Fig 2: OT and AVP are expressed in nociceptive DRG neurones. (a) Top panel: Confocal images DRG neurones cultured for 48 hours stained with antibodies against OT and TRPV1; the merged image shows co-localisation of both markers. Bottom panel: Confocal images of AVP-eGFP DRG neurones cultured for 48 hours and stained with TRPV1 antibody. The merged image shows co-localisation of both markers. Scale bars 50 µm. (b) AVP, OT and capsaicin-induced [Ca2+]i responses in cultured DRG neurones. Representative traces showing [Ca2+]i responses to AVP (100 nM), OT (100 nM), K+ (50 mM) and capsaicin (1 µM). (c) Pharmacology of AVP and OT-induced [Ca2+]i responses. Traces show AVP or OT-induced [Ca2+]i transients in control, in the presence of specific antagonist ([deamino-Pen1, O-Me-Tyr2, Arg8]-vasopressin or dOVT) and after washout. (d) Concentration dependence of AVP and OT-induced Ca2+ signals. The graph shows average peak amplitudes of [Ca2+]i transients at different concentrations of the agonist. (amplitudes are presented as mean ± SEM; experiments were performed on 6 AVP-eGFP neurones from 3 different cultures and 6 OT-mRFP neurones from 3 different cultures). (e) Average amplitudes (mean ± SEM), of [Ca2+]i transients triggered by AVP and OT in control conditions and in the presence of specific inhibitors of V1a receptors ([deamino-Pen1, O-Me-Tyr2, Arg8]-vasopressin; n = 6 AVP-eGFP neurones from 3 cultures; p = 0.0094, Friedman test) or OT receptors (dOVT; n = 5 OT-mRFP neurones from 3 cultures p = 0.0067, Friedman test).

Fig 3: Expression levels of P-LAP/IRAP and AVP in the (A) murine hippocampus and (B) pituitary gland. The expression level of P-LAP/IRAP was monitored by western blot analysis (top panel) and quantified by densitometric analysis (bottom panel). AVP levels (bottom panel) were monitored using a commercially available ELISA assay kit, as described in section “Materials and methods.” The LD cycle of the sacrificed animals is also shown in the figure.

Fig 4: Expression patterns of P-LAP/IRAP and AVP in several sub-regions of the murine brain. (A) Western blot analysis of P-LAP/IRAP distribution in several sub-regions of the murine brain. (B) Immunohistochemical localization of P-LAP/IRAP (green) and AVP (red) in several sub-regions of the murine brain. Higher-magnification views of the boxed areas in the merged images are shown at the bottom panel. CA2: cornu ammonis 2 area, CA3: cornu ammonis 3 area, 3V; 3rd ventricle, and opt; optic tract.

Fig 5: Effects of dehydration on AVP and OT expression in DRG Expression of endogenous fluorescence in DRG explants (lower panels) isolated from AVP-eGFP and OT-mRFP transgenic rats dehydrated for 3 (middle panels) and 5 days (right panels). The relative fluorescence expression in control preparations was taken as 100%. Dehydration affected fluorescence of neither AVP-eGFP nor OT-mRFP DRG explants, as quantified on bar graphs on the right. Scale bars 20 µm.