Colocalization of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus and hypothalamus

We investigated the distribution and colocalization pattern of the two corticosteroid receptors, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), in the hippocampus and hypothalamus, the main target regions of corticosterone in the rat brain, using double immunofluorescence histochemistry in conjunction with specific polyclonal antibodies against MR and GR. In the hippocampus, MR- and GR-immunoreactivity (ir) were colocalized in CA1 and CA2 pyramidal neurons and granule cells of the dentate gyrus, while only MR-ir was seen in the CA3 pyramidal neurons. Colocalization of MR- and GR-ir was also observed in the parvocellular region, but not in the magnocellular region of the paraventricular nucleus (PVN). Subcellular distribution of MR-ir was more cytoplasmic in comparison with that of GR-ir, while the ratio of cytoplasmic to nuclear distribution of these receptors was different among the regions. After adrenalectomy (ADX), the distribution pattern of both receptors was changed to cytoplasmic, although the degree of the change of distribution was different among each region. Replacement of corticosterone after ADX recovered the distribution pattern to that of the sham-operated animals. These results suggest that the balance of MR and GR in the cell underlies the potential regulation of corticosteroid through the hippocampus and hypothalamus.     

The importance of corticosterone in mediating restraint-induced weight loss in rats

I. J. Scherer, P. V. Holmes, R. B.S. Harris. The importance of corticosterone in mediating restraint-induced weight loss in rats. PHYSIOL BEHAV 00 (0) 000-000, 2010. Rats restrained for 3 h/day for 3 days (RR) lose weight and do not return to the weight of non-restrained controls once restraint has ended. This study tested the importance of restraint-induced corticosterone release in mediating the change in body weight by injecting ADX rats with 2.0 mg corticosterone/kg before each restraint to replicate the restraint-induced surge in circulating corticosterone. Restrained adrenalectomized (ADX) rats injected with corticosterone had the same initial weight loss as intact restrained rats, whereas corticosterone injection in non-restrained ADX rats and restraint of ADX rats injected with saline each produced only half as much initial weight loss. Sustained weight loss, measured for 14 days after the end of RR, was the same for restrained intact rats and restrained ADX rats injected with corticosterone whereas restrained ADX rats injected with saline achieved the same weight gain as their controls. Corticosterone injections had no effect on weight gain of non-restrained intact rats. In situ hybridization showed that corticotropin releasing factor (CRF) mRNA expression in the paraventricular nucleus of the hypothalamus (PVN) was increased by the same degree in ADX rats and restrained intact rats and was not modified by corticosterone injections. There was no significant effect of restraint, ADX or corticosterone injection on PVN arginine vasopressin (AVP) mRNA expression. These data indicate that a surge in corticosterone causes sustained weight loss in ADX rats through a mechanism that can be compensated for in intact rats and is independent of changes in PVN CRF or AVP mRNA expression.     

Blockage of glucocorticoid, but not mineralocorticoid receptors prevents the persistent increase in circulating basal corticosterone concentrations following stress in the rat

Exposure to a single session of intense inescapable stressors results in elevations of plasma corticosterone levels selective to the trough of the circadian rhythm that last for several days after stressor cessation. In the present study, we examined whether this stress-induced alteration in the regulation of the circadian trough is dependent upon glucocorticoid and/or mineralocorticoid receptor activation during stress. Pre-treatment with the mineralocorticoid receptor (MR) antagonist, spironolactone (RU-28318; 50 mg/kg, s.c.), and/or the glucocorticoid receptor (GR) antagonist, mifepristone (RU-38486; 50 mg/kg, s.c.) 1 h before inescapable stress (40, 2.0-mA tail-shocks delivered over a 1 h period) had no effect on the acute plasma corticosterone response to inescapable stress. Treatment with the MR antagonist alone did not affect the appearance of basal corticosterone elevations in stressed rats. However, the elevated trough plasma corticosterone levels were no longer evident in rats treated previously with the GR antagonist either alone or in combination with the MR antagonist. GR activation during stressor exposure appears to be necessary for the development of subsequent basal corticosterone elevations.     

The effects of adrenalectomy and aldosterone replacement in transgenic mice expressing antisense RNA to the type 2 glucocorticoid receptor

Bilateral adrenalectomy (ADX) either prevents or attenuates obesity in several animal models. Mice that express an antisense RNA to the glucocorticoid receptor (GCR) are obese. The present study was conducted to examine the effects of ADX and aldosterone (ALDO) replacement on the rate of weight gain and body composition of mice bearing an antisense GCR gene construct. Twenty-eight male transgenic mice bearing the antisense GCR construct and 16 male B6C/3F1 mice were either bilaterally ADX or given sham operations. At the time of surgery, some of the ADX mice and all of the sham-operated mice were implanted with 100-mg cholesterol (CHOL) pellets inserted subcutaneously in the subscapular region. The remaining ADX mice were implanted with 100-mg 1% w/w ALDO pellets using CHOL as vehicle. All mice were returned to their home cages for 2 weeks. They were then decapitated and the blood was collected for corticosterone, ALDO, insulin, and leptin radioimmunoassay. Carcasses were eviscerated and prepared for gravimetric analyses, including bomb calorimetry. ADX resulted in a significant drop in carcass fat in both transgenic and wildtype groups. ALDO prevented the decrease in carcass fat in both groups. Two weeks after ADX, transgenic mice were as fat as sham-operated wildtype controls, whereas both sham-operated and ALDO-treated transgenic groups were significantly fatter. Despite observing a reliable decrease in carcass fat following ADX, no corresponding decrease in circulating leptin was found.     

Excretion of electrolytes in Brown Norway and Fischer 344 rats: effects of adrenalectomy and of mineralocorticoid and glucocorticoid receptor ligands

Our previous studies showed that adrenalectomy (ADX) has surprisingly no effect on body weight and fluid intake in the Brown Norway rat strain, suggesting that mineralocorticoid receptor (MR)-mediated effects are present even in absence of corticosteroids in this strain. Moreover, glucocorticoid receptor (GR)-mediated mechanisms are more effective in Brown Norway than in Fischer 344 rats. Such functional differences in corticosteroid receptor pathways between Brown Norway and Fischer 344 rats led us to compare the effect of ADX and MR/GR-mediated actions on sodium and potassium excretion between these two rat strains. To this end, we first measured the effect of an acute high dose of aldosterone on the urinary Na+/K+ concentration ratio in intact and ADX Brown Norway and Fischer 344 rats. Second, to discriminate mineralocorticoid from glucocorticoid actions, we treated chronically ADX rats with increasing doses of aldosterone or RU28362, a pure GR agonist, in the drinking fluid. As sodium homeostasis involves salt appetite regulation, behaviour under mineralocorticoid control, we also measured saline preference in Brown Norway and Fischer 344 rats. Our data illustrate: (1) the very limited effect of ADX on body weight, food and fluid intake, diuresis, natriuresis, kaliuresis and salt appetite in Brown Norway rats, supporting the presence of MR signalling pathways in the absence of adrenal steroids in these rats; (2) the insensitivity of MR to aldosterone in intact Brown Norway rats, and the reduced sensitivity of MR to aldosterone in ADX Brown Norway rats compared with ADX Fischer 344 rats; and (3) the greater sensitivity of GR-related mechanisms to RU28362 in Brown Norway than in Fischer 344 rats in terms of body weight gain and electrolyte excretion. Considering that both MRs and GRs regulate hypothalamic-pituitary-adrenal axis processes, such functional differences in corticosteroid receptors could be at the origin, at least partly, of the strain differences in corticotropic activity/reactivity to stress previously described.     

Paradoxical sleep deprivation facilitates subsequent corticosterone response to a mild stressor in rats

The pituitary-adrenal responsiveness to a mild stressor was assessed in rats that were deprived of paradoxical sleep (PS) and in controls that were not deprived. Animals were either individually- or group-deprived for 96 h and hormone levels were assessed at 0, 5, 20 or 60 min after a saline injection+novelty and compared with rats which were not deprived. Both types of PS deprivation resulted in elevated adrenocorticotropin levels at 0 min, which peaked at 5 min in all animals. Individually-deprived rats exhibited the highest corticosterone (CORT) levels at 0 min. Peak levels were higher and occurred earlier in PS-deprived than in control rats (5 vs. 20 min, respectively). At 20 min, CORT levels had already returned to unstressed levels in PS-deprived rats, but not in control rats. These data indicate that PS deprivation induces facilitation of the adrenocortical response to a mild stressor, but do not suggest that PS deprivation changes the negative feedback sensitivity to CORT.     

Previous chronic ACTH administration does not protect against the effects of acute or chronic stress in male rats

The effect of previous chronic ACTH administration on the physiological response to acute and chronic immobilization stress was studied in adult male rats. Chronic ACTH administration slightly reduced food intake and drastically inhibited body weight gain. Serum corticosterone levels were similar in saline- and ACTH-treated rats twenty hours after the last administration. However, the corticosterone response to 1 hr immobilization was greatly reduced by previous ACTH administration. When the exposure to the stressor was prolonged up to 18 hours, the corticosterone response was similar in saline, and ACTH-treated rats. While body weight loss caused by starvation and acute stress was lower in ACTH-treated rats, stomach ulceration was greater in the latter animals. Although ACTH-treated rats showed higher body weight gain than saline-treated animals during chronic immobilization, this was probably due to catch-up growth as food intake inhibition caused by the stressor was similar in the two groups. Pituitary-adrenal adaptation to the repeated stressor was the same in saline- and ACTH-treated rats. Therefore, the effects of previous ACTH treated on the physiological response to either acute or chronic stress appear to be mixed, depending on the variable studied.     

Effect of neonatal respiratory infection on adult BALB/c hippocampal glucocorticoid and mineralocorticoid receptors

The current study investigated the effects of neonatal infection with Chlamydia muridarum bacteria on glucocorticoid (GR) and mineralocorticoid (MR) receptors in the adult mouse hippocampus. In male adults infected at birth, circulating corticosterone was significantly increased when compared to same sex controls; while neonatal infection resulted in female adults with significantly increased GR mRNA compared to same sex controls. When comparing males and females after neonatal infection, males had significantly less GR protein than females. Interestingly, after control treatment, males had significantly more GR mRNA, MR mRNA, and GR protein with significantly lower corticosterone than females. Neonatal respiratory infection significantly impacts adult hippocampal GR and MR, and circulating corticosterone in a sex-specific manner potentially altering stress responsivity.     

Strain differences in proliferation of progenitor cells in the dentate gyrus of the adult rat and the response to fluoxetine are dependent on corticosterone

This paper investigates the role of differences in adrenal cortical function on the proliferation rate of progenitor cells in the dentate gyrus of the hippocampus in adult Sprague-Dawley (SD) and Lister-Hooded (LH) male rats. SD rats had around 60% more cells labeled with Ki67 (an index of mitosis) than LH rats under basal conditions. Bilateral adrenalectomy (ADX) increased levels in both strains, but by unequal amounts, such that post-ADX numbers of Ki67-labeled cells were similar in both strains. Daily injections of 5 mg/kg corticosterone for 7 days reduced levels to similar values in ADX rats of both strains. The activity of progenitor cells in either strain did not respond to daily i.p. injections of fluoxetine (10 mg/kg) for 14 days, but an equivalent dose administered by osmotic minipump stimulated proliferation in both by a similar proportional amount, such that strain differences persisted. S.c. implantation of a corticosterone pellet (75 mg), which flattens the diurnal rhythm in corticosterone, prevented fluoxetine delivered by minipump from activating progenitor cell mitosis in SD rats, as it had in the LH strain in a previous study. These results show that much, if not all, of the marked strain differences between SD and LH rats in progenitor cell activity, and hence rates of neurogenesis in the dentate gyrus may be ascribed to corresponding differences in adrenal cortical activity.     

Chronic restraint or variable stresses differently affect the behavior, corticosterone secretion and body weight in rats

Organisms are constantly subjected to stressful stimuli that affect numerous physiological processes and activate the hypothalamo-pituitary-adrenal (HPA) axis, increasing the release of glucocorticoids. Exposure to chronic stress is known to alter basic mechanisms of the stress response. The purpose of the present study was to compare the effect of two different stress paradigms (chronic restraint or variable stress) on behavioral and corticosterone release to a subsequent exposure to stressors. Considering that the HPA axis might respond differently when it is challenged with a novel or a familiar stressor we investigated the changes in the corticosterone levels following the exposure to two stressors: restraint (familiar stress) or forced novelty (novel stress). The changes in the behavioral response were evaluated by measuring the locomotor response to a novel environment. In addition, we examined changes in body, adrenals, and thymus weights in response to the chronic paradigms. Our results showed that exposure to chronic variable stress increased basal plasma corticosterone levels and that both, chronic restraint and variable stresses, promote higher corticosterone levels in response to a novel environment, but not to a challenge restraint stress, as compared to the control (non-stressed) group. Exposure to chronic restraint leads to increased novelty-induced locomotor activity. Furthermore, only the exposure to variable stress reduced body weights. In conclusion, the present results provide additional evidence on how chronic stress affects the organism physiology and point to the importance of the chronic paradigm and challenge stress on the behavioral and hormonal adaptations induced by chronic stress.     

Gonadal and adrenal effects on the glucocorticoid receptor in the rat hippocampus,with special reference to regulation by estrogen from an immunohistochemical view-point

Focusing on the hippocampal CA1 region, effects of peripheral gonadal and adrenal steroids on the glucocorticoid receptor (GR) were immunohistochemically evaluated in male and female adult rat brains after adrenalectomy (ADX), gonadectomy (GDX), and administration of estradiol (E2) and/or corticosterone (CS). In ADXed male rats, the hippocampal nuclear GR decreased and turned back to the cytoplasm, whereas in females, nuclear localization persisted even after ADX. In GDX+ADXed female rats, the GR was dispersedly translocated from the nucleus to the cytoplasm as well as in GDX+ADXed males. The dispersed cytoplasmic GR was again translocated into the nucleus by administration of CS. In addition, administration of a small dose of E2 for 4-13 days was found to sufficiently recover the nuclear location of GR in GDX+ADXed rat brains, whereas medium-to-large doses could not do this. Also, a longer administration more strongly enhances the nuclear GR location and expression. The present study provided strong immunohistochemical evidence that the sexually dimorphic effects of ADX on hippocampal GR are attributable to gonadal hormones, and that E2 is implicated in the effects in inversely-dose- and directly-duration-dependent manner. Taken together, intriguing gonadal and adrenal crosstalk is considered to play some important role in regulating hippocampal GR morphology and to have a possibly crucial influence on stress-related disorders such as depression.     

Feeding and macronutrient selection patterns in rats: adrenalectomy and chronic corticosterone replacement

To analyze further the role of corticosterone (CORT) in the control of feeding behavior, we examined the impact of adrenalectomy (ADX) and chronic CORT implants on the food intake and macronutrient self-selection patterns of adult male rats at different periods of the diurnal cycle. Consistent with a separate study of acute CORT injection in ADX rats (Kumar and Leibowitz, 1988), the present findings indicate that ADX significantly attenuated the rats' daily (24 hr) ingestion of all three macronutrients, namely, protein, carbohydrate and fat. However, food intake in the dark cycle, specifically during the first few hours after dark onset, was significantly more affected (-70%) than feeding in the later dark and light periods (-25%). Moreover, during this early dark time when circulating CORT level normally peaks, ADX appeared to have its strongest suppressive effect on carbohydrate ingestion. Chronic subcutaneous CORT implants in the ADX animals reversed these effects of surgery and generally restored the rats' eating patterns to that of the cholesterol-implanted SHAM animals. These findings suggest that CORT exerts a decisive influence on caloric intake, on the diurnal pattern of feeding, and on appetite for specific macronutrients. The impact of CORT on carbohydrate intake is apparent specifically during the active eating period, particularly at dark onset when endogenous CORT levels normally peak and carbohydrate is exhibited as the preferred macronutrient.     

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

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.     

Pathophysiologic glucocorticoid elevations promote bacterial translocation after thermal injury.

Thermal injury results in transient elevations of plasma glucocorticoids and promotes translocation of bacteria from the gut to the mesenteric lymph nodes (MLN) in rats. Translocated organisms are quickly cleared following uncomplicated thermal injury. However, subsequent burn wound infection, in temporal association with sustained pathophysiologic elevations of plasma corticosterone, results in the continued presence of enteric bacteria in the MLN. To study the role of sustained pathophysiologic steroid elevations in the mediation of this prolonged bacterial translocation, Wistar rats were randomly placed in groups receiving one of the following: (i) a 30% total body surface area scald injury with placement of a subcutaneous corticosterone pellet, (ii) a 30% total body surface area scald and a sham pellet implantation, (iii) a sham burn and a corticosterone pellet implantation, or (iv) a sham burn and a sham pellet implantation. The animals were sacrificed on days 1 and 4 after injury, and cultures of the MLN, as well as the liver and spleen, were taken. Implantation of corticosterone pellets resulted in sustained elevations of plasma corticosterone compared with controls not receiving corticosterone pellets, similar to results seen in association with injury and infection. These pathophysiologic elevations were associated with the prolonged presence of organisms in the MLN (90% of burned rats with implanted corticosterone pellets versus 25% of rats with uncomplicated burns on postburn day 4; P less than 0.01), but only in the presence of burn injury. Pathophysiologic glucocorticoid elevations did not lead to progression of translocation to the viscera or blood. Thus, the pathophysiologic glucocorticoid response contributes to the translocation of enteric bacteria and their prolonged presence in the MLN after systemic injury.     

Prevention of adrenalectomy-induced brain growth stimulation by corticosterone treatment

Previous research in our laboratory has shown that adrenalectomizing rats on day 11 postnatal leads to subsequent increases in brain weight, cellularity (measured in terms of DNA content), and myelination. The present study was designed to determine whether these effects are due to removal of circulating glucocorticoids. Male and female albino rats were adrenalectomized (ADX) on day 11 and then injected daily with either 7.0 mg/kg corticosterone in a steroid suspension vehicle or vehicle alone. A third group of animals was sham-operated (SHAM) and given daily vehicle injections. Subjects were sacrificed at 60 days of age for measurement of brain and body weights, cerebral DNA content, and the activity of 2',3'-cyclic nucleotide phosphodiesterase (CNP), a myelin marker enzyme. As expected from our earlier findings, brain weights and DNA and CNP levels were all significantly elevated in ADX-untreated rats compared to SHAMs. More importantly, all of these changes were completely prevented by chronic corticosterone administration. These results are consistent with previous reports that corticosterone treatment blocks increased brain growth in ADX weanling rats and support our hypothesis that the brain growth stimulating effects of day-11 adrenalectomy are likewise mediated by the removal of glucocorticoids. It appears that glucocorticoid hormones exert a tonic inhibition of at least some growth related processes in the brains of intact developing organisms.     

Acute stressors increase plasma corticosterone and decrease locomotor activity in a terrestrial salamander (Desmognathus ochrophaeus)

Vertebrates respond to the onset of an acute stressor with an acute increase in plasma glucocorticoids. The increase in plasma glucocorticoids is believed to be adaptive, helping an animal cope until the stressful episode subsides. Although much is known about the effects of chronic elevation of glucocorticoids, far less is known about the role of acute increases in glucocorticoids in mediating stress responses. To better understand the regulation and function of acute increases in plasma glucocorticoids, we measured stress-induced increases in plasma corticosterone (CORT) and the effects of stressors and exogenous CORT on activity in male Allegheny dusky salamanders (Desmognathus ochrophaeus). Capture and handling of field-caught salamanders resulted in an acute elevation of plasma CORT during the nonmating season but not during the mating season. In laboratory-housed salamanders, a handling stressor that simulated capture resulted in decreased locomotor activity. Noninvasive elevation of plasma CORT via dermal patches did not replicate the handling-induced decrease in activity. Together, this work indicated that 1) the CORT response to the acute stressor of capture and handling was seasonally variable, 2) handling induced a decrease in locomotor activity in the laboratory, and 3) acute increases in plasma CORT did not contribute to stress-induced changes in locomotor activity. Future studies using noninvasive methods to elevate plasma CORT should illuminate the role of acute increases in plasma glucocorticoids in coordinating organismal responses to acute stressors.     

Restraint stress is associated with changes in glucocorticoid immunoregulation

Psychological stress has been associated with activation of the hypothalamic-pituitary-adrenal (HPA) axis and impaired cell-mediated immune (CMI) responses. There is also evidence suggesting that intermittent chronic stress differentially alters CMI across different immune compartments, but the mechanisms underlying this phenomenon have not been explored in detail. In the present study, we investigated (i) acute and chronic restraint stress effects in Sprague-Dawley rats on both peripheral blood lymphocyte (PBL) and splenocyte mitogen-induced proliferation and (ii) also determined whether differential stress effects within these immune compartments might reflect alterations in lymphocyte sensitivity to glucocorticoids. It was found that while acute stress exposure significantly raised plasma corticosterone levels (1048% vs. controls, P<.001), this response was attenuated in the animals previously exposed to chronic intermittent stress (-79.66% vs. acute; P<.001). Acute stress increased phytohemagglutinin (PHA)-induced lymphocyte proliferation in the spleen (69.04%, P=.01) and suppressed PBL proliferation (-45.52%, P<.001). Neither of these changes were observed following chronic stress. We also demonstrated that reexposure to the stressor rapidly increased splenocyte sensitivity to in vitro dexamethasone (P<.05) and corticosterone (P<.05) in chronically stressed rats. Our data (1) confirm that acute stress is associated with compartment-specific changes in CMI function, (2) indicate that chronic stress is associated with habituated endocrine and immune responses and (3) that stressor exposure rapidly alters splenocyte sensitivity to glucocorticoids and we suggest that the latter may contribute to differential stress effects across immune compartments.