Mild Restraint Stress Research Articles

Acute restraint stress produces behavioral despair in weanling rats in the forced swim test

Stressful experiences in the rat during early life increase the vulnerability to later signs of behavioral despair in adulthood, reflected in increased immobility in the forced swim test (FST). However, the possible immediate effects of stress in weanling rats have only been partially described. The present study tested whether a single session of mild restraint stress modifies immobility in the FST in 21-day-old Wistar rats. After evaluating any possible changes in locomotion using the open field test (OFT), the latency and total duration of immobility were assessed in a single FST session. Regardless of gender, mild restraint stress significantly reduced crossings in the OFT, shortened the latency to the first period of immobility, and increased immobility in the FST compared with a control group devoid of stress. We conclude that a single mild physical stress session, as early as postnatal day 21, produces signs of behavioral despair.

The Immune–Pineal Axis

The temporal organization of mammals presents a daily adjustment to the environmental light/dark cycle. The environmental light detected by the retina adjusts the central clock in the suprachiasmatic nuclei, which innervate the pineal gland through a polysynaptic pathway. During the night, this gland produces and releases the nocturnal hormone melatonin, which circulates throughout the whole body and adjusts several bodily functions according to the existence and duration of darkness. We have previously shown that during the time frame of an inflammatory response, pro-inflammatory cytokines, such as tumor necrosis factor-alpha, inhibit while anti-inflammatory mediators, such as glucocorticoids, enhance the synthesis of melatonin, interfering in the daily adjustment of the light/dark cycle. Therefore, injury disconnects the organism from environmental cycling, while recovery restores the light/dark information to the whole organism. Here, we extend these observations by evaluating the effect of a mild restraint stress, which did not induce macroscopic gastric lesions. After 2 h of restraint, there was an increase in circulating corticosterone, indicating activation of the hypothalamus-pituitary-adrenal (HPA) axis. In parallel, an increase in melatonin production was observed. Taking into account the data obtained with models of inflammation and stress, we reinforce the hypothesis that the activity of the pineal gland is modulated by the state of the immune system and the HPA axis, implicating the darkness hormone melatonin as a modulator of defense responses.

Cognitive and neuroinflammatory consequences of mild repeated stress are exacerbated in aged mice

Peripheral immune stimulation as well as certain types of psychological stress increases brain levels of inflammatory cytokines such as interleukin-1beta (IL-1beta), IL-6 and tumor necrosis factor alpha (TNFalpha). We have demonstrated that aged mice show greater increases in central inflammatory cytokines, as well as greater cognitive deficits, compared to adults in response to peripheral lipopolysaccharide (LPS) administration. Because aged mice are typically more sensitive to systemic stressors such as LPS, and certain psychological stressors induce physiological responses similar to those that follow LPS, we hypothesized that aged mice would be more sensitive to the physiological and cognitive effects of mild stress than adult mice. Here, adult (3-5 months) and aged (22-23 months) male BALB/c mice were trained in the Morris water maze for 5 days. Mice were then exposed to a mild restraint stress of 30 min before being tested in a working memory version of the water maze over a 3-day period. On day 4 mice were stressed and then killed for collection of blood and brain. In a separate group of animals, mice were killed immediately after one, two or three 30 min restraint sessions and blood was collected for peripheral corticosterone and cytokine protein measurement, and brains were dissected for central cytokine mRNA measurement. Stress disrupted spatial working memory in both adult and aged mice but to a much greater extent in the aged mice. In addition, aged mice showed an increase in stress-induced expression of hippocampal IL-1beta mRNA and MHC class II protein compared to non-stressed controls while expression in adult mice was unaffected by stress. These data show that aged mice are more sensitive to both the cognitive and inflammatory effects of mild stress than are adult mice and suggest a possible role for IL-1beta.

Pituitary‐Adrenal Response to Acute and Repeated Mild Restraint, Forced Swim and Change in Environment Stress in Arginine Vasopressin Receptor 1b Knockout Mice

Arginine vasopressin and corticotrophin-releasing hormone synthesised and released from the hypothalamic paraventricular nucleus are the prime mediators of the hypothalamic-pituitary-adrenal (HPA) axis response to stress. These neurohormones act synergistically to stimulate adrenocorticotophin (ACTH) secretion from the anterior pituitary, culminating in an increase in circulating glucocorticoids. Arginine vasopressin mediates this action at the arginine vasopressin 1b receptor (Avpr1b) located on pituitary corticotrophs. Arginine vasopressin is regarded as a minor ACTH secretagogue in rodents but evidence suggests that it has a role in mediating the neuroendocrine response to some acute and chronic stressors. To investigate the role of the Avpr1b in the HPA axis response to an acute and chronic (repeated) stress, we measured the plasma ACTH and corticosterone concentrations in three stress paradigms in both Avpr1b knockout and wild-type mice. Single acute exposure to restraint, forced swim and change in environment stressors elevated both plasma ACTH and corticosterone concentrations in wild-type animals. Conversely, the ACTH response to the acute stressors was significantly attenuated in Avpr1b knockout mice compared to their wild-type counterparts. Plasma corticosterone concentrations were reduced in Avpr1b knockout mice in response to change in environment but not to mild restraint or forced swim stress. Irrespective of genotype, there was no difference in the plasma ACTH or corticosterone concentrations in response to acute and repeated stressors. The data show that a functional Avpr1b is required for an intact pituitary ACTH response to the acute and chronic stressors used in this study. Furthermore, the normal corticosterone response to repeated exposure to change in environment stress also requires the Avpr1b to drive HPA axis responsiveness.

Stress blunts serotonin- and hypocretin-evoked EPSCs in prefrontal cortex: Role of corticosterone-mediated apical dendritic atrophy

Morphological studies show that repeated restraint stress leads to selective atrophy in the apical dendritic field of pyramidal cells in the medial prefrontal cortex (mPFC). However, the functional consequence of this selectivity remains unclear. The apical dendrite of layer V pyramidal neurons in the mPFC is a selective locus for the generation of increased excitatory postsynaptic currents (EPSCs) by serotonin (5-HT) and hypocretin (orexin). On that basis, we hypothesized that apical dendritic atrophy might result in a blunting of 5-HT- and hypocretin-induced excitatory responses. Using a combination of whole-cell recording and two-photon imaging in rat mPFC slices, we were able to correlate electrophysiological and morphological changes in the same layer V pyramidal neurons. Repeated mild restraint stress produced a decrement in both 5-HT- and hypocretin-induced EPSCs, an effect that was correlated with a decrease in apical tuft dendritic branch length and spine density in the distal tuft branches. Chronic treatment with the stress hormone corticosterone, while reducing 5-HT responses and generally mimicking the morphological effects of stress, failed to produce a significant decrease in hypocretin-induced EPSCs. Accentuating this difference, pretreatment of stressed animals with the glucocorticoid receptor antagonist RU486 blocked reductions in 5-HT-induced EPSCs but not hypocretin-induced EPSCs. We conclude: (i) stress-induced apical dendritic atrophy results in diminished responses to apically targeted excitatory inputs and (ii) corticosterone plays a greater role in stress-induced reductions in EPSCs evoked by 5-HT as compared with hypocretin, possibly reflecting the different pathways activated by the two transmitters.

Sex Differences in Neurotransmission Parameters in Response to Repeated Mild Restraint Stress Exposures in Intact Male, Female and Ovariectomised Female Rats

The present study determined whether a repeated mild restraint stress exposure would differentially alter neuronal activity in male and female rats to gain insights into neurobiological substrates involved in sex differences in stress-induced behavioural responses. In our first set of experiments, we used Western blot analysis to determine whether alterations in several synaptic proteins were elicited by the repeated stress treatment. We found bidirectional changes in synaptophysin levels in female cerebral cortex and hippocampus that diverged between intact and ovariectomised females. There were persistent elevations in spinophilin levels in the male, but not female, hippocampus following the repeated mild restraint stress exposure. By contrast, levels of the NMDA receptor scaffolding protein, PSD-95, were altered only in intact female cerebral cortex and ovariectomised female hippocampus. We next used immunohistochemical evaluation of Fos expression as a marker for neuronal activation. We found significant increases in Fos immunoreactivity in all sex conditions across multiple brain regions in response to the repeated mild stress. Fos protein induction was greatest in the frontal cortex, piriform cortex and amygdala, with the degree of induction varying by sex condition. Fos induction was dramatically higher in amygdala and piriform cortex only in intact females following repeated stress compared to a single restraint stress exposure, suggestive of sensitisation rather than habituation. By contrast, the frontal cortex of intact and ovariectomised females showed habituation to the repeated stressor. Males displayed modest sensitisation in both the frontal cortex and dentate gyrus with no changes in other brain areas. Taken together, these findings show that exposure to a mild repeated stress results in sex differences in synaptic adaptations and patterns of brain activation that likely contribute to observed sex differences in stress-induced behaviours. This approach provides valuable insights into interactions between the hormonal milieu and responses to a repeated mild stress, and further supports the importance of considering hormonal status in treatment of stress-related disorders.

Effects of repeated stress on regeneration of serotonergic and noradrenergic axons in the cerebral cortex of adult rats

We examined whether regeneration of serotonergic (5-HT) and noradrenergic (NA) axons might be affected by stress. Neurotoxins to 5-HT or NA axons were injected into the frontal cortex to cause partial denervation. Mild restraint stress (40 min/day) was started 16 days later and given for 14 consecutive days (30-day stressed group). Non-stressed animals were divided into two groups, animals sacrificed at 14 days (14-day control group) and those sacrificed at 30 days (30-day control group) after the toxin injection. In immunohistochemical study, the denervation area of 5-HT axons but not that of NA axons was significantly smaller in the 30-day control group than in the 14-day control group. However, there was no significant difference between the 14-day control and 30-day stressed groups. These findings suggested that regeneration of 5-HT axons occurred earlier than that of NA axons, and that stress exerted inhibitory influence on regeneration of 5-HT axons.

Mild prenatal stress enhances learning performance in the non-adopted rat offspring

The present study was designed to investigate whether mild stress during pregnancy affects offspring behaviors, including learning performance. Prenatal stress was induced by short-lasting, mild restraint stress, which had previously been shown to facilitate the morphological development of fetal brain neurons. Adult offspring whose dams had been restrained in a small cage for 30 min daily from gestation day 15 to 17 showed enhanced active avoidance and radial maze learning performance. In addition, the prenatally stressed rats showed weaker emotional responses than unstressed control, as indicated by decreases both in ambulation upon initial exposure to an open field and in Fos expression in the amygdala induced by physical stress. The observed effects of prenatal stress on learning performance and emotional behavior were attenuated by foster rearing by unstressed dams. Fos expression in the hypothalamic paraventricular nucleus following physical stress and corticosterone secretion during physical and psychological stress did not differ between the prenatally stressed and unstressed control rats. From these results we suggest that mild prenatal stress facilitates learning performance in the adult offspring. The enhancement of learning performance appears to be accompanied by reduced emotionality, but not by any apparent alterations in hypothalamic–pituitary–adrenal responses. In addition, the observation of differential behaviors in the adopted and non-adopted animals supports the notion that the postnatal environment modifies the behavioral effects of prenatal stress.

Exaggerated stress-induced release of nonesterified fatty acids in JCR:LA-corpulent rats

Studies were performed to test the hypothesis that the stress response is exaggerated in obesity and to identify which component of the response is modified. Chronically cannulated lean (+/+) and obese ( cp cp ) JCR:LA rats were subjected to mild restraint stress for 15 minutes. Blood pressure and serum glucose, insulin, and corticosterone responses did not differ significantly between genotypes before, during, or after restraint. Lean rats had a significantly greater plasma epinephrine (EPI) response but a similar norepinephrine (NE) response compared with obese rats. Serum nonesterified fatty acid (NEFA) concentrations were unchanged in lean rats, but increased from 0.86 to a mean of 1.48 mmol/L in obese rats within 10 minutes of restraint. All animals recovered to prestress values by 45 minutes postrestraint. In obese rats, handling increased NEFAs to greater than 2 mmol/L before or at 165 minutes after restraint. In lean rats, NEFAs increased when handling occurred at 165 minutes after restraint, but there was no significant NEFA response at the prerestraint point. The sensitivity of adipose tissue to NE-induced lipolysis was not significantly different between genotypes. It is concluded that the exaggerated accumulation of NEFAs in the blood of obese rats results from increased adipose tissue mass. These increases in NEFAs in obese rats resulting from mild stress reached levels normally associated with gross pathology such as ketotic diabetes.

Effects of quercetin and/or restraint stress on formation of aberrant crypt foci induced by azoxymethane in rat colons.

The present study examines the effect of dietary quercetin and/or restraint stress on the formation of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in the colon. Female F344 rats were divided into four groups. All animals were given intraperitoneal injections of AOM. The animals were fed a basal diet (group A, C), or a 2% quercetin-supplemented diet (group B, D). The animals were put individually to narrow wire cages for 1 h every day throughout the study to expose them to mild restraint stress (group C, D). At week 5, all the rats were killed and analyzed for ACF in the colon. The number of ACF increased significantly in the animals subjected to stress (p < 0.05). On the other hand, dietary quercetin significantly reduced the number of ACF in both the nonstress (p < 0.001) and stress groups (p < 0.05). These findings suggest that quercetin might have a potential as a chemopreventive drug for colon cancer despite stress.

Previous inflammation alters the response of the rat colon to stress

BACKGROUND & AIMS: Patients with inflammatory bowel disease have symptoms of irritable bowel syndrome (IBS) with a higher than expected prevalence. Stress is an important factor in the pathogenesis of IBS. Thus, previous inflammation may predispose to IBS by rendering the bowel more susceptible to the impact of stress. The aim of this study was to examine the effect of previous colitis on stress-induced responses in rats. METHODS: Acute colitis was induced in rats by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and the rats were allowed to recover for 6 weeks before application of mild restraint stress for 3 consecutive days. In vitro measurements included myeloperoxidase activity, plasma corticosterone levels, interleukin 1 beta messenger RNA expression, and [3H]noradrenaline release from the myenteric plexus. RESULTS: Six weeks after administration of TNBS, stress caused a significant increase in myeloperoxidase activity in TNBS-treated rats but not in stressed controls; plasma corticosterone responses were similar. Stress also caused an exaggerated and significant suppression of [3H]noradrenaline release in TNBS-treated stressed rats compared with stressed controls. This was accompanied by a significant decrease in interleukin 1 beta messenger RNA expression in the colon. CONCLUSIONS: Previous colitis rendered the colon more susceptible to effects of stress on enteric nerve function and also increased some parameters of inflammation in response to stress. (Gastroenterology 1996 Dec;111(6):1509-15)

The Gut-Brain Axis in IBD: An Investigator’s Perspective

Three clinical observations in inflammatory bowel disease (IBD) prompt an examination of the role of gut-brain interactions in the pathophysiology of IBD. The first is the recognition that anorexia is among several factors that lead to malnutrition in IBD patients. The second is the suspicion that stressful life events may precipitate exacerbations of IBD. The third is that IBD and irritable bowel syndrome (IBS) seem to be related. Recent work in animal models has provided insights into these clinical observations. Animal models of colitis exhibit a marked reduction in feeding during the acute phase of inflammation. Interestingly, thirst mechanisms are preserved and meal pattern analysis reveals that meal frequency is unchanged while meal size is reduced. These observations suggest that the anorexia does not reflect a general debility or malaise phenomenon. The anorexia is mediated in part by prostaglandins and interleukin-10. In another set of experiments, the authors evaluated the effect of stress on colitis induced by trinitrobenzene (TNB) in rats. Mild restraint stress had no effect on the histology or activity of myeloperoxidase in rats that had not previously had colitis. In contrast, stress caused an acute exacerbation of colitis, reflected histologically and by myeloperoxidase activity, in rats with TNB colitis six weeks previously. These results provide new evidence in support of a causal relationship between stress and reactivation of intestinal inflammation. The apparent relationship between IBD and IBS has prompted speculation of a causal relationship between inflammation and gut ‘irritability’. Studies in animal models of colitis and jejunitis have shown that mucosal injury and inflammation are associated with neuromuscular dysfunction and abnormal motility. Neuromuscular dysfunction often persists after the mucosa has healed. More recent studies indicate that cells in the muscularis externa, which includes smooth muscle, are capable of cytokine and other inflammatory mediator production. Thus, one may speculate that the local production of mediators outlasts the mucosal injury and maintains a state of persistent dysfunction in the tissue. This may contribute to postinflammatory irritability in the gut. In summary, recent studies provide a tangible basis for further investigation of the inter-relationships between behaviour and inflammation in the clinical expression of IBD.

The Application of Sub-Perception Electrical Stimuli Elicits a Temporally Distinct Response from Restraint Stress: I. Antinociceptive Characteristics

The antinociceptive action of a transcranially-applied, sub-perception, electrical stimulus was distinguished from the effect of mild restraint stress by comparing the temporal characteristics of the two analgesic responses in two experimental tests. Rats restrained in perspex tubes received either transcranial sub-perception electrostimulation treatment (TCET) at 12 μA, 10 Hz, or were sham-treated. Sham treatment for 5 or 10 but not 20, 40, 60 or 120 min produced an antinociceptive effect in the tail flick latency (TFL) test. The withdrawal latencies in the TFL test were significantly longer in the rats that received TCET while being restrained for 10, 20 and 40 but not 5, 60 or 120 min, compared with sham animals comparably restrained for the same periods of time. Rats restrained and receiving electrical stimuli for 20, 40 or 60 but not 5, 10 or 120 min exhibited a significantly decreased abdominal constriction (AC) response to intraperitoneal injection with hypertonic saline, compared with sham animals restrained for similar periods of time. These data indicate that the arousal induced by the obligatory handling and mild restraint produces a transient antinociceptive effect in the TFL test. Concomitant administration of a sub-perception current induced an antinociceptive effect which persisted and increased after the current had ceased. In the AC test, an antinociceptive effect was apparent only if the sub-perception current was administered, and this also increased after cessation of current.

Aging and stress-induced changes in choline and glutamate uptake in hippocampus and septum of two rat strains differing in longevity and reactivity to stressors

Stress induced changes in neurochemical indices of neurotransmission are more pronounced in the septohippocampal cholinergic system of Wistar Kyoto rats, which are behaviorally more reactive to Stressors and have a shorter life span, than in Brown Norway rats. Moreover, pronounced degeneration of septohippocampal cholinergic neurons occurs earlier in life in Wistar Kyoto rats. In the present study the high affinity synaptosomal uptakes of choline and glutamate were used as indices for cholinergic and glutamatergic systems respectively. Following 2 hr of mild restraint stress increases in both uptake systems were observed in all regions examined (hippocampus, septum and frontal cortex). The stress-induced increases were generally similar in young (3 months) and aged (20 months) rats of both strains. The noted exception was that choline uptake levels, which were reduced in the hippocampus of unhandled aged WKY rats, remained unchanged after stress. The results confirm the involvement of the septohippocampal cholinergic system in the response to acute stress and extend the findings to include the hippocamposeptal glutamatergic system activation as well. It is suggested that in spite of neuronal degeneration during aging, these responses to stress can be maintained by compensatory efforts of neurons that remain intact.