Inhibitory effects of GABA on CRH neurons Decreasing activity of the HPA axis Normal condition THDOC (Stress-derived neurosteroid) Stress condition Excitatory effects of GABA on CRH neurons Increasing activity of the HPA axis
Animals CRH-GFP mice which express GFP specifically in CRH neurons by crossing GFP mice and CRH-Cre mice (?) Gabrd(GABA receptor subunit delta) knocked out mice (control) If two loxP sites are in the DNA like the figure shows , the target gene will be excised. If two DNA contain single loxP sites, they will be fused.
Detection • How the Cre recombinase is expressed in CHR neurons • How many GFPs are linked with CRH • How the CRH is expressed in PVN
X-gal staining assay kit CRH-Cre mice X Rosa26 reporter mice fluorogold lacZ reactivity(black) fluorogold labeling( purple) GFP antibody CRH-GFP mice All the GFP and Cre recombinase are in CRH neurons
Methods Whole-cell patch clamp Perforated patch clamp
Results GABAA receptors δ subunit expression in the PVN GABAA receptors δ subunit largely exist in CRH neurons
Neurosteroid regulation of CRH neurons SR95531: GABAAR inhibitor Tonic currents IPSCs Average decay time of IPSCs Neurosteroid has effects on GABAAR δ subunit.
Spontaneous firing rate Binned firing rate (?) Neurosteroid makes CRH neurons harder to fire.
Neurosteroid actions on CRH neurons regulate corticosterone levels Under non-stress condition, neurosteroid decreased the corticosterone level (HPA axis activity). Under stress condition, by effects on δ subunit, neurosteroid increased the corticosterone level (HPA axis activity) and blockade of the neurosteroid seemed to turn off the HPA axis activity.
Neurosteroids increase the activity of CRH neurons following stress Perforated patch, current champ I=0 Spontaneous firing rates of CRH-GFP neurons Neurosteroid increased firing rates under stress and decreased under normal condition.
Animals were THDOC-treatment. Most CRH neurons depolarized following stress. When treated with the GABAAR inhibitor, neurons switched from excitatory to inhibitory.
Decreased phosphorylation of KCC2 residue Ser940 in the PVN following stress Biotinylated KCC2 = expression of KCC2 on cell surface The expression of KCC2 both in total and on surface and phosphorylation of KCC2 Ser940 decreased following stress.
Neurosteroidogenesis mediates stress-induced anxiety Blockade of neurosteroid by finasteride largely decreased animals’ anxiety behaviors.
谢谢 ！ Thank you!