Differential neuroendocrine responses to chronic variable stress in ...

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Charlotte O. Ladd, K.V. Thrivikraman, Rebecca L. Huot, Paul M. Plotsky* ... separation (HMS) in neonatal Long Evans hooded rats leads to stable phenotypes.
Psychoneuroendocrinology (2005) 30, 520–533

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Differential neuroendocrine responses to chronic variable stress in adult Long Evans rats exposed to handling-maternal separation as neonates Charlotte O. Ladd, K.V. Thrivikraman, Rebecca L. Huot, Paul M. Plotsky* Stress Neurobiology Laboratory, Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, 1639 Pierce Drive, Ste 4105, Atlanta, GA 30322, USA Received 21 November 2004; accepted 13 December 2004

KEYWORDS Chronic stress; CRF; Mineralocorticoid receptor; Glucocorticoid receptor; Maternal separation; HPA axis

Summary Burgeoning evidence supports a preeminent role for early- and late-life stressors in the development of physio- and psychopathology. Handling-maternal separation (HMS) in neonatal Long Evans hooded rats leads to stable phenotypes ranging from resilient to vulnerable to later stressor exposure. Handling with 180 min of maternal separation yields a phenotype of stress hyper-responsiveness associated with facilitation of regional CRF neurocircuits and glucocorticoid resistance. This study assessed whether or not prolonged HMS (180 min/day, HMS180) on post-natal days 2–14 sensitizes the adult limbic hypothalamo–pituitary–adrenal (LHPA) axis to chronic variable stress (CS) compared to brief HMS (15 min/day, HMS15). We examined regional mRNA densities of corticotropin-releasing factor (CRF), its receptor CRF1, glucocorticoid receptor (GR), and mineralocorticoid receptor (MR); regional CRF1 and CRF2a binding, and pituitary–adrenal responses to an acute airpuff startle (APS) stressor in four groups: HMS15, nonstressed; HMS15, stressed; HMS180, nonstressed; HMS180, stressed. As expected we observed exaggerated pituitary–adrenal responses to APS, increased regional CRF mRNA density, decreased regional CRF1 binding, and decreased cortical GR mRNA density in nonstressed HMS180 vs. HMS15 animals. However, in contrast to our hypothesis, CS decreased pituitary–adrenal reactivity and central amygdala CRF mRNA density in HMS180 rats, while increasing cortical GR mRNA density and CRF1 binding. CS had no effect on the pituitary–adrenal response to APS in HMS15 rats, despite tripling hypothalamic paraventricular CRF mRNA density. The data suggest that many effects of prolonged HMS are reversible in adulthood by CS, while the neuroendocrine adaptations imbued by brief HMS are sufficiently stable to restrain pituitary–adrenal stress responses even following CS. Q 2005 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: C1 404 727 8258; fax: C1 404 727 3233. E-mail address: [email protected] (P.M. Plotsky). 0306-4530/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.psyneuen.2004.12.004

Rearing effects on adult neuroendocrine responses to chronic stress

1. Introduction Throughout life the continuous interaction of genetic and environmental factors shapes the neurocircuitry of the mammalian central nervous system, giving rise to individual differences in stress responsiveness that may become adaptive or maladaptive over time depending on circumstances. Recent modifications of the stress diathesis theory of depression suggest that adverse experiences early in life may sensitize specific neurocircuits to subsequent acute stressors, thereby increasing individual vulnerability to the onset of physio- and psycho-pathology (Brown et al., 1987; Anisman et al., 1998; Heim and Nemeroff, 2001). We proposed that one such neurocircuit is that containing corticotropin-releasing factor (CRF), a 41-amino acid peptide which coordinates the mammalian endocrine, behavioral, autonomic, and immunological responses to stress via the HPA axis and reciprocal limbic-brainstem neurocircuits (Owens and Nemeroff, 1991; Herman et al., 1996; Herman and Cullinan, 1997; Arborelius et al., 1999; van de Kar and Blair, 1999). Regional expression of CRF and its receptors CRF1 and CRF2a is sensitive to external stressors (Imaki et al., 1991; Lightman and Harbuz, 1993; Gomez et al., 1996; Albeck et al., 1997; Brown and Sawchenko, 1997; Bonaz and Rivest, 1998; Stout et al., 2000), including those encountered in early life (Eghbal-Ahmadi et al., 1997, 1998, 1999). To explore the long-term consequences of early adverse experience on individual adult stress responsiveness, we have developed a rodent model of moderate handling-maternal separation (180 min/day; HMS180) during post-natal days (PND) 2–14 and have compared these animals to briefly handled (15 min/day, HMS15) rats that we consider an appropriate handling and cage-transfer comparison group (Ladd et al., 2000). As adults, these two rearing groups exhibit distinct and stable individual phenotypes in stress responsiveness (Sanchez et al., 2001). Compared to HMS15 animals, the HMS180 group exhibits increased basal tone of central CRFergic activity as well as sensitization of the HPA axis (Plotsky and Meaney, 1993; Ladd et al., 2000; Sanchez et al., 2001), which may be secondary to facilitation of ascending noradrenergic neurocircuits (Liu et al., 2000), dampened regional GABAergic tone (Caldji et al., 2000), and/or focal glucocorticoid resistance (Ladd et al., 2000). While several investigators have reported the effects of chronic stress on CRF neurocircuits (Imaki et al., 1991; Lightman and Harbuz, 1993; Gomez

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et al., 1996; Albeck et al., 1997; Brown and Sawchenko, 1997; Bonaz and Rivest, 1998; Stout et al., 2000, 2002) and regional corticosteroid receptor expression (Herman and Spencer, 1998; Lopez et al., 1998; Kim et al., 1999; Paskitti et al., 2000) in normal adult rats, little is known about the influence of rearing conditions on individual adult responses to chronic stress. Given the propensity for stress-related neurocircuits to be sensitized by perceived insults (Plotsky and Meaney, 1993; Heim et al., 1997; Bruijnzeel et al., 1999; Ladd et al., 2000; Heim and Nemeroff, 2001; Weaver et al., 2004), we evaluated the hypothesis that early adverse experience sensitizes CRF and glucocorticoid responsive neurocircuits toward later stressors by examining CRF, glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and CRF receptor expression in maternally separated rats exposed to chronic variable stress (CS).

2. Materials and methods 2.1. Animals Animal studies were approved by the Emory University Institutional Care and Use Committee and conformed to the NIH Guidelines for the Care and Use of Laboratory Animals. Timed-pregnant Long Evans hooded rats arrived at the Emory University vivarium on gestation day 11–12 from Charles Rivier (Portage, MI). All dams were housed in transparent, polypropylene cages (20!25 cm) with 2 cm wood shavings and ad libitum access to food (Purina Lab Chow) and water in a room on a 12 h:12 h light:dark cycle (lights on at 07:00 h). Day of birth marked post-natal day (PND) 0. On PND 2, all pups were removed from their home cages, randomized, and culled to eight male pups per dam. Each litter was then exposed to one of two rearing conditions from PND 2 to 14, inclusive: (a) brief handling-maternal separation, in which animals were removed from the home cage daily for 15 min periods (HMS15 group) or (b) prolonged handling-maternal separation in which pups were removed from the home cage for 180 min daily (HMS180 group). There were four litters of each rearing condition, totaling 64 animals. Prior to manipulation of the HMS15 and HMS180 pups, the foster mother was removed from her home cage and housed in a clean cage throughout the daily separation period. During the separation period, rat pups in the HMS180 and HMS15 groups were housed as individual litters in a temperaturecontrolled environment (incubator set at 30G0.5 8C

522 during PND 2–14) between 08:00 and 13:00 h. The transparent separation cages (10!20 cm) were lined with 3 cm bedding material so that pups could further thermoregulate by huddling with littermates and burrowing into the bedding. Previous studies indicate that, under similar conditions, pup core temperature is maintained at 36–37 8C for up to 6 h (Jans and Woodside, 1990). At the end of each separation period, both HMS15 and HMS180 pups were returned to the home cage, rolled in the soiled bedding, and then reunited with their foster mother. No manipulation of the rats occurred other than that described above; cages were not cleaned in the HMS180 and HMS15 group until after PND15. Investigators wore gloves while manipulating the animals to prevent rejection of pups by the dams. Litters were weaned on PND23, and rats were housed two per cage until PND74.

2.2. Chronic variable stress Beginning on PND74 (average body weightZ399 g), four animals from each litter were randomly selected for the study and housed individually throughout the experiment (NZ32 total). Each half litter was subdivided into two groups, stressed (CS) and nonstressed (NS), yielding four experimental groups of eight pups each: HMS15, HMS15CCS, HMS180, and HMS180CCS. Animals in the stressed groups were exposed to 14 days of CS and then sacrificed. The CS paradigm (Herman et al., 1995) has been used to interrogate rodent stress neurocircuitry through persistent exposure to multimodal random stressors. Our modified CS protocol (Stout et al., 2002) consisted of a rotating schedule of two psychophysical stressors per day, one presented in the morning and one in the evening, for 14 days. The stressors included overnight food deprivation, 0.5 cm3 subcutaneous 0.9% saline injection, 5 min cold swim, overnight strobe light exposure, 1 h of darkness during the light phase, transfer to a soiled cage for 60 min, 60 min 4 8C cold exposure, 24 h light exposure, or jugular cannulation under anesthesia (one time only). The time of each morning and evening stressor varied daily to ensure unpredictability of exposure. Morning stressors were presented between 07:00 and 11:00 h and evening stressors were initiated between 14:00 and 17:00 h. During this 2-week period the nonstressed groups were housed in a separate room maintained under identical holding conditions. Both stressed and nonstressed rats were handled by the investigator at least three times weekly, twice for weighing and once for cage cleaning in addition to handling

C.O. Ladd et al. necessary for stressor exposure. The degree of handling each group received was approximately equivalent, as only three of the eight rotating stressors required handling. To minimize further handling, cage cleaning and weighing of the animals in the stressed groups were conducted following one of the three stressors requiring handling.

2.3. Jugular cannulation On PND81, 1 week after initiation of the CS regimen, all animals were fitted with intra-atrial cannulae through the right jugular vein in order to facilitate repeated blood sampling with minimal stress under aseptic conditions as described (Thrivikraman et al., 2002). Two days later, the lines were refilled by flushing with 200–250 ml of a sterile Gentamicin solution (120 mg/ml) to keep the lines patent. Animals were allowed to recuperate and habituate to their environment for 4 days prior to the air-puff startle (APS) stressor.

2.4. Stressor presentation and blood collection The air puff startle (APS) stressor was administered 4 days after jugular venous cannulation on PND85 between 08:00 and 09:00 h. One and one-half hours prior to the baseline blood sample, 50 cm tubing extensions (PE50; VWR, Atlanta) containing heparinized (20 IU/ml) saline and weighted by 1-cm3 syringes were connected to the exteriorized cannula at the base of the neck and extended outside the test cages. This procedure minimized contact with the rat and allowed undisturbed repeated blood sampling. A baseline blood sample was taken immediately prior to air-puff startle (APS). The APS stressor is a nonnoxious, aversive psychophysical stimulus. APS was administered in three repeated blocks with a 1-min interval from a pressurized can (50–65 psi) to rats in their test cage. Each block consisted of three successive 1-s long air blasts applied over a 5-s period beginning at time 0. The air puffs, directed at the side of the head, were applied from a distance of 10–20 cm. Serial blood samples (300 ml) were withdrawn 5, 10, 15, 30, 45, and 60 min following initiation of APS using a clean syringe attached to the extension cannula. Blood was immediately dispensed into 1.5 ml microcentrifuge tubes containing 10 ml Na4EDTA solution (100 mg/ml) and stored on ice. Each sample was replaced with an equal volume of sterile heparinized saline (20 IU/ml). Samples were centrifuged for 15 min at 12,000g and plasma was removed

Rearing effects on adult neuroendocrine responses to chronic stress and frozen (K20 8C) until assay for ACTH and corticosterone.

2.5. Tissue collection Following the APS stressor, the polyethylene lines were flushed with sterile gentamicin solution (120 mg/ml) and capped. The animals in the unstressed groups were then left undisturbed for 4 days, while animals in the CS groups received an evening stressor on PND 85 and continued the stress regimen for the next 3 days. The CS groups received the last stressor at 16:00 h on the evening before sacrifice, which occurred on PND89 between 09:00 and 13:00 h. Each animal was brought into a separate room, deeply anesthetized with 0.1 ml IV bolus of a concentrated pentobarbital sodium solution (Euthanasia-5), and decapitated under stress-free conditions. The skull was quickly removed and the brain was frozen in isopentane cooled to K70 8C by a methanol/dry ice bath. The frozen brains were stored in scintillation vials in a K80 8C freezer until sectioning.

2.6. ACTH and corticosterone radioimmunoassay analysis ACTH and corticosterone radioimmunoassays (RIA) were performed using commercially available kits. The ACTH assay (Allegroe HS-ACTH, Nichols Institute, San Juan Capistrano, CA) has a sensitivity of 1 pg/ml and intra- and inter-coefficients of variation of less than 6%. The corticosterone assay (ImmunoCheme Double antibody, ICN Biomedicals, Costa Mesa, CA) has a sensitivity of 1 ng/ml and intra- and inter-coefficients of variation of less than 10%.

2.7. Hybridization probes In situ hybridization analysis was performed on adjacent coronal sections (15 mm) using specific antisense riboprobes complementary to CRF, CRF1, GR, and MR mRNA. The CRF riboprobe (specific activity: 6!108 cpm/mg) was constructed from a 1.2 kb EcoR1 fragment of a full length rat CRF cDNA ligated into pGEM4 (Dr K. Mayo, University of Minnesota). The CRF1 riboprobe (specific activity: 6.1!108 cpm/mg) was constructed from a 1.3 kb PstI–PstI fragment containing the full length coding region for the rat CRF1 receptor ligated into a pBluescript II-SKC plasmid (W. Vale, the Salk Institute). The rGR riboprobe (specific activity: 1.5!109 cpm/mg) was transcribed from a 2815 bp BamH1 fragment ligated into pGEM4. The cDNA

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insert, generously provided by Dr K.R. Yammamoto (UCSF), contains the full coding region of the rGR gene. The rMR probe (specific activity: 1.4!109 cpm/mg) was constructed from a 550 bp HindIII fragment ligated into pGEM4. The cDNA insert, provided generously by Dr P.D. Patel (University of Michigan Medical Center), corresponds to the 3 0 -translated and untranslated regions of the rMR gene. Both the MR and GR plasmids were linearized to expose the full cDNA insert. In vitro transcription was performed in a 20 ml transcription reaction (Ambion MAXIscript) containing 250 mCi 35S-UTP; 0.5 mM each ATP, CTP, GTP; 20 units of RNA polymerase (SP6 or T7), 5 units of ribonuclease inhibitor; and 1 mg linearized plasmid cDNA in a 10! transcription buffer containing 10 mM DTT. The reaction mixture was incubated in a 37 8C water bath for 1 h. Subsequently, 2 units of RNase-free DNase 1 were added to digest the DNA template. After incubation for 15 min at 37 8C, the volume was increased to 50 ml with diethylpyrocarbonate (DEPC)-treated water and ethanol precipitated with 7.5 mM ammonium acetate. Pellets were reconstituted in 50 mL DEPC water and immediately put into hybridization buffer.

2.8. In situ hybridization Adjacent coronal sections (15 mm) were cut on a cryostat at K12 8C, thaw-mounted on SuperfrostPlus coated slides (Fisher), and stored with desiccant in a K20 8C freezer under RNAse free conditions until assay. Prior to hybridization, slides were thawed to RT, post-fixed in 4% paraformaldehyde (pH 7.4) for 30 min at RT, then rinsed twice (3 min each) in 1! PBS. Slides were then predigested for 15 min at RT with proteinase K (0.001%) in 25 mM Tris (pH 8.0) and 12.5 mM EDTA (pH 8.0). Afterward, slides were rinsed briefly in RT sterile water, followed by a 3 min rinse in 0.1 M TEA. Next, slides were acetylated (0.25% acetic anhydride) in 0.1 M TEA buffer to remove positive charges resulting from the proteinase K digestion. Slides were then rinsed twice in 2! SSC, 2 min each, and dehydrated in graded alcohols (Simmons et al., 1989). Hybridization (Simmons et al., 1989) was performed at 58 8C for 18 h in buffer (100 ml/slide) containing 50% deionized formamide, 10% dextran sulfate, 500 mg/ml yeast tRNA, 300 mM NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0), 10 mM dithiothreitol (DTT), 0.2% Denhardt’s solution, and 1!107 cpm/ml of [35S]-UTP-labeled cRNA antisense probe. Slides were cooled to RT, rinsed four times

524 (5 min each) in 4! SSC, then treated with RNase A (0.002%) for 30 min at 37 8C. Slides were then rinsed in decreasing salt concentrations (2! SSC twice, 5 min each; 1! SSC, 10 min; 0.5! SSC, 10 min) in the presence of 1 mM DTT to a final stringency of 0.1! SSC at 60 8C (30 min). Slides were dehydrated in graded alcohols, dried at RT, and apposed to Kodak BioMax MR film (Eastman Kodak Co., Rochester, NY) for 1–7 days along with 14C standards (Amersham). Adjacent slides were treated with an [35S]-UTP-labeled cRNA sense probe under the same conditions for negative controls.

2.9. Autoradiography CRF receptor autoradiography was performed on fresh-frozen 15 mm coronal brain sections. Slides were warmed to room temperature, post-fixed in 0.1% paraformaldehyde, rinsed in receptor incubation buffer (50 mM Tris, 10 mM MgCl, 2 mM EGTA, 0.1% BSA, pH 7.5) for 15 min, and incubated for 2 h in 0.1 nM 125I sauvagine (NEN Dupont) with 0.0001% aprotinin to inhibit protease activity. 125I-sauvagine has a high affinity for both CRF1 and CRF2 receptors. In order to isolate CRF2a binding, adjacent sections were incubated in 0.1 nM 125Isauvagine and the selective CRF1 antagonist CP154, 546 (1 mM). To determine nonspecific binding, another set of adjacent sections was incubated in 0.1 nM 125I-sauvagine and 1 mM cold sauvagine. All slides were rinsed twice (5 min each) in PBSC1%BSA (pH 7.4) and ddH2O (1 min). Slides were then dried and exposed to Kodak BioMax MR film (Eastman Kodak Co., Rochester, NY) for 7–10 days.

2.10. Densitometry Film autoradiographs were digitized using a Dage-MTI CCD camera (Sony Corp., Tokyo, Japan) and images were captured using a Frame Grabber card (Data Translation, S. Natick, MA) equipped Quadra 950 computer (apple computer, Cupertino, CA) running NIH Image version 1.60 software (Wayne Rasband, NIH, Bethesda, MD) for semiquantification against 14C standards which were run on each film. Three to four sections were averaged to derive a single value for each animal. Sections from seven to eight rats per group were used for statistical analysis. Gray-level determinations were corrected for background and values were expressed as Bq/mg tissue.

2.11. Statistical analysis Three-way ANOVA (rearing, stress, and time as factors) with repeated measures on time was used

C.O. Ladd et al. for statistical comparisons of radioimmunoassays. For in situ hybridization and CRF receptor autoradiography analyses, two-way ANOVA with rearing and stress as factors was performed. Each ANOVA reporting significant effects was followed by a Student Newman Keuls (SNK) post-hoc test.

3. Results 3.1. Plasma hormones 3.1.1. Plasma ACTH Serial plasma ACTH concentrations following acute APS stressor were analyzed by three-way ANOVA, using rearing and stress as factors and time as a repeated measure (Fig. 1A). We observed significant effects of both rearing [F(1,26)Z3.96, P!0.05] and time [F(6,156)Z15.48, P!0.01] on plasma ACTH concentrations. In all four experimental groups, acute APS caused a marked increase in mean plasma ACTH concentration, peaking at 5 min and returning to baseline by 60 min post-stress. The plasma ACTH peak was 2.5-fold greater (P!0.05) in nonstressed HMS180 rats than in their HMS15 counterparts. CS reduced peak plasma ACTH concentrations by 50% (P!0.05) in HMS180 animals but had no effect on the ACTH response to acute APS in HMS15 animals. Two-way ANOVA of the integrated (area under the curve, AUC) ACTH response (Fig. 1C) revealed a significant effect of rearing [F(1,26)Z5.48, P!0.05] and a significant interaction between rearing and chronic stress [F(1,26)Z4.05, P!0.05], such that nonstressed HMS180 rats mounted a greater integrated response to APS than nonstressed HMS15 rats (P!0.05) that was abolished (P!0.05) following CS. Consonant with previous findings, we observed no effects of rearing or CS on basal plasma ACTH concentration. 3.1.2. Plasma corticosterone Three-way ANOVA with repeated measures on time revealed a significant effect of time [F(6,156)Z20.8, P!0.01], interactions between rearing and stress [F(1,26)Z9.10, P!0.01] and rearing, stress, and time [F(6,156)Z4.19, P!0.01] on the plasma corticosterone response to acute APS (Fig. 1B). Plasma corticosterone concentrations in the nonstressed HMS180 group were significantly greater at 15 (P!0.05) and 30 min (P!0.01) following APS than in nonstressed HMS15 rats. CS was associated with an 86% reduction in plasma corticosterone concentrations 30 min (P!0.01) after the APS in HMS180 rats. No effect of CS was observed on the plasma corticosterone

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response to acute APS in HMS15 rats. Two-way ANOVA of the integrated corticosterone response to APS (Fig. 1C) revealed a significant interaction between rearing and chronic stress [F(1,26)Z6.90, P!0.05], such that nonstressed HMS180 rats mounted a higher adrenocortical stress response than their HMS15 counterparts (P!0.05) that was decreased following chronic stress (P!0.05). There were no effects of rearing or CS on adult basal plasma corticosterone concentration.

3.2. CRFergic activity 3.2.1. CRF mRNA CRF mRNA density was measured in the PVN, CeA, and BNST in all four experimental groups (Fig. 2). In the PVN we observed significant effects of rearing [F(1,24)Z8.49, P!0.01] and stress [F(1,24)Z6.63, P!0.05]. As expected CRF mRNA density within the PVN was significantly (P!0.01) greater in nonstressed HMS180 vs. HMS15 animals. CS increased CRF mRNA density threefold in HMS15 rats (P!0.01) but had no effect on their HMS180 counterparts. In the CeA we observed a significant interaction between rearing and stress [F(1,26)Z4.26, P!0.05] on CRF mRNA density (Fig. 2). Nonstressed HMS180 rats exhibited a 47% greater density of CRF mRNA in the CeA compared to their HMS15 counterparts (P!0.05). CS was associated with a nonsignificant 26% increase in CRF mRNA in HMS15 rats and a 39% decrease in CRF mRNA density in HMS180 animals (P!0.05). No differences due to rearing [F(1,24)Z0.07, PZ0.79] or stress [F(1,24)Z0.205, PZ0.65] were observed in CRF mRNA density in the BNST.

Figure 1 (A) Mean (GSEM) plasma ACTH concentrations in four experimental groups: HMS15 (NZ7), HMS15CCS (NZ7), HMS180 (NZ8), and HMS180CCS (NZ8). *P!0.05 vs. nonstressed HMS15 animals. (B) Mean (GSEM) plasma corticosterone concentrations in four experimental groups: HMS15 (NZ7), HMS15CCS (NZ7), HMS180 (NZ8), and HMS180CCS (NZ8). a P!0.05 and bP!0.01 vs. nonstressed HMS15 animals; c P!0.01 vs. nonstressed HMS180 counterparts; and d P!0.01 vs. HMS15CCS animals. (C) Mean (GSEM) area under the curve (AUC) analysis of plasma ACTH and corticosterone in four experimental groups: HMS15 (NZ7), HMS15CCS (NZ7), HMS180 (NZ8), and HMS180CCS (NZ8). *P!0.05 vs. nonstressed HMS15 rats; #P!0.05 vs. HMS180CCS rats.

Figure 2 Densitometric analysis of mean (GSEM) levels of rat CRF mRNA in the hypothalamic paraventricular nucleus (PVN), central nucleus of the amygdala (CeA), and bed nucleus of the stria terminalis (BNST) in four experimental groups: HMS15 (NZ7), HMS15CCS (NZ7), HMS180 (NZ7), and HMS180CCS (NZ7). *P!0.05 vs. nonstressed HMS15 animals; **P!0.01 vs. nonstressed HMS15 rats; #P!0.05 vs. nonstressed HMS180 rats.

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3.2.2. CRF1 mRNA We observed a significant effect of rearing on CRF1 mRNA density in the cingulate gyrus [F(1,28)Z3.75, P!0.05], frontal cortex [F(1,28)Z7.4, PZ0.01], and parietal cortex [F(1,28)Z6.60, P!0.05] as well as a significant effect of CS (P!0.05) in each of these regions [F(1,28)Z6.20, 9.60, 5.50, respectively] (Fig. 3A). Moderate HMS was associated with a significant decrease (24–37%) in CRF1 mRNA density in the cingulate (P!0.01), frontal (P!0.01) and parietal (P!0.01) cortices of nonstressed HMS180 vs. HMS15 rats. In addition, CS decreased CRF1 mRNA density in the cingulate (P!0.01), frontal (P!0.01) and parietal (P!0.01) cortices of HMS15 rats and in the frontal (P!0.05) and parietal (P!0.05) cortices of HMS180 rats. Although CS caused a greater percentage decrease in CRF1 mRNA density in the cingulate gyrus and parietal cortex of HMS15 vs. HMS180 animals, the HMS180CCS group exhibited the lowest CRF1 mRNA density in all cortical regions, particularly within the frontal cortex (P!0.05). 3.2.3. CRF receptor binding CRF1 receptor binding (Fig. 3B) was determined in the cingulate gyrus, frontal and parietal cortices, and basolateral amygdala (BLA), while CRF2a binding (Fig. 3C) was measured in the ventromedial hypothalamus (VMH) and lateral septum (LS). A two-way ANOVA with rearing and stress as factors was performed for each region. In the cingulate gyrus (Fig. 3B) we observed a significant effect of rearing [F(1,28)Z5.78, P!0.05] on CRF1 binding, with nonstressed HMS180 animals exhibiting less binding than their HMS15 counterparts (P!0.05). CS increased cingulate gyrus CRF1 binding in the HMS180 group (P!0.05) but had no effect on HMS15 animals. In the frontal cortex we observed a significant effect of stress [F(1,28)Z4.66, P!0.05] and an interaction between rearing and stress [F(1,28)Z4.16, P!0.05] on CRF1 binding (Fig. 3B). Frontal cortical CRF1 binding was markedly reduced (38%, P!0.05) in nonstressed HMS180 rats compared to their HMS15 counterparts. As in the cingulate gyrus, CS increased CRF1 binding in the frontal cortex of HMS180 animals (P!0.05) but was without effect in HMS15 rats. In the parietal cortex (Fig. 3B), there was a significant effect of stress [F(1,28)Z4.2, P!0.05] and an interaction between rearing and stress [F(1,28)Z5.36, P!0.05]. Nonstressed HMS180 rats exhibited significantly less CRF1 binding than HMS15 rats (P!0.05) in this region, an effect that was selectively increased by CS in the HMS180 group (P!0.05).

Figure 3 (A) Densitometric analysis of mean (GSEM) levels of rat CRF1 mRNA in the cortex of four experimental groups: HMS15 (NZ8), HMS15CCS (NZ8), HMS180 (NZ8), and HMS180CCS (NZ8). *P!0.05 vs. nonstressed HMS180 animals; #P!0.05 vs. HMS15CCS rats; **P!0.01 vs. nonstressed HMS15 group. (B) Densitometric analysis of mean (GSEM) levels of rat CRF1 binding in the cortex and basolateral amygdala (BLA) of four experimental groups: HMS15 (NZ8), HMS15CCS (NZ8), HMS180 (NZ8), and HMS180CCS (NZ8). *P!0.05 vs. nonstressed HMS15 animals; #P!0.05 vs. nonstressed HMS180 rats. (C) Densitometric analysis of mean (GSEM) levels of rat CRF2a binding in the ventromedial hypothalamus (VMH) and lateral septum of four experimental groups: HMS15 (NZ8), HMS15CCS (NZ8), HMS180 (NZ8), and HMS180CCS (NZ8).

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In the basolateral amygdala (BLA) we observed a significant effect of rearing [F(1,26)Z5.43, P!0.05], with nonstressed HMS180 rats exhibiting significantly less CRF1 binding (P!0.05) than HMS15 animals (Fig. 3B). Chronic stress did not alter CRF1 binding in the BLA of either rearing group. There were no rearing or stress associated differences in CRF2a binding in the lateral septum or ventromedial hypothalamus (Fig. 3C).

3.3. Glucocorticoid receptor mRNA 3.3.1. MR mRNA No significant effects of rearing or stress on hippocampal MR mRNA density were observed in this study, although there was a trend toward decreased MR mRNA density following CS in both rearing groups in CA1 [F(1,24)Z2.89, PZ0.10], CA2 [F(1,24)Z3.98, PZ0.058], and DG [F(1,24)Z2.18, PZ0.15] (Fig. 4A). 3.3.2. GR mRNA Regional GR mRNA density was analyzed using a two-way ANOVA with rearing and stress as factors. No significant effects of rearing or stress were observed for hippocampal GR mRNA density (Fig. 4B). However, we did note a trend for CS to selectively decrease GR mRNA density in the HMS180 but not HMS15 animals in all hippocampal subfields examined. We observed a significant effect of rearing [F(1,24)Z4.9, P!0.05] and stress [F(1,24)Z7.5, P!0.05] on GR mRNA density in the frontal cortex but not in the cingulate gyrus or parietal cortex (Fig. 4C). Moderate HMS was associated with a 37% decrease (P!0.05) in GR mRNA density in the frontal cortex of nonstressed HMS180 vs. HMS15 rats. CS selectively increased GR mRNA in this region in the HMS180 rats (P!0.05) but had no significant effects on GR mRNA density in the cingulate gyrus or parietal cortex.

4. Discussion Consonant with previous studies (Plotsky and Meaney, 1993; Ladd et al., 1996), we report exaggerated pituitary–adrenal responses to stress and coincident alterations in regional CRFergic activity in animals exposed to prolonged early maternal separation. We observed a twofold increase in peak ACTH and corticosterone responses to acute APS in nonstressed HMS180 vs. HMS15 animals. Furthermore, nonstressed HMS180 rats

Figure 4 (A) Densitometric analysis of mean (GSEM) levels of rat GR mRNA in the hippocampal subfields CA1, CA2, and dentate gyrus (DG) of four experimental groups: HMS15 (NZ7), HMS15CCS (NZ7), HMS180 (NZ7), and HMS180CCS (NZ7). (B) Densitometric analysis of mean (GSEM) levels of rat GR mRNA in the cortex of four experimental groups: HMS15 (NZ7), HMS15CCS (NZ7), HMS180 (NZ7), and HMS180CCS (NZ7). (C) Densitometric analysis of mean (GSEM) levels of rat MR mRNA in the hippocampal subfields CA1, CA2, CA3 and dentate gyrus (DG) of four experimental groups: HMS15 (NZ7), HMS15CCS (NZ 7), HMS180 (NZ7), and HMS180CCS (NZ7). *P!0.5 vs. nonstressed HMS15 rats; #P!0.05 vs. nonstressed HMS180 rats.

528 exhibited increased CRF mRNA density in the PVN and CeA and a concomitant decrease in CRF1 binding in the cortex and basolateral amygdala compared to nonstressed HMS15 rats. No rearing effects were observed for the binding density of CRF2a, a receptor originally implicated in anorexic behavior (Lovenberg et al., 1995; Behan et al., 1996) but more recently linked to anxiogenic behavior (Bakshi et al., 2002). These results support the thesis that HMS180 rats experience heightened CRF release in the limbic system and cortex that may contribute to the behavioral and endocrinological stress hyperresponsiveness observed in these animals (Menzaghi et al., 1993; Heinrichs et al., 1995; Ladd et al., 1996; Herman and Cullinan, 1997; De Kloet et al., 1998; Koob, 1999; Ladd et al., 2000; Sanchez et al., 2001). Long-term adaptations in corticosteroid receptor expression may also subserve some of the endocrine changes associated with early adverse experience (Ladd et al., 2004). We observed a robust decrease in GR mRNA density in the frontal cortex of HMS180 vs. HMS15 rats. Although changes in central GR expression may be secondary to glucocorticoid tone, increasing evidence suggests that altered GR transcription may be a primary event in the development of individual stress responsiveness (Weaver et al., 2001, 2004; Francis et al., 2002). Van Oers et al. (van Oers et al., 1998, 1999) demonstrated that most acute neuroendocrine responses to prolonged maternal separation are independent of corticosterone and are instead associated with the absence of maternal care. In this vein, Meaney and colleagues have shown that pups reared by low ‘licking and grooming’ mothers exhibit a stress reactive phenotype as adults associated with decreased hippocampal GR expression via methylation of a cysteine residue at the 5 0 NGFI-A binding region in the promoter of exon 17 (McCormick et al., 1998, 2000; Bird, 2001; Weaver et al., 2001). Their hyperresponsive HPA responses to stress are reversed in adulthood by trichostatin A, a histone deacetylase inhibitor that ‘opens’ bound DNA to transcription (Eden et al., 1998; Weaver et al., 2004). Unlike the effects of licking, grooming, and arched back nursing alone (McCormick et al., 1998, 2000; Weaver et al., 2001), the phenotypic changes associated with moderate HMS appear to be associated with changes in cortical rather than hippocampal GR mRNA density. We have observed the phenotype both with (Ladd et al., 2000; Francis et al., 2002) and without (vide supra and unpublished data) a concurrent decrease in hippocampal GR mRNA density, while decreased GR transcript in the frontal cortex is a consistent finding in HMS180

C.O. Ladd et al. vs. HMS15 rats (Huot et al., 2004; Ladd et al., 2004) that is inversely related to pituitary stress responsiveness. In a multiple regression analysis of several datasets in our laboratory, cortical GR mRNA predicted 12% of the variability in peak ACTH concentrations in both rearing groups following APS (unpublished data). These observations are congruent with previous reports that handling increases, while prenatal stress decreases, GR binding in the frontal cortex of Long Evans rats (McCormick et al., 1995; Meaney et al., 1996). The interexperimental variability in hippocampal GR mRNA expression among animals exposed to moderate vs. brief HMS (Francis et al., 2002; Ladd et al., 2004) most likely results from subtle differences in genetic and environmental factors that influence maternal care (Liu et al., 1997, 2000; Francis and Meaney, 1999; Ladd et al., 2000). Up to 70% of the variability in adult endocrine stress responsiveness is attributed to the quality of maternal licking, grooming, and arched back nursing in infancy (Liu et al., 1997; Francis et al., 1999). Since we rely on rodent suppliers to breed and transport our animals as timed pregnant dams, both genetic heterogeneity (or lack thereof) and prenatal stress are variables which may influence the effects of HMS. In this study we tested the hypothesis that HMS180 rats, compared to their HMS15 counterparts, would exhibit even greater dysregulation of central CRF neurocircuits and HPA activity following CS. In marked contrast to our hypothesis, CS decreased the neuroendocrine stress response in HMS180 rats. A similar stress-induced decline in HPA responsiveness has been observed in rats genetically (Albeck et al., 1997; Edwards et al., 1999; Herman et al., 1999) or epigenetically (Hodgson and Knott, 2002) predisposed to anxiety or learned helplessness and in humans with the onset of post-traumatic stress disorder (PTSD) (Resnick et al., 1995; Heim et al., 1997, 1998; Yehuda et al., 1998). The latter is believed to be associated with enhanced glucocorticoid negative feedback of the HPA axis, maintaining a hypocortisolemic state even in the face of robust facilitatory drive (Yehuda, 2002). The neuroendocrine adaptations observed following CS in HMS180 animals also parallel those following electroconvulsive therapy (ECT) in depressed patients (Carroll et al., 1976; Ylikorkala et al., 1976; Papakostas et al., 1981; Aperia et al., 1984; Whalley et al., 1987; Amsterdam et al., 1988; Nemeroff et al., 1991). While ECT’s therapeutic mechanism of action is unknown, recent studies have implicated regional neurotrophin expression (Nibuya et al., 1995; Duman and Vaidya, 1998; Hiroi

Rearing effects on adult neuroendocrine responses to chronic stress et al., 1998; Duman et al., 1999) and activation of cortical and limbic GABAergic neurocircuits (Bowdler et al., 1983; Weilosz et al., 1985; Otero Losada, 1988; Acosta et al., 1992; Sanacora et al., 2003). Enhanced GABAergic activity in adult rats could help reverse the neuroendocrine adaptations to prolonged HMS in early life. We have previously reported a regional decrease in GABAA expression in HMS180 vs. HMS15 rats (Caldji et al., 2000) that may contribute to their exaggerated behavioral, endocrinological, and molecular responses to stress (Ladd et al., 2000) through disinhibition of ascending noradrenergic and CRFergic neurocircuits. We hypothesize that CS may increase regional GABAergic tone similar to ECT (Sanacora et al., 2003), thereby dampening stress responsiveness in HMS180 animals. In support of this hypothesis, Patchev et al. (1997) demonstrated that the maternal separation phenotype can be prevented by concurrent neonatal treatment with THDOC, a neuroactive steroid that enhances GABAergic transmission. Alternatively, CS may dampen the pituitary– adrenal response to APS in HMS180 rats by enhancing transcription of GR in the cortex. In HMS180 rats, diminished cortical GR mRNA is reversed by both cross fostering (Huot et al., 2004) and CS in a manner that is inversely related to pituitary stress responsiveness. CS may decrease GR expression via demethylation of specific GR promoters (Weaver et al., 2001), acetylation of associated chromatin (Weaver et al., 2004), or regulation of an active AP-1 site via induction of cAMP-dependent transcriptional regulators (Hope et al., 1994; Nibuya et al., 1996; Duman and Vaidya, 1998; Duman et al., 1999; Barret and Vedeckis, 1996; Wei and Vedeckis, 1997). In contrast to HMS180 rats, animals exposed to brief HMS showed few changes in the limbic hypothalamo–pituitary–adrenal (LHPA) axis following CS. This may be due to enhancement of inhibitory pathways regulating the HPA axis following brief HMS, thereby imparting some resilience to external manipulation in adulthood (Caldji et al., 2000; Ladd et al., 2004). It is plausible that global GR expression is sufficiently stable in HMS15 animals (perhaps via demethylation of specific GR promoters in early life) to restrain the pituitary–adrenal response to APS even in the face of CS (Meaney et al., 1993; Sanchez et al., 2001; Weaver et al., 2004). Alternatively, enhanced GABAergic neurotransmission associated with brief HMS may dampen the HPA axis well into adulthood (Caldji et al., 2000). In this study hypothalamic paraventricular CRF mRNA concentrations at the time of death were

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incongruent with the endocrine stress response 4 days prior in both rearing groups. CS increased PVN CRF mRNA density in HMS15 animals by threefold but had no effect on their pituitary–adrenal response to a heterologous stressor, APS. Conversely, CS had no effect on PVN CRF mRNA density in HMS180 animals but halved their endocrine stress response. Similar dissociation between CRF mRNA and ACTH responsiveness has been observed following environmental enrichment in HMS180 vs. HMS15 rats (Francis et al., 2002). These data indicate that there are other circuits, possibly involving cortical GR mRNA (Meaney et al., 1985; Diorio et al., 1993; Dallman et al., 1994) and/or regional GABAergic tone (Caldji et al., 2000), acting at the level of the hypothalamus and/or pituitary which are orchestrating a less-than expected pituitary–adrenal stress response in both rearing groups. Alternatively, CRF1 receptors in the anterior pituitary may be down-regulated following CS, thereby limiting corticotroph responsiveness to hypothalamic CRF release. Unexpectedly, we report no effect of mild chronic stress on GR mRNA density in the hippocampus of either rearing group, suggesting that some genetic or epigenetic factor(s) in both rearing groups kept hippocampal GR expression surprisingly resistant to external manipulations (Kim et al., 1999; Beck and Luine, 2002; Bielajew et al., 2002). Alternatively, a threshold of stressor intensity as well as duration may be required to observe hippocampal GR down regulation (Sapolsky et al., 1984; Herman and Spencer, 1998; Paskitti et al., 2000). Extrahypothalamic CRF neurocircuits were more affected by CS in HMS180 vs. HMS15 rats, likely secondary to changes in glucocorticoid tone. CRF mRNA expression in the CNA is positively regulated by circulating glucocorticoids (Makino et al., 1995; Gray and Bingaman, 1996). CS decreased adrenal stress responsiveness in HMS180 animals concomitant with decreased CRF mRNA density in the CNA, while not affecting either in the HSM15 group. We observed reciprocal changes in CNA CRF mRNA density and cortical CRF1 binding in response to both moderate HMS and CS in Long Evans rats. Since the CNA provides many CRFergic efferents to cortical, limbic, and brainstem areas (Herman and Cullinan, 1997; van de Kar and Blair, 1999), we postulate that alterations in CRF expression in this nucleus contribute to the regulation of cortical CRF1 binding. In support of this hypothesis, we have noted a significant inverse correlation between CRF mRNA density in the CNA and CRF1 binding in the cingulate gyrus of both rearing groups (r2Z

530 0.13, unpublished data) in a multiple regression analysis of several data sets. CS was associated with one main effect irrespective of rearing: decreased cortical CRF1 mRNA density, with a trend toward decreased hippocampal MR mRNA density as well. Similar results have been reported in normally reared animals (Iredale et al., 1996; Herman and Spencer, 1998; Lopez et al., 1998), and may reflect decreased gene transcription following repeated stressor exposure. Notably, cortical CRF1 binding was selectively increased following CS in HMS180 rats, implying that CS enhanced post-transcriptional processing (Nikodemova et al., 2002) in these animals. Collectively, these observations emphasize the necessity to carefully monitor the handling and treatment of animals included in studies investigating the mammalian stress response to avoid obscuring or nullifying significant results. Numerous studies have iterated the role of environmental experiences on behavior and neuroendocrinological functioning (Liu et al., 1997; Anisman et al., 1998; Kim et al., 1999; Cabibet al., 2000; Weaver et al., 2001; Beck and Luine, 2002; Francis et al., 2002). Heterogeneous conditions among animal facilities may explain the failure of some laboratories to observe neuroendocrine and behavioral hyperresponsiveness following neonatal maternal separation for periods of less than 5–6 h daily (Muneoka et al., 1994; Durand et al., 1998; Lehmann and Feldon, 2000). In summary, we have demonstrated that early experience, in the form of brief vs. moderate HMS, has long lasting effects on the susceptibility to CS as an adult. While glucocorticoid and CRF neurocircuits previously sensitized by moderate maternal separation were at least partially reversed by CS, the neuroendocrine adaptations imbued by brief HMS were sufficiently stable to restrain much of the LHPA axis during CS. We postulate that such inhibition inherent in HMS15 rats and acquired after CS in HMS180 rats may result from increased cortical glucocorticoid and/or regional GABAergic tone. Future studies in our laboratory will be directed toward testing these hypotheses.

Acknowledgements This study was funded by NIH grants MH50113 (PMP), MH12163 (COL) and the Emory University Silvio O. Conte Center for the Neuroscience of Mental Disease (MH58922).

C.O. Ladd et al.

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