Responses of the hypothalamopituitary adrenal axis and the sympathoadrenal system to isolation/restraint stress in sheep of different adiposity

There is evidence that levels of adipose tissue can influence responses of the hypothalamopituitary-adrenal (HPA) axis to stress in humans and rats but this has not been explored in sheep. Also, little is known about the sympathoadrenal responses to stress in individuals with relatively different levels of adipose tissue. We tested the hypothesis that the stress-induced activation of the HPA axis and sympathoadrenal system is lower in ovariectomized ewes with low levels of body fat (lean) than ovariectomized ewes with high levels of body fat (fat). Ewes underwent dietary manipulation for 3 months to yield a group of lean ewes (n = 7) with a mean (±SEM) live weight of 39.1 ± 0.9 kg and body fat of 8.9 ± 0.6% and fat ewes (n = 7) with a mean (±SEM) live weight of 69.0 ± 1.8 kg and body fat of 31.7 ± 3.4%. Fat ewes also had higher circulating concentrations of leptin than lean ewes. Blood samples were collected every 15 min over 8 h when no stress was imposed (control day) and on a separate day when 4 h of isolation/restraint was imposed after 4 h of pretreatment sampling (stress day). Plasma concentrations of adrenocorticotropic hormone (ACTH), cortisol, epinephrine and norepinephrine did not change significantly over the control day and did not differ between lean and fat ewes. Stress did not affect plasma leptin levels. All stress hormones increased significantly during isolation/restraint stress. The ACTH, cortisol and epinephrine responses were greater in fat ewes than lean ewes but norepinephrine responses were similar. Our results suggest that relative levels of adipose tissue influence the stress-induced activity of the hypothalamopituitary-adrenal axis and some aspects of the sympathoadrenal system with fat animals having higher responses than lean animals.

Isolation and restraint stress results in differential activation of corticontrophin-releasing hormone and arginine vasopressin neurones in sheep

This study investigated sex differences in the stress-induced activation of neurons containing corticotrophin-releasing hormone (CRH), arginine vasopressin (AVP) and enkephalin in the paraventricular nucleus (PVN) of gonadectomized male and female sheep. Groups (n=3) of both sexes were either subjected to 90 min isolation and restraint stress (stress group) or were not stressed. Blood samples were taken every 10 min for 90 min prior to and after stress to monitor cortisol levels in plasma. Brains were harvested after 90 min of stress. Stress caused elevation of plasma cortisol levels to a similar extent in both sexes. Double-labeling immunohistochemistry for Fos and either CRH, AVP or enkephalin was undertaken to quantify the numbers of neurons staining for CRH, AVP and enkephalin that also immunostained for Fos. Stress increased Fos immunostaining in all cell types. There was a greater proportion of CRH than AVP neurons activated in stressed animals. There were no sex differences in the activation of CRH and AVP neurons although females had a greater proportion of enkephalin cells staining for Fos than males in both control and stressed animals. There were no differences between control and stressed animals in the proportion of cells co-staining for CRH and AVP. We conclude that isolation and restraint stress activates neurons producing CRH, AVP and enkephalin in sheep and that CRH may play a greater role than AVP in regulating adrenocorticotrophic hormone secretion in response to this stressor in sheep. Finally, isolation and restraint stress does not influence co-localization of CRH and AVP in sheep.

Activation of the hypothalamo-pituitary-adrenal axis by isolation and restraint stress during lactation in ewes : effect of the presence of the lamb and suckling

We investigated the effect of the presence and absence of lambs and suckling by lambs to attenuate activation of the hypothalamo-pituitary-adrenal (HPA) axis to isolation and restraint stress in lactating sheep. In experiment 1, blood samples were collected every 10 min from nonlactating (n = 5) and lactating (n = 5) ewes for 4 h before and during stress. In experiment 2, ewes (n = 6) were allocated to 1) nonlactating, 2) lactating with lambs absent, 3) lactating with lambs present but unable to suckle, and 4) lactating with lambs present and able to suckle. Blood samples were collected over 8 h with no stress (control day) and for 4 h before and 4 h during stress (stress day). In experiment 1, the mean (±SEM) cortisol concentrations increased significantly (P < 0.05) in nonlactating ewes during stress but did not change in lactating ewes. In experiment 2, cortisol did not vary on the control day or pretreatment of the stress day but increased (P < 0.05) during stress in all groups except lactating ewes with lambs present and able to suckle. The greatest cortisol response occurred in nonlactating ewes followed by lactating ewes with lambs absent and lactating ewes with lambs present but unable to suckle. During stress, the ACTH concentrations increased (P < 0.05) in nonlactating ewes and lactating ewes with lambs absent but not in lactating ewes with lambs present. We conclude that the activity of the HPA axis during isolation and restraint is reduced in lactating ewes and that the presence of lambs increases this level of attenuation.

Seasonal differences in the effect of isolation and restraint stress on the luteinizing hormone response to gonadotropin-releasing hormone in hypothalamopituitary disconnected, gonadectomized rams and ewes

Stress responses are thought to act within the hypothalamopituitary unit to impair the reproductive system, and the sites of action may differ between sexes. The effect of isolation and restraint stress on pituitary responsiveness to GnRH in sheep was investigated, with emphasis on possible sex differences. Experiments were conducted during the breeding season and the nonbreeding season. In both experiments, 125 ng of GnRH was injected i.v. every 2 h into hypothalamopituitary disconnected, gonadectomized rams and ewes on 3 experimental days, with each day divided into two periods. During the second period on Day 2, isolation and restraint stress was imposed for 5.5 h. Plasma concentrations of LH and cortisol were measured in samples of blood collected from the jugular vein. In the second experiment (nonbreeding season), plasma concentrations of epinephrine, norepinephrine, 3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylglycol were also measured. In both experiments, there was no effect of isolation and restraint stress on plasma concentrations of cortisol in either sex. During the breeding season, there was no effect of isolation and restraint stress on plasma concentrations of LH in either sex. During the nonbreeding season, the amplitude of the first LH pulse after the commencement of stress was significantly reduced (P < 0.05) in rams and ewes. In the second experiment, during stress there was a significant increase (P < 0.05) in plasma concentrations of epinephrine in rams and ewes and significantly higher (P < 0.05) basal concentrations of norepinephrine in ewes than in rams. These results suggest that in sheep stress reduces responsiveness of the pituitary gland to exogenous GnRH during the nonbreeding season but not during the breeding season, possibly because of mediators of the stress response other than those of the hypothalamus-pituitary-adrenal gland axis.

Hypothalamic paraventricular nucleus neurons regulate medullary catecholamine cell responses to restraint stress

Both physical and psychological stressors recruit catecholamine cells (CA) located in the ventrolateral medulla (VLM) and the nucleus of the solitary tract (NTS). In the case of physical stressors, this effect is initiated by signals that first access the central nervous system at or below the level of the medulla. For psychological stressors, however, CA cell recruitment depends on higher structures within the neuraxis. Indeed, we have recently provided evidence of a pivotal role for the medial amygdala (MeA) in this regard, although such a role must involve a relay, as MeA neurons do not project directly to the medulla. However, some of the MeA neurons that respond to psychological stress have been found to project to the hypothalamic paraventricular nucleus (PVN), a structure that provides significant input to the medulla. To determine whether the PVN might regulate medullary CA cell responses to psychological stress, animals were prepared with unilateral injections of the neurotoxin ibotenic acid into the PVN (Experiment 1), or with unilateral injections of the retrograde tracer wheat germ agglutinin-gold (WGA-Au) into the CA cell columns of the VLM or NTS (Experiment 2). Seven days later, animals were subjected to a psychological stressor (restraint; 15 minutes), and their brains were subsequently processed for Fos plus appropriate cytoplasmic markers (Experiment 1), or Fos plus WGA-Au (Experiment 2). PVN lesions significantly suppressed the stress-related induction of Fos in both VLM and NTS CA cells, whereas tracer deposits in the VLM or NTS retrogradely labeled substantial numbers of PVN cells that were also Fos-positive after stress. Considered in concert with previous results, these data suggest that the activation of medullary CA cells in response to psychological stress may involve a critical input from the PVN.

Voluntary exercise does not affect stress-induced tachycardia, but improves resistance to cardiac arrhythmias in rats

1. It is currently unknown whether long-term voluntary exercise has enduring cardioprotective effects in animal models.

2. The present study was conducted in three groups of rats: (i) sedentary controls (n = 6); (ii) 24 h runners (n = 8; unlimited access to running wheels); and (iii) 2 h runners (n = 8; access to running wheels limited to 2 h daily). After termination of the 6 week exercise protocol, all rats were implanted with the telemetric electrocardiogram transmitters and were studied 1 week later.

3. Resting heart rate (HR) values in the control rats, 24 h runners and 2 h runners were 372 ± 7, 361 ± 9 and 298 ± 5 b.p.m., respectively (P < 0.05 for 2 h runners vs controls). The high-frequency spectral power in the control rats, 24 h runners and 2 h runners was 3.9 ± 0.2, 4.3 ± 0.3 and 5.3 ± 0.3 s2, respectively (P < 0.05 for 2 h runners vs controls), whereas intrinsic HR was 383 ± 3, 377 ± 2 and 346 ± 3 b.p.m., respectively (P < 0.001 for 2 h runners vs controls). Restraint stress provoked tachycardia of similar magnitude in all groups.

4. After completion of telemetric studies, haemodynamic indices and susceptibility to cardiac arrhythmias were assessed in anaesthetized animals, there were no major between-group differences in HR, arterial pressure, contractility indices or sensitivity to β-adrenoceptor stimulation (dobutamine) or blockade (atenolol). The effective refractory period in the control rats, 24 h runners and 2 h runners was 49 ± 2, 55 ± 2 and 60 ± 4 ms, respectively (P = 0.054 for 2 h runners vs controls). A significantly higher dose of aconitine was required to provoke ventricular arrhythmias in the 24 h and 2 h running groups compared with controls (489 ± 76, 505 ± 88 and 173 ± 33 μg, respectively; P < 0.05).

5. We conclude that, in rats, long-term voluntary exercise has enduring cardioprotective effects mediated at the level of both the central nervous system and the heart.

Influence of sex and gonadal status of sheep on cortisol secretion in response to ACTH and on cortisol and LH secretion in response to stress: importance of different stressors

There are sex differences in the response to stress and in the influence of stress on reproduction which may be due to gonadal steroids but the nature of these differences and the role of the gonads are not understood. We tested the hypotheses that sex and the presence/absence of gonads (gonadal status) will influence the cortisol response to injection of ACTH, insulin-induced hypoglycaemia and isolation/restraint stress, and that sex and gonadal status will influence the secretion of LH in response to isolation/restraint stress. Four groups of sheep were used in each of three experiments: gonad-intact rams, gonadectomised rams, gonad-intact ewes in the mid-luteal phase of the oestrous cycle and gonadectomised ewes. In Experiment 1 (n=4/group), jugular blood samples were collected every 10 min for 6 h; after 3 h, two animals in each group were injected (i.v.) with ACTH and the remaining two animals were injected (i.v.) with saline. Treatments were reversed 5 days later so that every animal received both treatments. Experiment 2 (n=4/group) used a similar schedule except that insulin was injected (i.v.) instead of ACTH. In Experiment 3 (n=5/group), blood samples were collected every 10 min for 16 h on a control day and again 2 weeks later when, after 8 h of sampling, all sheep were isolated and restrained for 8 h. Plasma cortisol was significantly (P<0.05) elevated following injection of ACTH or insulin and during isolation/restraint stress. There were no significant differences between the sexes in the cortisol response to ACTH. Rams had a greater (P<0.05) cortisol response to insulin-induced hypoglycaemia than ewes while ewes had a greater (P<0.05) cortisol response to isolation/restraint stress than rams. There was no effect of gonadal status on these parameters. Plasma LH was suppressed (P<0.05) in gonadectomised animals during isolation/restraint stress but was not affected in gonad-intact animals, and there were no differences between the sexes. Our results show that the sex that has the greater cortisol response to a stressor depends on the stressor imposed and that these sex differences are likely to be at the level of the hypothalamo-pituitary unit rather than at the adrenal gland. Since there was a sex difference in the cortisol response to isolation/restraint, the lack of a sex difference in the response of LH to this stress suggests that glucocorticoids are unlikely to be a major mediator of the stress-induced suppression of LH secretion.

Opposing roles for medial and central amygdala in the initiation of noradrenergic cell responses to a psychological stressor

Psychological stressors trigger the activation of medullary noradrenergic cells, an effect that has been shown to depend upon yet-to-be-identified structures located higher in the brain. To test whether the amygdala is important in this regard, we examined the effects of amygdala lesions on noradrenergic cell responses to restraint, and also looked at whether any amygdala cells that respond to restraint project directly to the medulla. Ibotenic acid lesions of the medial amygdala completely abolished restraint-induced Fos expression in A1 and A2 noradrenergic cells. In contrast, lesions of the central amygdala actually facilitated noradrenergic cell responses to restraint. Tracer deposits in the dorsomedial (but not ventrolateral) medulla retrogradely labelled many cells in the central nucleus of the amygdala, but none of these cells expressed Fos in response to restraint. These data suggest for the first time that the medial amygdala is critical to the activation of medullary noradrenergic cells by a psychological stressor whereas the central nucleus exerts an opposing, inhibitory influence upon noradrenergic cell recruitment. The initiation of noradrenergic cell responses by the medial amygdala does not involve a direct projection to the medulla. Accordingly, a relay through some other structure, such as the hypothalamic paraventricular nucleus, warrants careful consideration.

Psychosocial stress suppresses attractivity, proceptivity and pulsatile LH secretion in the ewe

Various stressors suppress pulsatile secretion of luteinizing hormone (LH) in ewes and cortisol has been shown to be a mediator of this effect under various conditions. In contrast, little is known about the impact of stress and cortisol on sexual behavior in the ewe. Therefore, we tested the hypothesis that both psychosocial stress and stress-like levels of cortisol will reduce the level of attractivity, proceptivity and receptivity in addition to suppressing LH secretion in the ewe. In Experiment 1, a layered stress paradigm of psychosocial stress was used, consisting of isolation for 4 h with the addition of restraint, blindfold and noise of a barking dog (predator stress) at hourly intervals. This stress paradigm reduced LH pulse amplitude in ovariectomized ewes. In Experiment 2, ovariectomized ewes were artificially induced into estrus with progesterone and estradiol benzoate treatment and the layered stress paradigm was applied. LH was measured and sexual behavior was assessed using T-mazes and mating tests. Stress reduced pulsatile LH secretion, and also reduced attractivity and proceptivity of ewes but had no effect on receptivity. In Experiment 3, ewes artificially induced into estrus were infused with cortisol for 30 h. Cortisol elevated circulating plasma concentrations of cortisol, delayed the onset of estrus and resulted in increased circling behavior of ewes (i.e. moderate avoidance) during estrus and increased investigation and courtship from rams. There was no effect of cortisol on attractivity, proceptivity or receptivity during estrus. We conclude that psychosocial stress inhibits LH secretion, the ability of ewes to attract rams (attractivity) and the motivation of ewes to seek rams and initiate mating (proceptivity), but cortisol is unlikely to be the principal mediator of these effects.

Testosterone manipulation post-castration does not alter cloacal gland growth differences in male quail selected for divergent plasma corticosterone stress response

Japanese quail selected for reduced (low-stress, LS) rather than exaggerated (high-stress, HS) plasma corticosterone response to brief restraint have consistently shown greater cloacal gland (CG) development, an androgen-dependent trait. In this study, the effects of testosterone implants on levels of plasma testosterone and CG development in castrated LS and HS quail were determined. Stress-line males were castrated and randomly allocated to 1 of 3 testosterone treatments: the empty testosterone (ET), low testosterone (LT), or high testosterone (HT) implant group. Cloacal gland volume was determined at 4 weekly intervals that represented ranges of 1 to 9 d, 8 to 17 d, 15 to 24 d, and 22 to 31 d after castration and testosterone implantation. Levels of plasma testosterone were also assessed at the end of the study. Development of the CG was affected by quail line (LS > HS), testosterone treatment (HT > LT > ET), and time of measurement (1 to 9 d < 8 to 17 d < 15 to 24 d = 22 to 31 d after castration and testosterone implantation). A significant interaction between testosterone treatment and time of measurement on CG volume was also detected (with CG volume generally increasing with time in LT- and HT-treated quail, but not in ET-treated quail). However, even though HT implant treatments induced higher CG development than did LT treatments beyond the first interval of CG volume measurement, and despite the finding of greater CG volumes in LS than HS quail during the last 2 measurement intervals within each of the LT and HT groups, no interaction was observed between testosterone implant dosages and quail stress line on CG volume. Thus, by the end of the study, regardless of testosterone dose, CG volume was consistently greater in LS quail than in their HS counterparts. In addition, although, as expected, the testosterone implant treatment significantly altered levels of plasma testosterone (HT > LT > ET), neither quail line nor its interaction with testosterone treatment affected plasma testosterone. The present findings suggest that the often-observed depressed CG development in the HS line may be independent of testosterone effects

Chronic stress alters the density and morphology of microglia in a subset of stress-responsive brain regions

The current study, in parallel experiments, evaluated the impact of chronic psychological stress on physiological and behavioural measures, and on the activation status of microglia in 15 stress-responsive brain regions. Rats were subjected, for 14 days, to two 30 min sessions of restraint per day, applied at random times each day. In one experiment the effects of stress on sucrose preference, weight gain, core body temperature, and struggling behaviour during restraint, were determined. In the second experiment we used immunohistochemistry to investigate stress-induced changes in ionized calcium-binding adaptor molecule-1 (Iba1), a marker constitutively expressed by microglia, and major histocompatibility complex-II (MHC-II), a marker often expressed on activated microglia, in a total of 15 stress-responsive nuclei. We also investigated cellular proliferation in these regions using Ki67 immunolabelling, to check for the possibility of microglial proliferation. Collectively, the results we obtained showed that chronic stress induced a significant increase in anhedonia, a decrease in weight gain across the entire observation period, a significant elevation in core body temperature during restraint, and a progressive decrease in struggling behaviour within and over sessions. With regard to microglial activation, chronic stress induced a significant increase in the density of Iba1 immunolabelling (nine of 15 regions) and the number of Iba1-positive cells (eight of 15 regions). Within the regions that exhibited an increased number of Iba1-positive cells after chronic stress, we found no evidence of a between group difference in the number of MHC-II or Ki67 positive cells. In summary, these results clearly demonstrate that chronic stress selectively increases the number of microglia in certain stress-sensitive brain regions, and also causes a marked transition of microglia from a ramified-resting state to a non-resting state. These findings are consistent with the view that microglial activation could play an important role in controlling and/or adapting to stress.