Footshock-induced changes in brain catecholamines and indoleamines are not mediated by CRF or ACTH

Stressful treatments have long been associated with increased activity of brain catecholaminergic and serotonergic neurons. An intracerebroventricular (icv) injection of the corticotropin-releasing factor (CRF) also activates brain catecholaminergic neurons. Because brain CRF-containing neurons appear to be activated during stress, it is possible that CRF mediates the catecholaminergic activation. This hypothesis has been tested by assessing the responses in brain catecholamines and indoleamines to footshock in mice pretreated icv with a CRF receptor antagonist, and in mice lacking the gene for CRF (CRFko mice). Consistent with earlier results, icv administration of CRF increased catabolites of dopamine and norepinephrine, but failed to alter tryptophan concentrations or serotonin catabolism. A brief period of footshock increased plasma corticosterone and the concentrations of tryptophan and the catabolites of dopamine, norepinephrine and serotonin in several brain regions. Mice injected icv with 25 microg alpha-helical CRF(9-41) prior to footshock had neurochemical responses that were indistinguishable from controls injected with vehicle, while the increase in plasma corticosterone was slightly attenuated in some experiments. CRFko mice exhibited neurochemical responses to footshock that were indistinguishable from wild-type mice. However, whereas wild-type mice showed the expected increase in plasma corticosterone, there was no such increase in CRFko mice. Similarly, hypophysectomized mice also showed normal neurochemical responses to footshock, but no increase in plasma corticosterone. Hypophysectomy itself elevated brain tryptophan and catecholamine and serotonin metabolism. Treatment with ACTH icv or peripherally failed to induce any changes in cerebral catecholamines and indoleamines. These results suggest that CRF and its receptors, and ACTH and other pituitary hormones, are not involved in the catecholamine and serotonin responses to a brief period of footshock.     

Comparison of the effects of CRF and stress on levels of pituitary cyclic AMP and plasma ACTH in vivo

We have previously reported that acute stress increases levels of rat pituitary cyclic AMP in vivo. The present study was conducted to test the hypothesis that stress-induced increases in pituitary cyclic AMP in vivo were mediated via CRF. We compared the effects of various stressors with the effects of CRF or epinephrine administration on pituitary cyclic AMP and plasma ACTH responses in vivo. Stressors, epinephrine or CRF increased levels of pituitary cyclic AMP. Pituitary cyclic AMP response to either immobilization or CRF was much greater at light onset than at lights off in rats maintained on at 12 hr light: 12 hr dark lighting regimen. In rats with pituitary stalk transections, footshock did not increase levels of pituitary cyclic AMP, suggesting that some factor of central origin was required for this stress response. Exogenous CRF administration did increase levels of pituitary cyclic AMP in stalk-transected rats, while epinephrine increased levels in sham-operated but not in stalk-transected rats. Antisera to CRF markedly decreased pituitary cyclic AMP response to exogenous CRF administered 6 min following antisera and partially attenuated pituitary cyclic AMP response to forced running. Taken as a whole these data support a major role for CRF in the pituitary cyclic AMP response to stress.     

“Green odor” inhalation by stressed rat dams reduces behavioral and neuroendocrine signs of prenatal stress in the offspring

Chronic maternal stress during pregnancy results in the “prenatally stressed” offspring displaying behavioral and neuroendocrine alterations that persist into adulthood. We investigated how inhalation of green odor (a mixture of equal amounts of trans-2-hexenal and cis-3-hexenol) by stressed dams might alter certain indices of prenatal stress in their offspring. These indices were depression-like behavior (increased immobility time in the forced-swim test) and acute restraint stress-induced changes in hypothalamo-pituitary-adrenocortical (HPA) axis activity [plasma corticosterone (CORT) and ACTH levels and the number of Fos-immunoreactive cells in the hypothalamic paraventricular nucleus (an index of neuronal activity)]. Pregnant rats were exposed to restraint stress for 60 min/day for 10 days (gestational days 10-19). The prenatally stressed offspring exhibited significant increases in depression-like behavior and in restraint stress-induced ACTH, CORT, and Fos responses, unless their dam had been exposed to green odor. The behavioral effect of the odor was also seen in offspring that were fostered by unstressed dams. The results obtained in the dams themselves were as follows. In vehicle-exposed stressed dams, but not in green odor-exposed ones, total body and adrenal weights were significantly decreased or increased, respectively. Depression-like behavior was not observed in the vehicle-exposed stressed dams themselves. Green odor inhalation prevented the impairment of maternal behavior induced by restraint stress. Thus, exposure of dams to stress may affect both the fetal brain and fetal HPA axis, and also maternal behavior, leading to altered behavioral and neuroendocrine responses in the offspring. Such effects may be prevented by the stressed dams inhaling green odor.     

Brain catecholaminergic and tryptophan responses to restraint are attenuated by nitric oxide synthase inhibition

Increases in the brain concentrations of tryptophan and in serotonin (5-HT) metabolism are commonly observed in animals under stress. Previous experiments indicated that the increase in brain tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) observed in response to administration of endotoxin (lipopolysaccharide, LPS) and interleukin-1 (IL-1) were largely prevented by pretreatment with N-nitro-L-arginine methylester (L-NAME), an inhibitor of NO synthase (NOS). Therefore we tested whether the increases in tryptophan and 5-HT metabolism observed following restraint and footsthock were similarly affected. Mice were injected with L-NAME (30 mg/kg) or saline and restrained for 40 min. Restraint caused increases in concentrations of tryptophan and the catabolites of dopamine (DA), norepinephrine (NE) and 5-HT in the medial prefrontal cortex, hypothalamus, and brain stem. The L-NAME pretreatment significantly attenuated, but did not prevent, the changes in tryptophan and catecholamine metabolism, with a very small effect on the increase in plasma corticosterone. When mice pretreated with L-NAME were subjected to 30 min footshock, the NOS inhibitor had no statistically significant effects on the increases in DA, NE and 5-HT metabolism, but tended to attenuate the increases in tryptophan. We interpret these results to indicate that NOS plays a relatively small role in the cerebral neurochemical responses to restraint and footshock, but the role in the restraint-induced changes was greater than that in the footshock-induced ones. The attenuation of the restraint-related effects on the catecholamines most probably reflects a contribution to the CNS responses from peripheral vascular changes which are likely to be limited by the inhibition of NOS.     

Restraint stress augments postprandial gastric contractions but impairs antropyloric coordination in conscious rats

Central corticotropin-releasing factor (CRF) plays an important role in mediating restraint stress-induced delayed gastric emptying. However, it is unclear how restraint stress modulates gastric motility to delay gastric emptying. Inasmuch as solid gastric emptying is regulated via antropyloric coordination, we hypothesized that restraint stress impairs antropyloric coordination, resulting in delayed solid gastric emptying in conscious rats. Two strain gauge transducers were sutured onto the serosal surface of the antrum and pylorus, and postprandial gastric motility was monitored before, during, and after restraint stress. Antropyloric coordination, defined as a propagated single contraction from the antrum to the pylorus within 10 s, was followed by > or = 20 s of quiescence. Restraint stress enhanced postprandial gastric motility in the antrum and pylorus to 140 +/- 9% and 134 +/- 9% of basal, respectively (n = 6). The number of episodes of antropyloric coordination before restraint stress, 2.4 +/- 0.4/10 min, was significantly reduced to 0.6 +/- 0.3/10 min by restraint stress. Intracisternal injection of the CRF type 2 receptor antagonist astressin 2B (60 microg) or guanethidine partially restored restraint stress-induced impairment of antropyloric coordination (1.6 +/- 0.3/10 min, n = 6). The restraint stress-induced augmentation of antral and pyloric contractions was increased by astressin 2B and guanethidine but abolished by atropine, hexamethonium, and vagotomy. Restraint stress enhanced postprandial gastric motility via a vagal cholinergic pathway. Restraint stress-induced delay of solid gastric emptying is due to impairment of antropyloric coordination. Restraint stress-induced impairment of antropyloric coordination might be mediated via a central CRF pathway.     

K—阿片受体拮抗剂MR—2266—BS对ACTH和催乳素释放的影响

The effect of intravenous injection of different doses of MR-2266-BS, a selective antagonist of kappa-opiate receptor, on plasma adrenocorticotropin (ACTH) and prolactin (PRL) in conscious male rats bearing an intrajugular cannulae was assessed. The results revealed that the MR-2266-BS of 3 mg/kg completely blocked the restraint stress-induced increase in plasma ACTH levels, and further elevated plasma PRL levels in these animals, while there were no effects on the resting levels of ACTH and PRL. MR-2266-BS of 6 mg/kg significantly increased the resting levels of plasma ACTH and also further elevated the restraint stress-induced increase of plasma ACTH and PRL. The present data suggest that kappa-opiate receptor and its endogenous ligand may be involved in the regulation of the resting and restraint stress-induced release of ACTH, and their action appears to be both stimulatory and inhibitory. Furthermore, kappa-opiate receptor and its endogenous ligand may only inhibit the stress-induced release of PRL.     

Differential effects of naloxone on basal and stress-induced release of ACTH and prolactin in the male rat

The effect of i.v. injection of various doses of naloxone (NAL) on plasma adrenocorticotropin (ACTH) and prolactin (Prl) in conscious animals bearing an indwelling intrajugular catheter was assessed. The effects were evaluated in animals which were left undisturbed and in others subjected to either restraint or ether stress. The results revealed that the dose of 3 mg/kg of NAL significantly reduced basal Prl levels, whereas a dose of 6 mg/kg of NAL was required to block completely either ether or restraint stress-induced release of Prl. The behavior of ACTH contrasted with that of Prl. There was no effect whatsoever of the 3 mg/kg dose of NAL on either resting or stress-induced ACTH levels, whereas a 6 mg/kg or 12 mg/kg dose of NAL elevated resting ACTH levels and only partially attenuated the further elevation induced by stress in these animals. The results clearly indicate a NAL sensitive step in the control of resting and stress-induced Prl release but indicate that the control of resting and stress-induced release of ACTH is different in that the predominantly millimicron receptor blocker, NAL, can elevate ACTH at high doses and can only partially block the response to stress. In contrast to Prl where opioid peptide control is solely stimulatory, this control of ACTH secretion appears to have both stimulatory and inhibitory features.     

Restraint stress stimulates colonic motility via central corticotropin-releasing factor and peripheral 5-HT3 receptors in conscious rats

Although restraint stress accelerates colonic transit via a central corticotropin-releasing factor (CRF), the precise mechanism still remains unclear. We tested the hypothesis that restraint stress and central CRF stimulate colonic motility and transit via a vagal pathway and 5-HT(3) receptors of the proximal colon in rats. (51)Cr was injected via the catheter positioned in the proximal colon to measure colonic transit. The rats were subjected to a restraint stress for 90 min or received intracisternal injection of CRF. Ninety minutes after the administration of (51)Cr, the entire colon was removed, and the geometric center (GC) was calculated. Four force transducers were sutured on the proximal, mid, and distal colon to record colonic motility. Restraint stress accelerated colonic transit (GC of 6.7 +/- 0.4, n=6) compared with nonrestraint controls (GC of 5.1 +/- 0.2, n=6). Intracisternal injection of CRF (1.0 microg) also accelerated colonic transit (GC of 7.0 +/- 0.2, n=6) compared with saline-injected group (GC of 4.6 +/- 0.5, n=6). Restraint stress-induced acceleration of colonic transit was reduced by perivagal capsaicin treatment. Intracisternal injection of CRF antagonists (10 microg astressin) abolished restraint stress-induced acceleration of colonic transit. Stimulated colonic transit and motility induced by restraint stress and CRF were significantly reduced by the intraluminal administration of 5-HT(3) antagonist ondansetron (5 x 10(-6) M; 1 ml) into the proximal colon. Restraint stress and intracisternal injection of CRF significantly increased the luminal content of 5-HT of the proximal colon. It is suggested that restraint stress stimulates colonic motility via central CRF and peripheral 5-HT(3) receptors in conscious rats.     

Dexamethasone suppresses ACTH release without attenuating pituitary cyclic AMP response to stress in vivo

Dexamethasone, a synthetic glucocorticoid, has been shown to decrease basal and stress-elevated levels of the pituitary hormone ACTH. Glucocorticoids are known to bind to multiple sites within the brain and pituitary and it is not known which site(s) is most important in mediating the observed inhibition of ACTH release. At the level of the corticotroph, there is contradictory data from in vitro studies regarding whether dexamethasone acts proximal or distal to the formation of the cyclic AMP second messenger that has been shown to be involved in CRF-stimulated ACTH release. In the present report, we have examined the effects of dexamethasone pretreatment on stress-induced elevations in pituitary cyclic AMP and the release of ACTH in vivo. Acute stress (15 min of intermittent footshock) elevated levels of pituitary cyclic AMP and plasma ACTH consistent with previous studies. Dexamethasone administration (0.4 mg/kg 24 hr prior to sacrifice plus 0.2 mg/kg 2 hr prior to sacrifice) inhibited stress-induced elevations in plasma ACTH but did not affect pituitary cyclic AMP response to acute stress. These findings suggest that dexamethasone inhibits the release of ACTH via an action distal to the generation of cyclic AMP.     

Effect of "rose essential oil" inhalation on stress-induced skin-barrier disruption in rats and humans

In stressed animals, several brain regions (e.g., hypothalamic paraventricular nucleus [PVN]) exhibit neuronal activation, which increases plasma adrenocorticotropic hormone (ACTH) and glucocorticoids. We previously reported that so-called "green odor" inhibits stress-induced activation of the hypothalamo-pituitary-adrenocortical axis (HPA axis) and thereby prevents the chronic stress-induced disruption of the skin barrier. Here, we investigated whether rose essential oil, another sedative odorant, inhibits the stress-induced 1) increases in PVN neuronal activity in rats and plasma glucocorticoids (corticosterone [CORT] in rats and cortisol in humans) and 2) skin-barrier disruption in rats and humans. The results showed that in rats subjected to acute restraint stress, rose essential oil inhalation significantly inhibited the increase in plasma CORT and reduced the increases in the number of c-Fos-positive cells in PVN. Inhalation of rose essential oil significantly inhibited the following effects of chronic stress: 1) the elevation of transepidermal water loss (TEWL), an index of the disruption of skin-barrier function, in both rats and humans and 2) the increase in the salivary concentration of cortisol in humans. These results suggest that in rats and humans, chronic stress-induced disruption of the skin barrier can be limited or prevented by rose essential oil inhalation, possibly through its inhibitory effect on the HPA axis.     

Role of hypothalamic inputs in maintaining pituitary-adrenal responsiveness in repeated restraint

The role of hypothalamic structures in the regulation of chronic stress responses was studied by lesioning the mediobasal hypothalamus or the paraventricular nucleus of hypothalamus (PVH). Rats were acutely (60 min) and/or repeatedly (for 7 days) restrained. In controls, a single restraint elevated the plasma adrenocorticotropin (ACTH), corticosterone, and prolactin levels. Repeated restraint produced all signs of chronic stress, including decreased body and thymus weights, increased adrenal weight, basal corticosterone levels, and proopiomelanocortin (POMC) mRNA expression in the anterior pituitary. Some adaptation to repeated restraint of the ACTH response, but not of other hormonal responses, was seen. Lesioning of the mediobasal hypothalamus abolished the hormonal response and POMC mRNA activation to acute and/or repeated restraint, suggesting that the hypothalamo-pituitary-adrenal axis activation during repeated restraint is centrally driven. PVH lesion inhibited the ACTH and corticosterone rise to the first restraint by approximately 50%. In repeatedly restrained rats with PVH lesion, the ACTH response to the last restraint was reduced almost to basal control levels, and the elevation of POMC mRNA level was prevented. PVH seems to be important for the repeated restraint-induced ACTH and POMC mRNA stimulation, but it appears to partially mediate other restraint-induced hormonal changes.     

Dietary tyrosine suppresses the rise in plasma corticosterone following acute stress in rats

Acute, uncontrollable stress increases norepinephrine (NE) turnover in the rat's brain (depleting NE) and diminishes the animal's subsequent tendency to explore a novel environment. Pre-treatment with tyrosine can reverse these adverse effects of stress, presumably by preventing the depletion of NE in the hypothalamus. Numerous studies suggest that NE inhibits the release of adrenocorticotropic hormone (ACTH) by suppressing corticotropic releasing factor (CRF) secretion in the hypothalamus. In the present study, we found that pre-treatment with supplemental tyrosine not only prevented the behavioral depression and hypothalamic NE depletion observed after an acute stress, but also suppressed the rise in plasma corticosterone. These results support a role for brain NE in stress-induced corticosterone secretion and demonstrate that supplemental tyrosine can protect against several adverse consequences of such stress.     

Characterization of CRF, AVT, and ACTH cDNA and pituitary-adrenal axis function in Japanese quail divergently selected for tonic immobility

Higher corticosterone (CORT) responses to acute stress have previously been reported in quail selected for short (STI) duration of tonic immobility (TI) than for long TI (LTI), although behavioral studies indicated that LTI quail were more fearful. To investigate adrenal and pituitary function in these quail lines and their possible involvement in the differences in hypothalamic-pituitary-adrenal (HPA) axis reactivity, we measured CORT responses to adrenocorticotropin (1-24 ACTH), corticotropin-releasing factor (CRF), and arginine vasotocin (AVT) after characterizing the nucleotide acid sequences of these peptides in quail. Although maximum adrenal responses, assessed by ACTH challenge, were higher in STI quail, adrenal sensitivity was comparable for the two genotypes. It is therefore unlikely that differences in HPA axis reactivity involved the adrenal level. AVT and ACTH induced comparable CORT responses in both genotypes, whereas those induced by CRF were much lower. AVT is thus more potent than CRF in quail, but the respective maximum pituitary capacity of both genotypes to secrete ACTH was similar, and it is doubtful that the AVT pathway is involved in the difference in HPA axis reactivity between genotypes. On the other hand, the higher CORT responses induced by CRF in STI quail suggest that CRF might be involved in the differences in HPA axis reactivity between LTI and STI genotypes.     

Immunoneutralization of oxytocin attenuates stress-induced corticotropin secretion in the rat

Recent studies have demonstrated that oxytocin (OT) is released during certain stresses and that OT can potentiate the activity of CRF in vitro. To better define the role of OT during stress, the effect of injections of anti-OT antiserum on stress-induced corticotropin (ACTH) secretion was studied in vivo. A dose of antiserum which completely neutralized the increase in plasma OT levels during tail-hang stress caused a 59% decrease in plasma ACTH concentrations (P less than 0.005). The data support a physiologic role for OT in the regulation of ACTH secretion.     

Acute and chronic restraint stress alter the incidence of social conflict in male rats

Stress and elevated stress hormone levels are known to alter cognition, learning, memory, and emotional responses. Three weeks of chronic stress or glucocorticoid exposure is reported to alter neuronal morphology in the hippocampus, the amygdala, and the prefrontal cortex, and to decrease neurogenesis in the dentate gyrus. Here we examine the effects of acute and chronic restraint stress exposure on the incidence of emotional responses throughout a 3-week period among adult rat conspecifics. Our data indicate that acute restraint stress (i.e., a single 6-h exposure) results in a significant reduction in aggressive conflicts among stressed males compared to experimental controls. In contrast, on Days 14 and 21, repeatedly restrained rats exhibited significantly more aggressive behaviors than controls. Blood samples taken 18 h after the last restraint session indicate that plasma concentrations of the stress hormone corticosterone (CORT) in stressed rats were equivalent to those of unstressed rats; however, the number of individually initiated aggressive acts observed positively correlated with plasma CORT measures taken at the end of the study. In contrast to studies of psychosocial stress or intruder paradigms, here we observe spontaneous emotional responses to an uncontrollable stressor in the homecage. This study provides a novel examination of the effects of chronic restraint stress on emotional responses in the home environment among cagemates. These results indicate that acute and chronic restraint stress alter the incidence of aggression, and emphasize the relevance of this model of chronic stress to studies of stress-responsive disorders characterized by aggressive behavior.     

Stress mediated changes in hypothalamic corticotropin releasing factor-like immunoreactivity

Hypothalamic corticotropin releasing factor-like immunoreactivity (CRF-LI), plasma ACTH and corticosterone levels were measured by radioimmunoassay over a two hour period of restraint stress. The results of this study demonstrate a significant decrease in hypothalamic CRF-LI levels 15 and 30 minutes after the start of restraint stress which is followed by a significant increase at 60 minutes that is abolished by cycloheximide pretreatment. Plasma ACTH and corticosterone levels were significantly elevated after 15, 30, 60, 90, and 120 minutes of restraint stress. These results are consistent with a release of CRF from the hypothalamus during stress. The cycloheximide-sensitive increase in hypothalamic CRF-LI indicates that synthesis of CRF-41 occurs during prolonged stress. These results suggest that the response of an organism to exposure to a long-term, high intensity stress involves both the release and synthesis of CRF-41.     

Effect of myiasis and acute restraint stress on plasma levels of immunoreactive beta-endorphin, adrenocorticotrophin (ACTH) and cortisol in the sheep

Cutaneous myiasis in sheep arising from the activity of Lucilia cuprina larvae can result in significant physiological changes in susceptible animals. The stress imposed on the pituitary-adrenal axis of the sheep in response to myiasis and acute restraint is the subject of this investigation. Merino wethers were exposed to handling restraint, and blood sampling, during examination for blowfly strike; where necessary, they were treated for cutaneous myiasis. Significant changes in the plasma concentrations of immunoreactive beta-endorphin (beta-EP), ACTH and cortisol were found in sheep with extensive myiasis, as compared with unstruck sheep or those with only localized myiasis. In five susceptible sheep with extensive cutaneous myiasis, mean plasma levels of beta-EP, ACTH and cortisol were 307 +/- 71 pg ml-1, 953 +/- 58 pg ml-1 and 232 +/- 46 nmol l-1 respectively, compared with 818 +/- 89 pg ml-1, 641 +/- 41 pg ml-1 and 107 +/- 17 nmol l-1 in six unstruck sheep handled similarly. Whereas significant increases in plasma ACTH and cortisol can result from pituitary-adrenal responses to acute emotional or surgical stress, and are usually accompanied by a concomitant release of beta-EP from the pituitary, the present findings indicate a marked reduction in beta-EP levels and a significant increase in ACTH and cortisol in sheep following blowfly strike and acute handling restraint. This result suggests that cutaneous myiasis in susceptible sheep can alter the pituitary-adrenal response to acute restraint stress, and this could occur either by an alteration of precursor processing in the pituitary or by the selective release of ACTH.     

Involvement of corticotropin-releasing factor in restraint stress-induced anorexia and reversion of the anorexia by somatostatin in the rat

The mechanism by which restraint stress induces suppression of food intake and the influence of intracerebroventricular (icv) administration of somatostatin on the anorexia induced by restraint stress were examined in the rat. Ninety minutes of restraint stress reduced food intake of rats to approximately 60% that of control. Anorexia induced by 90 min restraint stress was partially reversed by icv administration of alpha-helical CRF (9-41), a corticotropin-releasing factor (CRF) antagonist, and completely reversed by anti-CRF gamma-globulin. These results provide further evidence in support of the theory that CRF is involved in the inhibitory mechanism of food intake in restraint stress. ICV administration of somatostatin 14 and SMS 201-995, an analog of somatostatin, also reversed restraint stress-induced anorexia. It is, therefore, suggested that somatostatin may counteract the suppressive action of CRF on food intake in stress.     

Repeated handling, restraint, or chronic crowding impair the hypothalamic-pituitary-adrenocortical response to acute restraint stress.

The purpose of the present study was to assess whether, and to what extent prior handling, restraint or social crowding stress during 3-10 days affects the hypothalamic-pituitary-adrenocortical (HPA) response to an acute short-lasting restraint stress. Also the effect of a feedback inhibitory mechanism of corticosterone in the impairment of HPA axis by these stressors was investigated. Male Wistar rats were pretreated with handling 1 min/day for 3-10 days, restraint 2 times daily for 3-7 days and crowding stress for 7 days before exposure to acute restraint stress in metal tubes for 10 min. Some group of rats received exogenous s.c. corticosterone either once 25 mg/kg or 2 times daily 10 mg/kg for 3-10 days before restraint stress. After the last restraint the rats were decapitated and their trunk blood was collected for the measurement of plasma ACTH and serum corticosterone levels. Handling for 3-7 days, restraint for 3-7 days, and crowding for 7 days and a single pretreatment with corticosterone--all significantly and to a similar extent inhibited the restraint stress-induced increase in ACTH and corticosterone secretion. Chronic pretreatment with corticosterone blunted the restraint stress-induced increase in HPA axis activity. These results indicate that repeated short-lasting stress induced by handling, restraint, or crowding potently attenuates the acute restraint stress-induced stimulatory action of the HPA axis. They also indicate adaptive action of moderate stress on the HPA axis response to acute stress. The results also suggest that a short-lasting hypersecretion of corticosterone during psychological stress may induce a prolonged feedback inhibition of the HPA axis activity. The attenuation of HPA axis response by prior handling has also obvious methodological implications.