Negative feedback functions in chronically stressed rats: role of the posterior paraventricular thalamus

Physiology & Behavior 78 (2003) 365 – 373

Negative feedback functions in chronically stressed rats: role of the posterior paraventricular thalamus Azra Jaferi, Nathan Nowak, Seema Bhatnagar* Department of Psychology, University of Michigan, 525 East University, Ann Arbor, MI 48109-1109, USA Received 19 June 2002; received in revised form 18 October 2002; accepted 22 November 2002

Abstract A gradual decrement in hypothalamic – pituitary – adrenal (HPA) activity is observed following repeated exposure to the same stressor, such as repeated restraint. This decrement, termed habituation, may be partly due to alterations in corticosterone-mediated negative feedback inhibition of the HPA axis. We have previously found that the posterior division of the paraventricular thalamus (pPVTh) regulates habituated HPA activity without altering HPA responses to acute stress. Therefore, in the present study, we examined the role of the pPVTh in delayed feedback inhibition of plasma corticosterone responses to repeated restraint. Dexamethasone was administered subcutaneously 2 h prior to 30 min restraint to induce delayed negative feedback inhibition of the HPA axis. In the first experiment, we determined that a 0.05-mg/kg dose of dexamethasone produced submaximal suppression of corticosterone responses to acute restraint and used this dose in the remainder of the experiments. In Experiment 2, we examined dexamethasone-induced feedback inhibition to corticosterone responses to a single or eighth restraint exposure since negative feedback functions in chronically stressed rats are not well studied. We found that corticosterone levels following dexamethasone treatment were similar in repeatedly restrained compared to acutely restrained rats. In Experiment 3, we lesioned the pPVTh and examined dexamethasone-induced feedback inhibition of corticosterone responses to a single or eighth exposure to restraint. pPVTh lesions attenuated dexamethasone-induced inhibition of corticosterone at 30 min in chronically stressed rats but had no effect in acutely stressed rats. These data suggest that negative feedback functions are maintained in rats exposed to repeated restraint and implicate the pPVTh as a site that contributes to these negative feedback functions specifically under chronic stress conditions. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Hypothalamic – pituitary – adrenal; Corticosterone; Habituation; Paraventricular nucleus of the thalamus

1. Introduction Unlike the short-lived effects of a single acute exposure to stressful stimuli, chronic stress exposure exerts an enduring influence on the way in which the hypothalamic – pituitary– adrenal (HPA) axis will respond to subsequently experienced stressors. Changes in HPA responsiveness as a result of previous stress exposure are exemplified by the expression of facilitation and habituation. Chronically stressed animals can display enhanced or facilitated HPA responses to an acute, novel stressor compared to stressnaive animals only exposed to the novel stressor [1,2]. In contrast, animals that are repeatedly exposed to the same stressor can exhibit decreased or habituated HPA responses to that stressor [3,4].

* Corresponding author. Tel.: +1-734-615-3744; fax: +1-734-7637480. E-mail address: [email protected] (S. Bhatnagar).

Since habituation involves a gradual decrement in HPA activity following repeated stress exposure, habituation may be due, in part, to alterations in negative feedback inhibition of the HPA axis by elevated corticosterone levels produced by daily exposure to chronic stress. Negative feedback serves as a regulatory mechanism involved in inhibiting release of pituitary –adrenal hormones. Following stress-induced HPA activation, circulating corticosterone acts at multiple brain sites to exert negative feedback effects over different time domains on further adrenocorticotropin (ACTH) release [5,6]. For example, delayed negative feedback refers to the inhibitory effects of corticosterone exerted over hours, whereas fast feedback refers to inhibitory effects of corticosterone exerted over seconds and minutes [5]. Regulation of the HPA axis by corticosterone occurs via its actions on two receptor types in the brain: glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). MR and GR have been shown to display different properties and functions as well as different distribution patterns in the brain [7– 10]. However, the effects of chronic stress on negative feedback

0031-9384/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0031-9384(03)00014-3

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function have not been well characterized. While some studies have shown a decreased sensitivity to fast feedback signals following exposure to chronic stress [11,12], one recent study has shown that chronically stressed animals display enhanced delayed feedback inhibition compared to acutely stressed animals [13]. The first goal of the present studies was to determine delayed negative feedback sensitivity of the HPA axis as a consequence of repeated exposure to the same stressor, restraint. There is evidence that expression of habituation may indeed be dependent on the negative feedback effects of corticosterone. Spencer et al. [14] found that subcutaneous administration of MR or MR/GR antagonists prevents habituation of HPA responses to repeated restraint. The site of action of these negative feedback effects under chronic stress conditions remains unclear. Our recent findings pertaining to the neural circuitry that underlies chronic stressinduced HPA activity strongly suggest a critical role for the posterior paraventricular thalamus (pPVTh) in regulating habituation. Lesions of the pPVTh prevented habituation to repeated restraint but had no effect in acutely restrained rats [4]. These data suggest that, under normal conditions, the pPVTh inhibits HPA responses specifically in chronically stressed animals during habituation but has no functional effect in acutely stressed rats. Some evidence suggests the presence of GR and MR in the PVTh [15,16], but the pPVTh has not been studied with respect to a possible role in negative feedback inhibition of HPA activity. We hypothesized that the pPVTh is important in allowing corticosterone to exert its negative feedback effects in habituated rats. Therefore, our second goal in these studies was to examine if the pPVTh regulates negative feedback inhibition of the HPA axis in habituated rats. To summarize, our aims in the present studies were (1) to characterize delayed negative feedback function specifically under chronic stress conditions and (2) to examine the role of the pPVTh in delayed negative feedback functions in chronically stressed animals. To address the first aim, we investigated dexamethasone-induced inhibition of corticosterone in rats exposed to a single versus eighth restraint (rats that have habituated). Dexamethasone, a potent GR agonist, is commonly used to probe HPA sensitivity to negative feedback [17,18]. To address the second aim, we examined whether excitotoxic lesions of the pPVTh affect dexamethasone-induced inhibition of corticosterone in rats exposed to a single versus eighth restraint.

University of Michigan. Rats were individually housed in hanging metal cages and were allowed ad libitum access to rat chow and water. They were maintained on a 12-h light/ dark schedule (lights on at 0700 h), and all experiments took place during the trough of the diurnal rhythm. Animals were briefly handled the day before experiments were conducted. All experiments were approved by the University Committee on Use and Care of Animals at the University of Michigan.

2. Methods

Half of all rats were randomly assigned to treatment with ibotenic acid to induce lesions of the pPVTh while the other half received sham-lesions. Stereotaxic surgery was performed as described below. Sham- or pPVTh-lesioned rats were subjected to either 8 consecutive days of 30-min restraint or to 1 day of 30-min restraint. On day 1 or day 8, rats were injected subcutaneously with vehicle or 0.05 mg/kg dexamethasone 2 h prior to onset of acute restraint,

2.1. Animals All experiments used young adult male Sprague – Dawley rats supplied by Harlan Sprague Dawley (Indianapolis, IN). Body weights ranged from 200 to 220 g upon arrival at the animal housing facilities at the Department of Psychology,

2.2. Experiment 1: determination of a dose of dexamethasone The purpose of this experiment was to determine a dose of dexamethasone for testing in our subsequent experiments. Four groups of rats (n = 6 – 8 per group) were randomly assigned to treatment with one of three doses of dexamethasone (0.05, 0.2, and 2.0 mg/kg) or with vehicle. These doses of dexamethasone were based on previous studies [19,36]. Subcutaneous injections of dexamethasone or vehicle were administered 2 h prior to restraint exposure and blood collection. Each animal was placed in a cylindrical, Plexiglas restrainer and blood samples were taken from the tail vein at 0, 15, and 30 min during restraint. After collection of the 30min samples, animals were removed from their restrainers and replaced in their home cages. At 60 min, blood samples were collected again and animals subsequently returned to their home cages. All samples were collected within 60 s, since ACTH and corticosterone levels in plasma do not increase in response to manipulations within this time period. 2.3. Experiment 2: dexamethasone (0.05 mg/kg)-induced negative feedback in acutely or repeatedly restrained rats Based on the results of Experiment 1, a dose of 0.05 mg/ kg dexamethasone was chosen for Experiments 2 and 3. Two groups of rats were randomly assigned to treatment with 0.05 mg/kg dexamethasone or with vehicle. Half of each group was subjected to 8 consecutive days of 30-min restraint, while the other half underwent 1 day of 30-min restraint. On day 1 or day 8, rats were injected with dexamethasone or vehicle at 2 h prior to onset of acute restraint, and blood samples were collected according to the procedure described for Experiment 1. There were six to nine rats per group in this experiment, depending on the time point. 2.4. Experiment 3: effects of pPVTh lesions on dexamethasone (0.05 mg/kg)-induced negative feedback in acutely or repeatedly restrained rats

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and blood samples were collected according to the procedure described for Experiment 1. In order to have sufficient numbers of rats that met our criteria for proper pPVTh lesions, this experiment was conducted twice (final n = 7 – 12 per group, depending on time point). The results represent data pooled from both runs of this experiment. 2.5. Ibotenic acid lesions of the pPVTh Rats were anesthetized with a mixture of ketamine, xylazine, and acepromazine (77:1.5:1.5 mg/ml given ip at 0.1 ml/100 g body weight) and placed in a stereotaxic apparatus with the skull flat, and the tooth bar at 3.3 mm. pPVTh lesions were performed according to the following coordinates from bregma: AP: 2.8 mm, ML: 0.0 mm, DV: 6.2 mm. A Hamilton microsyringe containing 10 ng/250 nl of ibotenic acid (Sigma, St. Louis, MO) was lowered into the pPVTh. The drug was injected over a 1-min period, with the needle remaining in place for another 5 min before removal. In sham-lesioned animals, the needle was lowered into place and 250 nl of vehicle (0.1 M PBS/ 0.9% saline) was injected as described above. 2.6. Confirmation of pPVTh lesions At the end of Experiment 3, brains of all animals were collected, post-fixed in 4% formalin followed by 30% sucrose and sliced coronally at 30 mm on a sliding microtome. Sections were stained immunocytochemically for glial

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fibrillary acidic protein (GFAP) to assess the damage produced by ibotenic acid lesions. Free floating sections to be reacted with GFAP antibody were incubated with 10% normal horse serum in 0.1 M PBS solution for 20 min at 4 C. Sections were then incubated overnight at 4 C with a monoclonal mouse anti-GFAP (1:1500; Boehringer Mannheim, Indianapolis, IN) in 0.1 M PBS cocktail containing 1% normal horse serum (Vector, Burlingame, CA), 0.3% Triton X-100 (Sigma), and 0.25% BSA (Sigma). Sections were subsequently incubated with a biotinylated anti-mouse IgG (rat absorbed) in horse serum (Vector), diluted 1:200 in the above PBS cocktail for 2 h at room temperature, washed, and then incubated with an avidin –biotin-peroxidase complex (Vector) for 2 h at 4 C. The chromagen was diaminobenzidine in 0.3% hydrogen peroxide. Brain sections were mounted onto Superfrost Plus slides using tap water. Sections were coverslipped following immersion in water, ethanol (70%, 90%, 95%, 100%), and hemode. All brains were examined in a blind fashion. We only included those animals that exhibited damage as revealed by GFAP staining of, at least, approximately two-thirds of the posterior division of the PVTh. As previously defined by Bhatnagar and Dallman [20], the posterior division of the PVTh extends from 2.56 to 3.3 mm from bregma. All animals showing damage limited to the anterior and medial subdivisions were excluded, although animals exhibiting some damage to the medial subdivision were included if at least two-thirds of the posterior subdivision also exhibited damage. Although it

Fig. 1. Anterior, medial, and posterior divisions of the paraventricular nucleus of the thalamus (PVTh) are shown from left to right. Plates from Paxinos and Watson are shown in (a). A representative sham-lesioned rat is shown in (b), and a representative posterior PVTh-lesioned rat is shown in (c). Sections in (b) and (c) were immunocytochemically stained for glial fibrillary acidic protein to visualize the extent of the lesion. Rats with lesions limited to the anterior and medial divisions were eliminated from analysis. Only rats exhibiting damage to the posterior PVTh and limited damage to the medial PVTh were included in the analysis.

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is plausible that some ibotenic acid may have leaked into the ventricles, we did not see any damage, as assessed by increased GFAP staining above background, in any other regions of the brain in those animals that were included in the study. Our rates of successfully lesioning the pPVTh using the criteria described above were approximately 82% in these experiments. Fig. 1 shows GFAP staining in the pPVTh in a sham-lesioned rat and in a rat with representative ibotenic acid-induced lesions. 2.7. Corticosterone radioimmunoassays Blood was collected in tubes coated with sodium EDTA and kept on ice until centrifuged. After centrifugation, the plasma was aliquoted and kept frozen at 20 C until assay. Plasma corticosterone was measured using a kit from ICN Biomedicals (New York). The minimal detectable corticosterone concentration was 0.625 mg/dl. In some studies, we were not able to collect data for plasma ACTH. Therefore, only plasma corticosterone data are presented.

Fig. 2. Suppression of plasma corticosterone responses to acute restraint was observed following administration of 0.05, 0.2, and 2.0 mg/kg doses of dexamethasone compared to vehicle injection at 15 and 30 min time points. Doses of 0.2 and 2.0 mg/kg also significantly suppressed corticosterone at 60 min. We used the 0.05-mg/kg dose of dexamethasone in our subsequent experiments. * indicates all doses significantly different from vehicle ( P < .05). + indicates 0.2 and 2.0 mg/kg significantly different from vehicle ( P < .05).

2.8. Drugs Dexamethasone (Sigma) was initially dissolved in EtOH and brought up to volume in 0.9% saline. Saline/EtOH served as the vehicle injection. Subcutaneous injections of dexamethasone or vehicle were administered in a volume of 0.1 ml/100 g body weight. 2.9. Statistical analyses In Experiment 1, a one-way analysis of variance (ANOVA) was performed at each time point. Due to missing points, we were unable to perform repeated measures ANOVAs in any of these experiments. In Experiment 2, stress (control or chronic stress)  treatment (vehicle or dexamethasone) ANOVAs were performed at each time point. In Experiment 3, lesion (sham or lesion)  treatment (vehicle or dexamethasone) ANOVAs were carried out in acutely stressed and in chronically stressed rats at each time point. All significant main or interaction effects were followed by Fisher’s post hoc tests. The significance levels in all tests were set at P < .05.

3. Results 3.1. Experiment 1: determination of a dose of dexamethasone In order to determine a dose of dexamethasone that would effectively inhibit HPA responses to restraint in our lab, we evaluated plasma concentrations of corticosterone in response to acute restraint following administration of three different doses of dexamethasone or vehicle (Fig. 2). Compared to the vehicle, all three doses (0.05, 0.2, and 2.0 mg/kg) significantly inhibited plasma corticosterone concentrations

at 15-min [ F(3,30) = 12.4, P < .001] and 30-min time points [ F(3,30) = 12.2, P < .01]. The two highest doses also produced inhibition at 60 min [F(3,30) = 10.7, P < .001] and were the only doses to suppress plasma corticosterone to basal levels. Due to the submaximal activity of the lowest dose (0.05 mg/kg), it was used in the remainder of the studies. 3.2. Experiment 2: effects of repeated restraint on dexamethasone (0.05 mg/kg)-induced negative feedback Following pretreatment with vehicle or 0.05 mg/kg dexamethasone, we determined plasma concentrations of corticosterone in response to either one or eight exposures to restraint. At 0 min, there was a significant drug treatment effect [ F(1,28) = 13.2, P < .001]. Post hoc tests revealed that dexamethasone pretreatment significantly decreased corticosterone in chronically stressed rats. This effect of dexamethasone is only observed in chronically stressed rats because basal corticosterone is elevated in these rats due to repeated restraint exposure (evident in chronically stressed vehicle injected rats). At 15 min, there was a significant stress effect [ F(1,20) = 5.9, P < .02], a significant treatment effect [ F(1,20) = 28.3, P < .001] and a significant interaction [ F(1,20) = 5.1, P < .03] effect. Post hoc tests revealed that vehicle-treated acutely stressed rats had significantly higher corticosterone than vehicle-treated repeatedly restrained rats, demonstrating habituation of corticosterone responses to repeated restraint. Furthermore, both acute and chronically stressed rats injected with dexamethasone had significantly lower corticosterone compared to the acutely stressed rats injected with vehicle. At 30 min, there was a significant stress effect [ F(1,20) = 4.7, P < .04] with post hocs demonstrating that chronically stressed rats injected with dexamethasone had

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lower corticosterone than acutely stressed rats injected with dexamethasone. Furthermore, dexamethasone suppressed corticosterone compared to vehicle in chronically stressed rats but did not suppress corticosterone in acutely stressed rats. At 60 min, there was a significant treatment effect [F(1,21) = 13.3, P < .001]. Post hoc tests showed that chronically stressed rats injected with dexamethasone had significantly lower plasma corticosterone levels than acutely stressed rats injected with dexamethasone or vehicle. As at 30 min, dexamethasone suppressed corticosterone significantly in chronically stressed but not acutely stressed rats at 60 min. To summarize, chronically stressed rats exhibited lower plasma corticosterone at 15 min compared to acutely stressed rats, evidence of habituation. Dexamethasone suppressed corticosterone at all time points in chronically stressed rats but only at 15 min in acutely stressed rats. Plasma corticosterone in dexamethasone injected chronically stressed rats was lower at 30 and 60 min compared to all other groups.

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3.3. Experiment 3: effects of pPVTh lesions on dexamethasone (0.05 mg/kg)-induced negative feedback in acutely and repeatedly restrained rats Following pretreatment with vehicle or 0.05 mg/kg dexamethasone, we determined plasma concentrations of corticosterone in response to the first or eighth restraint in rats with sham or pPVTh lesions. 3.3.1. Acute stress groups Significant treatment (vehicle versus dexamethasone) effects (all P < .001) were observed at all time points in acutely stressed rats. Post hoc tests revealed that at each time point, acutely stressed rats injected with dexamethasone had significantly lower corticosterone levels in plasma compared to acutely stressed rats injected with vehicle. The only exception to this was the 0-min time point in shamlesioned rats. These effects occurred regardless of whether the rats were sham or pPVTh lesioned. No other significant effects were observed. Therefore, in acutely stressed rats,

Fig. 3. Plasma corticosterone responses to acute restraint following dexamethasone (DEX) or vehicle (VEH) treatment in acute and chronically restrained (CHRONIC) rats with sham (SHAM) or ibotenic acid lesion (LES) of the pPVTh. (a) In sham-lesioned acutely stressed rats, DEX-induced suppression of plasma corticosterone is seen at 15, 30, and 60 min compared to vehicle treatment. (b) In pPVTh-lesioned acutely stressed rats, DEX-induced suppression of plasma corticosterone is seen at 0, 15, 30, and 60 min compared to vehicle treatment. (c) In sham-lesioned rats that were chronically restrained, DEX-induced suppression of plasma corticosterone is seen at 15 and 30 min compared to vehicle treatment. (d) In pPVTh-lesioned rats that were chronically restrained, DEXinduced suppression of plasma corticosterone is seen at 0, 15, and 60, but not 30 min compared to vehicle treatment. Therefore, dexamethasone did not suppress corticosterone responses to restraint at 30 min in pPVTh-lesioned rats. However, pPVTh lesions did not alter responsivity in acutely restrained rats. * indicates dexamethasone-injected group is significantly lower than vehicle-injected counterpart ( P < .05).

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pPVTh lesions (1) did not significantly alter corticosterone responses to restraint in rats injected with vehicle and (2) did not alter dexamethasone-induced suppression of corticosterone. 3.3.2. Chronic stress Significant treatment (vehicle or dexamethasone) effects were observed at all time points (all P < .05). Post hoc tests revealed that in sham-lesioned rats, dexamethasone injections significantly reduced plasma corticosterone at 15 and 30 min and there was a tendency for a reduction at 60 min. In pPVTh-lesioned rats, plasma corticosterone was significantly lower in dexamethasone-injected rats versus vehicleinjected rats at 0, 15, and 60, but not 30 min. Therefore, pPVTh lesions prevented dexamethasone suppression of plasma corticosterone at 30 min in chronically stressed rats. At 0 min and 60 min, there were significant lesion and interaction effects (all P < .05). Post hoc tests revealed that pPVTh lesions elevated basal corticosterone in repeatedly restrained rats as they had in acutely restrained rats. Furthermore, at 60 min plasma corticosterone in vehicleinjected chronically stressed rats with pPVTh lesions was significantly higher than in vehicle-injected chronically stressed rats with sham-lesions. These data indicate that pPVTh lesions prevented complete habituation of plasma corticosterone in chronically stressed rats and support our previous findings [4]. 3.3.3. Chronic versus acute stress An examination of Fig. 3 highlights two points with respect to the effects of pPVTh lesions in acute and chronically stressed animals. First, pPVTh lesions increased basal 0-min corticosterone in both acute and repeatedly restrained rats injected with vehicle. Such elevations in basal HPA activity by pPVTh lesions are not always observed and when observed, are not always significant [2,37]. These effects might point to a role for the pPVTh in regulation of basal HPA activity, but this has not been directly studied. Second, the only group in which dexamethasone did not suppress restraint-induced corticosterone release was in chronically stressed rats with pPVTh lesions at the 30-min time point. These lesions had no effect on dexamethasone’s ability to suppress corticosterone in acutely stressed rats at any time point.

4. Discussion Based on a dose of dexamethasone found in Experiment 1 to produce submaximal suppression of plasma corticosterone in response to acute restraint, we used 0.05 mg/kg dexamethasone in our subsequent experiments. We demonstrated in Experiments 2 and 3 that chronically stressed rats display dexamethasone-induced negative feedback inhibition to a greater, or an equal extent as acutely stressed rats,

respectively. These data show that, following chronic stress, the HPA axis retains its ability to be inhibited in response to dexamethasone-induced feedback signals. We then showed in Experiment 3 that lesions of the pPVTh blocked dexamethasone-induced negative feedback inhibition at 30 min in chronically stressed rats compared to their vehicleinjected counterparts. These lesions had no effect in acutely stressed rats. Therefore, these data suggest that the pPVTh contributes to negative feedback inhibition of the HPA axis specifically under chronic stress conditions. In Experiment 2, we used the 0.05-mg/kg dose of dexamethasone to examine negative feedback inhibition of corticosterone in rats that had habituated to repeated restraint. Habituation of corticosterone responses to repeated restraint was evidenced by lower corticosterone responses to the eighth restraint in chronically stressed rats compared to the first restraint in previously stress-naive rats (Fig. 4). These data are consistent with others showing habituation to repeated restraint [4,21,22]. Relative to acutely stressed rats, repeatedly restrained rats displayed significantly suppressed plasma corticosterone in response to acute restraint following dexamethasone treatment at all four time points (Fig. 4b). In contrast, corticosterone was inhibited in acutely stressed rats at only the 15-min but not 30- or 60-min time point (Fig. 4a). Thus, a dose of dexamethasone that produces inhibition of corticosterone responses only at one time point in acutely restrained rats produces inhibition at all time points in chronically stressed rats that had habituated. It could be argued that the reason that repeatedly restrained rats are equally or more suppressed by dexamethasone as acutely stressed rats is because they are habituated and an escape from dexamethasone suppression would be difficult to observe. However, corticosterone levels at 0 and 15 min are equally suppressed in acutely and chronically stressed rats, but acutely stressed rats escape from dexamethasone suppression at 30 and 60 min while chronically stressed rats do not. Thus, regardless of how suppressed corticosterone levels are at 0 and 15 min, it is possible to escape from this suppression. Alternatively, these differences between acute and chronically stressed rats may be attributed to the fact that the central mechanisms that promote habituation prevent an escape from suppression. One way this alternative could be addressed is by examining negative feedback inhibition under conditions of facilitation in chronically stressed rats. We have done so and found that repeatedly socially stressed rats that facilitate to novel restraint are also as suppressed by dexamethasone (also 0.05 mg/kg) as acutely restrained rats [23]. This suggests that regardless of whether chronically stressed rats are examined under conditions of habituation or facilitation, they exhibit negative feedback inhibition of the HPA axis that is as effective as in acutely stressed rats. We were able to again address this question of negative feedback inhibition of corticosterone in acutely versus chronically stressed rats in Experiment 3 (by comparing the effects of dexamethasone in sham acute versus sham

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Fig. 4. Rats were either exposed to 7 days of repeated restraint (CHRONIC) or no stress. On day 8, rats were injected with either vehicle or dexamethasone (0.05 mg/kg) 2 h prior to acute restraint. Plasma corticosterone responses to acute restraint are shown following either dexamethasone (DEX) or vehicle treatment in previously nonstressed controls exposed to acute restraint (ACUTE) or in rats previously restrained for 7 days (CHRONIC). (a) In acutely stressed rats, suppression of plasma corticosterone by dexamethasone was seen only at 15 min compared to vehicletreated rats. (b) In repeatedly restrained, dexamethasone suppressed plasma corticosterone at all four time points compared to vehicle. Corticosterone levels in vehicle-injected chronically stressed rats were lower than in similarly treated acutely stressed rats at 15 min providing evidence of habituation by chronic stress. * indicates significantly different from dexamethasone-injected counterpart ( P < .05). + indicates chronically stressed group injected with vehicle is significantly lower than acutely stressed group injected with vehicle ( P < .05). + + indicates chronically stressed group injected with dexamethasone is significantly lower than vehicle or dexamethasone-injected acutely stressed group ( P < .05).

chronically stressed rats) with the important difference that these animals had undergone stereotaxic surgery. Shamlesioned acute versus chronically stressed rats were equally suppressed by dexamethasone in this experiment. We believe that the reason that chronically stressed rats were more inhibited than acutely stressed rats in Experiment 2 but only equally so in Experiment 3 is because of the stereotaxic surgery required in Experiment 3. We have found in

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previous studies that the magnitude of both facilitation and habituation is altered by prior surgery, and it is not surprising that stereotaxic surgery can affect the magnitude of negative feedback inhibition of the HPA axis as well. These findings of intact or even enhanced delayed negative feedback functions in chronically stressed rats are consistent with a recent study that reported a hypersuppression of plasma corticosterone in chronically stressed rats compared to controls following dexamethasone administration in response to acute restraint/water immersion [13]. Our findings seem to contrast with the results of several studies that have linked chronic stress with a decreased sensitivity to glucocorticoid receptor-mediated negative feedback signals. For example, rats exposed to chronic inescapable footshock do not display suppressed plasma corticosterone responses to acute footshock following dexamethasone treatment [11]. Rats exposed to previous footshock also do not exhibit fast feedback inhibition of betaendorphin to acute stress following corticosterone treatment [12]. However, the results of our study do not necessarily contradict the abovementioned findings. Reports of decreased feedback inhibition following repeated footshock and our findings of increased delayed feedback inhibition following repeated restraint may be explained in two ways. First, the studies describing impaired feedback in chronically stressed rats primarily study fast feedback and it is possible that the central mechanisms that govern fast versus delayed feedback are different. Second, the HPA axis, and the central neural mechanisms that regulate it, has the capacity to respond in a discriminative fashion to various stressors depending on the nature and intensity of the stressor [24 – 29]. Restraint, the stressor used in our study, is a relatively less intense stressor than footshock. While habituation is reliably observed in response to repeated restraint [4,14,21], there is evidence that habituation of the HPA axis often does not occur in response to intense physical stressors such as footshock or forced running [27,30,31]. Therefore, it is possible that negative feedback inhibition may function differently under intense versus mild stress conditions. The sites at which corticosterone exerts its negative feedback effects under chronic stress conditions have not yet been identified. Potential sites are abundant as studies of negative feedback under acute stress conditions suggest roles for the hippocampus, amygdala, and paraventricular hypothalamus [32 – 34]. We examined one such potential site in Experiment 3. The pPVTh has previously been shown to be inhibitory to HPA activity in repeatedly restrained rats, but not in acutely stressed rats [4]. Based on these data, we hypothesized that the pPVTh may be a site that mediates negative feedback effects of corticosterone specifically under chronic stress conditions. We found that in acutely restrained rats, lesions of the pPVTh did not affect dexamethasone’s capacity to inhibit the HPA response. However, in repeatedly restrained rats, lesions of the pPVTh blunted dexamethasone’s ability to inhibit the HPA response

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to acute restraint at the 30-min time point, the peak of the corticosterone response to restraint. Although these effects of pPVTh on negative feedback function are limited in their extent, these data nonetheless suggest that the pPVTh contributes to the negative feedback effects of corticosterone released by repeated stress exposure. The present data do not provide evidence for the pPVTh as a site of direct feedback action of corticosterone. A requirement that must be met for a feedback site is that delivery of steroid to that site should decrease HPA activity. We administered dexamethasone systemically which leaves open the possibility that the drug may have been binding to corticosteroid receptors anywhere in the brain. When injected peripherally, dexamethasone poorly crosses the blood – brain barrier [35] and its primary effects are probably at the pituitary [36]. Nonetheless, it is well accepted that the ability of dexamethasone to inhibit ACTH release from the pituitary is a function of the ACTH secretagogues released from the paraventricular hypothalamus. The release of these secretagogues, primarily corticotropin-releasing hormone and vasopressin are, in turn, controlled by central stress circuits. That lesions of the pPVTh affect dexamethasone’s ability to inhibit HPA acitvity suggests that the pPVTh is an important component of central circuits regulating negative feedback inhibition in chronically stressed rats. Negative feedback inhibition of HPA activity is an important component of habituation as demonstrated by the findings of Cole et al. [14] showing that blockade of MR or MR and GR prevented habituation to repeated restraint. Therefore, our data suggest that part of the mechanism by which the intact pPVTh inhibits HPA activity in chronically stressed animals is by altering negative feedback inhibition produced by stress-induced corticosterone release. However, we found that the extent of pPVTh lesion effects on negative feedback functions in chronically stressed rats was limited to the peak time of corticosterone release (30 min). These findings suggest that other mechanisms, such as activation of GABA-ergic circuitry, may play a more important role in the ability of the pPVTh to inhibit chronic stress-induced HPA activity. In summary, we found that repeatedly restrained rats have sensitivity to negative feedback signals that is similar to the sensitivity found in acutely stressed rats. Furthermore, we found that lesions of the pPVTh attenuated the ability of dexamethasone to inhibit corticosterone to restraint only in rats that had prior exposure to restraint but not in rats exposed to a single episode of restraint. These data confirm our previous findings that pPVTh inhibits HPA activity in both facilitated and habituated rats but does not seem to affect HPA activity in acutely stressed rats [2,4,37]. These data also suggest that part of the mechanism by which the pPVTh exerts its effects is by regulating negative feedback functions. Clearly, the pPVTh exerts its inhibitory effects on HPA activity only in part through negative feedback functions since its lesioning attenuated, but did not completely block, dexamethasone’s suppressive effects. However, fur-

ther investigation needs to be carried out in order to determine if the pPVTh itself is a site of direct feedback action. Ultimately, characterizing glucocorticoid negative feedback in chronically stressed animals and identifying brain sites where feedback inhibition occurs under chronic stress conditions may notably advance our current understanding of the dysregulation of the HPA axis seen in diseases such as depression.

Acknowledgements We would like to thank Courtenay Vining and Leslie Babich for their excellent technical assistance. This work was supported by the National Science Foundation (IBN0115212).

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