Dose-dependent effects of corticosterone on nuclear glucocorticoid receptors and their binding to DNA in the brain and pituitary of the rat

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Dose-dependent effects of corticosterone on nuclear glucocorticoid receptors and their binding to DNA in the brain and pituitary of the rat Francesca Spiga⁎, Stafford L. Lightman Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, UK

A R T I C LE I N FO

AB S T R A C T

Article history:

The glucocorticoid receptor (GR) is a transcription factor which regulates glucocorticoid-

Accepted 2 February 2009

mediated negative feed-back of the HPA axis. In this study, we have investigated the dose-

Available online 12 February 2009

dependent effects of corticosterone on nuclear GR and GR–DNA binding in the brain and pituitary of adult adrenalectomized male rats. Rats were injected with either saline (1 ml/kg,

Keywords:

i.p.) or corticosterone (0.1, 0.3, and 1 mg/kg, i.p.). Nuclear GR and GR–DNA binding were

Corticosterone

measured in nuclear extracts from hippocampus, hypothalamus, prefrontal cortex,

Glucocorticoid receptor

amygdala and pituitary using Western immunoblotting and ELISA, respectively. We found

HPA axis

that the dose-dependent effects of corticosterone on nuclear GR and GR–DNA binding are

Nuclear translocation

similar across all the areas we analyzed, although at lower levels of corticosterone changes

DNA binding

were observed only in the hippocampus. These data have important implications for our understanding of tissue specificity of glucocorticoid action including the corticosteroidmediated negative feed-back response of the HPA axis. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

The glucocorticoid receptor (GR) is a ligand activated transcription factor that regulates the expression of genes involved in the maintenance of homeostasis (Bagchi et al., 1992), including metabolic processes such as gluconeogenesis (Hers et al., 1970), inflammation (Adcock, 2003), and the response to stressors (De Kloet et al., 2005). In the absence of ligand, the inactive form of GR is found predominately in the cytoplasm (Pratt et al., 1990) and when activated by ligand (e.g. corticosterone and cortisol), GR undergoes conformational changes and translocates to the cell nucleus (Picard and Yamamoto, 1987). Within the nucleus, GR mediates transcriptional regulation through binding to specific sequences of DNA named glucocorticoid response elements (GREs), a process termed trans-activation. The transcrip-

tional outcome can be either enhancement or repression of gene expression depending on whether GR binds positive GREs or negative GREs (nGREs) (reviewed in Heitzer et al., 2007). GR also modulates gene expression through binding to other transcription factors (including NF-κB, AP-1, CREB) normally inhibiting gene transcription (trans-repression) although under certain conditions (depending on the transcription factor composition or cellular context) this mechanism can also promote gene expression (Diamond et al., 1990). The hypothalamic–pituitary–adrenal (HPA) axis is the main neuroendocrine system involved in the regulation of the stress response (De Kloet et al., 2005). The HPA axis responses are initiated by secretion of corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) from neurons of the medial parvocellular paraventricular nucleus (PVN) of the

⁎ Corresponding author. Fax: +44 117 3313169. E-mail address: [email protected] (F. Spiga). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.02.001

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hypothalamus promoting the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary into the systemic circulation. ACTH, in turn, stimulates the release of glucocorticoids from the adrenal cortex. The activity of the HPA axis is modulated by a mechanism of glucocorticoid-mediated negative feedback via activation of two types of receptors: mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) (Reul and De Kloet, 1985). Since glucocorticoid receptors have low affinity for glucocorticoids, GR activation occurs when glucocorticoid levels are elevated, such as during the diurnal peak of hormone secretion or following exposure to stress. Glucocorticoids activation of GR in the PVN and pituitary inhibit corticosterone release via inhibition of CRF/AVP and ATCH release, respectively. Moreover, glucocorticoids can also modulate HPA axis activity by GR mediated inhibition in the limbic hippocampus and medial prefrontal cortex, or activation in the amygdala, via projections to the PVN (reviewed in Herman et al., 1996). A difference in GR nuclear translocation, but not DNA binding during the circadian cycle and stress has previously been demonstrated in both hippocampus and prefrontal cortex (Kitchener et al., 2004). The same study also showed that both GR nuclear translocation and DNA binding were different in hippocampus and prefrontal cortex when measured during the circadian peak or during stress, supporting a region-specific difference on GR activity within brain regions. It is indeed known that GR sensitivity to corticosterone in rat brain is site-specific (Brinton, 2008). We have now investigated whether the effects of corticosterone on GR activity in areas of the brain and pituitary involved in the modulation of the HPA axis are dose- and region-dependent.

2.

Results

2.1.

Plasma corticosterone

Plasma corticosterone levels were not detectable in ADX rats treated with saline, whereas injection of corticosterone increased plasma corticosterone levels (ANOVA: F(3,21) = 111.861; P < 0.00001, Fig. 1). Specifically a significant effect was observed for all the corticosterone doses used (P = 0.0011

for 0.1 mg/kg, and P < 0.00001 for both 0.3 and 1 mg/kg). A positive linear correlation between corticosterone doses and plasma corticosterone levels (r = 0.968; P < 0.00001, data not shown) revealed a dose-dependent effect of exogenous corticosterone on circulating corticosterone levels.

2.2.

In the present study nuclear GR was measured using Western immunoblotting. Previous investigations using supraphysiological concentration of corticosterone have shown a reduction of cytoplasmic GR and enhanced nuclear GR in cell culture (Pariante et al., 2001) and in vivo (Spencer et al., 2000). However, both our group (Conway-Campbell et al., 2007) and others (Kitchener et al., 2004) have been unable to demonstrate a significant depletion of cytoplasmic GR following administration of lower doses of corticosterone in vivo. Quantitative evaluation of GR in nuclear extracts from specific brain areas and the pituitary of rats treated with saline and corticosterone is shown in Fig. 2. Data are expressed as mean fold induction ± SEM over rats treated with saline. Analysis of repeated measures showed a significant effect of corticosterone (treatment effect: F(1,18) = 32.823; P < 0.00001) and this effect was region specific (treatment × region effect: F(4,18) = 3.568; P = 0.0019). Corticosterone increased nuclear GR in all the regions analyzed: hippocampus (F(3,21) = 9.582; P = 0.0005), hypothalamus (F(3,21) = 5.677; P = 0.0064), prefrontal cortex (F(3,21) = 3.381; P = 0.041), amygdala (F(3,21) = 5.817; P = 0.0058) and pituitary (F(3,21) = 30.548; P < 0.0001). No significant effect of the low dose of corticosterone (0.1 mg/kg) was observed in any of the areas analyzed, although a trend was found in the hippocampus (P = 0.0685). In contrast, the medium dose of corticosterone (0.3 mg/kg) significantly increased nuclear GR in hippocampus (P = 0.0052), hypothalamus (P = 0.0226), prefrontal cortex (P = 0.021) and amygdala (0.0347) but only a trend in the pituitary (P = 0.067). The high dose of corticosterone (1 mg/kg) increased nuclear GR in all areas (P < 0.0001, P = 0.0019, P = 0.01, P = 0.0012 and P < 0.00001, respectively for hippocampus, hypothalamus, prefrontal cortex, amygdala and pituitary).

2.3.

Fig. 1 – Corticosterone administration dose-dependently increases circulating corticosterone levels. Adrenalectomized rats were treated with saline (0.1 ml/lk, i.p., n = 6) or corticosterone (0.1 mg/kg, n = 6; 0.3 mg/kg, n = 6; 1 mg/kg, n = 4, i.p.). Plasma corticosterone levels were measured 30 min after corticosterone injection using radioimmunoassay (RIA). ⁎P < 0.005; ⁎⁎P < 0.00001; significant difference vs saline.

Nuclear GR

GR–DNA binding

GR–DNA data are expressed as mean fold induction ± SEM above that of rats treated with saline (Fig. 3). In accordance with the nuclear GR data, there was an effect of corticosterone on GR–DNA binding (treatment effect: F(1,18) = 33.613; P < 0.00001) and this effect was region specific (treatment × region effect: F(4,18) = 8.246; P < 0.00001). Corticosterone induced GR–DNA binding in the hippocampus (F(3,21) = 28.166; P < 0.00001), hypothalamus (F(3,21) = 5.719; P = 0.0062), the prefrontal cortex (F(3,21) = 14.997; P < 0.0005), amygdala (F(3,21) = 11.601; P < 0.0005) and pituitary (F(3,21) = 11.645 P < 0.0005). Specifically, there was a significant effect of the low dose of corticosterone (0.1 mg/kg) on GR–DNA binding in the hippocampus (P = 0.014). Moreover, a significant effect of the medium dose of corticosterone (0.3 mg/kg) was observed only in the hippocampus (P = 0.0001), although a trend towards significance was observed in the hypothalamus, amygdala

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Fig. 2 – Effect of corticosterone on GR nuclear translocation in rat brain and pituitary. Adrenalectomized rats were treated with saline (Sal, 0.1 ml/lk, i.p., n = 6) or corticosterone (CORT, 0.1 mg/kg, n = 6; 0.3 mg/kg, n = 6; 1 mg/kg, n = 4, i.p.). Brain and pituitary were collected 30 min after corticosterone injection and GR nuclear translocation was measured as increased GR content in the nuclear fraction of hippocampus, hypothalamus, prefrontal cortex, amygdala and pituitary. Data are expressed as fold induction of nuclear translocation of GR of rats treated with saline, and are presented as mean ± SEM. Data were analyzed as adjusted volume OD for the GR as well as the Histone H1 loading control (data not shown). Each data point was then normalized relative to the Histone H1 present in that sample. ⁎P < 0.05; ⁎⁎P < 0.01; ⁎⁎⁎P < 0.005; ⁎⁎⁎⁎P < 0.00001; significant difference vs saline.

and pituitary (P = <0.1). The high dose of corticosterone (1 mg/ kg) induced GR–DNA binding in all areas (P = 0.005 for the hypothalamus and P < 0.00001 for the hippocampus, prefrontal cortex, amygdala and pituitary).

3.

Discussion

Corticosterone increased nuclear GR and induced GR–DNA binding in all the regions analyzed. Using three different doses of corticosterone we found that these responses were region specific. We chose doses of corticosterone to reproduce plasma corticosterone levels found in rats at their diurnal hormonal peak (0.1 and 0.3 mg/kg), or following exposure to a mild (0.3 mg/kg) or severe (1 mg/kg) stress. The effect of corticosterone treatment on nuclear GR levels and on GR–DNA binding was dose-dependent in all the areas analyzed. At the lowest dose (0.1 mg/kg) corticosterone had no significant effect on nuclear GR in any area, whereas the same dose did induce GR–DNA binding in the hippocampus but not in any other area analyzed. As this dose of corticosterone produced plasma levels compatible to those measured in rat during the diurnal peak (Spiga et al., 2007), our data are consistent with previous data showing a lower GR activation in the prefrontal cortex, compared to the hippocampus, during the circadian peak (Kitchener et al., 2004;Reul et al., 1987a).

The higher dose of corticosterone (1.0 mg/kg) was able to increase nuclear GR and to induce GR–DNA binding in all the areas analyzed. As this dose of corticosterone produced plasma corticosterone levels comparable to those induced by exposure to a severe stressor (i.e. restraint) (Spiga et al., 2007), our data are consistent with other reports showing GR activation during stress (De Kloet et al., 1987; Kitchener et al., 2004; Reul et al., 1987b,a). Interestingly, the medium dose of corticosterone (0.3 mg/ kg), significantly increased nuclear GR in hippocampus, hypothalamus, prefrontal cortex and amygdala, but not in the pituitary. Moreover, the same dose induced significant GR DNA binding only in the hippocampus with a non-significant trend seen in the hypothalamus, amygdala and pituitary. No effect of this dose was observed in the prefrontal cortex. The lack of effect of this dose of corticosterone on nuclear GR in the pituitary could be due to the presence of CBG in this tissue (De Kloet et al., 1977). In addition to the activity of circulating CBG to reduce the active level of free corticosterone in the blood (Pardridge, 1981) tissue CBG within the pituitary gland could also act to limit nuclear translocation of GR and subsequent GR-mediated effects (Sakly and Koch, 1981). Another mechanism that could contribute to the different responses in different areas is differential kinetics of corticosterone distribution within the pituitary and brain. It has been recently shown that subcutaneous administration of corticosterone

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Fig. 3 – Effect of corticosterone on GR–DNA binding in rat brain and pituitary. Adrenalectomized rats were treated with saline (Sal, 0.1 ml/lk, i.p., n = 6) or corticosterone (CORT, 0.1 mg/kg, n = 6; 0.3 mg/kg, n = 6; 1 mg/kg, n = 4, i.p.). Brain and pituitary were collected 30 min after corticosterone injection and DNA binding of GR was analyzed in the nuclear fraction of hippocampus, hypothalamus, prefrontal cortex, amygdala and pituitary using ELISA. Data are expressed as fold induction of GR–DNA binding of rats treated with saline and are presented as mean ± SEM. ⁎P < 0.05; ⁎⁎P < 0.001; ⁎⁎⁎P < 0.0005; ⁎⁎⁎⁎P < 0.00001; significant difference vs saline.

results in maximal plasma levels after 25 min, whereas maximal brain corticosterone levels were only achieved after 40 min (Droste et al., 2008). This suggests that peak tissue levels of corticosterone in the pituitary for instance might have occurred at different and probably earlier time point. However, it is possible that the timing of the peak of corticosterone in the pituitary and brain regions may relate to the absolute levels of corticosterone. Indeed, it is evident from our data that the higher dose of corticosterone had a much greater effect on nuclear GR levels in the pituitary than in the brain regions analyzed. A similar mechanism could account for the differential effect of the medium dose of corticosterone across different brain areas. Using radioimmunoassay, McEwen et al. (1980) have shown higher levels of corticosterone in the hippocampus, compared to other brain areas, in adrenalectomized rats treated with [3H] corticosterone. However, more recent studies in intact rats have shown identical endogenous levels of corticosterone within different brain regions (Droste et al., 2008; Kitchener et al., 2004). Intracellular levels of corticosterone in the brain are regulated by 11 beta-hydroxysteroid dehydrogenase (11 betaOHSD), which metabolizes corticosterone to inactive 11dehydrocorticosterone (Lakshmi and Monder, 1985). A differential distribution of this enzyme within the brain could account for the different pattern of GR–DNA binding found in hippocampus compared to hypothalamus and amygdala. High levels of this enzyme have been found in pituitary, hippocampus and cortex whereas a lower activity was found in the hypothalamus and amygdala (Moisan et al., 1990). This would

not, however, explain the differences in GR–DNA binding between the hippocampus and the prefrontal cortex. An essential regulator of GR translocation to the nucleus (and subsequent DNA binding) is the chaperone molecule HSP90. Although a widespread distribution of HSP90 has been described, regional variation in HSP9O mRNA density does occur. Specifically HSP90 mRNA was found to be most abundant in structures related to the limbic system, such as the hippocampus and amygdala (Izumoto and Herbert, 1993). Furthermore, a differential diurnal regulation of GR-HSP90 interaction has been described between the hippocampus and the hypothalamus (Furay et al., 2006). A further possibility is that differential kinetics of GR– DNA binding activation within different brain areas could also contribute to the difference between hippocampus and the other brain areas analyzed. Further studies with additional time points would be necessary to characterize this possibility. Finally, the potential role of corticosterone-mediated MR activation within the hippocampus needs attention. Due to lack of adequate nuclear extract, we were not able to measure nuclear MR or MR–DNA binding in the hippocampus. However, due to the higher affinity of corticosterone for MR, compared to GR (N10 fold) we could predict that both nuclear MR and MR– DNA binding levels will be saturated at the lower dose of corticosterone used in the present study. A further complication is that GR binding to DNA can also be induced by MR–GR heterodimers, and in view of the abundance of MR in the hippocampus, this mechanism could have contributed to the differential pattern of the dose–response found in this area.

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The fact that in this study we used adrenalectomized rats (without glucocorticoids replacement) also needs to be taken into consideration. There are several studies reporting an increase in GR (and MR) after adrenalectomy and the pattern of those changes appears to be similar across brain regions (Kalman and Spencer, 2002). Thus, increased GR density could effect the sensitivity of the response to corticosterone, although a similar corticosterone-induced translocation pattern of GR and MR between intact and adrenalectomized rats in hippocampus has recently been reported (Sarabdjitsingh et al., 2009). Adrenalectomy will of course also increase AVP and CRH levels (Sawchenko et al., 1984) which in turn could induce changes in both GR and MR (Hugin-Flores et al., 2003). Our data support the concept of GR activation in the hippocampus during both basal and stress-induced HPA axis activity. On the other hand, it would appear that GR located in the hypothalamus and in other limbic structures, such as amygdala and prefrontal cortex, might only be activated when circulating corticosterone levels are elevated, for instance during stress.

4.

Experimental procedures

4.1.

Animals

Male Sprague Dawley adults rats (Harlan-Olac, Bicester, UK) weighing 180–200 g upon arrival were housed in groups of four animals per cage under standard environmental conditions (21 ± 1 °C) under a 14 h light, 10 h dark schedule (lights on at 5:15) and food and water (or saline when specified) were provided ad libitum throughout the experiment. Animals were allowed to acclimatize to the facility for one week before surgery was performed. All procedures were approved by the University of Bristol Ethical Review Group and were conducted in accordance with Home Office guidelines and the UK Animals (Scientific Procedures) Act, 1986. All efforts were made to minimize the number of animals used and their suffering.

4.2.

Surgery

Rats were anesthetized with isoflurane (Merial Animal Health, Ltd, UK) and bilateral adrenalectomy was performed to deplete endogenous corticosteroids. After surgery, ADX rats were returned to their home cage, and allowed to recover for 5 days prior to the experiment with 0.9% NaCl in drinking water provided ad libitum.

4.3.

Drugs and experimental procedures

On the day of the experiment, rats were injected with either vehicle (saline, 0.9% NaCl, 1 ml/kg, i.p., n = 6) or three doses of corticosterone (CORT): low (0.1 mg/kg, i.p., n = 6), medium (0.3 mg/kg, i.p., n = 6) and high (1 mg/kg, i.p., n = 4) in the form of a preformed water-soluble complex of corticosterone and 2hydroxypropyl-β-cyclodextrin (Cort-HBC, Cat. No. C-174; Sigma-Aldrich, St. Louis, MO). Thirty minutes after treatment, rats were anesthetized with isoflurane and killed by decapitation. Trunk blood was collected into heparinized tubes and the plasma obtained by centrifugation was stored at − 20 °C until

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measurement of corticosterone. After decapitation, the brain and pituitary were removed from the skull and the former dissected for hippocampus, hypothalamus, prefrontal cortex and amygdala. All tissue was rapidly frozen in liquid nitrogen and stored at −80 °C until nuclear extracts were prepared.

4.4.

Corticosterone measurement

To assess circulating levels of corticosterone following the treatment, plasma corticosterone levels were measured by radioimmunoassay as previously described (Spiga et al., 2007). Intra- and inter-assay coefficients of variation of the assay were 16.65% and 13.30% respectively.

4.5.

Nuclear extract preparation

Nuclear fractions were prepared according to the method of Vallone et al. (1997). All procedures were performed at 4 °C and on ice. Each area was homogenized in 1 ml/100 mg tissue in S1 buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA (pH 8), supplemented with 0.5 mM dithiothreitol, 0.2 mM Na orthovanadate, 2 mM NaF, and Complete Protease Inhibitor (Roche Diagnostics Ltd., Burgess Hill, UK)] using a Dounce homogenizer (Jencons, Leeds, UK). Nuclear proteins were extracted in 1.2 pellet volume of S2 buffer [10 mM HEPES (pH 7.9), 400 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA (pH 8), and 5% glycerol, supplemented with 0.5 mM dithiothreitol, 0.2 mM Na orthovanadate, 2 mM NaF, and Complete Protease Inhibitor] and stored at −80 °C. Protein concentrations were determined by bicinchoninic acid assay (BCA) (Pierce, Rockford, IL, US).

4.6.

Western immunoblotting

Western blot was performed according to the original method of Laemmli (1970). Aliquots of each sample (10 μg for pituitary, 20 μg for hippocampus, aprefrontal cortex and amygdala, 15 μg for hypothalamus) were run on a 8–15% polyacrylamide gel, and transferred to polyvinylidene fluoride (PVDF) membrane (Amersham Biosciences, Uppsala, Sweden). The upper portion of each membrane (above 75 kDa) was probed with anti-GR antibody M20 at 1:10,000 dilution (Cat. No. sc-1004, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, US). To determine the lack of cytoplasmic contamination, the middle part of each membrane (between 75 and 37 kDa) was probed with antiMEK1/2 antibody at 1:1000 dilution (Cat. No. 9122, New England Biolabs, Inc. Ipswich, MA, US). Loading was determined by probing the lower portion of each membrane (below 37 kDa) with anti-Histone H1 at 1:1000 dilution (Cat. No. sc-8030, Santa Cruz Biotechnology). Membranes were then probed with antirabbit IgG-HRP (Cat. No. NA934V; Amersham Biosciences) for GR and MEK1/2, or anti-mouse IgG-HRP (Cat. No. NA931V; Amersham Biosciences) for Histone H1 (all at 1:10,000 dilution). The signal was detected using enhanced chemiluminescence (ECL Plus) reagent and enhanced chemiluminescence hyperfilm (Amersham Biosciences, Uppsala, Sweden). The Western blot bands were quantified by densitometry using an Epson perfection scanner (Epson Europe, Amsterdam, The Netherlands) in transmission mode and the associated Quantity One software (Bio-Rad Laboratories, Hercules,

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CA, US). Data were analyzed as adjusted volume optical density (OD) for the GR (97 kDa) band as well as the Histone H1 (31/33 kDa) band (data not shown). Each data point was then normalized relative to the Histone H1 present in that sample. For each blot, samples from rats treated with corticosterone were analyzed as fold inductions relative to vehicle. Only samples run on the same gel were compared. Fold inductions from all data sets were then collected for graphic representation and statistical analysis.

4.7.

GR–DNA binding assay

A commercially available ELISA-based transcription factor binding assay kit (TransAM GR, Cat. No. 45496, Active Motif, Carlsbad, CA, US) was used to measure GR–DNA binding activity for each nuclear sample. The protein concentration of each sample was determined by BCA, and, as an additional normalization control to more accurately measure the integrity of each sample, an aliquot of nuclear extract from each sample was also processed for nuclear factor-YA (NF-YA)-DNA binding activity (TransAM NF-YA, Cat. No. 40396, Active Motif). NF-YA is a ubiquitous transcription factor that showed no significant difference between the treatment groups (data not shown). DNA binding assay kits were used in accordance with the manufacturer's instructions. Briefly, nuclear extracts (15–20 μg for GR, 3–5 μg for NF-YA) from hippocampus, hypothalamus, prefrontal cortex amygdala and pituitary of rats treated with the vehicle (n = 6) or corticosterone (n = 5–6/ dose) were incubated in 96-well plates coated with GR or NF-YA binding consensus oligonucleotide sequence for 1 h, then incubated with the supplied primary anti-GR or NF-YA antibody (1:1000) for 1 h, then with a peroxidase-conjugated second antibody (1:1000) for 1 h. After the substrate was added, color development was read at 450 nm, and the OD of both GR and NF-YA were recorder. The results in OD obtained from the GR assay were normalized to the results obtained from the NF-YA assay. Data were then expressed as fold induction relative to vehicle.

4.8.

Statistical analysis

Data were analyzed using ANOVA, followed by Fisher protected least-significant difference (LSD) post hoc test. GR nuclear translocation and DNA binding data were analyzed for overall effect of corticosterone treatment using repeated measure ANOVA with area as within subject factor. Values of P < 0.05 were considered significant.

Acknowledgments We are grateful to Susanne K Droste, Yvonne Kershaw, David N Egbosimba and Becky L Conway-Campbell for technical assistance.

REFERENCES

Adcock, I.M., 2003. Glucocorticoids: new mechanisms and future agents. Curr. Allergy Asthma Rep. 3, 249–257.

Bagchi, M.K., Tsai, M.J., O'Malley, B.W., Tsai, S.Y., 1992. Analysis of the mechanism of steroid hormone receptor-dependent gene activation in cell-free systems. Endocr. Rev. 13, 525–535. Brinton RE MB1 (2008) Brinton, R.E., McEwen, B.S. 1988 Regional distinctions in the regulation of Type I and Type II adrenal steroid receptors in the central nervous system. Neurosci. Res. Commun. 2:37–45. Conway-Campbell, B.L., McKenna, M.A., Wiles, C.C., Atkinson, H.C., De Kloet, E.R., Lightman, S.L., 2007. Proteasome-dependent down-regulation of activated nuclear hippocampal glucocorticoid receptors determines dynamic responses to corticosterone. Endocrinology 148, 5470–5477. De Kloet, E.R., Burbach, P., Mulder, G.H., 1977. Localization and role of transcortin-like molecules in the anterior pituitary. Mol. Cell. Endocrinol. 7, 261–273. De Kloet, E.R., Ratka, A., Reul, J.M., Sutanto, W., van Eekelen, J.A., 1987. Corticosteroid receptor types in brain: regulation and putative function. Ann. N.Y. Acad. Sci. 512, 351–361. De Kloet, E.R., Joels, M., Holsboer, F., 2005. Stress and the brain: from adaptation to disease. Nat. Rev., Neurosci. 6, 463–475. Diamond, M.I., Miner, J.N., Yoshinaga, S.K., Yamamoto, K.R., 1990. Transcription factor interactions: selectors of positive or negative regulation from a single DNA element. Science 249, 1266–1272. Droste, S.K., de, G.L., Atkinson, H.C., Lightman, S.L., Reul, J.M., Linthorst, A.C., 2008. Corticosterone levels in the brain show a distinct ultradian rhythm but a delayed response to forced swim stress. Endocrinology 149, 3244–3253. Furay, A.R., Murphy, E.K., Mattson, M.P., Guo, Z., Herman, J.P., 2006. Region-specific regulation of glucocorticoid receptor/HSP90 expression and interaction in brain. J. Neurochem. 98, 1176–1184. Heitzer, M.D., Wolf, I.M., Sanchez, E.R., Witchel, S.F., DeFranco, D.B., 2007. Glucocorticoid receptor physiology. Rev. Endocr. Metab. Disord. 8, 321–330. Herman, J.P., Prewitt, C.M., Cullinan, W.E., 1996. Neuronal circuit regulation of the hypothalamo-pituitary-adrenocortical stress axis. Crit. Rev. Neurobiol. 10, 371–394. Hers, H.G., De, W.H., Stalmans, W., van den, B.G., 1970. The control of glycogen synthesis in the liver. Adv. Enzyme Regul. 8, 171–190. Hugin-Flores, M.E., Steimer, T., Schulz, P., Vallotton, M.B., Aubert, M.L., 2003. Chronic corticotropin-releasing hormone and vasopressin regulate corticosteroid receptors in rat hippocampus and anterior pituitary. Brain Res. 976, 159–170. Izumoto, S., Herbert, J., 1993. Widespread constitutive expression of HSP90 messenger RNA in rat brain. J. Neurosci. Res. 35, 20–28. Kalman, B.A., Spencer, R.L., 2002. Rapid corticosteroid-dependent regulation of mineralocorticoid receptor protein expression in rat brain. Endocrinology 143, 4184–4195. Kitchener, P., Di, B.F., Borrelli, E., Piazza, P.V., 2004. Differences between brain structures in nuclear translocation and DNA binding of the glucocorticoid receptor during stress and the circadian cycle. Eur. J. Neurosci. 19, 1837–1846. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lakshmi, V., Monder, C., 1985. Evidence for independent 11-oxidase and 11-reductase activities of 11 beta-hydroxysteroid dehydrogenase: enzyme latency, phase transitions, and lipid requirements. Endocrinology 116, 552–560. McEwen, B.S., Stephenson, B.S., Krey, L.C., 1980. Radioimmunoassay of brain tissue and cell nuclear corticosterone. J. Neurosci. Methods 3, 57–65. Moisan, M.P., Seckl, J.R., Edwards, C.R., 1990. 11 beta-hydroxysteroid dehydrogenase bioactivity and messenger RNA expression in rat forebrain: localization in hypothalamus, hippocampus, and cortex. Endocrinology 127, 1450–1455. Pardridge, W.M., 1981. Transport of protein-bound hormones into tissues in vivo. Endocr. Rev. 2, 103–123.

BR A I N R ES E A RC H 1 2 9 3 ( 2 00 9 ) 1 0 1 –1 07

Pariante, C.M., Pearce, B.D., Pisell, T.L., Su, C., Miller, A.H., 2001. The steroid receptor antagonists RU40555 and RU486 activate glucocorticoid receptor translocation and are not excreted by the steroid hormones transporter in L929 cells. J. Endocrinol. 169, 309–320. Picard, D., Yamamoto, K.R., 1987. Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 6, 3333–3340. Pratt, W.B., Dallman, F.C., Meshinchi, S., Scherrer, L.C., 1990. The relationship between glucocorticoid receptor binding to Hsp90 and receptor function. Nippon Naibunpi Gakkai Zasshi 66, 1185–1197. Reul, J.M., De Kloet, E.R., 1985. Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation. Endocrinology 117, 2505–2511. Reul, J.M., van den Bosch, F.R., De Kloet, E.R., 1987a. Differential response of type I and type II corticosteroid receptors to changes in plasma steroid level and circadian rhythmicity. Neuroendocrinology 45, 407–412. Reul, J.M., van den Bosch, F.R., De Kloet, E.R., 1987b. Relative occupation of type-I and type-II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications. J. Endocrinol. 115, 459–467. Sakly, M., Koch, B., 1981. Ontogenesis of glucocorticoid receptors in anterior pituitary gland: transient dissociation among

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cytroplasmic receptor density, nuclear uptake, and regulation of corticotropic activity. Endocrinology 108, 591–596. Sarabdjitsingh, R.A., Meijer, O.C., Schaaf, M.J., De Kloet, E.R., 2009. Subregion-specific differences in translocation patterns of mineralocorticoid and glucocorticoid receptors in rat hippocampus. Brain Res. 1249, 43–53. Sawchenko, P.E., Swanson, L.W., Vale, W.W., 1984. Co-expression of corticotropin-releasing factor and vasopressin immunoreactivity in parvocellular neurosecretory neurons of the adrenalectomized rat. Proc. Natl. Acad. Sci. U. S. A. 81, 1883–1887. Spencer, R.L., Kalman, B.A., Cotter, C.S., Deak, T., 2000. Discrimination between changes in glucocorticoid receptor expression and activation in rat brain using Western blot analysis. Brain Res. 868, 275–286. Spiga, F., Harrison, L.R., Wood, S.A., Atkinson, H.C., Macsweeney, C.P., Thomson, F., Craighead, M., Grassie, M., Lightman, S.L., 2007. Effect of the glucocorticoid receptor antagonist Org 34850 on basal and stress-induced corticosterone secretion. J. Neuroendocrinol. 19, 891–900. Vallone, D., Battista, S., Pierantoni, G.M., Fedele, M., Casalino, L., Santoro, M., Viglietto, G., Fusco, A., Verde, P., 1997. Neoplastic transformation of rat thyroid cells requires the junB and fra-1 gene induction which is dependent on the HMGI-C gene product. EMBO J. 16, 5310–5321.