Fig. 5: The transcription factor Sp1 conveys OXT-induced Gem expression, and astrocytic OXTR signaling within the PVN is required for the local anxiolytic effect of OXT.
From: OXTR-mediated signaling in astrocytes contributes to anxiolysis
a Successful knockdown of Sp1 by transfection of primary astrocytes with Sp1 siRNA assessed by immunoblotting (t4 = 4.548, *p = 0.01 at 24 h, and t4 = 4.548, *p = 0.009 at 48 h siRNA vs. scrRNA). Representative bands are shown below. b Effects of Sp1 knockdown on OXT-induced prolongation of the longest primary process. Primary astrocytes were transfected with either Sp1 siRNA or a control oligonucleotide (scrRNA) 48 h hrs prior to OXT exposure (500 nM, 10 min). (Interaction: F1,1106 = 16.51, p < 0.001; *p = 0.012 scrRNA/Veh vs. scrRNA/OXT; *p = 0.031 for siRNA/Veh vs. siRNA/OXT). c Effects of Sp1 knockdown on Gem mRNA expression (t9 = 2.818, *p = 0.02 vs. scrRNA). d Representative images from Sp1 siRNA or scrRNA-transfected astrocytes subsequently exposed to OXT (500 nM, 10 min) and stained for GFAP (green), DAPI (blue) and Gem (red) showing Sp1-dependency of OXT-induced Gem expression. Scale bar = 10 µm. Quantification of above threshold (Gem+) cells was defined as single cells marked by DAPI/GFAP staining displaying at least one maximum of above threshold Gem fluorescence (interaction: F1,832 = 22.32, p < 0.001; ***p < 0.001 scrRNA/Veh vs. scrRNA/OXT; scale bar = 30 µm). e Targeting of astrocytes within the rat PVN with AAV6-GFAP::shRNA vectors to induce either Oxtr (Oxtr shRNA) or Gem (Gem shRNA) knockdown in a cell type-specific manner. Illustration adapted from Paxinos and Watson (2006) to show anatomical structures of the rat PVN. Representative image taken from the PVN of rats that received bilateral microinfusions of either Oxtr- or Gem- knockdown vector (280 nl per side) 21 d before. Successful transfection is indicated by the expression of the fluorescent reporter mCherry, while cell-type specificity is indicated by double positive immunostaining for mCherry and GFAP (scale bar = 20 µm). f Quantification of perturbance and specificity of AAV6-GFAP::shRNA constructs (n = 3). g Percentage of time spent in open arms of the EPM of rats pre-treated with either Oxtr shRNA or Gem shRNA for astrocyte-specific knockdown of OXTR or Gem, or with scrRNA within the left and right PVN, and locally infused with either Veh or OXT (0.01 nmol/0.5 µl) 10 min prior testing (genotype F2,37 = 0.443, p = 0.639; treatment F1,37 = 0.021, p = 0.885, *p = 0.045 scr/Veh vs. scr/OXT, *p = 0.030 shRNA/Veh vs. shRNA/OXT; n = 6–11/group). h Time spent in the light box of the LDB of rats treated as described above (g), (genotype F2,41 = 3,965, p = 0.027; treatment F1,41 = 6.165, p = 0.017, *p = 0.016 scr/Veh vs. scr/OXT; n = 6–11/group). Data represent mean relative or absolute values +/−SEM.
