Fig. 4: The Notch1 signalling pathway negatively regulated the GABA_B1 receptor expression of METH-induced sensitized mice.

A GABA receptors and transporters expression changes following METH sensitization in the mPFC. One-way ANOVA showed the significant downregulation effects of METH on GABA_Aα3 [F _{(2, 17)} = 9.17, P < 0.05], GABA_Aβ1 [F _{(2, 17)} = 10.38_, P < 0.05], GABA_B1 [F _{(2, 18)} = 11.66, P < 0.05_], GABA_B2 [F _{(2, 17)} = 5.36, P < 0.05], GAT1 [F _{(2, 17)} = 25.79, P < 0.05] and GAT3 [F _{(2, 16)} = 13.48, P < 0.05]. Subsequent post hoc LSD comparisons found higher expression changes of GABA_Aβ1, GABA_B1 and GAT1 between the METH-sensitized group and acute METH group. *P < 0.05, **P < 0.01. ***P < 0.001 vs. saline group. #P < 0.05 METH-sensitized group vs. acute METH group. 4B-4F. GABA_B1 receptor expression changes following regulation of the Notch1 signalling pathway in the mPFC of MIP mice. B, F Two-way ANOVA followed by LSD test showed significant upregulation of the GABA_B1 receptor in the syn-NICD-shRNA group at both the mRNA and protein levels in the mPFC of MIP mice [B main effect of AAV, F _{(1, 20)} = 23.29, P < 0.001; METH, F _{(1, 20)} = 10.27, P < 0.01; AAV × METH, P > 0.05] and protein level [F Main effect of AAV, F _{(1, 20)} = 13.09, P < 0.01; METH, F _{(1, 20)} = 4.78, P < 0.05; AAV×METH, P > 0.05]. C, E Two-way ANOVA followed by LSD test showed significant downregulation of the GABA_B1 receptor at both the mRNA [C main effect of AAV, F _{(1, 20)} = 12.03, P < 0.01; METH, F _{(1, 20)} = 13.28, P < 0.01; AAV × METH, P > 0.05] and protein [E main effect of AAV, F _{(1, 20)} = 15.31, P < 0.01; METH, F _{(1, 20)} = 8.44, P < 0.01_; AAV × METH, P > 0.05] levels in the mPFC of MIP mice in the syn-NICD-OE group. D DAPT in the mPFC induced increased GABA_B1 receptor mRNA levels in the mPFC of MIP mice [Two-way ANOVA followed by LSD test, main effect of DAPT, F _{(1, 20)} = 17.38, P < 0.01; main effect of METH, F _{(1, 20)} = 9.88, P < 0.01; DAPT × METH, P > 0.05]. G The co-localization of GABA_B1 receptor (Green) and Notch1 receptor (Red) were observed in the mPFC of native mice by double-label immunofluorescence staining assay. The areas in the rectangles were enlarged respectively in the bottom of the image. Arrows indicate expressed both receptors, and the scale bar represent 50 μm (images in the first row) and 20 μm (images in the second row). *P < 0.05, **P < 0.01 vs. control saline group. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. paired METH group. The results are expressed as the mean ± SEM, n = 6–7.