstaining. Scale bar, 40 μm. C, Schematic of the tetracycline-inducible shRNAmir expression cassette. EGFP and rtTA3 are constitutively expressed under the control of promoters, the 5′ LTR, and human ubiquitin-C promoter (pUbiC), respectively. The rtTA3 binds to TRE and activates the transcription of shRNAmir in the presence of doxycycline. Ψ, Packaging signal; rtTA3, reverse tetracycline transactivator; TRE, tetracycline response element; pCMVmin, mini-CMV promoter; WPRE, wood-chuck hepatitis B posttranscriptional regulatory element. D, shNRG2mir was cotransfected with NRG2-myc into HEK293 cells. When Dox (2 μg/ml, 48 h) was applied to HEK293 cells, shNRG2mir almost depleted NRG2-myc, but shNTmir did not. The blot of β-actin is shown as a loading control. TF, Transfection. E1, F1, NRG2 and NRG1 immunofluorescence (magenta) of the dentate GC layer (7 dpi) in organotypic cultures infected with retrovirus encoding EGFP plus Tet-On/shNRG2mir. Slice cultures were treated (Tx.) with Dox started at 4 dpi (bottom) or untreated (top) and then fixed and immunostained at 7 dpi. Somata of EGFP-positive GCs are indicated by arrowheads. Scale bar, 20 μm. E2, F2, Mean values for the NRG2 and NRG1 immunofluorescence intensity in the EGFP-positive regions of Dox-treated and untreated slices were quantified at 7 or 14 dpi. Dox treatment was from 4–7 dpi or from 10–14 dpi, respectively.
- Figure 2.
Developmental changes of GPSCs and EPSCs in newborn GCs. A, Dentate gyrus of a hippocampal organotypic slice culture at 10 dpi. Newborn GCs infected by retroviruses expressed EGFP. The borders between the GC layer (GCL) and hilus or between the GCL and the IML are marked by dashed lines. Scale bar, 50 μm. B, Schema for excitatory and feedforward inhibitory synapses innervating a newborn GC. Stimulating electrode was located in IML (blue double lines). GPSCs or EPSCs were recorded from EGFP-positive newborn GCs in GCL using whole-cell patch-clamp techniques. Stim, Stimulation electrode; Rec, recording pipette; IN, interneuron. C, Representative GPSCs recorded in newborn GCs. Note that feedforward GPSCs were blocked not only by bicuculline (20 μm, C1) but also by CNQX (20 μm, C2). Scale bar, 200 pA and 200 ms. D, Representative EPSCs recorded at the holding potentials of −60 mV (D1) and at +40 mV (D2) in newborn GCs. Scale bar, 500 pA and 200 ms. D1, AMPA-EPSCs were abolished by CNQX (20 μm). D2, When APV (50 μm) was bath-applied to the slice, NMDA-EPSCs were and the outward AMPA-EPSCs were unaffected. The amplitude of NMDA-EPSCs was measured 50 ms after the stimulation (red dashed line and arrow in the inset). Insets, Traces in an expanded time scale. Scale bars, 500 pA and 50 ms. E, Averaged input–output curves of GPSCs (top), AMPA-EPSCs (middle), and NMDA-EPSCs (bottom) from newborn GCs. The amplitudes of evoked GABAergic or glutamatergic PSCs were plotted as a function of stimulus intensity at different dpi's. F, Developmental changes in amplitudes of GPSCs, AMPA-EPSCs, and NMDA-EPSCs recorded in newborn GCs grown in organotypic slice cultures under control conditions. All PSCs were evoked by stimulation of IML at 10 V. The age of GCs are shown as dpi, which represents the number of days elapsed from the day when the slice was infected by EGFP-encoding retrovirus. The number on each symbol indicates number of cells studied (n).
- Figure 3.
NRG2 is essential for GABAergic synapse formation in newborn GCs. A1, Averaged input–output curves of GPSCs in control (black) and NRG2-depleted (red, Dox-treated from 4 dpi) newborn GCs at 7 dpi (open circles, dotted line) or at 11 dpi (solid circles, solid line). For both GPSCs at 7 and 11 dpi, amplitudes were significantly lower in Dox-treated GCs (control vs Dox; 7 dpi, F(1,35) = 12.03, p = 0.001; 11 dpi, F(1,15) = 24.04, p < 0.001 by RM one-way ANOVA). A2, Effects of NRG2 knock-down on the developmental changes of feedforward GPSCs in newborn GCs. To deplete NRG2 hippocampal slices were treated (Tx.) with Dox from 4 dpi. GPSCs in Dox-treated GCs were significantly lower than control cells as early as 7 dpi. A3, Effects of NRG2 knock-down on GPSCs after the critical period of GABAergic synapse formation (6–9 dpi). The slice cultures were treated with Dox from 7 dpi (blue) or 10 dpi (purple). GPSCs of the treated slice were not significantly different from the untreated control slices at all ages (p > 0.1). In A2 and A3, mean amplitudes of GPSCs evoked by 10 V stimulation of IML were plotted as a function of dpi. A4, Mean amplitudes of GPSCs in newborn GCs older than 9 dpi, at which GPSCs reached a plateau under different Dox treatment conditions. B1, Monosynaptic GPSCs were evoked by minimal stimulation in the presence of AMPA and NMDA receptor antagonists using a monopolar electrode positioned 30–100 μm apart from the soma of a newborn GC. Rec, Recording pipette; IN, interneuron. B2, Representative trace of monosynaptic GPSCs, which was completely blocked by 20 μm bicuculline, a GABAA receptor blocker. Scale bar, 50 pA and 100 ms. B3, Representative input–output curve for monosynaptic GPSCs in newborn GCs. As the stimulation intensity was gradually increased from 1 V, the synaptic responses were all-or-none at a distinct threshold (6 V) and were kept stable for further increase by 5 V from the threshold. Such stable range of stimulation intensity was adopted as the voltage intensity for minimal stimulation (marked with yellow). C, Mean amplitudes of monosynaptic GPSCs in newborn GCs grown in slice cultures (ex vivo, C1) or acute slices (in vivo, C2). The starting dates for Dox treatment or feeding (Dox Tx.) are indicated in the abscissa. The recording dates are indicated above the bar graphs. Monosynaptic GPSCs were suppressed by early Dox Tx (from 4 or 5 dpi), but not by late Dox Tx (from 7 or 9 dpi), in both ex vivo and in vivo experiments.
- Figure 4.
NRG2 is essential for maturation of glutamatergic synapses. A1, A2, Averaged input–output curves of AMPA-EPSCs (A1) and NMDA-EPSCs (A2) in control (black) and NRG2-depleted (red, Dox-treated from 7 dpi) newborn GCs at 12 dpi (open circles, dotted lines) or at 15 dpi (solid circles, solid lines). Amplitudes of both AMPA-EPSCs and NMDA-EPSCs were significantly lower in Dox-treated GCs at 15 dpi but not at 12 dpi. B, Developmental increases of AMPA-EPSCs (B1) and NMDA-EPSCs (B2) in control and NRG2-depleted (Dox-treated from 7 dpi) newborn GCs. Mean amplitudes of EPSCs evoked by 10 V stimulation of IML were plotted as a function of dpi. C1, Representative traces of AMPA-EPSCs and NMDA-EPSCs under control (left) and Dox-treated conditions (right) at 15 dpi. C2, Ratio of AMPA-EPSC to NMDA-EPSC amplitude in control and NRG2-depleted (Dox-treated from 7 dpi) newborn GCs.
- Figure 5.
Impairment in dendritic growth precedes that in EPSCs in NRG2-depleted GCs. A, Representative dendritic arbors reconstructed from confocal images of control and Dox-treated newborn GCs at 10 and14 dpi. Scale bar, 50 μm. B, Sholl analysis of dendritic complexity at 10 and 14 dpi showing the intersection numbers per 10 μm radial unit distance. The complexity was significantly reduced by NRG2 depletion at both 10 and 14 dpi (control vs Dox, F(1,35) = 19.1 at 10 dpi, F(1,67) = 42.5 at 14 dpi, p < 0.001 for both dpi's, RM one-way ANOVA). C, Cumulative distributions of dendrites at 10 and 14 dpi. **p < 0.01; ***p < 0,001 by K-S test. D, Developmental increases of total dendritic lengths in control and Dox-treated GCs. ***p < 0.001 (t test).
- Figure 6.
Effects of ErbB4 inhibition on GPSCs and EPSCs. A, B, Effects of ErbB4 inhibitors on GPSCs of newborn GCs. A1, A2, Mean amplitudes of feedforward GPSCs in control (black), Dox-treated (red), and AG-1478-treated (10 μm, green, denoted as “AG”) newborn GCs. Dox and AG-1478 were added in culture media from 4 dpi (A1) or 10 dpi (A2) and feedforward GPSCs were recorded at 7 dpi (A1) or 14 dpi (A2), respectively. B, Mean amplitudes of monosynaptic GPSCs at 14 dpi in newborn GCs grown under control conditions or under the condition in the presence of Dox, AG-1478, or anti-ErbB4 antibody (10 μg/ml, purple, denoted as “Ab-3”). Note that the three kinds of treatment reduced the GPSCs to a similar extent when the starting date of treatment was 4 dpi. C, D, Effects of AG-1478 on EPSCs in newborn GCs. Mean amplitudes (C1, D1) and PPR (C2, D2) of AMPA-EPSCs (C) and those of NMDA-EPSCs (D) in control, Dox-treated, or AG-1478-treated newborn GCs. Dox and AG-1478 were added in culture media from 7 dpi and EPSCs were recorded at 14 dpi. Tx., Treatment.
- Figure 7.
Effects of NRG2 depletion on glutamatergic synaptic transmission and dendritic arborization are rescued by NRG2-ICD overexpression. A, Retrovirus for overexpression of intracellular domain of NRG2 (NRG2-ICD) in newborn GCs. A1, Schematic of the retroviral cassette for tetracycline-inducible expression of NRG2-ICD in parallel with constitutive expression of TagRFP. A2, Only under the Dox-treated conditions (Dox Tx.) did the TagRFP-positive newborn GCs express c-myc-tagged NRG2-ICD. NRG2-ICD was immunostained with anti-myc IgG. Scale bar, 10 μm. A3, Fluorescence image of a hippocampal slice culture infected with two kinds of retroviruses: shNRG2mir virus and NRG2-ICD virus. Newborn GCs infected with one of the two viruses express EGFP (green) or TagRFP (magenta). Open arrowheads indicate the newborn GCs infected with both viruses simultaneously. Scale bar, 100 μm. A4, Notations for experimental conditions of virus types and Dox treatment. B, C, Concurrent overexpression of NRG2-ICD in the NRG2-depeleted newborn GCs rescued the knock-down effects of shNRG2mir on AMPA-EPSCs (B1), NMDA-EPSCs (B2), and the AMPA/NMDA ratio (B3), but not GPSCs (C). The amplitudes of AMPA-EPSCs or NMDA-EPSCs of newborn GCs overexpressing only NRG2-ICD were not significantly different (B1–B3, gray bars) from the control GCs. Dox was applied from 7 to 14 dpi (B1–B3) or from 4 to 9 dpi (C). D, Overexpression of NRG2-ICD rescued the impaired dendritic arborization caused by NRG2 knock-down (control, n = 13; NRG K/D, n = 27; rescue, n = 10). D1, Representative morphology of control, NRG2 K/D, and rescued newborn GCs at 14 dpi. Scale bar, 50 μm. D2, Dendritic arbor complexity evaluated by Sholl analysis was not different between control and rescued GCs (control vs rescued, F(1,21) = 2.45, p = 0.774 by RM one-way ANOVA). D3, D4, Cumulative distribution (D3) and averages (D4) of total length of dendrites were rescued by NRG2-ICD to the control level (control vs rescue; D3, p = 0.943, K-S test; D4, p = 0.476). Results for control and NRG2-depleted newborn GCs were reproduced from those in Figure 5, B–D.
