Extended Data Fig. 1: No leak expression of pHRed in BGs.
From: Synaptic pruning through glial synapse engulfment upon motor learning
a, Representative images of pHRed expression and PV-IHC in PV-tTA; tetO-pHRed mice (12 weeks old; 4 weeks after Dox off). Most PCs expressed pHRed. We have done 1 independent experiment (n = 3 mice). b, PV-tTA mouse was crossed with tetO-FRET-sensor (PV-Venus) mouse. This mouse was generated in the same way as the tetO-pHRed mouse. As with the pHRed in PV-pHRed mouse, Venus expression was observed in PCs and interneurons. Venus was not found in BGs, Iba1 positive microglia, and CD31 positive endothelial cells. This result suggests that the gene expression is induced selectively in neurons with PV-tTA. pHRed found in glia is due to internalization of pHRed into the glia. We have done 1 independent experiment (n = 2 mice). c, Red and green fluorescence merged images from WT, 4w Dox off, and no Dox of PV-pHRed mice (all with 3PGDH staining for BGs). Dashed square areas are enlarged in inset with BG outline shown with yellow line. Quantification of the red fluorescence signal is shown on the right. pHRed gene expression in PC and pHRed internalization into BG are increased after Dox off (n = 17, 25, 29, 37, 29 PCs from 4, 3, 4, 3, 3 mice; 35, 45, 52, 42, 30 BGs from 5, 3, 4, 5, 3 mice). One-way ANOVA followed by Dunnett’s multiple comparison test (F (4, 13) = 80.93, p < 0.0001; **** p < 0.0001 for PC; F(4, 15) = 10.51, p = 0.0003; * p = 0.02, *** p = 0.0004 for BG). d, Magnified images of the dashed white square in a (Arrowhead: BG soma; dashed white circles: pHRed signals in BG; Asterisk: PC soma). Right graph, line profiles of the normalized fluorescence intensities at the yellow line shown in the left panel image. pHRed signals are found, but no PV-IHC signals are found in BG soma. e, PC and BG soma images from a 12-week-old, 4 weeks off Dox PV-pHRed mouse. No Calbindin-IHC signals in BG. f, Images from a 6-week-old, no Dox PV-FRET mouse. Venus (GFP antibody)-IHC signals were seen only in PC soma and not in BG. g, No membrane GFP-IHC signal in BG in PV-tTA; tetO-LckE2GFP mice (12 weeks old, 4 weeks off Dox). h, Images from PV-tTA; tetO-pHRed; tetO-LckE2GFP trigenic mice (12 weeks old, 3 weeks off dox). Punctate pHRed signals were found in BG soma, but no GFP-IHC signals in BG. i, Representative images from WT, Mlc1-tTA; tetO-pHRed, PV-tTA; tetO-pHRed mice. Yellow dashed lines and white dashed lines demarcate PC and BG soma, respectively. pHRed signals were found in BGs of the Mlc1-pHRed mouse and in BGs of the PV-pHRed mouse, but pHRed signals were not found in PCs of the Mlc1-pHRed mouse. This shows that inter-cell material transfer is unidirectional between PC and BG (n = 19, 31, 23, 32 PCs, 28, 37, 23, 30 BGs from 4 mice). One-way ANOVA followed by Dunnett’s multiple comparison test (F (3, 12) = 26.46, p < 0.0001; **** p < 0.0001 for PC; F(3, 12) = 23.23, p < 0.0001; * p = 0.040, **** p < 0.0001 for BG; NS p > 0.05). Data are shown as mean ± s.e.m.
