Figure 11.
Tamoxifen-induced RORα knock-out in mature PCs by CRISPR/Cas9. A, Schematic diagram of the experimental procedure. L7-ERT2-Cre-ERT2 and chimeric guide RNA against LacZ (gR-LacZ) or RORα (gR-RORα) are cotransfected with CAG-flex-EGFP and CAG-flex-SpCas9. Expression of SpCas9 was induced by 4-OHT at P21–P23 and mice were killed for analyses at P34–P43. B, Representative confocal images of immunofluorescent signals for endogenous RORα (red) of the EGFP-positive PCs (green, arrowheads) cotransfected with gR-LacZ (control) or gR-RORα (KO) at P35. 4-OHT was administered at P21. The asterisk indicates a neighboring untransfected PCs. Scale bar, 20 μm. C, Distribution of relative RORα levels in the PCs cotransfected with control gR-LacZ (blue, control) or gR-RORα (magenta, KO). n = 23 cells from 2 mice (control) and n = 45 cells from 4 mice (KO). D, Averaged RORα levels in control PCs and PCs expressing gR-RORα. According to the RORα fluorescent intensity, PCs were classified into low (when the RORα intensity was < 20% of the nearby EGFP-negative PCs) and high (≥ 20%) groups. The RORα fluorescent intensity was averaged in each group. All control PCs fall into the high group, but KO PCs were divided into low and high groups. Bars represent the mean ± SEM. ***p = 1.27 × 10−6 and ###p = 1.27 × 10−6 (ANOVA followed by Tukey's test). E–H, Representative confocal z-stacked EGFP images of control and RORα-knock-out (KO) PCs at P35. Cas9-dependent RORα deletion in mature PCs caused dendritic atrophy and abnormal spine formation. E, Dendritic morphology. The arrow indicates axon swelling. Scale bar, 20 μm. F, Spines on distal dendrites. Knock-out PCs showed extraordinarily thin spineless dendrites. Scale bar, 1 μm. G, Abnormal spines on knock-out PC soma. Scale bar, 10 μm. H, Confocal z-stacked images of immunofluorescent signals for vGluT2 (red) on the EGFP-positive control or knock-out PC soma (green) at P35. Note that somal and axonal swelling is observed in the knock-out PC. Scale bar, 10 μm. I, Histograms showing the percentage of PCs contacted by >15 vGluT2-immunopositive puncta. n = 28 cells from 2 mice (control) and n = 33 cells from 3 mice (KO), **p = 0.00509 (χ2 test). J–M, CF-EPSCs recorded from PCs clamped at −10 mV. J, Representative CF-EPSCs in control PCs and knock-out PCs at various stimulus intensities (J). L, Histograms showing the mean amplitude (***p = 0.000249 by the Mann–Whitney U test) and the PPR (at ISI = 50 ms, p = 0.541 by the Mann–Whitney U test) of CF-EPSCs. Error bars indicate SEM. M, Percentage of PCs innervated by single and multiple CFs. The number of CF-EPSCs induced by different stimuli thresholds (0–200 μA) was counted. n = 22 cells from 2 mice for control and n = 30 cells from 3 mice for KO (J–M).