Bosch et al.S1

Supplemental Material

Competitive receptor autoradiography for CRF receptor antagonist selectivity.

Since the specificity of the CRF-R1 and CRF-R2 antagonistisCP-154,526 and Astressin-2B has not been previously established in the prairie vole model, we performed competitive receptor binding experiments to examine specificity of these compounds for the prairie vole CRF receptors.Fresh frozen prairie vole brains were cut on a cryostat in 1:6 series of 20 µm thick coronal sections. Slides were thawed at room temperature until dry, briefly fixed in 0.1 % paraformaldehyde-PBS solution (2 min, pH 7.4), and then rinsed twice in 50 mM Tris base (pH 7.4) solution for 10 min each. Slides were then incubated in tracer buffer for 2 h. The tracer consisted of 50 mM Tris base, 10 mM MgCl, 0.1 % bovine serum albumin, and 0.2 nM [125I-Tyr0]-sauvagine (PerkinElmer/NEN, Boston, MA), a ligand with high affinity for CRF-R1 and CRF-R2 receptors. In the competition assays, unlabeled CP-154,526 or Astressin-2B was also included in the tracer buffer. Post-incubation, slides were rinsed with 50 mM Tris-base/10 mM MgCl (pH 7.4) for 4 x 5 min, plus 30 min with stirring on a magnetic stir plate. Finally, slides were dipped in dionized H2O, dried with cool air, and apposed to Kodak MR film for 89 h. [125I]-microscale standards (GE/Amersham Biosciences) were included to allow quantification.

To demonstrate that CP-154,526 and Astressin-2B have high affinity for the CRF-R1 and CRF-R2 receptors, respectively, we co-incubated these compounds (50 nM) separately (Fig. S1b,c) and together (Fig. S1d) in the [125I-Tyr0]-sauvagine binding buffer. To determine the specificity of the CRF receptor antagonists, competition binding assays were performed. Brains from two groups of animals (n = 4-5) were used to generate antagonist specificity curves. To analyze CRF-R1 receptor specificity, the tracer buffer contained 0.2 nM [125I-Tyr0]-sauvagine plus0, 0.5, or 25 Mof cold CP-154,526, a selective CRF-R1 antagonist(Schulz et al., 1996). The selectivity of Astressin-2B was determined by incubating the sections in tracer buffer containing 0.2 nM [125I-Tyr0]-sauvagine plus0, 0.05, 0.5, 5, or 25 M of cold Astressin-2B. CRF-R1 receptor binding was quantified in the cingulate cortex, while CRF-R2 binding was quantified in the choroid plexus.Binding density was quantified as described previously(Lim et al., 2005).Optical density measurements were taken bilaterally and averaged for each brain region across two or three sections. All density readings were converted to nano-curries per milligram tissue (nCi/mg) based on a known set of standard values ([125I]-microscale; GE/Amersham Biosciences). The means for each brain region were then averaged across 3-5 voles for each of the antagonist concentrations. Finally, mean binding densities across antagonist concentrations were normalized such that the regional densities obtained in the presence of 0 M of antagonist serve as 100 % bindingfor each brain region (Fig. S1e, f).

CP-154,526 is selective for CRF-R1 receptors since 50 nM eliminates binding at CRF-R1 binding sites (Cing.; factor CP-154,526 concentration: F2,11 202.6, p < 0.001; Fig. S1e) but does not decrease tracer binding at CRF-R2 binding sites even at 25 M (C.Plexus;1-way ANOVA, factor CP-154,526 concentration, F2,11 1.77, p = 0.216). Astressin-2B is selective for CRF-R2 since 50 nM of this compound significantly reduces radioligand binding in the choroid plexus (C.Plexus), but does not significantly reduce binding in the cingulate (Cing.) CRF-R1 receptors until 5 M. (C.Plexus (CRF-R2);1-way ANOVA, factor Astressin-2B concentration: F4,15153.9, p < 0.001; Fig. S1f).

These results indicate that CP-154,526 and Astressin-2B have similar specificities at the CRF-R1 and CRF-R2 as previously reported in the rat.

References

Lim MM, Nair HP,Young LJ (2005).Species and sex differences in brain distribution of corticotropin-releasing factor receptor subtypes 1 and 2 in monogamous and promiscuous vole species.J Comp Neurol487:75-92.

Schulz DW, Mansbach RS, Sprouse J, Braselton JP, Collins J, Corman M, et al(1996)CP-154,526: a potent and selective nonpeptide antagonist of corticotropin releasing factor receptors. Proc Natl Acad Sci93:10477-10482.

Figure Legends

Figure S1Competitive receptor autoradiography to show the selectivity of CP-154,526 and Astressin-2B for prairie vole CRF-R1 and CRF-R2 receptors, respectively. (a) Total CRF-R1 and CRF-R2 binding after incubation with 0.2 nM of [125I-Tyr0]-sauvagine. (b)CRF-R2 binding after competition at CRF-R1 receptors by 50 nM of cold CP-154,526. (c) CRF-R1 binding after competition at CRF-R2 by 50 nM of cold Astressin-2B. (d) [125I-Tyr0]-sauvagine binding after competition at both CRF-R1 and CRF-R2 by 50 nM of CP-154,526 and 50 nM Astressin-2B. (e, f) Dose-response curve for CP-154,526 (e) and Astressin-2B (f) in both the cingulate (Cing.; black square) and the choroid plexus (C.Plexus; gray triangle). Group size n = 3-5. Data are mean±SEM.* p < 0.001 vs. 0 nM binding density; † p < 0.05 vs. 0 nM binding density. Scale bar = 1mm.