Fig. 5: Increased sCRFR2α release underlies the anxiogenic effect of chronic OXT in combination with mild stress.

A Representative picture of Dot Blot analysis (for detailed methodology see Supplementary Methods) of CSF (VEH and GapmeR) and control tissue protein lysates (cerebellum = CB as negative control, PVN as positive control, H₂O). For detection, the sCRFR2α-specific antibody (provided by Joan Vaughan [57]) was used at a dilution of 1:20.000 in 5% BSA. B Schematic representation of the rat Crfr2α gene, the transmembrane domain is indicated by a blue bar, including Exon 4 to Exon 11. Target site blocker (TSB) binding sites are indicated by green bars. Multiple MEF2 binding sites are indicated as red arrows. Exon 5/7 boundary GapmeR binding site in the sCrfr2α mRNA is indicated in yellow. C Infusion of GapmeRs targeting sCrfr2α bilaterally into the PVN of male rats reduced sCRFR2α expression by ~50% as assessed by Dot Blot in CSF samples. Data are shown as fold change in protein expression ±SEM. One-tailed Student’s t test, t = 1.828, df = 12, *p = 0.046; n(scr) = 8, n(GapmeR) = 6. D Individual CSF sCRFR2α signal intensity and % time spent in LB correlate negatively (R = −0.54, ANOVA F(1;13) = 4.946, p = 0.046), suggesting that sCRFR2α is anxiogenic. E GapmeR-induced reduction in sCRFR2α increased the time spent in LB and inner zone of the OF, indicating anxiolysis. Anxiety-like behavior was determined 7 (LDB) or 8 days (OF) after GapmeR infusion. Data are represented as mean percentage of time spent in the LB ± SEM (one-tailed t-test *p = 0.0486), and the mean percentage of time in the inner zone of the OF (Mann–Whitney Rank Sum Test U = 16.000, *p = 0.037) ±SEM; n(scr) = 10, n(GapmeR) = 8. F Representative heat maps showing the distribution of rat presence in the light and dark compartment of the LDB or inner and outer zone in the OF test. G Infusion of TSBs bilaterally into the PVN of male rats and local induction of alternative splicing of Crfr2α enhanced anxiety-like behavior of male rats in the LDB 7 days after infusion, demonstrating an anxiogenic effect of sCRFR2α. Data are represented as mean percentage of time spent in the LB ± SEM. t = 1.843; one-tailed t-test *p = 0.0409; n(scr) = 10, n(TSB) = 10. H Representative heat map of the distribution of rat presence in the LDB after VEH or TSB treatment.