Laboratories of 1Stress Research and 2Molecular Neuroendocrinology, Institute of Experimental Medicine, Hungarian Academy of Sciences, and 3Laboratory of Neuromorphology, Semmelweis University Medical School, 1450 Budapest, Hungary
Submitted 15 May 2003 ; accepted in final form 22 July 2003
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ABSTRACT |
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adrenocorticotropin; corticosterone; mediobasal hypothalamus; paraventricular nucleus; proopiomelanocortin; rat
Chronic stress can be elicited by repeated exposure to short-acting stressors or by action of a sustained or persistent stimulus, e.g., a disease. Chronic stress is characterized by the classical symptoms of decreased body and thymus weight and a parallel increase in adrenal size and width of the adrenal cortex, consistent with chronic activation of the HPA axis (6, 12). The sustained activation of the CRH/AVP cells in the PVH is suggested to have a central role during chronic stress-induced HPA axis activation, and this hypothesis is also consistent with the changes of CRH and AVP mRNA in the PVH of chronically stressed rats (1, 17, 25, 26, 32, 36, 41). However, extrapolation from acute to chronic stress requires careful consideration of many factors. The work of Rivest and Rivier (40) showed that PVH lesion only diminished and did not block the footshock-induced ACTH and corticosterone elevation after either a single or repeated (7-day) exposure. Our group has shown that, during adjuvant-induced arthritis, the PVH has a minimal role in the maintenance of HPA axis activation, which may involve peripheral immune mediators (28). These data provide an example of chronic stress in which the mediators from the PVH have a limited role.
The aim of the present study was to test the role of the PVH and other parts of the hypothalamus in acute and repeated restraint-induced changes by use of hypothalamic lesions. To separate the anterior pituitary from direct hypothalamic influence, we used mediobasal hypothalamus (MBH) lesion instead of stalk transsection, because in this case the possibility of restitution is smaller. We produced chronic physical-psychological stress by repeating daily 1-h restraint. Such chronic stress stimulated CRH and AVP mRNA in the PVH (25, 26, 36), which suggests that PVH lesion would considerably inhibit HPA axis activation by chronic stress.
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MATERIALS AND METHODS |
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Hypothalamic Surgeries
Hypothalamic lesions were placed 5 days before starting the chronic stress, and the lesioned animals were housed singly postsurgery. Anesthesia was induced by intraperitoneal injection of ketamine (50 mg/kg, SelBruHa Állatgyógyászati Kft, Hungary)-xylazine (20 mg/kg, Spofa, Czech Republic)-promethazinium chloratum (0.2 ml/kg, EGIS, Budapest, Hungary). The lesions were placed as described previously, using wire knives fashioned from the stainless steel mandrel of a 20-gauge spinal needle and manipulated with the help of a stereotaxic frame (D. Kopf, Tujunga, CA). MBH lesion transected all hypothalamic axons, terminating in the median eminence region by a cone-shaped lesion (20). PVH lesion destroyed all CRH-containing cell bodies in the PVH by use of an inverted cone-shaped lesion (3.03.8 mm in diameter at its base) (28, 30, 31). Control rats were subjected to a sham operation consisting of opening the skull and introducing a knife into the midline in the brain to a position 5 mm below the brain surface, i.e., just above the hypothalamus.
For control of the lesions, the brains from each animal were removed. They were fixed in picric acid-formaldehyde solution and embedded in paraffin (MBH lesion) or frozen for cryostat sectioning (PVH lesion), and the hypothalamus was serially sectioned in the coronal plane. Every fifth 10-µm section was stained and examined. In the MBH-lesioned rats, water intake, plasma prolactin levels, and AVP and oxytocin content of the neurointermediate lobe of the hypophysis were also used for lesion verification. Only rats showing a distinct and complete lesion were retained for later analysis. In four rats of each group in the PVN lesion series, CRH and AVP mRNA in situ hybridization was also done.
Restraint Stress
We used transparent plastic tubes (56 cm ID) having a 4-cm-long conical head part ending with a large breathing hole (2 cm ID). The animals were tightly restrained by loosely packing with paper towels the rear end of the tube behind the body. This restraint procedure minimized the space around the animal, prevented turning, and provided a rather strong stressful stimulus without being harmful in the long run. Restraint sessions lasted for 1 h in the morning (09001200) and were repeated daily for 7 or 8 days.
Controls were nonstressed rats. An acute-restraint group was killed at the end of the first restraint; a repeated-restraint group at rest was killed on day 8, 24 h after the 7th restraint; an acute-restraint-after-repeated-restraint group was killed at the end of the 8th restraint.
Experimental Groups
MBH lesion series. The MBH lesion series consisted of four sham-operated groups: control (n = 11), acute restraint (n = 12), repeated restraint (n = 10), and acute restraint after repeated restraints (n = 10). MBH-lesioned groups were control (n = 18), acute restraint (n = 18), repeated restraint at rest (n = 15), and acute restraint after repeated restraints (n = 21).
PVH lesion series. In the PVH lesion series were four sham-operated groups: control (n = 18), acute restraint (n = 13), repeated restraint (n = 20), and acute restraint after repeated restraint (n = 12). PVH-lesioned groups were control (n = 17), acute restraint (n = 10), repeated restraint at rest (n = 11), and acute restraint after repeated restraint (n = 9).
Hormone Measurements
Trunk blood was collected into ice-cold tubes containing 50 µl of 20% K-EDTA. Hormones were measured by radioimmunoassay. Plasma ACTH was measured as described earlier (46). The intra- and interassay coefficients of variation (CVs) were 4.7 and 7%, respectively. Plasma corticosterone was measured from 10 µl of unextracted plasma with an RIA by use of a specific antiserum developed in our institute in rabbits against the corticosterone-3-carboxymethyloxim-BSA. An 125I-labeled carboxymethyloxim-tyrosine methyl ester derivative was used as tracer (catalog no. I-RBO-36, Institute for Isotopes, Budapest, Hungary). The corticosterone antibody cross-reactivity with other naturally occurring adrenal steroids was <0.05%, except desoxycorticosterone (1.5%) and progesterone (2.3%). Final dilution of the antibody was 1:40,000. Incubation time was 24 h at 4°C, and a second antibody (anti-rabbit from goat) and 6% polyethylene glycol solution was used for separation. A calibration curve was prepared from corticosterone (Calbiochem) and ranged from 0.27 to 40 pmol/tube. The intra- and interassay CVs were 12.3 and 15.33%, respectively. For prolactin RIA, we used materials donated by the Pituitary Program (National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD). The intra-assay and interassay CVs were 15.8 and 24.5%, respectively.
Northern Blot
To assess proopiomelanocortin (POMC) mRNA levels, pituitaries were rapidly removed, and anterior lobes were carefully dissected under microscope to avoid contamination of intermediate lobe cells. Samples were stored at 80°C until RNA isolation. The tissue samples were homogenized and lysed in RNA-Clean solution (AGS, Heidelberg, Germany), and total RNA was extracted with chloroform according to the manufacturer's instructions.
For preparation of Northern blots, 20 µg of total RNA samples were size-fractionated on a 1% agarose-8% formaldehyde denaturing gel and transferred onto Hybond N membranes (Amersham, UK) with the capillary transfer method. The POMC 48-mer synthetic oligonucleotide probe (kindly provided by G. Aguilera, National Institute of Child Health and Human Development, Bethesda, MD) was labeled by terminal transferase (Boehringer Mannheim), and the -actin riboprobe was labeled by the random primer method (HexaLabel DNA Labeling Kit, MBI Fermentas) using 32P-labeled dCTP (Izinta, Hungary). Prehybridizations (42°C, 4 h) and hybridizations (42°C, overnight) were carried out in a solution containing 50% (vol/vol) formamide, 6x SSC, 5x Denhardt's solution, 0.5% SDS, 50 mM Na-phosphate buffer, 100 µg/ml tRNA, 10 µg/ml polyU-homopolymer, and 7.5 µg/ml denatured salmon sperm DNA. The labeled probes were added to the hybridization solution at 1 x 106 counts · min1 · ml1. Filters were washed at high-stringency conditions (RT in 2x SSC-0.1% SDS for 5 min, 68°C, in 2x SSC-0.1% SDS for 30 min, 68°C, and in 0.2x SSC-0.1% SDS for 30 min). Blots were exposed to X-ray films (Kodak XAR, Sigma) for 1 or 2 days at 70°C with intensifying screens. Between hybridizations, filters were washed in a solution containing 5 mM Na-phosphate-0.1% SDS at 100°C for 30 min to remove the labeled probe. Quantification of the hybridization signals was achieved by use of an image analysis system (ScionImage, Scion) to obtain plot profiles of autora-diograms. All comparisons were made from RNA samples hybridized on the same filter and normalized to the content of
-actin RNA detected in each individual sample.
In Situ Hybridization Histochemistry
To determine CRH and AVP mRNA expression in the hypothalamus, frozen sections were hybridized with 35S-UTP-labeled riboprobes. Plasmids containing CRH and AVP cDNA were generously provided by Dr. W. S. Young (National Institutes of Health). Hybridization and autoradiographic techniques were performed according to a protocol described by Simmons (42) and as reported previously (35). After decapitation, brains were quickly frozen in isopentane, and frontal sections were cut on a cryostat, mounted on poly-L-lysine-coated slides, and stored at 70°C until hybridization. Hybridized sections were dipped into NTB-3 emulsion and exposed for 1014 days.
Statistical Analysis
Data were analyzed by analysis of variance using two- or three-way ANOVA or MANOVA models in the STATISTICA software package (Tulsa, OK). The models included lesion, repeated restraint, and (in case of the hormonal measurement) single restraint as experimental variables. Main effects and all interactions were tested for significance. To achieve homogeneity of the variances, the hormonal data were transformed using logarithms in the analysis. Analysis factors were identified as follows: acute stress, chronic stress, and lesion. Multiple pairwise comparisons where appropriate were made by the Newman-Keuls method. In the absence of statistical interaction, only the main treatment effects were evaluated, and they are described in legends to Figs. 1, 2, 3, 4 and Tables 1 and 2. Values are presented as means ± SE.
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RESULTS |
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Lesioned rats recovered from the adverse effects of surgery before the beginning of repeated restraint. All of the MBH and PVH lesions were verified by histological examination. In the case of MBH lesion, verification included histology as well as measurement of well-known physiological and biochemical consequences. Enhanced water consumption [ml/day; sham: 30.6 ± 6.2 (n = 42), MBH lesion: 139 ± 8 (n = 70)]; elevated plasma prolactin levels (see Table 2) and reduced AVP [ng/gland; sham: 645 ± 33 (n = 43), MBH lesion: 22 ± 4.7 (n = 71)] and oxytocin [ng/gland; sham: 1,456 ± 82 (n = 43), MBH lesion: 29 ± 4.7 (n = 71)] content of the neurointermediate lobe were also measured (31).
Weight Changes
One-week restraint caused consistent weight changes typical for chronic stress (body and thymus weight loss, adrenal mass increase, Table 1). Lesions stimulated weight gain during the first 5 postoperative days [in g, MBH lesion series: initial body wt 263 ± 4 (n = 93), increment in sham: +31.1 ± 3.3 (n = 37); increment in MBH lesion: +45.6 ± 3.3 (n = 56); and PVH lesion series: initial body wt 362 ± 9 (n = 156), increment in sham: +13.5 ± 2.0 (n = 81); increment in PVH lesion: +24.3 ± 3.2 (n = 75)]. Lesions did not block the effects of repeated restraint on body weight loss but did reduce repeated restraint-induced adrenal hypertrophy. The MBH lesion resulted in an enlargement of the thymus, but the reduction after repeated restraint could still be seen. Repeated restraint resulted in a significant reduction of thymus weight. PVH lesion prevented this effect; however, PVH lesion itself reduced the thymus weight.
Hormone Levels
Plasma ACTH. Plasma ACTH levels were elevated only in acutely stressed animals, but not at 24 h after the last stress session in repeated-stress paradigms (Fig. 1A). Previous repeated restraint slightly reduced ACTH elevation observed after the 8th restraint [significant chronic x acute interaction in 3-way ANOVA (P = 0.02), pairwise comparison to the single restraint (P = 0.056)]. As expected, MBH lesion abolished the stress-induced ACTH elevations except the small rise to the acute restraint (Fig. 1B). PVH lesion significantly reduced the acute stress-induced elevation by 50%, but after repeated restraint the inhibitory effect of the PVH lesion on restraint-induced ACTH elevation was stronger (Fig. 1C).
Plasma corticosterone. Plasma corticosterone levels were elevated by repeated restraint at baseline conditions (P < 0.01), but the 8th restraint session caused further elevation (Fig. 2A). Corticosterone response to acute restraint was similar in restraint-naive and repeatedly restrained rats (Fig. 2A). MBH lesion markedly reduced the corticosterone response (P < 0.01) to the first restraint and abolished the acute response to the 8th bout of restraint (Fig. 2B). PVH lesions prevented the repeated restraint-induced increase in basal plasma corticosterone and reduced the acute response to the 1st and 8th bouts of restraint by 50% (Fig. 2C).
Plasma prolactin. Plasma prolactin levels were increased by 1 h of restraint (Table 2). The chronic stress had no effect on basal or restraint-induced elevated prolactin values. The extensive lesioning (MBH lesion) elevated the prolactin levels in all groups to acute stress level because of the loss of hypothalamic dopaminergic inhibition. The PVH lesion did not change the stress responses of prolactin.
POMC mRNA
Anterior pituitary POMC mRNA levels (Fig. 3) were increased in chronically and in chronically + acutely stressed sham-operated rats compared with their appropriate controls, whereas acute restraint alone did not cause any changes (POMC mRNA is known to rise with a latency of several hours after a single exposure to strong stress). MBH or PVH lesion had no effect on POMC mRNA in the anterior pituitary in the absence of repeated restraint, but both lesions prevented the repeated restraint-induced elevation of POMC mRNA.
Hypothalamic CRH and AVP mRNA Expression
In accord with previous studies, on sections hybridized for CRH mRNA, repeated restraint elevated the number of autoradiographic silver grains, as well as the number of CRH-expressing cells in the medial dorsal parvocellular subdivision of the PVH (Fig. 4, A and B). In addition, cells in this subdivision also express AVP mRNA in chronically stressed rats. The AVP mRNA signal in the supraoptic nucleus (SON) was not affected by the stress or by the PVH lesion. Neurons expressing CRH mRNA were revealed in the region just below the fornix in PVH-lesioned rats (Fig. 4C). These cells, however, do not express a detectable AVP mRNA signal in chronically restrained rats.
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DISCUSSION |
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In sham-operated rats, the repeated-restraint stress resulted in long-term, sustained elevation of the HPA axis, as it decreased body and lymphoid organ weights and increased the baseline corticosterone level in the plasma and POMC mRNA levels in the anterior pituitary in a way typical of chronic stress (6, 12, 24).
Lesioning of the MBH abolishes all connections between hypothalamus and hypophysis, resulting in a hypophysis without direct central regulation. MBH-lesioned animals show only minimal response to acute, and no response to chronic, restraint, showing that the hypothalamo-hypophysial connection is necessary to maintain HPA axis activation in repeated-restraint stress.
Not only the MBH lesion but also the PVH lesioning prevented the elevation of basal corticosterone levels, the increased POMC mRNA expression in the anterior pituitary, the increase in adrenal weight, and the decrease in thymus weight that are normally seen after repeated restraint. All of these changes suggest that PVH lesioning, like MBH lesion, is likely to block the sustained release of CRH, which might be needed for the POMC mRNA increase and chronic adrenal hyperactivity. It is of interest that PVH lesion also blocked adrenalectomy-induced pituitary POMC mRNA and plasma ACTH increase (23, 30, 39). In contrast, the PVH lesion and also the MBH lesion failed to block the POMC rise accompanying an immune-related chronic situation, adjuvant-induced arthritis (28). The difference between the MBH and PVH lesion effects on repeated restraint and adjuvant-induced arthritis suggests that some, but not all, chronic stressful stimuli activate the HPA axis centrally through stimulating the hypophysiotropic CRH and/or AVP neurons in the PVH.
In different models of chronic stress, basal corticosterone levels are often elevated without any significant increase in baseline ACTH concentrations, perhaps because of the different timing characteristic of the two hormones (1, 3, 33). Moreover, basal ACTH levels were normal after 14 days of intraperitoneal hypertonic saline, when pituitary POMC mRNA levels were increased (21). Because we killed the animals at the end of 1-h restraint (as this is optimal for single hormone measurements), we did not find POMC mRNA changes in the groups subjected to a single stress only (15, 45), where changes usually appear only at 4 h or later.
The moderate adaptation of the HPA system to repeated restraint is shown by the inhibition of the plasma ACTH rise after the last restraint in the sham-operated rats, when the plasma prolactin and corticosterone responses to the last restraint were unchanged. These findings suggest that only some of the hormonal responses habituate to the stressful stimulation and that the stimulus remained effective after seven repetitions. According to Aguilera (1), repeated painful stimuli (footshock or intraperitoneal hypertonic saline) or metabolic disturbances do not result in adaptation to the primary repeated stress, but physical/psychological stress paradigms, such as immobilization or cold exposure, are characterized by adaptation of the ACTH response to the persistent primary stress. Restraint and immobilization are similar stimuli, differing in stimulus strength and technical details. Previous reports on repeated restraint used different strain, conditions, and timing, which might explain the variable adaptations in ACTH and corticosterone previously reported (9, 11, 19, 26, 43). Similar to our observations, Popovic (38) and Pitman et al. (37) reported no adaptation to restraint during 1821 days. Hauger et al. (16) also described a difference between ACTH and corticosterone inhibition with 2.5 h of daily immobilization. In considering adaptation to repeated stress, it is also possible that various measures may show different results. Peak levels of plasma ACTH and corticosterone might be almost normal, but time course and area under the curve might be influenced more by adaptation. We suggest that adaptation of the hormonal responses may be stimulus and intensity dependent and that responses adapt mainly to stimuli weaker and/or psychological in nature. It should be noted that, in our study, the method of restraint left no room for the movement of the animals and probably provided a stronger stimulus than that in other reports. Whether altered feedback plays a role in adaptation to restraint is not known.
PVH lesions are known to inhibit acute stress-induced ACTH and corticosterone responses to many stimuli by variable degrees. After PVH lesion, the plasma ACTH response to short and relatively weak neurogenic stressors may be completely blocked, but most, if not all, medium-to-strong, acute, stressful stimuli will elicit an almost normal ACTH and corticosterone response (7, 8, 10, 27, 29, 40). The present findings are in agreement with these data.
After repeated restraint in the PVH-lesioned rats, a further homologous stimulus was almost ineffective on ACTH release, but the plasma corticosterone response from the same rats showed only partial inhibition. Plasma corticosterone might be less impaired than ACTH, because either maximal adrenocortical stimulation occurs with plasma ACTH levels in the low-stress range in the PVN-lesioned group or the sensitivity of the adrenal cortex may rise and maintain corticosterone secretion even in the presence of plasma ACTH levels well below their acute stress peak. It is also possible that a direct neural influence on the adrenal cortex may play a role in elevating plasma corticosterone in restraint. Because the effects of adaptation to repeated restraint (50%) and PVH lesion (
50%) seemed to be additive, the marked inhibition of ACTH secretion in chronically stressed PVH-lesioned rats might reflect a stronger inhibition by corticosterone feedback in those few hypophysiotropic CRH neurons that are outside the PVH (e.g., in the perifornical region) and might have escaped lesioning. In addition, the hypothalamic mechanisms, including CRH neurons outside the PVH and AVP/CRH from the SON (18), may not receive all of the stimulatory influences in chronic stress normally supposed to be relayed, at least in part, through the paraventricular thalamic nucleus (4, 5).
In a PVH-lesioned rat, 90% of the hypophysiotropic CRH cells are eliminated, with some CRH-expressing neurons remaining in the medial preoptic area and the dorsal lateral hypothalamus near the fornix, just behind the PVH (22, 34), and with CRH appearing in the supraoptic neurons (35). The extraparaventricular CRH mRNA changes were also illustrated in the present study. It seems likely that extraparaventricular hypothalamic mechanisms are able to maintain a remarkably acute adrenocortical response, even in the absence of PVH.
In summary, our findings suggest a complex role for the hypothalamic mechanisms in chronic stress. The PVH seems to be essential for the repeated restraint-induced POMC mRNA stimulation, but it has less of a role in dynamic corticosterone changes. In repeated restraint, the regulation may involve not only the CRH/AVP neurons in the PVH but also mechanisms outside the PVH, such as CRH neurons in the hypothalamic areas outside the PVH (for example in the perifornical region). However, the CRH-releasing neurons in and outside the PVH seem to behave differently, because PVH lesion strongly inhibited the restraint-related ACTH elevation only in repeatedly stressed animals.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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