Aging attenuates glucocorticoid negative feedback in rat brain

Neuroscience 159 (2009) 259 –270

AGING ATTENUATES GLUCOCORTICOID NEGATIVE FEEDBACK IN RAT BRAIN K. MIZOGUCHI,a* R. IKEDA,a H. SHOJI,a Y. TANAKA,a W. MARUYAMAb AND T. TABIRAc

neuroendocrine systems associated with aging. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

a Section of Oriental Medicine, Department of Geriatric Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan

Key words: hypothalamic-pituitary-adrenal axis, prefrontal cortex, hippocampus, hypothalamus, pituitary, glucocorticoid receptor.

b Department of Geriatric Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan

Aging is considered as a complex multifactorial process that results in heterogeneous patterns of progressive morbidity and disability (Rowe and Kahn, 1987; Seeman and Robbins, 1994). This process is influenced by multiple homeostatic mechanisms. The hypothalamic–pituitary–adrenal (HPA) axis is one of the important hormonal systems that fulfils homeostatic function. Aged rats as well as elderly humans show progressive loss of control of the HPA axis, resulting in hypersecretion of glucocorticoids during times of stress (Sapolsky et al., 1983; Born et al., 1995). Abnormal glucocorticoid secretion is thought to be involved in many aging-related diseases, including depression, cognitive deficits, and Alzheimer’s disease (Sapolsky et al., 1986; Dodt et al., 1991; Martignoni et al., 1992; Lupien et al., 1999). Thus, understanding and controlling the factors underlying glucocorticoid signaling are of considerable importance to the treatment and prevention of aging-related diseases. Several reports have demonstrated that dysregulation of the HPA system presenting as dexamethasone (DEX)mediated negative feedback resistance to cortisol secretion is observed in approximate half of human depressives (Carroll et al., 1981; Kalin et al., 1982; Holsboer, 1983; Arana et al., 1985). This resistance also exists in elderly humans (Georgotas et al., 1986; Weiner, 1989; Ferrari et al., 1995) as well as aged monkeys (Sapolsky and Altman, 1991; Brooke et al., 1994; Goncharova and Lapin, 2002) and rats (Sapolsky et al., 1985, 1986; Landfield et al., 1987; Jacobson and Sapolsky, 1991). Although glucocorticoid secretion is negatively regulated by glucocorticoids at the level of the anterior pituitary (Miller et al., 1992), several regions of the brain, such as the hypothalamus, hippocampus, and prefrontal cortex (PFC), that have abundant glucocorticoid receptors (GRs), are also involved. For example, electrical stimulation or lesions of the PFC or glucocorticoids placed locally within the PFC, hippocampus, or hypothalamus can alter plasma corticosterone (CORT) levels (Feldman and Conforti, 1985; Magarinos et al., 1987; Kovács and Makara, 1988; Diorio et al., 1993; Feldman and Weidenfeld, 1999; Mizoguchi et al., 2003). Glucocorticoids are implicated in many physiological and pathological processes associated with the aging pro-

c

National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan

Abstract—Aging is thought to be a risk factor to develop vulnerability of the neuroendocrine system, including the hypothalamic-pituitary–adrenal (HPA) axis, and dysregulation of this axis characterized by dexamethasone (DEX)-mediated negative feedback resistance is sometimes observed in elderly humans and animals. However, the influence of aging on the feedback system including an involvement of the brain is not fully understood. In the present study, we examined the suppressive effects of DEX by the systemic injection or the intracranial infusion into the prefrontal cortex (PFC), hippocampus, and hypothalamus on circulating corticosterone levels, and compared between young (3-monthold) and aged (24-month-old) rats. Moreover, we examined expression levels of glucocorticoid receptors (GRs) and their translocation from the cytoplasm to the nucleus using immunohistochemical and Western immunoblot techniques in the pituitary in addition to three brain regions. When DEX was injected systemically, the suppressive response was significantly enhanced in aged rats, compared with young rats. When DEX was infused into three brain regions, the suppressive response to DEX was abolished in aged rats. The immunohistochemical analysis revealed that the number of GR positive cells in the PFC, hippocampus, and hypothalamus was decreased, but that in the pituitary was increased, in aged rats, compared with young rats. The Western immunoblot analysis confirmed these results. Thus, basal expression levels of GRs in three brain regions were decreased, but those in the pituitary were increased, in aged rats. After the injection or infusion of DEX, the translocation of GRs in three brain regions was reduced, but that in the pituitary was enhanced, in aged rats. These results suggest that aging in rats enhances the feedback ability at the systemic level, which mainly involves the pituitary, but it attenuates the ability in the brain. These mechanisms may underlie the vulnerable *Corresponding author. Tel: ⫹81-562-46-2311x5553; fax: ⫹81-562-482373. E-mail address: [email protected] (K. Mizoguchi). Abbreviations: ACTH, adrenocorticotropic hormone; CORT, corticosterone; DEX, dexamethasone; EDTA, ethylenediamine-N,N,N=,N=-tetraacetic acid; EGTA, ethylene glycol-bis-(2-aminoethyl ether)-N,N,N=,N=tetraacetic acid; GR, glucocorticoid receptor; Hepes, N-(2-hydroxyethyl) piperazine-N=-(2-ethanesulfonic acid); HPA, hypothalamic–pituitary–adrenal; hsp, heat shock protein; NDS, normal donkey serum; PBS-T, phosphate-buffered saline containing Triton X-100; PFC, prefrontal cortex; SDS, sodium dodecyl sulfate. 0306-4522/09 © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.12.020

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cess, including calcium metabolism, cognitive function, and stress integration (Sapolsky et al., 1986; Porter et al., 2001). The effect of glucocorticoids on brain functions and aging-related changes in glucocorticoid actions is well documented in humans and animals, in particular, on hippocampus-related memory performance, which has generally been described as showing an inverted U-shape fashion, with too low and too high levels being associated with impairment, while moderate levels enhance the performance (Sapolsky et al., 1986; Lupien et al., 1994, 1996, 1998; O’Brien et al., 1994; Seeman et al., 1997; Kalmijn et al., 1998; Greendale et al., 2000; Wolf et al., 2002; Erickson et al., 2003). On the other hand, expression levels and signals through GRs are likely to decrease generally in the aged hippocampus. For example, aged rats exhibiting impaired spatial memory show decreases in GR density and its mRNA in the hippocampus (Issa et al., 1990; Bizon et al., 2001). Also, nuclear transport and subsequent binding of GRs to the consensus sequence on genes, i.e. glucocorticoid responsive element, are decreased in the aged hippocampus, suggesting the reduced ability of GRs to transduce glucocorticoid signals (Murphy et al., 2002). Therefore, it is possible that decreased GRs in the brain feedback sites, such as the hippocampus, due to aging may cause attenuation of the glucocorticoid negative feedback to DEX. These findings from basic research have had a major impact on theories that the attenuated glucocorticoid negative feedback may involve altered GR regulation in several brain regions that play an important role in the agingrelated brain dysfunctions such as cognitive deficits. Considering this theory, we hypothesized that aging would affect GR levels in the brain regions associated with cognitive deficits, which in turn attenuates the feedback. In the present study, to test this hypothesis, we examined the suppressive response to DEX on circulating CORT levels by systemic administration or by local application to the PFC, hippocampus, and hypothalamus of aged rats. Next, to explore the involvement of the aging-related changes in GR levels on the feedback system, we analyzed basal expression levels of GRs and their translocation from the cytoplasm to the nucleus in the pituitary in addition to three brain regions by using an immunohistochemical or Western immunoblot technique.

EXPERIMENTAL PROCEDURES Animals All animal experiments were performed in accordance with ethical guidelines from the Ministry of Health, Labor and Welfare of Japan and were approved by the Laboratory Animal Committee at the National Center for Geriatrics and Gerontology. The experiments in the present study were designed to minimize the number of animals used and to minimize their suffering. Naive male F344/N rats were used. Twelve-week-old rats weighing 250 –300 g were purchased from Japan SLC (Shizuoka, Japan) and used as the young rat. Twenty-four-month-old rats weighing 400 – 450 g were obtained from the Aging Farm that produces aged rats and was established at our institute in 2000 (Tanaka et al., 2000), and used as the aged rat. They were housed two per cage in a temperature- (23⫾1 °C) and light- (12-h

light/dark schedule; lights on at 8:00 a.m. and off at 8:00 p.m.) controlled environment and were fed laboratory food and water ad libitum.

Systemic injection of DEX The systemic injection experiment was performed according to our previous report (Mizoguchi et al., 2001). Briefly, the rats received a single i.p. injection of DEX (Sigma, MO, USA) dissolved in sterile saline containing 1% ethanol at a dose of either 0 (vehicle, 1 ml/kg), 10, 30, or 100 ␮g/kg. The injection was performed between 10:00 a.m. and 11:00 a.m. These doses of DEX were determined according to our previous report (Mizoguchi et al., 2001). Four hours after the injection, the animals were sacrificed by decapitation, and the trunk blood was collected in a 15-ml polypropylene tube containing 250 ␮l of 100 mM EDTA-2Na (pH 7.4). The plasma was separated by centrifugation at 1800⫻g for 20 min, and stored at ⫺20 °C until radioimmunoassay. Immediately after the collection of blood, the pituitary gland of the rats treated with 10 ␮g/kg of DEX was removed, immediately frozen on dry ice, and stored at ⫺80 °C. Subsequently, the changes in GR proteins in the fractions of cytoplasm and nucleus of the pituitary were analyzed by using a Western immunoblot technique, described below.

Intracranial infusion of DEX The intracranial infusion experiment was performed according to the method described previously (Mizoguchi et al., 2003). Briefly, the animals were stereotaxically and bilaterally implanted with a guide cannula (9-mm-long, 0.8-mm-outer diameter; Bioanalytical Systems, IN, USA), which was anchored firmly to the skull by dental adhesive and acrylic resin under pentobarbital anesthesia (45 mg/kg, i.p.). The brain atlas of Paxinos and Watson (2007) was used to determine the coordinates. The following coordinates relative to the bregma were used for the cannula implantation: the PFC, anteroposterior, ⫹3.2; lateral, ⫾1.0; vertical, ⫺2.5; the hippocampus, anteroposterior, ⫺4.2; lateral, ⫾2.4; vertical, ⫺2.6; the hypothalamus, anteroposterior, ⫺1.8; lateral, ⫾0.4; vertical, ⫺7.0. Fig. 2 shows the infusion sites in the PFC, hippocampus, or hypothalamus. The animals were initially treated with Xylocaine (AstraZeneca PLC, London, UK) to minimize pain, and were monitored on a daily basis for signs of distress or infection. Animals were adapted to a mock infusion protocol to minimize any stress associated with the procedure before the start of infusion experiments. After an 8-day recovery period from the surgery, the animals were gently restrained while the stylets were removed and replaced with an infusion cannula (PC-12; Bioanalytical Systems) that extended 1 mm below the guide cannula. The animals received bilateral infusions of DEX at a concentration of either 0 (vehicle) or 10 ng in 1 ␮l sterile saline containing 0.2% ethanol at a rate of 0.2 ␮l/min for 5 min using a microinfusion pump. The infusion was performed between 10:00 a.m. and 11:30 a.m. The dose of DEX was determined according to our previous study (Mizoguchi et al., 2003). The cannula remained in place for 2 min after the completion of the infusion. Stylets were inserted back into the guide cannula, and the animals were returned to the home cage. Four hours after the infusion, the animals were sacrificed by decapitation, the trunk blood was collected, and the plasma was separated, and stored at ⫺20 °C. Immediately after the collection of blood, each brain was removed, immediately frozen on dry ice, and stored at ⫺80 °C. Subsequently, the changes in GR proteins in the PFC, hippocampus, and hypothalamus were analyzed by using a Western immunoblot technique, described below.

CORT radioimmunoassay The [125I]-labeled CORT (46.3 kBq) double antibody radioimmunoassay kit for rat (GE Healthcare UK, Buckinghamshire, UK) was

K. Mizoguchi et al. / Neuroscience 159 (2009) 259 –270 used to measure the plasma CORT concentration. To displace CORT from corticosteroid-binding globulin, the plasma was heated for 30 min at 60 °C. The assay was performed in duplicate at room temperature, using rabbit anti-CORT serum as the first antibody and donkey anti-rabbit serum coated on magnetizable polymer particles as the second antibody. According to the manufacturer, the cross-reactivity is low. The highest cross-reactivity is found with 11-deoxycorticosterone (2.4% in contrast to 100% for CORT). The possible range of the assay is between 0.78 and 200 ng/ml.

Immunohistochemistry Immunostaining for GR was performed according to our previous report (Mizoguchi et al., 2000, 2004) with some modifications. Briefly, the rats were perfused transcardially with 100 ml of 0.1 M phosphate-buffered and heparinized saline (pH 7.4), followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) under pentobarbital anesthesia (45 mg/kg, i.p.). The brains were post-fixed for 1 h at 4 °C in the same fixative, cryoprotected in 30% sucrose before being frozen in powdered dry ice, and stored at ⫺80 °C. Sections (30 ␮m) were cut using a freezing microtome. Free-floating tissue sections were rinsed three times with phosphate-buffered saline (PBS, pH 7.4) containing 0.1% Triton X-100 (PBS-T). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS-T, followed by incubation with 10% normal donkey serum (NDS) in PBS-T for 30 min. The sections were then incubated overnight at room temperature with polyclonal rabbit antibody against GR (M-20; Santa Cruz Biotechnology, CA, USA), diluted to 1:3000 in PBS-T containing 1% NDS. After the incubation, the sections were washed three times with PBS-T, and were incubated for 1 h at room temperature with peroxidase-linked donkey anti-rabbit immunoglobulin G (GE Healthcare) diluted to 1:200 in PBS-T. The sections were washed three times with PBS-T, and the peroxidase was visualized using 0.05% diaminobenzidine hydrochloride containing 0.005% hydrogen peroxide. Control sections, in which anti-GR antibody was replaced with NDS at the same dilution, were routinely processed together with the sections of interest. The number of GR-positive cells was counted on microphotographs that were taken from three sections for each individual animal, with an interval of 120 ␮m between sections, and the mean value of three counting data equaled the mean number of cells of each animal. Data are expressed as the number of cells per area (mm2) for the PFC and pituitary, length (mm) for the hippocampal CA1 subfield, or whole section for the hypothalamus.

Western immunoblot analysis Western immunoblot analysis was performed according to the method of Kitchener et al. (2004) with minor modifications. The stored brain and pituitary tissues described above were thawed, and the PFC, hippocampus, and hypothalamus were quickly dissected from the brain on an ice plate. The following sample preparation was performed at a temperature below 4 °C. Briefly, each tissue was homogenized in S1 buffer [10 mM Hepes (pH 7.6) containing 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 1 mM dithiothreitol, and 1% protease inhibitor cocktail (Sigma)] in a 1.5-ml tube with a micro homogenizer (Physcotron NS-310E; Microtec, Chiba, Japan). The homogenates were centrifuged at 20,000⫻g for 20 min to yield a cytoplasmic fraction (the supernatant) and a fraction containing nuclei (the pellet). The pellets were re-homogenized in S1 buffer containing 0.32 M sucrose, and were centrifuged at 1000⫻g for 3 min. Pelleted nuclei were washed with S1 buffer two times by centrifugation. Resultant pellets were extracted with S1 buffer containing 0.5 M NaCl and 5% glycerol for 1 h on ice, and then centrifuged at 20,000⫻g for 20 min to yield soluble nuclear extracts. An aliquot of each cytoplasmic or nuclear fraction was used for the determination of protein concentrations

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according to Lowry’s methods (Lowry et al., 1951). The samples were denatured in 50 mM Tris–HCl (pH 7.4) containing 10 mM EDTA-2Na, 2% sodium dodecyl sulfate (SDS), 1% 2-mercaptoethanol, and 0.001% Bromophenol Blue, and subjected to gel electrophoresis. For immunoblot analysis, the proteins (10 ␮g/lane) were separated by electrophoresis on 12.5% SDS–polyacrylamide gels (ExcelGel; GE Healthcare), and electroblotted onto nitrocellulose filters. The filters were incubated with 10% NDS in PBS-T for 1 h, followed by incubation with 1⫻ Block Ace solution (Dainippon Pharmaceutical, Osaka, Japan) for 1 h. After the blocking procedure, the anti-rabbit GR polyclonal antibody (M-20; Santa Cruz Biotechnology) diluted to 1:1200 in PBS-T containing 0.1⫻ Block Ace was applied for 2 h at room temperature. After washing three times with PBS-T, the filters were incubated for 1 h at room temperature with peroxidase-linked donkey anti-rabbit immunoglobulin G (GE Healthcare) diluted to 1:50,000 in PBS-T. The filters were washed three times with PBS-T, and the peroxidase was visualized on X-ray film using an Enhanced Chemiluminescence system (GE Healthcare). Semiquantitative densitometric analyses of GR signals were performed on X-ray film using Macintosh-driven image-processing and analysis software (version 1.61; W. Rasband, National Institutes of Health, MD, USA). The immunoreactive GR was quantified by linear regression of the values obtained by the quantitative study, according to our previous study (Mizoguchi et al., 2003). In brief, to confirm the quantifiability of our Western immunoblot analysis system for GR, the cytosolic preparations derived from the hippocampus with protein concentrations ranging between 0.2 and 10 ␮g/lane were analyzed by the same procedure, and the intensity of GR signals was increased linearly with increasing protein concentration (data not shown). Data are expressed as relative change (%) for GR levels in each cytoplasmic or nuclear fraction of young rats.

Statistics All data were initially analyzed using one-way analysis of variance (ANOVA). The comparisons of plasma CORT levels following systemic administration of DEX in young or aged rats were made using the Fisher’s protected least significant difference test. The comparisons of plasma CORT levels between young and aged rats following systemic administration of DEX or intracranial infusions of DEX, the number of GR-positive cells, and GR protein levels were analyzed using the unpaired t-test.

RESULTS Plasma CORT levels after systemic injection of DEX The decreasing response of plasma CORT levels induced by systemic injections of DEX was compared between young and aged rats, and the results are shown in Fig. 1. Basal CORT level (0 ␮g/kg of DEX) in young rats was significantly suppressed by the DEX injection at 30 or 100 ␮g/kg [30 ␮g/kg, F(3,15)⫽3.355, P⬍0.01; 100 ␮g/kg, F(3,15)⫽3.355, P⬍0.01], but not at 10 ␮g/kg. Basal CORT level in aged rats was also significantly suppressed by the DEX injection at 10, 30, or 100 ␮g/kg [F(3,15)⫽16.091, P⬍0.001, respectively]. There was a significant difference in CORT levels following 10 or 100 ␮g/kg of DEX injection between young and aged rats [10 ␮g/kg, F(1,7)⫽14.213, P⬍0.01; 100 ␮g/kg, F(1,7)⫽4.933, P⬍0.05]. Note that there was no significant difference in basal CORT levels between young and aged rats.

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these regions, it seems that the intensity of GR immunoreactivity was weak in aged rats. In the anterior pituitary, the expression was predominantly distributed in adrenocorticotropic hormone (ACTH) cell-like cells, and it seemed that the cells were dense in aged rats. The number of GR-positive cells was quantified from the immunohistochemical results, as shown in Fig. 5. The number of GR-positive cells was significantly decreased in the medial PFC, CA1 subfield of the hippocampus, and the paraventricular nucleus of the hypothalamus of aged rats, compared with young rats [PFC, F(1,8)⫽10.472, P⬍0.05; hippocampus, F(1,8)⫽23.141, P⬍0.01; hypothalamus, F(1,8)⫽10.886, P⬍0.01]. In the anterior pituitary, the number tended to be increased in aged rats, compared with young rats [F(1,8)⫽4.189, P⫽0.07]. Basal expression levels of GR protein The results of immunoblot detections of GR proteins were shown in Fig. 6. As shown in Fig. 6A, one major band Fig. 1. Changes in plasma CORT levels induced by systemic injection of DEX in young and aged rats. DEX was injected (0, 10, 30, or 100 ␮g/kg, i.p.), and plasma CORT levels were measured at 4 h after the injection (see Experimental Procedures). Each column is the mean⫾S.E.M. of five rats per group. Asterisks indicate a significant difference from vehicletreated (0 ␮g/kg) rats in each group: ** P⬍0.01; *** P⬍0.001; daggers, a significant difference from DEX-injected young rats: † P⬍0.05; †† P⬍0.01.

Plasma CORT levels after intracranial infusion of DEX The decreasing response of plasma CORT levels induced by bilateral infusions of DEX into the PFC (A), hippocampus (B), or hypothalamus (C) was compared between young and aged rats, and the results are shown in Fig. 3. In the PFC, basal CORT level of young rats was significantly suppressed by the DEX infusion, compared with vehicle infusion [F(3,20)⫽4.911, P⬍0.01]. However, basal CORT level in aged rats was not suppressed by DEX, and the level was significantly higher than that of DEX-infused young rats [F(3,20)⫽4.911, P⬍0.01]. Similarly, in the hippocampus and hypothalamus, basal CORT levels of young rats were significantly suppressed by the DEX infusions [hippocampus, F(3,20)⫽8.654, P⬍0.001; hypothalamus, F(3,20)⫽5.626, P⬍0.01]. However, basal CORT levels in aged rats were not suppressed by DEX, and the levels were significantly higher than those of DEX-infused young rats [hippocampus, F(3,20)⫽8.654, P⬍0.01; hypothalamus, F(3,20)⫽5.626, P⬍0.001]. GR-positive cells The distribution of GR-positive cells in the PFC, hippocampus, hypothalamus, and pituitary was analyzed between young and aged rats by using an immunohistochemical technique, and the results are shown in Fig. 4. In the PFC (medial region, cingulate gyrus) (A and B), the GR expression was mainly distributed in large cells that seem to be neurons. In the hippocampus (CA1 subfield) (C and D), the expression was predominantly distributed in the pyramidal neurons. In the hypothalamus (E and F), the expression was mainly distributed in the paraventricular nucleus. In

Fig. 2. Location of the DEX infusion sites. Asterisks indicate DEX infusion sites in the PFC (A, anteroposterior ⫹3.2 mm, lateral ⫾1.0 mm, vertical ⫺3.5 mm), hippocampus (B, anteroposterior ⫺4.2 mm, lateral ⫾2.4 mm, vertical ⫺3.6 mm), or hypothalamus (C, anteroposterior ⫺1.8 mm, lateral ⫾0.4 mm, vertical ⫺8.0 mm).

K. Mizoguchi et al. / Neuroscience 159 (2009) 259 –270

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(molecular weight: 97 kDa) was detected in cytoplasmic preparations derived from the PFC in the presence of anti-GR antibody (lane 1), but no band was detected in the absence of the antibody (lane 2). According to a previous report (Kitchener et al., 2004) and data sheet of the antibody (Santa Cruz Biotechnology), this immune-specific band was identified as GR. We next compared basal expression levels of cytoplasmic GRs in the PFC (B), hippocampus (C), hypothalamus (D), and pituitary (E) between young and aged rats. Note that basal levels of cytoplasmic GRs in the brain regions of aged rats were lower than those of young rats (B, C, and D), and the levels in the pituitary of aged were higher than those of young (E). The results of densitometric analyses of the immunoblot detections, including the data shown in Fig. 6, were shown in Fig. 7, as below. Changes in GR protein levels after DEX treatments The changes in GR protein levels in the cytoplasmic and nuclear fractions induced by bilateral infusions of DEX (10 ng/site) into the PFC, hippocampus, or hypothalamus were examined and compared between young and aged rats. The changes in GRs in the pituitary following systemic injection of DEX (10 ␮g/kg) were also examined and compared. In the PFC (Fig. 7A), basal levels of cytoplasmic GRs showed a significant decrease in aged rats, compared with young rats [F(3,20)⫽6.077, P⬍0.01], but those of nuclear GRs did not. After the DEX infusions, a significant decrease in cytoplasmic GRs [F(3,20)⫽6.077, P⬍ 0.01] and a significant increase in nuclear GRs [F(3,20)⫽ 51.681, P⬍0.001] were observed in young rats, but these decreasing and increasing responses of GRs were abolished in aged rats. Similar results were obtained in the hippocampus (Fig. 7B) and hypothalamus (Fig. 7C). Thus, basal levels of cytoplasmic GRs were significantly decreased in aged rats, compared with young rats [hippocampus, F(3,20)⫽34.159, P⬍0.001; hypothalamus, F(3,20)⫽16.337, P⬍0.001], but those of nuclear GRs were not. After the DEX infusions, significant decreases in cytoplasmic GRs [hippocampus, F(3,20)⫽34.159, P⬍0.01; hypothalamus, F(3,20)⫽16.337, P⬍0.01] and significant increases in nuclear GRs [hippocampus, F(3,20)⫽12.068, P⬍0.001; hypothalamus, F(3,20)⫽8.699, P⬍0.01] were observed in young rats, but not in aged rats. In the pituitary (Fig. 7D), different results from the brain were obtained. Thus, basal levels of cytoplasmic GRs were significantly increased in aged rats, compared with young rats [F(3,20)⫽10.231, P⬍0.05], but those of nuclear GRs were not. After the DEX injections to young and aged rats, significant decreases in cytoplasmic GRs [young, F(3,20)⫽ 10.231, P⬍0.05; aged, F(3,20)⫽10.231, P⬍0.001] and

Fig. 3. Changes in plasma CORT levels induced by intracranial infusion of DEX into the PFC (A), hippocampus (B), or hypothalamus (C) of young and aged rats. DEX (10 ng/site) was infused into each site, and plasma CORT levels were measured at 4 h after the infusion (see

Experimental procedures). Each column is the mean⫾S.E.M. of six rats per group. Asterisks indicate a significant difference from vehicle (Veh)-infused rats: ** P⬍0.01; *** P⬍0.001; daggers, a significant difference from DEX-infused young rats: †† P⬍0.01; ††† P⬍0.001.

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Fig. 4. Representative photomicrographs showing immunohistochemical analysis of GRs in young and aged rats. (A, B) Medial PFC (cingulate gyrus); (C, D) CA1 subfield of the hippocampus; (E, F) paraventricular nucleus of the hypothalamus; (G, H) anterior pituitary. (A, C, E, G) Young; (B, D, F, H) aged. Original magnification: PFC, ⫻200; hippocampus, ⫻400; hypothalamus, ⫻100; pituitary, ⫻400.

significant increases in nuclear GRs [young, F(3,20)⫽ 23.498, P⬍0.01; aged, F(3,20)⫽23.498, P⬍0.001] were observed in both groups, and the nuclear GR level of aged rats was significantly higher than that of young rats [F(3,20)⫽23.498, P⬍0.05].

DISCUSSION The present study indicated that aging differentially regulated the feedback ability and GR levels between the brain and the pituitary in rats. Thus, aging abolished the feed-

K. Mizoguchi et al. / Neuroscience 159 (2009) 259 –270

Fig. 5. The number of GR-positive cells in the medial PFC (cingulate gyrus) (A), CA1 subfield of the hippocampus (B), paraventricular nucleus of the hypothalamus (C), and anterior pituitary (D). Quantification was performed from the results presented in Fig. 4. Each column is the mean⫾S.E.M. of five rats per group. Asterisks indicate a significant difference from young rats: * P⬍0.05; ** P⬍0.01.

back ability, decreased cytoplasmic GRs, and weakened the translocation of GRs from the cytoplasm to the nucleus in the PFC, hippocampus, and hypothalamus; oppositely, aging enhanced the feedback ability at the systemic level, increased cytoplasmic GR proteins, and strengthened the GR translocation in the pituitary. In a general way, this paper is simply a study comparing differences between young and old rats, because we did not include middleaged rats in the experiments. As shown in Fig. 1, we did not find the difference in basal CORT levels between young and aged rats (0 ␮g/kg of DEX). Although there are inconsistent findings showing a progressive age-related increase in basal CORT levels in rats (Sapolsky et al., 1983, 1986; De Kosky et al., 1984; Meany et al., 1992; Haugert et al., 1994), our data are consistent with the data obtained in humans (Ohashi et al., 1986; Pavlov et al., 1986; Born et al., 1995), monkeys (Goncharova and Lapin, 2002), and rats as well (Kasckow et al., 2005; Meijer et al., 2005). Most human studies have indicated that either no change or insignificant increase in basal cortisol levels in older ages. After systemic injection of DEX, plasma CORT was suppressed at a dose of over 30 ␮g/kg in young rats, but of 10 ␮g/kg in aged rats, indicating the enhanced feedback at the systemic level in aged rats. These data are also consistent with a previous report (Kasckow et al., 2005). The glucocorticoid negative feedback system is regulated at the level of the pituitary (Miller et al., 1992), as well as the brain, such as the hypothalamus, hippocampus, and

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PFC (Feldman and Conforti, 1985; Magarinos et al., 1987; Kovács and Makara, 1988; Diorio et al., 1993; Feldman and Weidenfeld, 1999). Indeed, we previously indicated that, in chronically stressed rats, glucocorticoid negative feedback resistance observed when DEX was administered systemically was caused partially by the attenuated feedback ability in the PFC and hippocampus, suggesting that the brain feedback ability influences the systemic feedback system (Mizoguchi et al., 2001, 2003). Therefore, to explore the involvement of the brain feedback ability in the enhanced feedback observed in aged rats, we examined the suppressive response to the DEX infusions into the PFC, hippocampus, or hypothalamus on circulating CORT levels. As shown in Fig. 3, plasma CORT was suppressed by the DEX infusions into each brain region of young rats, which confirms that each region has a role as the feedback site in the brain and is consistent with our previous report (Mizoguchi et al., 2003). However, when DEX was infused into each region of aged rats, no suppressive response to DEX was observed, indicating that the feedback ability at the brain level is abolished in aged rats. Taken, in aged rats, the glucocorticoid negative feedback was enhanced at the systemic levels, but it was attenuated oppositely at the brain level. Tritiated DEX can be distributed in the frontal cortex, hippocampus, and hypothalamus when it is injected systemically (Birmingham et al., 1993). Therefore, the enhanced feedback observed when DEX was administered systemically in aged rats is thought to be the total sum of the feedback ability in the feedback sites, i.e. the pituitary and brain. Since the brain feedback ability was attenuated, the enhanced feedback involves predominantly the augmented pituitary function. Although the mechanisms underlying the augmented pituitary function on the feedback ability are unknown, some pathogenic abnormalities may be involved. Individual strains of rats are susceptible to tissue- or organ-specific pathologies that can affect feedback parameters. For example, the F344 strain is prone to development of hypertrophy or tumors in the pituitary (Lipman et al., 1996), which can directly (e.g. ACTH-secreting adenomas) or indirectly (e.g. space-occupying lesions in the sella turcica/basal hypothalamus) affect negative feedback functioning. Several previous studies had indicated that aged rats showed prolonged elevation of plasma CORT after acute stress exposure, suggesting that attenuation of the feedback at the systemic level (Sapolsky et al., 1986; Issa et al., 1990; Bizon et al., 2001). However, a recent study has indicated the rather enhanced feedback in aged rats as evidenced by lesser plasma CORT levels after systemic administration of DEX (Kasckow et al., 2005). According to our data, this inconsistency may be caused by different involvement of the pituitary. Thus, it is possible that small or no involvement of the pituitary induces the attenuated feedback that mainly reflects the brain feedback ability; however, large involvement causes the enhanced feedback that mainly reflects the pituitary. In particular, the attenuated brain feedback ability in the present study might be a reasonable explanation as a mechanism of dysregu-

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Fig. 6. Western immunoblot analysis of GR proteins and comparison of expression levels between young and aged rats. (A) Cytoplasmic preparations derived from the PFC were analyzed in the presence (lane 1) or absence (lane 2) of anti-GR antibody. (B–E) Basal expression levels of GRs in the PFC (B), hippocampus (C), hypothalamus (D), and pituitary (E) were compared between young (lane 1) and aged (lane 2) rats. Molecular weight markers are indicated in kDa to the right of each panel.

lation of the HPA axis characterized by negative feedback resistance to DEX frequently observed in elderly humans (Georgotas et al., 1986; Weiner, 1989; Ferrari et al., 1995). A molecule that mediates actions of DEX is GR, which is abundantly distributed in the PFC, hippocampus, and hypothalamus (Feldman and Conforti, 1985; Magarinos et al., 1987; Kovács and Makara, 1988; Diorio et al., 1993; Feldman and Weidenfeld, 1999; Mizoguchi et al., 2003). GRs are translocated from the cytoplasm to the nucleus through an active transport process (Guiochon-Mantel et al., 1991; Dauvois et al., 1993; Madan and DeFranco, 1993; Galigniana et al., 1999). Hormone binding is transient, and the loss of hormone from the receptor leads to a recycling of the receptor (Scherrer et al., 1990). Therefore, we analyzed distribution of the cells having GRs in the PFC, hippocampus, hypothalamus, and pituitary of aged rats. As shown in Figs. 4 and 5, immunohistochemical data revealed that the number of GR-positive cells in the medial PFC, CA1 subfield of the hippocampus, and the paraventricular nucleus of the hypothalamus was decreased and that in the anterior pituitary tended to be increased in aged rats, compared with young rats. In addition to the changed number of cells, it seemed that intensity of immunoreac-

tivity for the GR was weak in each brain region of aged rats. The number of GR positive cells differed among the PFC, hippocampus, and hypothalamus of young rats, despite that DEX infusions into these regions induced a similar degree of the negative feedback. Regarding its mechanism, it is possible that there is a dose-response relationship in each brain region and the region has individual sensitivity to DEX to decrease plasma CORT levels. In addition, stimulation with DEX of a part of cells having GRs in each region might be enough to produce maximum decreasing response for plasma CORT. Indeed, in our preliminary experiment using young rats, we examined the decreasing response of DEX infused into the PFC or hippocampus on plasma CORT, at a dose of 1, 3, 10, 30, or 100 ng/site, and obtained the results showing that DEX decreased plasma CORT levels in a dose-dependent manner, and 10 ng/site produced maximum response in both regions (data not shown). Since the results of immunohistochemical analysis are generally influenced by the limits of detection afford by the procedure, we next examined the changes in GR levels using a Western immunoblot technique. As a result, the

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immunohistochemical changes in GRs were confirmed in the Western immunoblot analysis. Thus, basal levels of cytoplasmic GRs were decreased in each brain region and were increased in the pituitary of aged rats, compared with young rats. Note that nuclear GRs levels were not affected in all regions by age. Thus, it is suggested that agingrelated decreases in GRs in each brain region occur in the cytoplasm. Although several factors can regulate expression levels of GRs, the ligand, glucocorticoid, negatively regulates GR levels at the transcription level (Rosewicz et al., 1988). In particular, GRs in the hippocampus as well as in the PFC are sensitive to changes in endogenous glucocorticoid secretion. For example, suppression of circulating CORT by adrenalectomy increases GR densities in both regions, and CORT replacement reverses the increases (Lowy, 1991; O’Donnell et al., 1995). However, basal levels of plasma CORT were not changed between young and aged rats (Fig. 1), suggesting that decreased GR proteins in the PFC, hippocampus, and hypothalamus of aged rats is not caused by increased plasma CORT levels. Therefore, other mechanisms for the decreased GR levels, such as aging-related neuron loss, should be foreseen. Indeed, aging-related neuron loss has been shown to occur in the CA1 subfield of the hippocampus, one of the regions firstly affected in Alzheimer’s disease and also a region displaying many other age-related changes (Haigler et al., 1986; Kerr et al., 1991; West and Gundersen, 1990; West, 2002). After infusions or injections of DEX, cytoplasmic GRs were decreased and nuclear GRs were increased in all regions tested of young rats, indicating that cytoplasmic GRs were transported to the nucleus after the DEX treatments. However, in the aged brain, cytoplasmic and nuclear GRs were not changed, suggesting that nuclear transporting mechanisms of GRs were disturbed and that this disturbance is involved in the attenuated feedback in each brain region. On the other hand, in the aged pituitary, cytoplasmic GRs were decreased and nuclear GRs were increased after the DEX treatments, and this increase was greater than that in young rats. These results suggest that nuclear transporting activity of GRs is facilitated in the aged pituitary and that this facilitation is implicated in the enhanced feedback at the systemic level (Fig. 1). These are consistent partially with a previous report indicating that aging-related hippocampal GR deficits are associated with decreased nuclear transport of GRs (Murphy et al., 2002). In addition, our results suggest that the agingrelated decrease in the GR translocation reduces physiological impact of GRs by attenuating entry into the nucleus and subsequent transcriptional regulation. This is consistent with decreases in GR-related processes with aging. Fig. 7. Changes in immunoreactive GRs in the cytoplasmic or nuclear fraction of the PFC (A), hippocampus (B), or hypothalamus (C) following intracranial infusion of DEX in each brain region or of the pituitary (D) following systemic injection of DEX. DEX (10 ng) was infused bilaterally into each brain region or DEX (10 ␮g/kg) was injected systemically. At 4 h after the infusion or injection, GR levels were measured (see Experimental Procedures). Cytoplasmic or nuclear GR levels of vehicle (Veh)-treated young rats were used as a control

(100%). Each column is the mean⫾S.E.M. of five rats per group. Asterisks indicate a significant difference from vehicle-treated young (control) rats: * P⬍0.05; ** P⬍0.01; *** P⬍0.001; daggers, a significant difference from vehicle-treated young or aged rats in each group: † P⬍0.05; †† P⬍0.01; ††† P⬍0.001; a double dagger, a significant difference from DEX-treated young rats, ‡ P⬍0.05.

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For example, hippocampus-mediated inhibition of activation of the HPA axis in response to stress is attenuated in aged rats (Herman et al., 2001). Moreover, aging-related impairment of spatial memory parallels that observed after GR deficiency or blockade (Oitzl and de Kloet, 1992; Oitzl et al., 1997). The mechanisms of the aging-related decreases in the GR translocation in the brain are unknown. The changes in several GR-associated proteins during aging may be implicated. For example, Murphy et al. (2002) have demonstrated that expression of heat shock protein (hsp) 90 reduces in the hippocampus of aged rats. Since hsp 90 is an essential protein for ligand binding and transport into the nucleus of steroid receptors (Pratt, 1997; Rajapandi et al., 2000), decreased hsp 90 may reduce glucocorticoid binding to GRs and subsequent translocation. In the present study, we focused on the PFC, hippocampus, and hypothalamus as the feedback site in the brain. These regions, in particular, the PFC and hippocampus have an important role for maintaining cognitive and memory functions that are frequently impaired in elderly subjects. A recent report has shown that dysregulation of the HPA axis is associated with smaller volume of medial PFC in elderly humans (MacLullich et al., 2006). In addition, PFC dysfunction (Drevets et al., 1997, 2000) or hippocampal atrophy (Sheline et al., 1996) is observed in depressive patients, and both animal and human studies challenging the HPA system have suggested that some of depressive symptoms can be attributed to the dysregulated HPA axis (Steckler et al., 1999). Indeed, shutdown of GR functions by suppression of circulating CORT impairs PFC-dependent cognitive functions in rats (Mizoguchi et al., 2004). Moreover, HPA dysregulation increases with aging in depressive patients (Asnis et al., 1981; Oxenkrug et al., 1983; Alexopoulos et al., 1984; Halbreich et al., 1984; Lewis et al., 1984; Whiteford et al., 1987; Brown et al., 1988; von Bardeleben and Holsboer, 1991; Akil et al., 1993). These findings have suggested that HPA dysregulation in aged subjects is related to dysfunction of the PFC or hippocampus. The attenuated glucocorticoid actions suggested from the present results that the suppressive responses to DEX were abolished and the GR translocations were not observed in the PFC, and hippocampus of aged rats might help to understand this relationship.

CONCLUSION In conclusion, our present results revealed that the feedback ability between the brain and pituitary is differentially regulated in aged rats. The different regulatory mechanism is thought to be caused by the different regulations of cytoplasmic GR proteins and their nuclear transport. Although the mechanisms of GR regulation in the brain during aging remain to be studied and it is unclear whether the altered responses observed in aged rats precede or result from aging, the present findings provide important information for understanding the aging pathology of the brain.

Acknowledgments—This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (18590663), a Research Grant for Longevity Sciences (18C-8) from the Ministry of Health, Labor and Welfare of Japan, and Takeda Science Foundation.

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(Accepted 12 December 2008) (Available online 24 December 2008)