Chronic Variable Stress Paradigm Research Articles

RGS4 Actions in Mouse Prefrontal Cortex Modulate Behavioral and Transcriptomic Responses to Chronic Stress and Ketamine.

The signal transduction protein, regulator of G protein signaling 4 (RGS4), plays a prominent role in physiologic and pharmacological responses by controlling multiple intracellular pathways. Our earlier work identified the dynamic but distinct roles of RGS4 in the efficacy of monoamine-targeting versus fast-acting antidepressants. Using a modified chronic variable stress (CVS) paradigm in mice, we demonstrate that stress-induced behavioral abnormalities are associated with the downregulation of RGS4 in the medial prefrontal cortex (mPFC). Knockout of RGS4 (RGS4KO) increases susceptibility to CVS, as mutant mice develop behavioral abnormalities as early as 2 weeks after CVS resting-state functional magnetic resonance imaging I (rs-fMRI) experiments indicate that stress susceptibility in RGS4KO mice is associated with changes in connectivity between the mediodorsal thalamus (MD-THL) and the mPFC. Notably, RGS4KO also paradoxically enhances the antidepressant efficacy of ketamine in the CVS paradigm. RNA-sequencing analysis of naive and CVS samples obtained from mPFC reveals that RGS4KO triggers unique gene expression signatures and affects several intracellular pathways associated with human major depressive disorder. Our analysis suggests that ketamine treatment in the RGS4KO group triggers changes in pathways implicated in synaptic activity and responses to stress, including pathways associated with axonal guidance and myelination. Overall, we show that reducing RGS4 activity triggers unique gene expression adaptations that contribute to chronic stress disorders and that RGS4 is a negative modulator of ketamine actions. SIGNIFICANCE STATEMENT: Chronic stress promotes robust maladaptation in the brain, but the exact intracellular pathways contributing to stress vulnerability and mood disorders have not been thoroughly investigated. In this study, the authors used murine models of chronic stress and multiple methodologies to demonstrate the critical role of the signal transduction modulator regulator of G protein signaling 4 in the medial prefrontal cortex in vulnerability to chronic stress and the efficacy of the fast-acting antidepressant ketamine.

Repeated neonatal isoflurane exposure facilitated stress-related fear extinction impairment in male mice and was associated with ΔFosB accumulation in the basolateral amygdala and the hippocampal dentate gyrus

Volatile anesthetics elicit neurodevelopmental toxicity in rodents and primates and lead to more exaggerated anxiety-like behavior in response to future stress. Anxiety and fear are closely correlated and maladaptive fear-associated learning is regarded as the core mechanism underlying anxiety-related disorders. However, little is known about the interaction between early-life anesthetic exposure and future stress and the accompanying effect on fear-associated learning. In the present study, we evaluated the alterations in fear-associated learning (fear acquisition and extinction) occurring in mice receiving repeated neonatal isoflurane exposure and chronic variable stress (CVS) successively through a series of fear conditioning, fear reinforcing, and fear extinction paradigms. The corticosterone (CORT) response during CVS and the immunohistochemical levels of ΔFosB and c-Fos expression in the basolateral amygdala (BLA) and the hippocampal dentate gyrus (DG) after the extinction retrieval test were also investigated. The results showed that neonatal isoflurane exposure could increase CORT levels following the first diurnal CVS procedure, but not after completion of the whole CVS paradigm. Neonatal isoflurane exposure exerted a repressive effect on fear acquisition, in contrast to that seen with CVS. Neonatal isoflurane exposure and CVS both exerted suppressive effects on fear extinction and there was a significant synergy between them. Furthermore, neonatal isoflurane exposure facilitated CVS-mediated ΔFosB accumulation in the BLA and the hippocampal DG, which may have been responsible for c-Fos expression deficits and fear extinction impairment. Collectively, these findings contribute to the understanding of the interaction between early-life anesthetic exposure and future stress, as well as the accompanying behavioral alterations.

Repeated Neonatal Isoflurane Exposure is Associated with Higher Susceptibility to Chronic Variable Stress-induced Behavioural and Neuro-inflammatory Alterations

Numerous studies have reported that prolonged or multiple exposures to anaesthetics in early life lead to detrimental effects on brain function, most having focused on neurocognitive function, and relatively few on long term neuropsychiatric performance. The present study investigated the impact of repeated neonatal isoflurane exposure on chronic variable stress (CVS)-induced psychiatric and behavioural outcomes together with CVS-related neuronal activity and neuro-inflammatory reactivity in relevant brain circuits. In the present study, C57BL/6J mice received either three exposures to isoflurane at postnatal days 7, 8, and 9 or a control exposure. From postnatal day 45, mice were exposed to a mild, 3-week, CVS paradigm or none and the CVS-related neuropsychiatric performance was evaluated using a series of behavioural tests. The neuronal activity in relevant brain regions was measured by ΔFosB immunopositivity and CVS-related neuroinflammation was assessed by analysing levels of pro-inflammatory cytokines IL-1α, IL-1β, IL-6, and TNF-α. In mice experiencing serial neonatal isoflurane exposure, we detected a significant enhancement in anxiety levels following CVS procedures, together with enhanced neuronal activity, and exacerbated neuroinflammation in the basolateral amygdaloid nuclei (BLA) and hippocampal dentate gyrus (DG) regions. No such change was found in control mice. These results indicate an association between early multiple isoflurane exposures in infant mice and susceptibility to a CVS-evoked anxious phenotype accompanied by enhanced neuronal activity in BLA and DG regions and high inflammatory reactivity in response to CVS.

Sex-dependent effects of chronic variable stress on discrete corticotropin-releasing factor receptor 1 cell populations

Anxiety and depression are strikingly more prevalent in women compared with men. Dysregulation of corticotropin-releasing factor (CRF) binding to its cognate receptor (CRFR1) is thought to play a critical role in the etiology of these disorders. In the present study, we investigated whether there were sex differences in the effects of chronic variable stress (CVS) on CRFR1 cells using CRFR1-GFP reporter mice experiencing a 9-day CVS paradigm. Brains were collected from CVS and stress naïve female and male mice following exposure to the open field test. This CVS paradigm effectively increased anxiety-like behavior in female and male mice. In addition, we assessed changes in activation of CRFR1 cells (co-localization with c-Fos and phosphorylated CREB (pCREB)) in stress associated brain structures, including two sexually dimorphic CRFR1 cell groups in the anteroventral periventricular nucleus (AVPV/PeN; F>M) and paraventricular hypothalamus (PVN; M>F). CVS increased CRFR1-GFP cell number as well as the number of CRFR1/pCREB co-expressing cells in the female but not male AVPV/PeN. In the PVN, the number of CRFR1/pCREB co-expressing cells was overall greater in males regardless of treatment and CVS resulted in a male-specific reduction of CRFR1/c-Fos cells. In addition, CVS induced a female-specific reduction in CRFR1/c-Fos cells within the anteroventral bed nucleus of the stria terminalis and both sexes exhibited a reduction in CRFR1/c-Fos co-expressing cells following CVS within the ventral basolateral amygdala. Overall, these sex-specific effects of CVS on CRFR1 populations may have implications for sex differences in stress-induction of mood disorders.

The Effects of Chronic Stress on Migraine Relevant Phenotypes in Male Mice.

Migraine is a disabling neurological disorder affecting 12% of the world’s population. Stress is a major reported trigger and exacerbator of migraine. We evaluated the effects of two chronic stress paradigms on migraine relevant phenotypes in male C57Bl/6 mice.Methods: Fifty six mice were used in a 14 day social defeat stress (SDS) and twenty three mice were used in a 40 day chronic variable stress (CVS) paradigm. Anxiety measures were evaluated using the open field and elevated plus maze (EPM) tests. Migraine relevant phenotypes were evaluated using the nitroglycerin (NTG) and cortical spreading depression (CSD) models.Results: Stress sensitive SDS mice and chronically stressed CVS mice showed decreased exploration in the open field and reduced time spent in the open arms of the EPM compared to controls. Stress sensitive and resilient SDS mice had increased serum corticosterone levels, and stressed mice in the CVS paradigm had decreased weight gain compared to controls, providing combined behavioral and physiological evidence of a stress response. In the CVS paradigm but not the SDS paradigm, the stressed group showed a significant decrease in baseline mechanical withdrawal threshold compared to controls. All groups showed a significant reduction in withdrawal threshold after treatment with NTG, but the reduction was not larger in SDS or CVS than in controls. Interestingly, stress resilient SDS mice showed a rapid recovery from NTG effects that was not seen in other groups. No difference in CSD frequency or velocity was seen between stress and control mice in either stress paradigms.Conclusion: We observed distinct effects of stress on generalized pain response, migraine relevant pain, and migraine relevant excitability. CVS but not SDS was associated with a reduced mechanical withdrawal threshold, consistent with a generalized pain response to chronic stress. Neither SDS nor CVS exacerbated phenotypes considered specifically relevant to migraine - withdrawal to NTG, and susceptibility to CSD. However, the significantly reduced response of stress resilient mice to the NTG stimulus may represent a specific migraine-resistant phenotype.

Higher susceptibility of somatostatin 4 receptor gene-deleted mice to chronic stress-induced behavioral and neuroendocrine alterations

The somatostatin 4 receptor (sst4) is widely expressed in stress-related brain areas (e.g. hippocampus, amygdala) and regulates the emotional behavior in acute situations. Since its importance in chronic stress-induced complex pathophysiological alterations is unknown, we investigated the involvement of sst4 in the responsiveness to chronic variable stress (CVS). Sstr4 gene-deficient (Sstr4−/−) mice and their wildtype counterparts (Sstr4+/+) were used to examine the behavioral and neuroendocrine alterations as well as chronic neuronal activity (FosB expression) changes in response to CVS. In Sstr4+/+ mice, there was no behavioral response to the applied CVS paradigm. In contrast, immobility time in the tail suspension test increased after the CVS in the knockouts. In the forced swim test, Sstr4−/− animals showed increased baseline immobility and then it decreased after the CVS. Light–dark box and open field test behaviors and sucrose preference did not respond to the stress in the knockouts. Adrenal weights increased and thymus weights decreased in both Sstr4+/+ and Sstr4−/− mice demonstrating the effect of chronic stress. The relative adrenal weight of stressed knockouts increased to a greater extent, while relative thymus and body weights decreased only in the Sstr4−/− mice. Basal plasma corticosterone concentrations did not change after the CVS in either genotype. FosB immunopositivity in the central and basolateral amygdaloid nuclei was enhanced in stressed knockouts, but not in wild types. This is the first evidence that sst4 activation is involved in the behavioral and neuroendocrine alterations induced by chronic stress with a crucial role of plastic changes in the amygdala.

A cafeteria diet modifies the response to chronic variable stress in rats

Stress is known to lead to metabolic and behavioral changes. The aim of this study was to investigate the effect of long‐term nutrient intake on the response to chronic variable stress (CVS). Adult Sprague‐Dawley rats were fed one of three diets for 6 weeks: high carbohydrate (HC), high fat (HF), or “Cafeteria” (CAF) (Standard HC and a choice of highly palatable cafeteria foods: chocolate, biscuits and peanut butter). After the first 3 weeks, half of the animals from each group were stressed daily using a CVS paradigm while the others (controls) were kept undisturbed. Rats were sacrificed at the end of the 6‐week period. Abdominal fat, serum lipids and corticosterone were measured. Results showed higher total cholesterol and LDL, and lower HDL associated with both chronic stress and the CAF diet; as well as an increase in body weight (P =0.03), food intake (P <0.001) and intra‐abdominal fat (P <0.001) in the CAF group compared to the other dietary groups. In addition, there was a significant interaction between stress and diet on serum corticosterone levels (P=0.012). More precisely, there was an increase in corticosterone levels of stressed rats compared to non‐stressed rats in the HC and HF groups but not in the CAF group. These results show that a highly palatable diet offering a choice of food items is associated with a reduction in the response to CVS and could validate a stressor‐induced preference for comfort food.

Enhanced fear recall and emotional arousal in rats recovering from chronic variable stress

Emergence of posttraumatic-like behaviors following chronic trauma is of interest given the rising prevalence of combat-related posttraumatic stress disorder (PTSD). Stress associated with combat usually involves chronic traumatization, composed of multiple, single episode events occurring in an unpredictable fashion. In this study, we investigated whether rats recovering from repeated trauma in the form of chronic variable stress (CVS) express posttraumatic stress-like behaviors and dysregulated neuroendocrine responses. Cohorts of Long–Evans rats underwent a 7 day CVS paradigm followed by behavioral and neuroendocrine testing during early (16 h post CVS) and delayed (7 day) recovery time points. A fear conditioning–extinction–reminder shock paradigm revealed that CVS induces exaggerated fear recall to reminder shock, suggestive of potentiated fear memory. Rats with CVS experience also expressed a delayed expression of fearful arousal under aversive context, however, social anxiety was not affected during post-CVS recovery. Persistent sensitization of the hypothalamic-pituitary-adrenocorticotropic response to a novel acute stressor was observed in CVS exposed rats. Collectively, our data are consistent with the constellation of symptoms associated with posttraumatic stress syndrome, such as re-experiencing, and arousal to fearful contexts. The CVS–recovery paradigm may be useful to simulate trauma outcomes following chronic traumatization that is often associated with repeated combat stress.

The Anteroventral Bed Nucleus of the Stria Terminalis Differentially Regulates Hypothalamic-Pituitary-Adrenocortical Axis Responses to Acute and Chronic Stress

The anteroventral region of the bed nucleus of the stria terminalis (BST) stimulates hypothalamic-pituitary-adrenocortical (HPA) axis responses to acute stress. However, the role of the anterior BST nuclei in chronic drive of the HPA axis has yet to be established. Therefore, this study tests the role of the anteroventral BST in physiological responses to chronic drive, using a chronic variable stress (CVS) model. Male Sprague-Dawley rats received either bilateral ibotenate lesions, targeting the anteroventral BST, or vehicle injection into the same region. Half of the lesion and control rats were exposed to a 14-d CVS paradigm consisting of twice-daily exposure to unpredictable, alternating stressors. The remaining rats were nonhandled control animals that remained in home cages. On the morning after the end of CVS exposure, all rats were exposed to a novel restraint stress challenge. CVS induced attenuated body weight gain, adrenal hypertrophy, thymic involution, and enhanced CRH mRNA in hypophysiotrophic neurons of the hypothalamic paraventricular nucleus, none of which were affected by anteroventral BST lesions. In the absence of CVS, lesions attenuated the plasma corticosterone and paraventricular nucleus c-fos mRNA responses to the acute restraint stress. In contrast, lesions of the anteroventral BST elevated plasma ACTH and corticosterone responses to novel restraint in the rats previously exposed to CVS. These data suggest that the anterior BST plays very different roles in integrating acute stimulation and chronic drive of the HPA axis, perhaps mediated by chronic stress-induced recruitment of distinct BST cell groups or functional reorganization of stress-integrative circuits.