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Glucocorticoids (GCs) play essential roles in physiology, development, and behavior that are mediated largely by the glucocorticoid receptor (GR). Although the GR has been intensively studied in mammals, very little is known about the GR in nonmammalian tetrapods. We analyzed the distribution and GC regulation of GR in the brain of the frog Xenopus laevis by immunohistochemistry. GR-immunoreactive (GR-ir) cells were widely distributed, with the highest densities in the medial pallium (mp; homolog of the mammalian hippocampus), accumbens, anterior preoptic area (POA; homolog of the mammalian paraventricular nucleus), Purkinje cell layer of the cerebellum, and rostral anterior pituitary gland (location of corticotropes). Lower but distinct GR-ir was observed in the internal granule cell layer of the olfactory bulbs, dorsal and lateral pallium, striatum, various subfields of the amygdala, bed nucleus of the stria terminalis (BNST), optic tectum, various tegmental nuclei, locus coeruleus, raphe nuclei, reticular nuclei, and the nuclei of the trigeminal motor nerves. Treatment with corticosterone (CORT) for 4 days significantly decreased GR-ir in the POA, mp, medial amygdala (MeA), BNST, and rostral pars distalis. Treatment with the corticosteroid synthesis inhibitor metyrapone (MTP) also significantly reduced GR-ir in the POA, mp, MeA and BNST, but not in the rostral pars distalis. Replacement with a low dose of CORT in MTP-treated animals reversed these effects in brain. Thus, chronic increase or decrease in circulating corticosteroids reduces GR-ir in regions of the frog brain. Our results show that the central distribution of GR-ir and regulation by corticosteroids are highly conserved among vertebrates.
Figure 1.
Affinity-purified antiserum to X. laevis GR recognizes full-length xGR synthesized in vitro. A: A major product of â¼96 kD was synthesized in the in vitro transcription/translation reaction using the p6xGR plasmid as template. [35S]methionine + cysteine was included in the in vitro transcription-translation reaction, and the reaction was analyzed by 10% SDS-PAGE, followed by fluorography. B: A protein of â¼96 kD was detected by Western blotting of the in vitro transcription/translation reaction programmed with the p6xGR vector but not in unprogrammed rabbit reticulocyte lysate. The arrow indicates the xGR protein band. The two lower MW bands are the rabbit IgG heavy and light chains present in the lysate and recognized by the goat anti-rabbit secondary antibody.
Figure 2.
The affinity-purified anti-xGR IgG recognizes a protein in A6 cells that shows nuclear translocation following treatment with the GR agonist dexamethasone. A6 cells were fixed and immunostained with anti-xGR IgG (A) or anti-xGR IgG preabsorbed with 50 μg/ml of the antigenic xGR peptide (B; Ã20 magnification). xGR immunoreactivity in A6 cells treated with (C,E) or without (D,F) dexamethasone for 1.5 hours (100 nM). C and D are at Ã10 magnification, and E and F are at Ã40 magnification. Scale bars = 100 μm in A (applies to A,B); 100 μm in C (applies to C,D); 100 μm in E (applies to E,F). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 3.
The anti-xGR IgG specifically stains a subset of cells in the X. laevis brain. Shown are photomicrographs of transverse sections through the anterior preoptic area (POA) of a juvenile X. laevis. Adjacent sections were immunostained with anti-xGR IgG (A), anti-xGR IgG preabsorbed with 50 μg/ml of the antigenic xGR peptide (B), or anti-xGR IgG preabsorbed with 50 μg/ml of a mixture of three unrelated peptides that corresponded to regions of the frog MR (C). All three images were captured at the same magnification. Scale bar = 120 μm.
Figure 4.
AâJ: Schematic coronal illustration of glucocorticoid receptor (GR) immunoreactivity (-ir) distribution in the brain of juvenile X. laevis. The drawing at the top of the figure shows a dorsal view of the X. laevis brain. Letters correspond to the rostrocaudal location of sections as depicted in the whole-brain drawing. Large circles represent large cells that exhibited robust GR-ir, and small circles represent smaller GR-ir cells. The anatomical drawings are from Tuinhof et al. (1998), with modifications of basal ganglia subdivisions according to MarÃn et al. (1998).
Figure 5.
Photomicrographs of transverse sections through the forebrain and part of the midbrain of juvenile X. laevis showing the distribution of glucocorticoid receptor (GR)-immunoreactive (-ir) cells. A: Internal granule cell layer of the olfactory bulb (igl). B: Dorsal pallium (dp) and lateralpallium (lp). C: Nucleus accumbens (Acc) and lateralseptum (ls). D: Medialpallium (mp). E: Medialamygdala (MeA). F: Bed nucleus of the stria terminalis (BNST). G: Anterior preoptic area (POA). H: Posterior preoptic area (POA). I: Ventral hypothalamic nucleus (VH). lv, Lateralventricle, IIIv, third ventricle. Scale bars = 120 μm.
Figure 6.
Photomicrographs of transverse sections through the midbrain, hindbrain, and pituitary gland of juvenile X. laevis showing the distribution of glucocorticoid receptor (GR)-immunoreactive cells. A: Suprachiasmatic nucleus (SC). B: Tegmental nuclei (Tn). C: Cerebellum (Cb). D: Motor nucleus of the trigeminal nerve (Vm) and raphe nucleus (Ra). E: Pars distalis of the anteriorpituitary gland (pd with arrow; ME, median eminence). Scale bars = 120 μm.
Figure 7.
Plasma corticosterone concentrations in X. laevis juveniles following pharmacological treatments and exposure to shaking/confinement stressor. Juvenile frogs were reared in water containing vehicle (0.0005% ethanol; control and stressed) for 4 days, corticosterone (CORT; 500 nM) for 4 days, metyrapone (MTP; 110 μM) for 5 days, or MTP for 1 day followed by MTP + CORT (50 nM) for 4 days (MTP CORT 50 nM). During the 4-day treatment period, all groups were exposed to 0.0005% ethanol. The âstressedâ group was exposed to 6 hours of shaking/confinement stressor before sacrifice; animals in the other treatments were left undisturbed, and all animals were killed in the afternoon of the same day. Data presented are the mean ± SEM. Significant differences from control are indicated (n = 5â6/treatment; *P < 0.05, ANOVA).
Figure 8.
Effects of changes in plasma corticosteroid concentration on glucocorticoid receptor (GR) immunoreactivity (-ir) in the anterior preoptic area (POA) of juvenile X. laevis. A: The photomicrographs are of representative transverse sections in the same anatomical plane of the POA; vehicle control (B; 0.0005% ethanol), corticosterone (C; CORT; 500 nM for 4 days), metyrapone (D; MTP; 110 μM for 5 days), MTP + CORT (110 μM MTP for 1 day followed by MTP + 50 nM CORT for 4 days). The graph shows the densitometric analysis of GR-ir in the POA following hormone or drug treatments (treatments as described above). Bars represent the mean ± SEM. Significant differences from the control are indicated (n = 5â6/treatment, *P < 0.05; ANOVA). Scale bar = 120 μm.
Figure 9.
Effects of changes in plasma corticosteroid concentration on glucocorticoid receptor (GR) immunoreactivity (-ir) in the medial amygdala, bed nucleus of the stria terminalis (BNST), medial pallium, and rostral pars distalis of the anterior pituitary of juvenile X. laevis. Treatments are described in the legend to Figure 8. Bars represent the mean GR-ir density (see Materials and Methods) ± SEM. Significant differences from the control are indicated (n = 5â6/treatment; *P < 0.05, ANOVA).
Figure 10.
Expression of GR mRNA in frog brain as analyzed by real-time quantitative PCR (RTqPCR). A: Distribution of GR mRNA in adult frog brain. Brain regions were microdissected from five male and five female adult X. laevis. There were no sex differences in GR mRNA expression, so we pooled the data (n = 8â10 per brain region). B: Effects of manipulation of circulating corticosteroids on mRNA expression in the telencephalon/preoptic area of juvenile X. laevis. Frogs were treated as described in the legend to Figure 8. Total RNA was extracted and gene expression analyzed by RTqPCR. A relative quantitation method was used, and GR mRNA was normalized to the expression of the housekeeping gene rpL8; therefore, the data are presented as arbitrary units. Bars represent the mean ± SEM. Letters indicate significant differences among brain regions or treatment groups (P < 0.05, Fisher's LSD multiple-comparisons test).
