Persistent neuroendocrine changes in multiple hormonal axes after a single or repeated stressor exposures

Many researchers have studied acute responses to stress in animals and how they are modified by prior stressor exposure, but relatively few have examined whether responses to stressors might last for prolonged periods of time. We have previously demonstrated that trough plasma corticosterone levels in rats are elevated for three to five days after single or repeated exposures to mild restraint and inescapable tailshock. The current study measured other aspects of the adrenal axis, and activity in other neuroendocrine systems, 24 hours after one or three consecutive exposures to the same stress paradigm. The data indicated persistent activation of the adrenal axis and prolactin levels, whereas the thyroid and reproductive hormone axes were inhibited after either one or three stress sessions. These changes are remarkable in that one would have expected acute responses to even intense stressors to have ended within hours after the end of the stressor. It will be important to understand the interactions among these responding neuroendocrine systems and to know how long such persistent changes last. Finally, it will be critical to understand the relative contributions of neuroendocrine and psychological factors in maintaining these persistent neuroendocrine changes after exposure to intense stressors.     

Ontogeny of the adrenal response to (+)-methamphetamine in neonatal rats: the effect of prior drug exposure

We examined the ontogeny of the corticosterone response to (+)-methamphetamine in neonatal rats. In experiment-1, animals were injected with 10 mg/kg of (+)-methamphetamine or saline and plasma corticosterone levels were examined in separate groups 30 or 105 min later on postnatal day (P) 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19. The adrenal response to methamphetamine was best described by a U-shaped function with the nadir of corticosterone release occurring between P7 and P13. Experiment-2 was similar except that the effect of four consecutive days of exposure to (+)-methamphetamine (four times daily at 2 h intervals with 10 mg/kg) was assessed with a single final dose early on the fifth day (i.e. P1-5, 3-7, 5-9, 7-11, 9-13, 11-15, 13-17, 15-19). The 30 min corticosterone response after multiple methamphetamine doses was augmented compared to single exposures, with the exception of the two earliest dosing intervals ending on P5 and P7, where the responses were lower. In addition, at 105 min, the levels of corticosterone were attenuated relative to a single drug administration. With the exception of animals receiving methamphetamine from P15 to P19, thymus weights were unaffected. The data demonstrate that (+)-methamphetamine is a robust activator of corticosterone release in developing animals and this release is extensively modified by age and previous drug exposure.     

Acute stress rapidly and persistently enhances memory formation in the male rat

Previous studies, as well as the present one, report that acute exposure to intermittent tailshocks enhances classical eyeblink conditioning in male rats when trained 24 h after stressor cessation. In Experiment 1, it was determined that the facilitating effect of stress on conditioning could also be obtained in response to a stressor of acute inescapable swim stress but not inescapable noise or the unconditioned stimulus of periorbital eyelid stimulation. These selective responses arose despite comparable enhancements of the stress-related hormone corticosterone in response to tailshocks, periorbital eyelid stimulation, noise stress, and supraelevation in response to swim stress. Although corticosterone is necessary for the enhanced learning in response to stress (Beylin & Shors, 1999), these results suggest that it is not sufficient. In addition, the results suggest that the enhancement is not dependent on common characteristics between the stressor and the conditioning stimuli (stimulus generalization). In Experiment 2, it was determined that the facilitating effect of the stressor on conditioning occurs within 30 min of stressor cessation. Thus, the mechanism responsible for facilitating memory formation is rapidly induced as well as persistently expressed. In Experiment 3, it was determined that exposure to the stressor does not enhance performance of the conditioned response after the response has been acquired. Thus, exposure to the stressor enhances the formation of new associations rather than affecting retention or performance of the motor response. These studies extend the circumstances under which stress is known to enhance associative learning and implicate neural mechanisms of memory enhancement that are rapidly induced and persistently expressed.     

A single exposure to severe stressors causes long-term desensitisation of the physiological response to the homotypic stressor

Although some laboratories have reported that a single session of stress is able to induce a long-lasting sensitisation of the hypothalamic-pituitary-adrenal (HPA) response to further exposures to stress, we have found that a single exposure to severe emotional (immobilisation, restraint or shock) or systemic (endotoxin) stressors reduces the responsiveness of the HPA to the same, but not to a novel (heterotypic), stressor, in which case a slight sensitisation was observed. Long-term desensitisation has been found to reduce not only secretion of peripheral HPA hormones (ACTH and corticosterone), but also to reduce responses of central components of the HPA axis (c-fos and CRF gene expression at the level of the paraventricular nucleus of the hypothalamus, PVN). In addition, desensitisation also applies to the impact of the stressor on food intake and, probably, to stress-induced hyperglycaemia. The development of long-term desensitisation of the HPA axis does not appear to be a universal consequence of exposure to severe stressors as it was not observed in response to insulin-induced hypoglycaemia. Whether or not the development of long-term effects of stress depend on the specific pathways activated by particular stressors remains to be tested. The observed desensitisation of the HPA axis in response to the homotypic stressor shows two special features which makes it difficult to be interpreted in terms of an habituation-like process: (a) the effect increased with time (days to weeks) elapsed between the first and second exposure to the stressor, suggesting a progressive maturational process; and (b) the stronger the stressor the greater the long-term desensitisation. Therefore, it is possible that desensitisation of the HPA axis is the sum of two different phenomena: long-term effects and habituation-like processes. The contribution of the former may be more relevant with severe stressors and longer inter-stress intervals, and that of the latter with mild stressors and repeated exposures. Long-term stress-induced changes may not take place at the level of the PVN itself, but in brain nuclei showing synaptic plasticity and putatively involved in the control of the HPA axis and other physiological responses. As for the precise areas involved, these remain to be characterized.     

Influence of Regularity of Exposure to Chronic Stress on the Pattern of Habituation of Pituitary-Adrenal Hormones, Prolactin and Glucose

The effect of regularity of exposure to two different chronic stressors (noise or immobilization (IMO)) on the pattern of habituation of pituitary-adrenal (PA) hormones, prolactin and glucose was evaluated in adult male rats. Animals were chronically subjected to either regular or irregular time schedule of noise (30 min/day) or IMO (2 h/day) for two weeks. The day after the last stress session the rats were killed without stress or after having been subjected to 30 min of the homotypic stressor. Whereas regular noise did not affect food intake, body weight gain or adrenal weight, irregular noise decreased body weight gain and induced a moderate adrenal hypertrophy. In addition, previous daily exposure to regular but not to irregular noise reduced both prolactin and corticosterone responses to acute noise. In contrast, glucose response to acute noise was reduced after both regular and irregular exposure to chronic noise. Either regular or irregular exposure to chronic IMO decreased food intake and body weight and increased adrenal weight to the same extent. Likewise, no influence of regularity of exposure to chronic IMO on corticosterone and prolactin responses to acute IMO was observed. However, habituation of the ACTH response to acute IMO was observed in rats subjected to chronic regular IMO, but not in rats subjected to chronic irregular IMO. Finally, acute IMO-induced hyperglycemia diminished to the same extent after regular and irregular IMO. From these results we can conclude that: first, the process of habituation of the PA axis to chronic stress is greatly dependent upon factors such as regularity of exposure to the stressor and stressor intensity, and second, the influence of regularity on the pattern of habituation to a repeated stressor is dependent on the physiological variable we are dealing with.     

Acute exposure to a novel stressor further reduces the habituated corticosterone response to restraint in rats

The present study sought to identify dishabituation of the hypothalamic-pituitary-adrenal(HPA) axis response to different psychological stressors. Young adult male Sprague Dawley rats were exposed to five, 1 h sessions of restraint stress on five consecutive days. On the sixth day, and 2 h before additional exposure to restraint, animals were subjected to 30 min of a small (27cm square), elevated open field stressor (pedestal), which served as the dishabituating stimulus. We predicted HPA axis response dishabituation in chronically restrained rats exposed to the novel pedestal. Rats which underwent five days of restraint stress showed significantly blunted plasma corticosterone levels to restraint (habituation) as compared to restraint-nai've rats. However, rats which underwent five sessions of restraint responded with an enhanced habituation response when confronted with restraint shortly after exposure to the novel pedestal. Instead of HPA axis response dishabituation, we observed enhanced habituation. Subsequent experiments determined that a 1.25 mgkg corticosterone injection could substitute for pedestal exposure to produce enhanced restraint habituation.Combined treatment with both the glucocorticoid receptor antagonist RU40555 (30 mgkg)and the mineralocorticoid receptor antagonist RU283 18 (50 mgkg) blocked the expression of enhanced habituation after pedestal exposure. Thus, the delayed corticosterone negative feedback produced by novel stress led to enhanced expression of corticosterone response habituation.     

Chronic stress and environmental enrichment as opposite factors affecting the immune response in Japanese quail (Coturnix coturnix japonica)

Procedures in the commercial production of animals involve stressful situations which lessen the animal's welfare. This study on Japanese quail evaluated whether an environmental enrichment manipulation can affect avian immune responses and if combined with a chronic stressor exposure can help to counteract the negative effects of stress on the immune system. Potential gender effects were also considered. After hatch, half of the birds were housed in non-enriched boxes and half were housed in environmentally enriched boxes. From day 33 to 42 of age, all birds within half of the non-enriched and enriched boxes remained undisturbed while the other half were daily exposed to a 15 min restraint stressor (chronic stressor). The inflammatory response (lymphoproliferation after phytohemagglutinin-p), percentage of lymphocytes, heterophil/lymphocyte (H/L) ratio and primary antibody response against sheep red blood cells were assessed. The chronic stressor application and the enrichment procedure, respectively, either increased or reduced the four immunological parameters evaluated and always in opposite directions. Males consistently showed lower antibody titres than females and presented the highest H/L ratio in response to the stressor when reared in the non-enriched environment. The findings indicate that submitting these animals to an enriched environment can be effectively used to improve their immune response and to reduce the detrimental effects of a stressor exposure.     

Effects of motherless rearing on basal and stress-induced corticosterone secretion in rat pups

Rearing of rat pups without a mother, artificial rearing (AR), produces substantial changes in the pups' behavior in later life. These changes are similar to those produced by the stress of repeated mother-pup separations. The predominant interpretation is that the long-term effects of disruptions to the mother-pup relationship are mediated by exposure to elevated levels of corticosterone which affect the development of neurobiological systems underlying cognition and behavior. Indeed, repeated separation of pups from the mother sensitizes the pups' corticosterone response to stress. This study examined basal and stress-induced corticosterone release in AR pups. Corticosterone levels were increased immediately following implantation of feeding cannulae. One day after the start of AR, circulating concentrations of corticosterone were not increased unless AR pups were challenged with an additional stressor (injection). Corticosterone levels were lowest when cannulation and AR started on postnatal day (PND) 5 compared with earlier PNDs. On PND 12, there was no evidence of increased corticosterone levels in AR pups at baseline or in response to stress, indicating that AR did not result in persistent sensitization of corticosterone release. The long-term effects of motherless rearing on rat behavior are mediated by mechanisms that are independent of sustained early corticosterone exposure.     

The ulcerogenic effect of a rest period after exposure to water-restraint stress in rats

Shock-induced gastric ulceration in rats is exacerbated by allowing animals to rest between the period of stress and sacrifice. Conflicting data have been reported for similar post-stress rest effects using restraint stress. In Experiment 1, rats restrained in water for 2 h and then allowed a 2-h rest period exhibited significantly greater gastric ulceration than rats sacrificed immediately after the 2-h restraint-in-water stress period. The strength of this phenomenon is confirmed by Experiment 2, in which rats challenged with restraint in water for 1.25 h and then allowed a 1.25-h rest period before sacrifice exhibited greater ulceration than animals maintained in the restraint-in-water stress for the full 2 1/2 h. Corticosterone levels for these two treatment groups were similar at the time of sacrifice. Post-stressor rest therefore actively contributes to the ulcerogenic processes initiated by this form of stress challenge.     

Adaptation of anterior pituitary hormones to chronic noise stress in male rats

We have studied the effects of acute and chronic noise on serum levels of pituitary hormones in male Wistar rats. Acute noise increased serum levels of corticosterone, prolactin, and luteinizing hormone and decreased serum GH. FSH was unaffected by this stressor. Chronic noise did not modify basal levels of any hormone studied, however responsiveness of some hormones to the same stimuli was altered. Reduced corticosterone, prolactin, and GH responses to noise was observed after previous chronic exposure to this stimuli. LH response followed the same pattern although it did not reach statistical significance. It might be concluded that adaptation to a repeated stress stimulus is not confined to the pituitary-adrenal axis, however, the degree of adaptation could vary between different hormones.     

Neuronal nitric oxide synthase gene inactivation reduces the expression of vasopressin in the hypothalamic paraventricular nucleus and of catecholamine biosynthetic enzymes in the adrenal gland of the mouse

The impact of a lifelong absence of the neuronal nitric oxide synthase (nNOS) in the neuroendocrine stress response was investigated in nNOS knockout (KO) and wild type (WT) mice under basal conditions and in response to forced swimming. In the hypothalamic paraventricular nucleus oxytocin and corticotropin-releasing-hormone mRNA levels did not differ between these genotypes under resting conditions, whereas vasopressin mRNA levels were significantly lower in nNOS KO than in WT animals. Also, in the adrenal glands basal levels of tyrosine hydroxylase protein, the rate-limiting enzyme for catecholamine biosynthesis, and of phenylethanolamine N-methyltransferase, which converts norepinephrine to epinephrine, were significantly reduced in nNOS KO mice. Plasma adrenocorticotropin, corticosterone, norepinephrine and epinephrine levels were similar in the KO and WT genotypes under resting conditions. In response to forced swimming, a similar increase in plasma adrenocorticotropin and corticosterone was observed in KO and WT animals. Stressor exposure triggered also an increased epinephrine release in WT animals, but did not significantly alter plasma epinephrine levels in KO mice. These data suggest that the chronic absence of nNOS reduces the capacity of epinephrine synthesising enzymes in the adrenal gland to respond to acute stressor exposure with an adequate epinephrine release.     

Effect of neonatal handling on adult rat spatial learning and memory following acute stress

Brief neonatal handling permanently alters hypothalamic-pituitary-adrenal axis function resulting in increased ability to cope with stress. Since stress is known to affect cognitive abilities, in the present study we investigated the effect of brief (15 min) handling on learning and memory in the Morris water maze, following exposure to an acute restraint stress either before training or recall. Exposure of non-handled rats to the acute stress prior to training resulted in quicker learning of the task, than in the absence of the stressor. When acute stress preceded acquisition, male handled rats showed an overall better learning performance, and both sexes of handled animals were less impaired in the subsequent memory trial, compared to the respective non-handled. In addition, the number of neurons immunoreactive for GR was higher in all areas of Ammon's horn of the handled rats during the recall. In contrast, the number of neurons immunoreactive for MR was higher in the CA1 and CA2 areas of the non-handled males. When the acute restraint stress was applied prior to the memory test, neonatal handling was not effective in preventing mnemonic impairment, as all animal groups showed a similar deficit in recall. In this case, no difference between handled and non-handled rats was observed in the number of GR positive neurons in the CA2 and CA3 hippocampal areas during the memory test. These results indicate that early experience interacts with sex and acute stress exposure in adulthood to affect performance in the water maze. Hippocampal corticosterone receptors may play a role in determining the final outcome.     

Environmental enrichment in male CD-1 mice promotes aggressive behaviors and elevated corticosterone and brain norepinephrine activity in response to a mild stressor

Housing rodents in an enriched environment (EE) has been typically considered to have positive effects on well-being and cognitive functioning of the animals. However, in some strains of mice, EEs have also been reported to elicit aggression and to promote stress-related outcomes. In the current investigation, we examined whether environmental enrichment would elicit aggression among CD-1 male mice and thus sensitize responses to a subsequent mild stressor. It was first observed that mice housed in an EE for 2 weeks displayed more aggressive behaviors than did mice that had been housed in a standard environment (SE). In the second experiment, it was noted that after 4 weeks of EE or SE housing, mice exhibited comparable plasma corticosterone concentrations as well as levels of brain norepinephrine and its metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), in the absence of a challenge. However, upon exposure to mild stressor (placement in a novel cage), relative to their SE counterparts, EE mice were more active and displayed higher plasma corticosterone concentrations and enhanced MHPG accumulation in the prefrontal cortex and hippocampus. It seems that enrichment in male CD-1 mice promotes aggression, and may sensitize biological processes, possibly increasing vulnerability to stressor-related outcomes.     

Benefit of social support for resilience-building is contingent on social context: examining cardiovascular adaptation to recurrent stress in women

Previous work on social support and stress tolerance using laboratory-based cardiovascular stress response paradigms has suggested that perceived social support may be effective in building resilience in recipients. However, such paradigms are often socially de-contextualized insofar as they fail to take account of the social aspects of stress itself. Using 90 healthy college women, the present study sought to examine the association between self-reported perceived social support and cardiovascular stress tolerance. Participants underwent two consecutive exposures to a mental arithmetic task. On second exposure to the stressor, participants completed the task under either social threat or control conditions. Social threat was manipulated using socially salient instructions, to create a high social context. Adaptation to stress was established in terms of comparisons between cardiovascular responses to successive exposures. Results showed that cardiovascular responses tended to habituate across time, with perceived social support associated with the degree of habituation, but only under certain contextual conditions; high perceived support was associated with effective habituation under control conditions only. This response pattern is consistent with the view that high perceived social support buffers against stress in healthful ways, but only in asocial contexts.     

Maternal separation in early life impairs tumor immunity in adulthood in the F344 rat

Neonatal stress alters the hypothalamic-pituitary-adrenal (HPA) axis in rodents, such that, when these animals are exposed to stress as adults they hypersecrete corticosterone. Given that glucocorticoids are immunosuppressive, we examined the impact of maternal separation on HPA axis reactivity, natural killer (NK) cytotoxicity, and tumor growth in Fischer 344 rats following chronic restraint stress in adulthood. Pups underwent a chronic stress protocol whereby they were separated from their dams for 3 h on postnatal days 1-21. In adulthood, corticosterone responses were assessed following exposure to chronic (6 days for 10 h) restraint stress. Rats allocated to the chronic stress condition were inoculated with MADB106 tumor cells on day 4 of the restraint protocol. Blood was assessed for NK cytotoxicity on the final day of the chronic restraint protocol, and tumor colonization was assessed 3 weeks thereafter. Maternal separation impaired developmental weight gain (P < 0.05), depressed NK cytotoxicity (P < 0.05), and increased tumor colonization in the presence of chronic restraint stress in adulthood (P < 0.00 l). These findings occurred independently of circulating plasma corticosterone as only adult stress exposure potentiated corticosterone responses (P < 0.05). Our findings indicate that maternal separation and chronic stress can impair NK cytotoxicity and hence tumor immunity, but these effects are not directly mediated by perturbations in HPA axis function.     

Exposure to War Trauma, War-Related PTSD, and Psychological Impact of Subsequent Hurricane

This study explored the impact of psychological outcomes to war on response to subsequent natural disaster. Participants were 312 military personnel, 66% of whom saw Gulf War duty. All were exposed to the 1992 Hurricane Andrew. Troops were compared on reported traumatic events, hurricane impact responses, and psychological symptoms in subgroups defined by war or no war exposure prior to hurricane and by presence or absence of war-related posttraumatic stress disorder (PTSD). Data were gathered in face-to-face clinical assessments. War trauma prior to hurricane was associated with more reported traumatic events, greater fears for safety during the hurricane, and heightened psychological symptoms. Troops with preexisting war-related PTSD showed more adverse psychological hurricane sequelae and reported more traumatic events, higher depression, anxiety, anger, PTSD symptoms, and physical symptoms, and lower self-esteem than those free of diagnoses. Results point to the negative influence of exposure to one traumatic event on the experience of and response to a subsequent stressor.     

Dexamethasone and stressor-magnitude regulation of stress-induced transcription of HPA axis secretagogues in the rat

Regulation of the production of hypothalamic-pituitary-adrenal (HPA) axis secretagogues, corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), may be differentially sensitive to the negative feedback effects of glucocorticoids. We chose to study this phenomenon by examining the ability of dexamethasone to influence CRH and AVP heteronuclear RNA (hnRNA) levels in an escapable/inescapable (ES/IS) foot-shock stress paradigm. On Day 1, adult male rats were subjected to either ES or IS foot-shock; on Day 2, saline or dexamethasone (100 microg/kg) was administered 2 h prior to the stressor. We found that ES/IS foot-shock stimulated similar robust increases in plasma adrenocorticotrophic hormone (ACTH) and corticosterone concentrations, and medial parvocellular division of the paraventricular nucleus (mpPVN) AVP and CRH hnRNA and c-fos mRNA levels in saline-treated ES/IS rats. Dexamethasone pretreatment suppressed ACTH and corticosterone levels similarly in IS and ES animals. Dexamethasone pretreatment also suppressed mpPVN CRH and AVP hnRNA levels at 30 min. However, by 120 min, the mpPVN AVP hnRNA levels in dexamethasone-treated rats were similar to those measured in the saline group. We also found that rats that received the most shocks on Day 1 had greater HPA axis activation on Day 2. We conclude that the magnitude of the foot-shock stressor, determined by learned and immediate cues, is important in determining the magnitude of the HPA response.     

Gastric ulceration and adrenocortical activity after inescapable and escapable pre-shock in rats

Two experiments are reported which investigated the effects of pre-shock treatment in rats on adrenocortical activity and susceptibility to later restraint-induced ulceration. In Experiment I, animals were exposed to a single inescapable 3 minute shock on 5 consecutive days. These shocked animals exhibited sensitisation of the adrenocortical response over days, increased adrenocortical reactivity to open field testing one week later and a trend for more severe ulceration after 20 hours of restraint. In Experiment II, animals were given 20 escapable or yoked pre-shock trials on 5 consecutive days. No sensitisation of the adrenocortical response was observed. Escape animals however were found to exhibit less gastric ulceration and a reduced adrenocortical responses after 20 hours of restraint 9 days after the last preshock session.     

Proactive sensitizing effects of acute stress on acoustic startle responses and experimentally induced colitis in rats: relationship to corticosterone

In humans, some individuals develop a syndrome after trauma (post-traumatic stress disorder, PTSD) characterized by increased startle responses and lower than normal cortisol secretion. We explored a rat model using the acoustic startle response (ASR) as a behavioral indicator of the effect of a short series of shocks. Because gastrointestinal disorders have been associated with prior stress, we also studied the rats' vulnerability to a chemically-induced colitis. After initial blood sampling, 12 rats were exposed to ten 1 mA 5 s foot-shocks while 12 rats served as controls. Nineteen days later the rats were tested for ASR. Thirty trials (10 trials at each of 95, 105, and 115 dB, pseudo-randomized) were given. After exposure for 6 days to dextran sulphate sodium in their drinking water, the rats were killed and the colons examined for erosions. Shocked rats showed greater startle responses and more colonic erosion than unshocked rats, but the shock effects were significant only for animals with low initial plasma corticosterone levels. Shocked rats also showed higher levels of granulocyte marker protein (GMP) in their faeces. These results suggest that low corticosterone secretion may represent a marker for vulnerability to long term effects of shocks as indicated by increased startle responses and colonic pathology.