Daily Restraint Stress Research Articles

Effects of daily and acute restraint stress during lactation on maternal aggression and behavior in mice

A decreased reactivity to stressors during lactation might heighten the expression of maternal care (including defense of offspring) by minimizing the extent to which stress can impact maternal care. Although stressors applied during pregnancy have variable effects on maternal aggression (or defense of offspring), to date no study has examined the effects of stress applied during the postpartum period on maternal aggression. In this study, we examined the effects of both daily and acute restraint stress (30 min) applied postpartum on maternal aggression and other maternal behaviors. Daily restraint (ending 2 h before testing) did not alter any measure of maternal behavior, including nursing, licking and grooming of pups and pup retrieval, or any measure of maternal aggression. In contrast, acute stress significantly impaired total time aggressive and number of attacks, but pup retrieval was normal. c-Fos levels were significantly elevated in a number of brain regions in association with acute stress, including lateral septum (LS), caudal periaqueductal gray and medial amygdala (MeA), suggesting possible sites where stress reactivity could alter aggression. Together, the results indicate that acute restraint stress impairs maternal aggression and provide a starting point for future studies examining how stress reactivity pathways may intersect with maternal aggression pathways.

Repeated Stress Induces Dendritic Spine Loss in the Rat Medial Prefrontal Cortex

The prefrontal cortex (PFC) plays an important role in higher cognitive processes, and in the regulation of stress-induced hypothalamic-pituitary-adrenal (HPA) activity. Here we examined the effect of repeated restraint stress on dendritic spine number in the medial PFC. Rats were perfused after receiving 21 days of daily restraint stress, and intracellular iontophoretic injections of Lucifer Yellow were carried out in layer II/III pyramidal neurons in the anterior cingulate and prelimbic cortices. We found that stress results in a significant (16%) decrease in apical dendritic spine density in medial PFC pyramidal neurons, and confirmed a previous observation that total apical dendritic length is reduced by 20% in the same neurons. We estimate that nearly one-third of all axospinous synapses on apical dendrites of pyramidal neurons in medial PFC are lost following repeated stress. A decrease in medial PFC dendritic spines may not only be indicative of a decrease in the total population of axospinous synapses, but may impair these neurons' capacity for biochemical compartmentalization and plasticity in which dendritic spines play a major role. Dendritic atrophy and spine loss may be important cellular features of stress-related psychiatric disorders where the PFC is functionally impaired.

Mild, Short-term Stress Alters Dendritic Morphology in Rat Medial Prefrontal Cortex

Prefrontal cortex is a target for glucocorticoids, shows neurochemical changes in response to stress and mediates many of the behaviors that are altered by chronic corticosterone administration. Three weeks of either daily corticosterone injections or 3 h daily restraint stress result in dendritic changes in pyramidal neurons in medial prefrontal cortex. Interestingly, vehicle injection results in similar but less pronounced changes. Thus, the mild stress of daily injections alone may alter morphology of medial prefrontal cortex, suggesting an exquisite sensitivity to chronic stress. To further examine this morphological sensitivity, we assessed the effect of 1 week of daily brief restraint stress on dendritic morphology in medial prefrontal cortex. Male rats were restrained 10 min per day for one week, handled daily or left unhandled. Rats were then overdosed and brains were stained using a Golgi-Cox procedure. Layer II-III pyramidal neurons in medial prefrontal cortex were drawn and dendritic morphology was quantified. One week of daily brief restraint resulted in selective remodeling of apical dendrites, with atrophy of up to 22-35% in distal branches and sparing of proximal branches. This pattern of reorganization is similar to that seen after either corticosterone injections or 3 weeks of daily 3 h restraint stress. Thus, the stress-induced dendritic changes in medial prefrontal cortex occur rapidly, and in response to a mild stressor.

Photoperiod and stress affect wound healing in Siberian hamsters

Changes in day length alter several indices of immune function in Siberian hamsters. These experiments tested the hypothesis that photoperiodic changes in immune function are integrated at an organismal level as reflected by the ability to heal a cutaneous wound. Given the well-documented effects of psychological stressors on immune function, we also tested the hypothesis that photoperiod modulates the effects of acute stress on wound healing. Male hamsters were housed in long (16L:8D; LD) or short (8L:16D; SD) day lengths for 8±2 weeks. SD-treatment was sufficient to induce winter reproductive status. Hamsters then received a dermal punch wound. Hamsters were subjected to either 2 h of daily restraint stress or a control treatment for 3 days prior to and 5 days after wounding. Wounds were digitally photographed daily, and wound size was measured to quantify healing. Wounds of LD hamsters healed significantly faster than did those of SD hamsters. Restraint stress significantly accelerated healing in SD hamsters. The results suggest that the enhancing effects of short-term psychological stressors on immune function are apparent only when reproductive function is suppressed. In nature, enhanced wound healing coincident with the breeding season and territorial defense may be adaptive.

Mu-opioid receptor mediates chronic restraint stress-induced lymphocyte apoptosis.

Psychological stress is associated with immunosuppression in both humans and animals. Although it was well established that psychological stressors stimulate the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system, resulting in the release of various hormones and neurotransmitters, the mechanisms underlying these phenomena are poorly understood. In this study, mu-opioid receptor knockout (MORKO) mice were used to investigate whether the mu-opioid receptor mediates the immunosuppression induced by restraint stress. Our results showed that wild-type (WT) mice subjected to chronic 12-h daily restraint stress for 2 days exhibited a significant decrease in splenocyte number with a substantial increase in apoptosis and CD95 (Fas/APO-1) expression of splenocytes. The effects are essentially abolished in MORKO mice. Furthermore, inhibition of splenic lymphocyte proliferation, IL-2, and IFN-gamma production induced by restraint stress in WT mice was also significantly abolished in MORKO mice. Interestingly, both stressed WT and MORKO mice showed a significant elevation in plasma corticosterone and pituitary proopiomelanocortin mRNA expression, although the increase was significantly lower in MORKO mice. Adrenalectomy did not reverse restraint stress-induced immunosuppression in WT mice. These data clearly established that the mu-opioid receptor is involved in restraint stress-induced immune alterations via a mechanism of apoptotic cell death, and that the effect is not mediated exclusively through the glucocorticoid pathway.

Effects of chronic restraint stress and estradiol on open field activity, spatial memory, and monoaminergic neurotransmitters in ovariectomized rats

Twenty-one days of chronic restraint stress impairs male rat performance on the radial arm maze [Luine et al. (1994) Brain Res. 639, 167–170], but enhances female rat performance [Bowman et al. (2001) Brain Res. 904, 279–289]. To assess possible ovarian hormone mechanisms underlying this sexually dimorphic response to stress, we examined chronic stress effects in ovariectomized rats. Ovariectomized rats received Silastic capsule implants containing cholesterol or estradiol and were assigned to a daily restraint stress (21 days, 6 h/day) or non-stress group. Following the stress period, subjects were tested for open field activity and radial arm maze performance. Stress and estradiol treatment affected open field activity. All stressed animals, with or without estradiol treatment, made fewer total outer sector crossings. In contrast, estradiol-treated animals, with or without stress, made more inner sector visits, an indication that estradiol decreased anxious behavior on the open field across time. As measured by the total number of visits required to complete the task, stress did not affect radial arm maze performance in ovariectomized rats, but estradiol-treated animals, with or without stress, performed better than non-treated animals on the radial arm maze. Stressed subjects receiving estradiol showed the best radial arm maze performance. Following killing, tissue samples were obtained from various brain regions known to contribute to learning and memory, and monoamine and metabolite levels were measured. Several changes were observed in response to both stress and estradiol. Most noteworthy, stress treatment decreased homovanillic acid levels in the prefrontal cortex, an effect not previously observed in stressed intact females. Estradiol treatment increased norepinephrine levels in CA3 region of the hippocampus, mitigating stress-dependent changes. Both stress and estradiol decreased dentate gyrus levels of 5-hydroxyindole acetic acid. In summary, the current study provides novel information showing that estradiol alters behavioral and neurochemical responses to stress in ovariectomized rats. Estradiol treatment decreased anxious behavior on the open field and stressed animals receiving estradiol had enhanced radial arm maze performance. In relation to interactions between stress and estradiol on cognition and anxiety, changes in the prefrontal cortex dopaminergic system, dentate gyrus serotonergic system, and norepinephrine levels in the CA3 region appear important. Results show that estradiol may moderate stress effects on cognition and anxiety through both organizational and activation effects.

Acute and chronic restraint stress: effects on [ 125I]-galanin binding in normotensive and hypertensive rat brain

The neuropeptide galanin (GAL) has been implicated in the neural response to a number of stressors including restraint; however, the effect of restraint stress on GAL receptor density in the central nervous system (CNS) has not been investigated. Normotensive (Wistar-Kyoto; WKY) and hypertensive (spontaneously hypertensive; SHR) rats were subjected to a daily 60-min restraint stress paradigm for 0 (control), 1, 3, 5 or 10 consecutive days, and the density of [ 125I]-GAL binding sites following exposure to restraint was compared between strains using quantitative autoradiography. Significant differences in basal (no stress) levels of GAL receptor density between WKY and SHR were detected in regions such as the central nucleus of the amygdala (Ce) and ventromedial hypothalamus (VMH) ( P<0.05). In WKY, restraint stress (1 day) induced significant decreases in GAL receptor density in forebrain regions such as the Ce (−41%) and medial nucleus of the amygdala (−41%) ( P<0.05). Chronic restraint (10 days) did not induce significant decreases in these nuclei in WKY, indicating that forebrain neurons containing GAL receptors in WKY possessed a functional ability to adapt to repeated restraint. In addition, restraint stress induced significant decreases in GAL receptor density in SHR in regions such as the lateral parabrachial nucleus (−43%; 5 days of restraint) and hypoglossal nucleus (∼−18% for entire restraint period) ( P<0.05). In conclusion, restraint stress resulted in region- and strain-specific alterations in GAL receptor density, some of which may contribute to the altered stress response previously observed in hypertensive rats. The results clearly support the hypothesis that neuropeptides such as GAL are an integral component of the neural response to psychological stress, although the functional significance of the changes in GAL receptor density described in this study awaits elucidation.

Restraint stress-induced alterations in the levels of biogenic amines, amino acids, and AChE activity in the hippocampus.

Inspite of large number of studies on the neurochemical changes in the stress, an equivocal case is yet to be made for the role of a specific neurotransmitter in this important neurobiological disorder. The difficulty arises from the fact that there is no single neurotransmitter system appears to be responsible for the stress induced damage to the hippocampal neurons. The present study evaluates the effect of restraint stress on the alterations in the levels of biogenic amines, aminoacids and acetylcholinesterase activity in the hippocampus. Male Wistar rats of 45 days old were subjected to 6 hours of daily restraint stress over a period of 21 days. Immediately after the last session of stress, rats were sacrificed and neurotransmitter levels were estimated in the hippocampus. A significant (p < 0.001) decrease in the levels of noradrenaline, dopamine, 5-hydroxytryptamine and acetylcholinesterase activity in the stressed rats was observed compared to controls. However, levels of glutamate was significantly (p < 0.001) increased in stressed rats. These results indicate that chronic restraint stress decreases aminergic and cholinergic neurotransmission, and increases the glutamatergic transmission in the hippocampus.

CRH-deficient mice have a normal anorectic response to chronic stress

Many studies have implicated corticotropin-releasing hormone (CRH) as a mediator of stress-induced decreases in food intake. However, urocortin, sauvagine, and urotensin, other members of the family of CRH-like molecules, have also been shown to be potent inhibitors of food intake. This raises the possibility that a CRH-related molecule might also be responsible for stress-induced anorexia. We therefore examined the effects of three chronic stressors, repetitive daily restraint, turpentine abscess, and surgical stress, upon food intake in wildtype and CRH-deficient mice created by targeted inactivation of the CRH gene. We have found that both genotypes have similar basal food intake which initially decreases to the same degree following initiation of each stress paradigm. Food intake also recovers following the same time course and to the same degree in both genotypes. Therefore, CRH is not necessary for decreases in food-intake induced by the chronic stressors examined in this study.

Electroshock seizures protect against apoptotic hippocampal cell death induced by adrenalectomy

Seizures evoked by electroshock induce rapid changes in the expression of several genes in the adult brain, including those encoding for neurotrophic factors. Some of the neurotrophic factors induced by brief seizures such as basic fibroblast growth factor and nerve growth factor have been shown to have neuroprotective action. We reasoned therefore that these seizures may protect against neural injury. To test this hypothesis, we examined the effect of electroshock-induced seizures on the vulnerability to cell death in the hippocampus. Cell death was induced by adrenalectomy, which results in a highly selective apoptotic neuronal death in the dentate granule cell layer of the hippocampus. Daily electroshock seizures were administered for seven days to sham-operated and adrenalectomized rats. Neuronal degeneration was evaluated by the highly sensitive and reliable cupric-silver impregnation method. Animals experiencing electroshock seizures were completely protected against adrenalectomy-induced cell death, whereas adrenalectomized animals not exposed to electroshock seizures exhibited substantial neuronal cell degeneration in the dentate granule cell layer. Daily restraint stress did not prevent the adrenalectomy-induced neuronal death, indicating that the neuroprotective effect of the seizure treatment is not accounted for by stress. We conclude that brief controlled seizure-evoked neural activation may allow the sparing of otherwise vulnerable neuronal populations in the injured adult brain. This prompts a need to explore the possibility that controlled administration of electroshock seizures may have therapeutic potential in treating neurodegenerative disorders.

Neonatal Nonhandling andIn UteroPrenatal Stress Reduce the Density of NADPH-Diaphorase-Reactive Neurons in the Fascia Dentata and Ammon’s Horn of Rats

The density of nitric oxide (NO)-producing neurons in the fascia dentata and Ammon's horn was assessed in 6-month-old male rats using NADPH-diaphorase (NADPH-d) histochemistry. Two separate experiments investigated whether (1) the complete absence of neonatal handling or (2) the administration of periodic prenatal stress could affect the expression and distribution of NADPH-d reactivity in the hippocampus, when compared with rats raised in normal standard laboratory conditions. Experiment 1 demonstrated that adult rats that received no handling during neonatal development (from birth to postnatal day 22) showed a very substantial reduction in NADPH-d-positive neurons per unit area throughout the entire hippocampus when compared with rats that received regular daily handling in this period. Quantitative analysis further revealed that this effect was significantly more pronounced in Ammon's horn than in the fascia dentata, and within Ammon's horn the dorsal region was selectively more affected. Experiment 2 showed that prenatal stress, which involved the administration of daily restraint stress to pregnant dams throughout the gestation period, also led to a reduction in NADPH-d reactivity in the hippocampus of the offspring of these dam when they reached adulthood. The present results suggest that behavioral manipulations in the early neonatal or prenatal period can significantly alter the neurodevelopment of the hippocampal NO system and these changes might be related to some of the behavioral abnormalities that emerge later in adulthood.

Effect of stress on cochlear glucocorticoid protein. II. Restraint

The effect of restraint stress via immobilization on rat cochlear glucocorticoid receptor (GR) levels was determined using an enzyme-linked immunosorbent assay (ELISA). Results demonstrated that GR levels in cochlear tissues exhibited tissue-specific and time-dependent responses to immobilization (6 hours daily). Similar responses of the GR were observed in rats restrained during two different times of the day. A significant quadratic trend ( P = 0.019, R 2 = 0.58) was observed in levels of GR in spiral ligament tissues of rats restrained from 10:00 to 16:00 h; levels of GR were elevated by day 2, and by day 21 GR levels had returned to near normal levels. GR levels in the spiral ligament tissues also were found to increase significantly after 2 days in response to repeated restraint stress administered from 06:00 to 12:00 h ( P = 0.017, R 2 = 0.34). Interestingly, a subtle, but statistically significant, decreasing trend in the organ of Corti's GR levels was detected when the daily restraint stress was applied from 06:00 to 12:00 h for up to 7 days. No significant trends ( P > 0.05) were observed in GR levels of stria vascularis tissues regardless of the time of the restraint protocol. Stress has been implicated as an etiological factor in Ménière's disease and other ear pathologies. The data presented here indicate that the effect of stress is specific to tissue region and that, as in tissues of other systems, the GR of cochlear tissues are responsive to stress.

Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: Involvement of glucocorticoid secretion and excitatory amino acid receptors

Repeated restraint stress of rats for 21 days causes atrophy of apical dendrites of hippocampal CA3c pyramidal neurons. This effect is mimicked by daily corticosterone treatment for 21 days and is prevented y the anti-epileptic drug, phenytoin, known to interfere with excitatory amino acid release and action. The present study was designed to investigate the involvement of endogenous corticosterone secretion and excitatory amino acid receptors in the stress-induced hippocampal dendritic atrophy. Treatment of chronically stressed rats with the steroid synthesis blocker cyanoketone prevented stress-induced dendritic atrophy. Cyanoketone-treated animals showed an impaired corticosterone secretion in response to the stressor, while basal levels were maintained. Besides the involvement of endogenous corticosterone secretion, N-methyl-D-aspartate receptors also play a role, since the competitive receptor antagonist, CGP 43487, blocked stress-induced dendritic atrophy. In contrast, NBQX, a competitive inhibitor of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, was ineffective at a dose that blocks ischemic damage. These results indicate that the reversible atrophy induced by 21 days of daily restraint stress requires corticosterone secretion and that excitatory mechanisms involving N-methyl-D-aspartate receptors play a major role in driving the atrophy.

Effects of adrenal steroid manipulations and repeated restraint stress on dynorphin mRNA levels and excitatory amino acid receptor binding in hippocampus

Adrenal steroid and stress effects were determined in hippocampus on levels of dynorphin (DYN) mRNA, expressed in dentate gyrus, and excitatory amino acid receptors, measured in Ammon's horn and dentate gyrus. Adrenalectomy (ADX) decreased DYN mRNA levels in dentate gyrus and replacement with aldosterone (ALDO), a specific type I adrenal steroid receptor agonist, prevented the decrease. Ru28362, a specific type II receptor agonist, had no effect. Likewise, kainate receptor binding to the stratum lucidum and hilus region of dorsal hippocampus was decreased after ADX and this decrease was prevented by ALDO but not by Ru28362 treatment. Similar though smaller effects were found for CNQX binding to AMPA receptors but only in the dentate gyrus molecular or infra- and supragranular layers. Although corticosterone (CORT) treatment of intact rats (40 mg/kg for 3 weeks) elevated DYN mRNA levels in dentate gyrus, up to 14 days of daily restraint stress (1 or 6 h/day) had no significant effect. Neither CORT treatment nor repeated restraint stress altered NMDA and non-NMDA glutamate receptors in hippocampus. The results of this study showing ADX-induced decreases of DYN mRNA and CNQX binding in dentate gyrus and decreased kainate binding in mossy fiber terminal regions are consistent with morphological evidence showing that adrenal steroids maintain normal integrity and structure of dentate gyrus neurons and do so via type I adrenal steroid receptors. These same parameters are apparently not sensitive to chronic restraint stress although the effects of other stressors must be examined.

The habituation of brainstem catecholaminergic groups to chronically daily restraint stress is stress specific like that of the hypothalamo-pituitary-adrenal axis

It has previously been shown that immobilization and ether stress induce activation of the hypothalamo-pituitary-adrenal (HPA) axis and that this occurs subsequent to activation of brain stem catecholaminergic neurones. In the present study we have investigated whether the brain stem catecholaminergic (CA) neurons show habituation to chronic daily intermittent exposure to the same restraint stress comparable to that of the HPA axis. The level of activity of the brainstem CA groups was estimated by measurement in this tissue punches of content of 3,4-dihydroxyphenylacetic acid (DOPAC), a side metabolite of noradrenaline and adrenaline biosynthesis which has been shown to be a reliable index of the stress-induced activation of the CA groups. The level of activity of the HPA axis was determined by measurement of plasma corticosterone and adrenocorticotrophic hormone (ACTH) levels. The animals were submitted to a 15 min restraint stress daily. They were sacrified at the end of the stress session on day 3, 5 and 10. The ACTH response to the acute restraint stress whilst unchanged on day 3 was significantly decreased on day 5 (−54%) and day 10 (−70%) compared to the response in naive rats. The approximately twofold increase in DOPAC level induced by acute restraint stress in the so-called CA medullary group A1/C1 of naive rats was reduced in daily restraint rats on day 5 (−22%) and day 10 (−30%) but was unchanged on day 3. A small (−20%) decrease of the stress-induced DOPAC response in the A2/C2 CA group and locus coeruleus was also observed on day 10. In animals submitted to four daily sessions of restraint stress, the increase in ACTH response to a novel (ether) stress was not reduced but rather was enhanced by 30% as compared to the response obtained in naive rats. Similarly the DOPAC response in the A1/C1 group to an acute ether stress was enhanced by 25% in chronically stressed animals while the DOPAC response inthe A2/C2 group and the locus coeruleus were unchanged. Our present data indicate that brain stem CA group show habituation to chronic intermittent stress. Habituation of the A1/C1 group and habituation of HPA axis have similar time-courses and are similarly stress-specific. The latter results suggest that habituation observed in these experimental conditions is not due to intrinsic biochemical changes in the brain stem CA neurones but indirectly reflects decreased stimulatory inputs to the brain stem CA groups resulting from behavioural adaptation at higher brain centers.

Tianeptine attenuates stress-induced morphological changes in the hippocampus

Repeated 6-h daily restraint stress over 21 days reduces length and number of branch points of hippocampal CA3c pyramidal dendrites in the hippocampal formation of adult male rats. This effect is mimicked by daily injections of 40 mg/kg corticosterone. Daily treatment with tianeptine (15 mg/kg) prior to stress sessions or the corticosterone treatment prevented these effects of stress or corticosterone, respectively. Tianeptine treatment did not prevent the effects of stress to increase adrenal/body weight ratio, nor did it prevent the effects of stress to decrease body weight gain, indicating that its actions are not mediated solely by effects on stress-induced secretion of corticosterone. Because tianeptine is known to enhance neural uptake of serotonin, these results suggest that the serotonergic system may be involved in modulating stress and corticosterone effects on dendritic morphology.

Chronic stress induces strain-dependent sensitization to the behavioral effects of amphetamine in the mouse

Following 10 days of daily restraint stress, sensitization developed to the stimulatory effect of amphetamine on locomotion in DBA/2 but not in C57BL/6 mice tested 24 h after the last stressful experience regardless of their being naive or habituated to the test cages. Saline-injected C57BL/6 mice, however, showed an increase of locomotion 24 h after chronic stress treatment. Chronically stressed mice of the two strains did not exhibit any alteration of dopamine and metabolites (3-4-dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine) levels in the frontal cortex, caudatus putamen, or nucleus accumbens septi, thus ruling out that stress-induced alteration of basal dopamine metabolism affected the behavioral response to amphetamine challenging in DBA/2 mice. Ten daily amphetamine injections (5 mg/kg) did not significantly modify the behavioral response to amphetamine in either strain of mice tested 24 h after the end of the chronic treatment and did not increase locomotion in saline-injected C57BL/6 mice. Finally, chronically stressed hybrids B6D2F1 did not show sensitization to the locomotor effects of amphetamine, suggesting a dominant mode of inheritance in the response to chronic stress of the C57BL/6 strain.

Repeated stressful experiences differently affect brain dopamine receptor subtypes

The binding of tritiated spiperone (D2 antagonist) and tritiated SCH 23390 (D1 antagonist), in vivo , was investigated in the caudatus putamen (CP) and nucleus accumbens septi (NAS) of mice submitted to ten daily restraint stress sessions. Mice sacrificed 24 hr after the last stressful experience presented a 64% decrease of D2 receptor density (Bmax) but no changes in D1 receptor density in the NAS. In the CP a much smaller (11%) reduction of D2 receptor density was accompanied by a 10% increase of D1 receptors. These results show that the two types of dopamine (DA) receptors adapt in different or even opposite ways to environmental pressure, leading to imbalance between them.