Fig. 1. Reduced dendritic spine density in high-expressing NRG1 neurons.

a Representative images of neuronal morphology and spine density in hippocampal pyramidal neurons. Neurons were isolated at embryonic 18 (E18) rat to culture for 9 days and transfected with 1.5 µg control (empty HA vector) or HA-NRG1 construct, and fixed for staining at DIV17. Scale bar, 10 μm. Statistical analysis of data in a for total (b), mature (c) and immature (d) spine density. N = 32 neurons for control, N = 45 neurons for HA-NRG1 (p = 0.0066 for total spine density; p = 0.0048 for mature spine density; p = 0.0109 for immature spine density). *p < 0.05, and **p < 0.01; Student’s t-test. e Representative images of spine density in hippocampal neurons transfected with HA-NRG1 in gradient. Scale bar, 10 μm. f–h The statistical results for total (f), mature (g) and immature (h) spine density. N = 28 neurons for control, N = 32 neurons for 0.5 μg HA-NRG1, N = 31 neurons for 1.5 μg, N = 34 neurons for 4.5 μg (p = 0.0169 for 0.5 μg, p < 0.001 for 1.5 μg and 4.5 μg for total spines; p = 0.0251 for 0.5 μg, p < 0.001 for 1.5 and 4.5 μg for mature spines; p = 0.6044 for 0.5 μg, p = 0.0446 for 1.5 μg and p < 0.001 for 4.5 μg for immature spines). Data were shown as mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA. i Representative images of time-lapse imaging from hippocampal neurons transfected with HA-NRG1 or control taken at five adjacent time points during the 30-min live-imaging period. Cultured neurons were transfected with indicated constructs at DIV9 and imaged every minute for 30-min at DIV17. N = 10 neurons for control, N = 11 neurons for HA-NRG1. j–l Quantitative analysis for percentages of stable (red arrow), newborn (yellow arrow) and eliminated (green arrow) spines. p = 0.003 for stable spines, p = 0.571 for newborn spines, and p = 0.07 for eliminated spines. Data were shown as mean ± SEM; **p < 0.01; ns, p > 0.05; Student’s t-test.