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. 2022 May 19;119(21):e2122544119. doi: 10.1073/pnas.2122544119

Fig. 4.

Fig. 4.

Increased neuronal activity in the cerebellum during postnatal development reproduces the transient increase in Ctgf expression but not the long-lasting synaptic effects. (A) A chemogenetic strategy was used to test whether an increase in neuronal activity in the cerebellum during the second postnatal week would reproduce the gene expression and synaptic changes seen in the neonatal subchronic PCP model. For this, knock-in mice allowing Cre-dependent expression of the excitatory hM3Dq, along with mCitrine, were crossed with the NeuroD1CRE line that drives Cre recombinase expression in the cerebellar GC lineage (ND1Cre/hM3Dq). CNO or vehicle was injected intraperitoneally at P7, P9, and P11 to reproduce the time course of neonatal PCP injections. Control littermates lacking hM3Dq expression in cerebellar GCs were also analyzed (ND1Cre/WT). Expression of the hM3Dq was monitored using anti-GFP immunostaining to detect mCitrine in slices from the cerebellum of ND1Cre/hM3Dq mice at P7. (B) Digital droplet PCR quantifications were used to monitor the gene expression changes induced by increased activity in cerebellar extracts from P11 animals. Subchronic CNO administration leads to a significant increase in cFos and Ctgf expression in extracts from ND1Cre/hM3Dq mice but not in ND1Cre/WT littermates. Gal and Rab7L1 expression levels remain unchanged. Vehicle treatment does not modify gene expression in either genotype. Gene expression is normalized to Rpl13a. Data are presented as mean ± SEM. ND1Cre/WT + CNO: n = 6; ND1Cre/HM3Dq + CNO: n = 4; ND1Cre/WT + vehicle: n = 3; ND1Cre/HM3Dq + vehicle: n = 4. Mann–Whitney U test. (C, Upper) CF presynaptic boutons were immunostained with an anti-VGLUT2 antibody (green) and PCs and their dendritic tree were immunostained with an anti-calbindin antibody (magenta) in parasagittal cerebellar sections from ND1Cre/hM3Dq and ND1Cre/WT control mice. Insets represent high magnification of VGLUT2 staining. All mice were injected with CNO at P7, P9, and P11. (Scale bars: 50 µm; Insets, 12.5 µm.) (C, Lower) Quantification of the mean density and the mean volume of VGLUT2 puncta revealed no difference in lobules VI and VIII of ND1Cre/hM3Dq mice compared with control mice (mean ± SEM; ND1Cre/WT: n = 4 animals; ND1Cre/hM3Dq: n = 4 to 5 animals; unpaired Student’s t test).