Figure 3
Astrocytic Best1 is responsible for PAR1 activation-induced synaptic potentiation. A, A schedule for tamoxifen and lentivirus injection into hGFAP-CreERT2, wild-type mice (naïve), or CaMKIIα-Cre mice. For acute Cre expression in hGFAP-CreERT2 mice, tamoxifen was pre-injected for 5 days before lentivirus injection. Sunflower oil, a control for tamoxifen. B, Diagram showing Cre-loxP regulation of shRNA expression in cell-type specific gene silencing system. In hGFAP-Cre-ERT2 mice, pretreatment of tamoxifen induces Cre expression, leading to neither shRNA nor EGFP expression in astrocytes through Cre/loxP-mediated deletion. Due to the same mechanism, CA1 pyramidal neurons cannot express both lentiviral shRNA and EGFP expression in CaMKIIα-Cre mice. C, Left: Representative immunohistochemistry results showing Cre-dependent regulation of lentiviral shRNA expression. Cre-dependent shRNA expression in astrocytes or neurons was indicated by co-labeling of EGFP with GFAP or NeuN, respectively. s.p., stratum pyramidale; s.r., stratum radiatum. Scale bar = 50 μm. Right: Bar graphs demonstrate the % of GFAP/EGFP double positive cells (hGFAP-CreERT2) or EGFP positive cells from GFAP- or NeuN positive cells (CaMKIIα-Cre) among total EGFP expressing cells. Mean (%) ± s.e.m. ***, p < 0.001, unpaired t-test. Numbers of counted slices at least from three mice were indicated within each bar. D, eEPSP responses from each recording shown as in Figure 2. Graphs represent averaged eEPSP responses (mean ± s.e.m.) recorded from loxP-floxed scrambled-shRNA expressing slices from all genotypes. (Sc-sh, empty circles), loxP-floxed Best1-shRNA expressing slices from hGFAP-CreERT2 without [Best1-shRNA (−Tam), black circles] or with tamoxifen [Best1-shRNA (+Tam), light grey circles], and loxP-floxed Best1-shRNA expressing slices from CaMKIIα-Cre mice [Best1-shRNA (CamKIIα-Cre), dark gray circles]. E, Bar graph representing mean eEPSPs (mean ± s.e.m), analyzed during the period covered by the gray box in D. Same bar colors as in D. *, P < 0.01, one-way ANOVA with post-hoc test. n.s., not significant.