Chronic Adolescent Restraint Stress Downregulates miRNA-200a Expression in Male and Female C57BL/6J and BALB/cJ Mice.

Adolescence is a critical developmental period when the brain is plastic, and stress exposure can have lasting physiological consequences. One mechanism through which adolescent stress may have lasting effects is by altering microRNAs (miRNAs), leading to wide-scale gene expression changes. Three prior independent studies used unbiased approaches (RNA sequencing or microarray) to identify miRNAs differentially expressed by chronic variable stress in male rodents. In all three studies, miRNA-200a was differentially expressed in areas of the brain associated with emotion regulation. The current study extends this research to determine if chronic non-variable adolescent stress downregulates miRNA-200a expression by looking at two strains (BALB/cJ and C57BL/6J) of male and female mice. We utilized a 14-day (2 h/day) restraint stress protocol and verified stress effects on adolescent body weight gain and circulating corticosterone concentrations relative to non-restraint controls. Mice were then left undisturbed until they were euthanized in adulthood, at which time brains were collected to measure miRNA-200a in the ventral hippocampus. Three weeks after adolescent stress ended, differences in body weight between groups were no longer significant; however, animals exposed to stress had less miRNA-200a expression in the ventral hippocampus than control animals. These data implicate miRNA-200a expression as a potential mechanism by which adolescent stress can have persistent impacts on multiple outcomes in both male and female mice.

Effects of limited bedding and nesting on postpartum mood state in rats.

This study examined the effect of limited bedding and nesting (LBN) stress on postpartum anhedonia, maternal behaviors, anxiety-like behaviors, and neuroendocrine and neuroimmune function as a potential model of postpartum depression. Dams underwent sucrose preference tests prior to breeding, during gestation and again postpartum, to examine the potential onset of anhedonia. On embryonic day 19, dams were placed into either a LBN or control housing condition. Contrary to our predictions, LBN stress had no effect on postpartum sucrose preference. We also found no effect of LBN condition on fecal estradiol or corticosterone levels, both of which increased at birth and decreased postpartum. Regardless of housing conditions, approximately 40% of new mothers exhibited a decrease in sucrose preference, while others show no change, suggesting an individual susceptibility to postpartum anhedonia. In a separate cohort of LBN and control dams, we measured pup retrieval, hoarding behavior, elevated plus maze (EPM), and marble burying. LBN dams exhibited increased anxiety, associated with decreased time spent in the open arms of the EPM. We also measured a significant increase in IL-6 expression in the dorsal hippocampus and medial prefrontal cortex of postpartum dams compared to nonpregnant dams. These findings suggest that while LBN stress has effects on anxiety and maternal care, it does not induce postpartum anhedonia. Rather, there are inherent differences in susceptibility to anhedonia in individual dams, and future studies should be conducted to better understand individual vulnerability and resilience to postpartum anhedonia.

The effect of stress on opioid addiction-related behaviors: A review of preclinical literature.

Opioid misuse is a critical public health crisis in the United States that results in over 50,000 deaths per year and a substantial economic burden to society. Human epidemiological data suggest that exposure to stress is one of many risk factors for opioid misuse; however, opioid abusers tend to have multiple risk factors and use other drugs in addition to opioids. To identify causal mechanisms by which stress may increase risk, preclinical animal experiments provide a means to conduct experimental manipulations and maintain precise controls over environmental and drug exposures. The current review examines how stressful experiences alter opioid addiction-related behaviors in animal models, with a focus on how age of stress exposure affects drug outcomes. The findings summarized here suggest that neonatal or adult stress increase behaviors indicative of opioid intake and reward in rodent models, but that adolescent social stress may protect against later opioid addiction-related behaviors, which contradicts human epidemiological literature. We highlight three important areas to consider across this body of literature: the species and/or strain used, stressor type, and inclusion of both sexes. Finally, we suggest areas where additional research is warranted. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Chronic Sleep Deprivation Blocks Voluntary Morphine Consumption but Not Conditioned Place Preference in Mice.

The opioid epidemic remains a significant healthcare problem and is attributable to over 100,000 deaths per year. Poor sleep increases sensitivity to pain, impulsivity, inattention, and negative affect, all of which might perpetuate drug use. Opioid users have disrupted sleep during drug use and withdrawal and report poor sleep as a reason for relapse. However, preclinical studies investigating the relationship between sleep loss and substance use and the associated underlying neurobiological mechanisms of potential interactions are lacking. One of the most common forms of sleep loss in modern society is chronic short sleep (CSS) (<7 h/nightly for adults). Here, we used an established model of CSS to investigate the influence of disrupted sleep on opioid reward in male mice. The CSS paradigm did not increase corticosterone levels or depressive-like behavior after a single sleep deprivation session but did increase expression of Iba1, which typically reflects microglial activation, in the hypothalamus after 4 weeks of CSS. Rested control mice developed a morphine preference in a 2-bottle choice test, while mice exposed to CSS did not develop a morphine preference. Both groups demonstrated morphine conditioned place preference (mCPP), but there were no differences in conditioned preference between rested and CSS mice. Taken together, our results show that recovery sleep after chronic sleep disruption lessens voluntary opioid intake, without impacting conditioned reward associated with morphine.

A physiological profile approach to animal temperament: How to understand the functional significance of individual differences in behaviour.

Animal behaviour research has experienced a renewed interest in consistent individual differences (i.e. animal personality or temperament). Recent ecological studies have identified environmental conditions that give rise to the development and evolution of temperaments and to fitness-related outcomes of temperament. Additional literature has also described relationships between temperaments and physiological regulation. However, one-to-one relationships between one behavioural trait and one physiological system do not account for co-selection of behavioural and physiological traits, nor the complex signalling among physiological systems. In the current paper, we review the literature on multiple physiological processes associated with temperament, propose temperament-specific physiological profiles, and focus on next steps to understand the functional significance, evolution and maintenance of temperaments. We propose that to understand causes and consequences of temperament we need to characterize integrative physiological profiles associated with different temperaments.

The impact of adolescent stress on nicotine use and affective disorders in rodent models.

Recent findings indicate that stress exposure during adolescence contributes to the development of both nicotine use and affective disorders, suggesting a potential shared biological pathway. One key system that may mediate the association between adolescent stress and nicotine or affective outcomes is the hypothalamic-pituitary-adrenal (HPA) axis. Here we reviewed evidence regarding the effects of adolescent stress on nicotine responses and affective phenotypes and the role of the HPA-axis in these relationships. Literature indicates that stress, possibly via HPA-axis dysfunction, is a risk factor for both nicotine use and affective disorders. In rodent models, adolescent stress modulates behavioural responses to nicotine and increases the likelihood of affective disorders. The exact role that the HPA-axis plays in altering nicotine sensitivity and affective disorder development after adolescent stress remains unclear. However, it appears likely that adolescent stress-induced nicotine use and affective disorders are precipitated by repetitive activation of a hyperactive HPA-axis. Together, these preclinical studies indicate that adolescent stress is a risk factor for nicotine use and anxiety/depression phenotypes. The findings summarized here suggest that the HPA-axis mediates this relationship. Future studies that pharmacologically manipulate the HPA-axis during and after adolescent stress are critical to elucidate the exact role that the HPA-axis plays in the development of nicotine use and affective disorders following adolescent stress.

Next Steps in Integrative Biology: Mapping Interactive Processes Across Levels of Biological Organization.

Emergent biological processes result from complex interactions within and across levels of biological organization, ranging from molecular to environmental dynamics. Powerful theories, database tools, and modeling methods have been designed to characterize network connections within levels, such as those among genes, proteins, biochemicals, cells, organisms, and species. Here, we propose that developing integrative models of organismal function in complex environments can be facilitated by taking advantage of these methods to identify key nodes of communication across levels of organization. Mapping key drivers or connections among levels of organization will provide data and leverage to model potential rule-sets by which organisms respond and adjust to perturbations at any level of biological organization.

Adolescent Stress Reduces Adult Morphine-Induced Behavioral Sensitization in C57BL/6J Mice.

Deaths related to opioid use have skyrocketed in the United States, leading to a public health epidemic. Research has shown that both biological (genes) and environmental (stress) precursors are linked to opioid use. In particular, stress during adolescence-a critical period of frontal lobe development-influences the likelihood of abusing drugs. However, little is known about the biological mechanisms through which adolescent stress leads to long-term risk of opioid use, or whether genetic background moderates this response. Male and female C57BL/6J and BALB/cJ mice were exposed to chronic variable social stress (CVSS) or control conditions throughout adolescence and then tested for morphine locomotor sensitization or morphine consumption in adulthood. To examine possible mechanisms that underlie stress-induced changes in morphine behaviors, we assessed physiological changes in response to acute stress exposure and prefrontal cortex (PFC) miRNA gene expression. Adolescent stress did not influence morphine sensitization or consumption in BALB/cJ animals, and there was limited evidence of stress effects in female C57BL/6J mice. In contrast, male C57BL/6J mice exposed to adolescent CVSS had blunted morphine sensitization compared to control animals; no differences were observed in the acute locomotor response to morphine administration or morphine consumption. Physiologically, C57BL/6J mice exposed to CVSS had an attenuated corticosterone recovery following an acute stressor and downregulation of twelve miRNA in the PFC compared to control mice. The specificity of the effects for C57BL/6J vs. BALB/cJ mice provides evidence of a gene-environment interaction influencing opioid behaviors. However, this conclusion is dampened by limited locomotor sensitization observed in BALB/cJ mice. It remains possible that results may differ to other doses of morphine or other behavioral responses. Long-term differences in stress reactivity or miRNA expression in C57BL/6J mice suggests two possible biological mechanisms to evaluate in future research.

Gaps to Address in Ecological Studies of Temperament and Physiology.

Ecology is a diverse field with many researchers interested in drivers and consequences of variability within populations. Two aspects of variability that have been addressed are behavioral and physiological. While these have been shown to separately influence ecological outcomes such as survival, reproductive success, and fitness, combined they could better predict within-population variability in survival and fitness. Recently there has been a focus on potential fitness outcomes of consistent behavioral traits that are referred to as personality or temperament (e.g., boldness, sociability, and exploration). Given this recent focus, it is an optimal time to identify areas to supplement in this field, particularly in determining the relationship between temperament and physiological traits. To maximize progress, in this perspective paper, we propose that the following two areas be addressed: (1) increased diversity of species and (2) increased number of physiological processes studied, with an eye toward using more representative and relatively consistent measures across studies. We first highlight information that has been gleaned from species that are frequently studied to determine how animal personality relates to physiology and/or survival/fitness. We then shine a spotlight on important taxa that have been understudied and that can contribute meaningful, complementary information to this area of research. And last, we propose a brief array of physiological processes to relate to temperament, and that can significantly impact fitness, and that may be accessible in field studies.

Inhaled corticosteroids as treatment for adolescent asthma: effects on adult anxiety-related outcomes in a murine model.

RATIONALE: Allergic asthma, typically controlled with inhaled corticosteroids (ICS), is the leading chronic health condition for youth under 18 years of age. During this peri-adolescent period, significant brain maturation occurs. Prior studies indicate that both chronic inflammation and corticosteroid medications increase risk for developing an internalizing disorder like anxiety. OBJECTIVES: To determine if chronic ICS treatments exacerbate or alleviate anxiety symptoms associated with developmental allergic asthma, we used a mouse model to isolate the influence of ICS (fluticasone propionate, FLU) vs. airway inflammation (induced with house dust mite extract, HDM). METHODS: During development, male and female BALB/cJ mice were repeatedly exposed to HDM or saline plus one of four FLU doses (none/vehicle, low, moderate, or high). In adulthood, we assessed lung inflammation, circulating and excreted corticosteroids, anxiety-like behavior, and gene expression in stress and emotion regulation brain regions. RESULTS: FLU treatment decreased body weight and anxiety-like behavior and increased fecal corticosterone metabolite concentrations and Crhr2 gene expression in ventral hippocampus. FLU effects were only observed in saline/non-HDM-exposed mice, and the FLU doses used did not significantly decrease HDM-induced airway inflammation. Females had greater serum and fecal corticosterone concentrations, less anxiety-like behavior, and lower Crhr1 gene expression in ventral hippocampus and prefrontal cortex than males. CONCLUSIONS: These findings suggest that steroid medications for youth with allergic asthma may not exacerbate anxiety-related symptoms, and that they should be avoided in children/adolescents without a health condition. The results are informative to future work on the use of corticosteroid medications during childhood or adolescent development.

Peri-adolescent asthma: Acute impacts on innate immune response, corticosterone, and microglia in mice.

Asthma is highly comorbid with anxiety in youth. We investigated the hypothalamic-pituitary-adrenal (HPA) axis and microglia as mechanisms underlying asthma and anxiety comorbidity. We induced asthma symptoms in developing BALB/cJ mice with house dust mite (HDM) for airway inflammation and methacholine (MCH) for bronchoconstriction. On the last day of exposure, we analyzed samples at six timepoints. Lung IL-5 and IL-1ß expression peaked 4 h after final HDM exposure. Circulating corticosterone was blunted in a sex- and treatment-specific temporal pattern. Hippocampal IL-1ß expression and microglial area were marginally increased 24 h after MCH exposure. These results provide a foundation for further work investigating asthma-anxiety mechanisms.

Divergent immune responses in behaviorally-inhibited vs. non-inhibited male rats.

Stable behavioral traits (temperament, personality) often predict health outcomes. Temperament-specific differences in immune function could explain temperament-specific health outcomes, however, we have limited information on whether immune function varies by personality. In the present study, we examined the relationship between a basic behavioral trait (behavioral-inhibition vs. non-inhibition) and two immune responses (innate inflammation and delayed-type hypersensitivity, DTH) in a rodent model. In humans, behavioral inhibition (fearful temperament) is associated with altered stress physiology and allergies. In laboratory rats, the trait is associated with elevated glucocorticoid production. We hypothesized that behavioral inhibition is associated with glucocorticoid resistance and dampened T-helper 1 cell responses often associated with chronic stress and allergies. Further, this immune profile would predict poorly-regulated innate inflammation and dampened DTH. In male Sprague-Dawley rats, we quantified consistent behavioral phenotypes by measuring latency to contact two kinds of novelty (object vs. social), then measured lipopolysaccharide(LPS)-induced innate inflammation or keyhole limpet hemocyanin(KLH)-induced DTH. Behaviorally-inhibited rats had heightened glucocorticoid and interleukin-6 responses to a low/moderate dose of LPS and reduced DTH swelling to KLH re-exposure compared to non-inhibited rats. These results suggest that behavioral inhibition is associated with a glucocorticoid resistant state with poorly regulated innate inflammation and dampened cell-mediated immune responses. This immune profile may be associated with exaggerated T-helper 2 responses, which could set the stage for an allergic/asthmatic/atopic predisposition in inhibited individuals. Human and animal models of temperament-specific immune responses represent an area for further exploration of mechanisms involved in individual differences in health.

Individual differences in glucocorticoid regulation: Does it relate to disease risk and resilience?

Glucocorticoid (GC) signaling varies among individuals, and this variation may relate to individual differences in health outcomes. To determine if and which aspects of signaling (basal, circadian, integrative, or reactivity) are associated with specific health outcomes, we reviewed recent studies that relate GCs to health outcomes. We identified papers through PubMed and reviewed 100 original research articles related to mental health, cardiovascular health, cancer, diabetes, obesity, pulmonary health, sleep, and fitness. Many studies reported elevated GC secretion associated with worse health, but this was only particularly true for integrative GC measures. On the other hand, accentuated cortisol awakening response and a steeper circadian rhythm were both associated with positive health outcomes. Overall, relationships between GC secretion and health outcomes were relatively weak. This systematic review of relationships between GC metrics and health outcomes highlights the importance of careful consideration when selecting methods to measure GC regulation in health research.

Genetic Factors Mediate the Impact of Chronic Stress and Subsequent Response to Novel Acute Stress.

Individual differences in physiological and biobehavioral adaptation to chronic stress are important predictors of health and fitness; genetic differences play an important role in this adaptation. To identify these differences we measured the biometric, neuroendocrine, and transcriptional response to stress among inbred mouse strains with varying degrees of genetic similarity, C57BL/6J (B), C57BL/6NJ (N), and DBA/2J (D). The B and D strains are highly genetically diverse whereas the B and N substrains are highly similar. Strain differences in hypothalamic-pituitary-adrenal (HPA) axis cross-sensitization were determined by plasma corticosterone (CORT) levels and hippocampal gene expression following 7-weeks of chronic mild stress (CMS) or normal housing (NH) and subsequent exposure to novel acute restraint. Fecal CORT metabolites and body and organ weights were also measured. All strains exposed to CMS had reduced heart weights, whereas body weight gain was attenuated only in B and N strains. Acute stress alone produced larger plasma CORT responses in the D and N strains compared to the B strain. CMS paired with acute stress produced cross-sensitization of the CORT response in the N strain. The N strain also had the largest number of hippocampal transcripts with up-regulated expression in response to stress. In contrast, the D strain had the largest number of transcripts with down-regulated expression following CMS and acute stress. In summary, we observed differential responses to CMS at both the physiological and molecular level among genetically diverse strains, indicating that genetic factors drive individual differences in experience-dependent regulation of the stress response.

Impact of Genetic Variation on Stress-Related Ethanol Consumption.

BACKGROUND: The effect of stress on alcohol consumption in humans is highly variable, and the underlying processes are not yet understood. Attempts to model a positive relationship between stress and increased ethanol (EtOH) consumption in animals have been only modestly successful. Our hypothesis is that individual differences in stress effects on EtOH consumption are mediated by genetics. METHODS: We measured alcohol consumption, using the drinking-in-the-dark (DID) paradigm in females from 2 inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2), and 35 of their inbred progeny (the BXD family). A control group was maintained in normal housing and a stress group was exposed to chronic mild stress (CMS), consisting of unpredictable stressors over 7 weeks. These included predator, social, and environmental perturbations. Alcohol intake was measured over 16 weeks in both groups during baseline (preceding 5-week period), CMS (intervening 7-week period), and post-CMS (final 4-week period). RESULTS: We detected a strong effect of CMS on alcohol intake. A few strains demonstrated CMS-related increased alcohol consumption; however, most showed decreased intake. We identified 1 nearly significant quantitative trait locus on chromosome 5 that contains the neuronal nitric oxide synthase gene (Nos1). The expression of Nos1 is frequently changed following alcohol exposure, and variants in this gene segregating among the BXD population may modulate alcohol intake in response to stress. CONCLUSIONS: The results we present here represent the first study to combine chronic stress and alcohol consumption in a genetic reference population of mice. Differences in susceptibility to the effects of stressful environments vis-à-vis alcohol use disorders would suggest that the differences have at least some basis in genetic constitution. We have also nominated a likely candidate gene underlying the large individual differences in effects of stress on alcohol consumption.

Population history with invasive predators predicts innate immune function response to early-life glucocorticoid exposure in lizards.

Early-life stress can suppress immune function, but it is unclear whether transgenerational stress exposure modulates the immune consequences of early stress. In populations where, historically, the immune system is frequently activated, e.g. persistent stressors that cause injury, it may be maladaptive to suppress immune function after early-life stress. Thus, the relationship between early-life stress and immune function may vary with population-level historical stressor exposure. We collected gravid fence lizards (Sceloporus undulatus) from populations that naturally differ in long-term exposure to invasive fire ants (Solenopsis invicta). We manipulated early-life stress in the resulting offspring via weekly exposure to fire ants, application of the stress-relevant hormone corticosterone or control treatment from 2 to 43 weeks of age. We quantified adult immune function in these offspring with baseline and antigen-induced hemagglutination and plasma bacterial killing ability. Early-life corticosterone exposure suppressed baseline hemagglutination in offspring of lizards from populations not exposed to fire ants but enhanced hemagglutination in those from populations that were exposed to fire ants. This enhancement may prepare lizards for high rates of wounding, toxin exposure and infection associated with fire ant attack. Adult bacterial killing ability and hemagglutination were not affected by early-life exposure to fire ants, but the latter was higher in offspring of lizards from invaded sites. A population's history of persistent stress may thus alter individual long-term immunological responses to early-life stressors. Further consideration of historical stressor exposure (type and duration) may be important to better understand how early-life stressors affect adult physiology.

Genetic Contribution to Initial and Progressive Alcohol Intake Among Recombinant Inbred Strains of Mice.

We profiled individual differences in alcohol consumption upon initial exposure and during 5 weeks of voluntary alcohol intake in female mice from 39 BXD recombinant inbred strains and parents using the drinking in the dark (DID) method. In this paradigm, a single bottle of 20% (v/v) alcohol was presented as the sole liquid source for 2 or 4 h starting 3 h into the dark cycle. For 3 consecutive days mice had access to alcohol for 2 h followed by a 4th day of 4 h access and 3 intervening days where alcohol was not offered. We followed this regime for 5 weeks. For most strains, 2 or 4 h alcohol intake increased over the 5-week period, with some strains demonstrating greatly increased intake. There was considerable and heritable genetic variation in alcohol consumption upon initial early and sustained weekly exposure. Two different mapping algorithms were used to identify QTLs associated with alcohol intake and only QTLs detected by both methods were considered further. Multiple suggestive QTLs for alcohol intake on chromosomes (Chrs) 2, 6, and 12 were identified for the first 4 h exposure. Suggestive QTLs for sustained intake during later weeks were identified on Chrs 4 and 8. Thirty high priority candidate genes, including Entpd2, Per3, and Fto were nominated for early and sustained alcohol intake QTLs. In addition, a suggestive QTL on Chr 15 was detected for change in 2 h alcohol intake over the duration of the study and Adcy8 was identified as a strong candidate gene. Bioinformatic analyses revealed that early and sustained alcohol intake is likely driven by genes and pathways involved in signaling, and/or immune and metabolic function, while a combination of epigenetic factors related to alcohol experience and genetic factors likely drives progressive alcohol intake.

Asthma Induction During Development and Adult Lung Function, Behavior and Brain Gene Expression.

In developing youth, allergic asthma is the most common chronic condition, with 9%-10% of youth affected. Asthma onset during childhood and adolescence is further associated with other health issues, particularly psychiatric conditions. To understand causal mechanisms by which developmental asthma may lead to altered behavior, brain and health trajectories, we developed a mouse model of developmental allergic asthma. In the current study, we tested for potential long-term effects of developmental asthma on adult lung function and behavior and brain gene expression associated with emotion and stress regulation. We manipulated airway inflammation (AI) and methacholine (MCH)-induced bronchospasm (resulting in labored breathing, LB) in young male and female BALB/cJ mice and measured adult outcomes 3 months after final asthma manipulations. Results indicated that allergen exposure, used to cause AI, and which ended on post-natal day 56 (P56), led to persistent lung AI, mucus buildup and gene expression related to allergic asthma 3 months after final allergen exposure. In addition, at this same age, early allergen exposure led to altered brain gene expression related to stress regulation (prefrontal corticotropin releasing hormone receptor 1, Crhr1 and hippocampal glucocorticoid receptor, GR) and serotonin function (brainstem serotonin transporter, SERT). On the other hand, LB events during development led to altered anxiety-related behavior. Importantly, sex and pre-asthma fear-related behavior (ultrasonic vocalization, USV rates) modulated these adult outcomes. Asthma that develops during childhood/adolescence may have long-term impacts on emotion and stress regulation mechanisms, and these influences may be moderated by sex and pre-asthma temperament.

Timing matters: the interval between acute stressors within chronic mild stress modifies behavioral and physiologic stress responses in male rats.

Chronic mild stress can lead to negative health outcomes. Frequency, duration, and intensity of acute stressors can affect health-related processes. We tested whether the temporal pattern of daily acute stressors (clustered or dispersed across the day) affects depression-related physiology. We used a rodent model to keep stressor frequency, duration, and intensity constant, and experimentally manipulated the temporal pattern of acute stressors delivered during the active phase of the day. Adult male Sprague-Dawley rats were exposed to one of three chronic mild stress groups: Clustered: stressors that occurred within 1 hour of each other (n = 21), Dispersed: stressors that were spread out across the active phase (n = 21), and Control: no stressors presented (n = 21). Acute mild stressors included noise, strobe lights, novel cage, cage tilt, wet bedding, and water immersion. Depression-related outcomes included: sucrose preference, body weight, circulating glucocorticoid (corticosterone) concentration after a novel acute stressor and during basal morning and evening times, and endotoxin-induced circulating interleukin-6 concentrations. Compared to control rats, those in the Clustered group gained less weight, consumed less sucrose, had a blunted acute corticosterone response, and an accentuated acute interleukin-6 response. Rats in the Dispersed group had an attenuated corticosterone decline during the active period and after an acute stressor compared to the Control group. During a chronic mild stress experience, the temporal distribution of daily acute stressors affected health-related physiologic processes. Regular exposure to daily stressors in rapid succession may predict more depression-related symptoms, whereas exposure to stressors dispersed throughout the day may predict diminished glucocorticoid negative feedback.