Blocking Metabotropic Glutamate Receptor Subtype 7 via the Venus Flytrap Domain Promotes a Chronic Stress-Resilient Phenotype in Mice.

Chronic psychosocial stress participates prominently in the etiology of various psychiatric conditions and comorbid somatic pathologies; however, suitable pharmacotherapy of these disorders is still of high medical need. During the last few decades, research on mGlu receptors advanced remarkably and much attention was given to the mGlu7 subtype. Here, genetic mGlu7 ablation, short-term pharmacological mGlu7 blockade, as well as siRNA-mediated knockdown of mGlu7 were shown to result in an acute anti-stress, antidepressant- and anxiolytic-like phenotype in mice. Moreover, we recently revealed a prominent stress-protective effect of genetic mGlu7 ablation also with respect to chronic psychosocial stress. In addition, we are able to demonstrate in the present study that the chronic pharmacological blockade of mGlu7 interferes with various chronic stress-induced alterations. For this, we used the chronic subordinate colony housing (CSC), a mouse model of chronic male subordination, in combination with chronic treatment with the mGlu7-selective orthosteric-like antagonist XAP044 (7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one). Interestingly, XAP044 dose-dependently ameliorates hypothalamic-pituitary-adrenal axis dysfunctions, thymus atrophy, as well as the CSC-induced increase in innate anxiety. Taken together, our findings provide further evidence for the role of mGlu7 in chronic psychosocial stress-induced alterations and suggests the pharmacological blockade of mGlu7 as a promising therapeutic approach for the treatment of chronic stress-related pathologies in men.

Metabotropic glutamate receptor subtype 7 controls maternal care, maternal motivation and maternal aggression in mice.

The group III metabotropic glutamate receptor subtype 7 (mGlu7) is an important regulator of glutamatergic and GABAergic neurotransmission and known to mediate emotionality and male social behavior. However, a possible regulatory role in maternal behavior remains unknown to date. Adequate expression of maternal behavior is essential for successful rearing and healthy development of the young. By understanding genetic and neural mechanisms underlying this important prosocial behavior, we gain valuable insights into possible dysregulations. Using genetic ablation as well as pharmacological modulation, we studied various parameters of maternal behavior in two different mouse strains under the influence of mGlu7. We can clearly show a regulatory role of mGlu7 in maternal behavior. Naïve virgin female C57BL/6 mGlu7 knockout mice showed more often nursing postures and less spontaneous maternal aggression compared to their heterozygous and wildtype littermates. In lactating C57BL/6 wildtype mice, acute central activation of mGlu7 by the selective agonist AMN082 reduced arched back nursing and accelerated pup retrieval without affecting maternal aggression. In addition, in lactating CD1 wildtype mice the selective mGlu7 antagonist XAP044 increased both pup retrieval and maternal aggression. With respect to receptor expression levels, mGlu7 mRNA expression was higher in lactating vs virgin C57BL/6 mice in the prefrontal cortex, but not hypothalamus or hippocampus. In conclusion, these findings highlight a significant role of the mGlu7 receptor subtype in mediating maternal behavior in mice. Region-dependent studies are warranted to further extend our knowledge on the specific function of the brain glutamate system in maternal behavior.

Adrenal gland plasticity in lactating rats and mice is sufficient to maintain basal hypersecretion of corticosterone.

Increased basal glucocorticoid secretion and a reduced glucocorticoid response during acute stress, despite only minor changes in the secretion of the major secretagogue adrenocorticotropic hormone (ACTH), have been documented in the peripartum period in several species. We recently showed that the adrenal gland, the site of glucocorticoid synthesis, undergoes substantial postpartum-associated plasticity in the rat at mid-lactation. Here, we asked the question whether adrenal changes already take place around parturition in the rat and in another species, namely the mouse. After demonstrating that several components of the adrenal machinery mediating cholesterol supply for steroidogenesis, including protein levels of hormone-sensitive lipase, low-density lipoprotein receptor (LDLR) and scavenger receptor class-B type-1 (SRB1), are upregulated, while hydroxymethylglutaryl coenzyme A reductase (HMGCR) is downregulated in the lactating rat one day after delivery, as previously observed at mid-lactation, we demonstrated profound changes in the mouse. In detail, protein expression of LDLR, SRB1, HMGCR and adrenal lipid store density were increased in the mouse adrenal one day after parturition as tested via western blot analysis and oil-red lipid staining, respectively. Moreover, using in vitro culture techniques, we observed that isolated adrenal explants from lactating mice secreted higher levels of corticosterone under basal conditions, but showed impaired responsiveness to ACTH, mimicking the in vivo scenario. These results suggest that mechanisms of adaptation in the maternal adrenal after delivery, namely increased cholesterol availability and decreased ACTH sensitivity, are crucial for the basal increase in circulating glucocorticoids and maternal stress hyporesponsiveness that are typical of this period.

Individual differences in stress vulnerability: The role of gut pathobionts in stress-induced colitis.

Chronic subordinate colony housing (CSC), an established mouse model for chronic psychosocial stress, promotes a microbial signature of gut inflammation, characterized by expansion of Proteobacteria, specifically Helicobacter spp., in association with colitis development. However, whether the presence of Helicobacter spp. during CSC is critically required for colitis development is unknown. Notably, during previous CSC studies performed at Regensburg University (University 1), male specific-pathogen-free (SPF) CSC mice lived in continuous subordination to a physically present and Helicobacter spp.-positive resident. Therefore, it is likely that CSC mice were colonized, during the CSC procedure, with Helicobacter spp. originating from the dominant resident. In the present study we show that employing SPF CSC mice and Helicobacter spp.-free SPF residents at Ulm University (University 2), results in physiological responses that are typical of chronic psychosocial stress, including increased adrenal and decreased thymus weights, decreased adrenal in vitro adrenocorticotropic hormone (ACTH) responsiveness, and increased anxiety-related behavior. However, in contrast to previous studies that used Helicobacter spp.-positive resident mice, use of Helicobacter spp.-negative resident mice failed to induce spontaneous colitis in SPF CSC mice. Consistent with the hypothesis that the latter is due to a lack of Helicobacter spp. transmission from dominant residents to subordinate mice during the CSC procedure, colonization of SPF residents with Helicobacter typhlonius at University 2, prior to the start of the CSC model, rescued the colitis-inducing potential of CSC exposure. Furthermore, using SPF CSC mice and H. typhlonius-free SPF residents at University 1 prevented CSC-induced colitis. In summary, our data support the hypothesis that the presence or absence of exposure to certain pathobionts contributes to individual variability in susceptibility to stress-/trauma-associated pathologies and to reproducibility of stress-related outcomes between laboratories.

Blocking metabotropic glutamate receptor subtype 5 relieves maladaptive chronic stress consequences.

Etiology and pharmacotherapy of stress-related psychiatric conditions and somatoform disorders are areas of high unmet medical need. Stressors holding chronic plus psychosocial components thereby bear the highest health risk. Although the metabotropic glutamate receptor subtype 5 (mGlu5) is well studied in the context of acute stress-induced behaviors and physiology, virtually nothing is known about its potential involvement in chronic psychosocial stress. Using the mGlu5 negative allosteric modulator CTEP (2-chloro-4-[2-[2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]imidazol-4yl]ethynyl]pyridine), a close analogue of the clinically active drug basimglurant - but optimized for rodent studies, as well as mGlu5-deficient mice in combination with a mouse model of male subordination (termed CSC, chronic subordinate colony housing), we demonstrate that mGlu5 mediates multiple physiological, immunological, and behavioral consequences of chronic psychosocial stressor exposure. For instance, CTEP dose-dependently relieved hypothalamo-pituitary-adrenal axis dysfunctions, colonic inflammation as well as the CSC-induced increase in innate anxiety; genetic ablation of mGlu5 in mice largely reproduced the stress-protective effects of CTEP and additionally ameliorated CSC-induced physiological anxiety. Interestingly, CSC also induced an upregulation of mGlu5 in the hippocampus, a stress-regulating brain area. Taken together, our findings provide evidence that mGlu5 is an important mediator for a wide range of chronic psychosocial stress-induced alterations and a potentially valuable drug target for the treatment of chronic stress-related pathologies in man.

Type-7 metabotropic glutamate receptors negatively regulate α1-adrenergic receptor signalling.

We studied the interaction between mGlu7 and α1-adrenergic receptors in heterologous expression systems, brain slices, and living animals. L-2-Amino-4-phosphonobutanoate (L-AP4), and l-serine-O-phosphate (L-SOP), which activate group III mGlu receptors, restrained the stimulation of polyphosphoinositide (PI) hydrolysis induced by the α1-adrenergic receptor agonist, phenylephrine, in HEK 293 cells co-expressing α1-adrenergic and mGlu7 receptors. The inibitory action of L-AP4 was abrogated by (i) the mGlu7 receptor antagonist, XAP044; (ii) the C-terminal portion of type-2 G protein coupled receptor kinase; and (iii) the MAP kinase inhibitors, UO126 and PD98059. This suggests that the functional interaction between mGlu7 and α1-adrenergic receptors was mediated by the ßγ-subunits of the Gi protein and required the activation of the MAP kinase pathway. Remarkably, activation of neither mGlu2 nor mGlu4 receptors reduced α1-adrenergic receptor-mediated PI hydrolysis. In mouse cortical slices, both L-AP4 and L-SOP were able to attenuate norepinephrine- and phenylephrine-stimulated PI hydrolysis at concentrations consistent with the activation of mGlu7 receptors. L-AP4 failed to affect norepinephrine-stimulated PI hydrolysis in cortical slices from mGlu7-/- mice, but retained its inhibitory activity in slices from mGlu4-/- mice. At behavioural level, i.c.v. injection of phenylephrine produced antidepressant-like effects in the forced swim test. The action of phenylephrine was attenuated by L-SOP, which was inactive per se. Finally, both phenylephrine and L-SOP increased corticosterone levels in mice, but the increase was halved when the two drugs were administered in combination. Our data demonstrate that α1-adrenergic and mGlu7 receptors functionally interact and suggest that this interaction might be targeted in the treatment of stress-related disorders.

Relief from detrimental consequences of chronic psychosocial stress in mice deficient for the metabotropic glutamate receptor subtype 7.

Chronic stress-related psychiatric conditions and comorbid somatic pathologies are an enormous public health concern in modern society. The etiology of these disorders is complex, with stressors holding a chronic and psychosocial component representing the most acknowledged risk factor. During the last decades, research on the metabotropic glutamate receptor (mGlu) system advanced dramatically and much attention was given to the role of the metabotropic glutamate receptor subtype 7 (mGlu7) in acute stress-related behavior and physiology. However, virtually nothing is known about the potential involvement of mGlu7 in chronic psychosocial stress-related conditions. Using the chronic subordinate colony housing (CSC, 19 days) in male mice, we addressed whether central mGlu7 is altered upon chronic psychosocial stressor exposure and whether genetic ablation of mGlu7 interferes with the multitude of chronic stress-induced alterations. CSC exposure resulted in a downregulation of mGlu7 mRNA transcript levels in the prefrontal cortex, a brain region relevant for stress-related behaviors and physiology. Interestingly, mGlu7 deficiency relieved multiple chronic stress-induced alterations including the CSC-induced anxiety-prone phenotype; mGlu7 ablation also ameliorated CSC-induced physiological and immunological consequences such as hypothalamo-pituitary-adrenal (HPA) axis dysfunctions and colonic inflammation, respectively. Together, our findings provide first evidence for the involvement of mGlu7 in a wide range of behavioral and physiological alterations in response to chronic psychosocial stressor exposure. Moreover, the stress-protective phenotype of genetic mGlu7 ablation suggests mGlu7 pharmacological blockade to be a relevant option for the treatment of chronic stress-related emotional and somatic dysfunctions. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

The Emerging Role of Metabotropic Glutamate Receptors in the Pathophysiology of Chronic Stress-Related Disorders.

Chronic stress-related psychiatric conditions such as anxiety, depression, and alcohol abuse are an enormous public health concern. The etiology of these pathologies is complex, with psychosocial stressors being among the most frequently discussed risk factors. The brain glutamatergic neurotransmitter system has often been found involved in behaviors and pathophysiologies resulting from acute stress and fear. Despite this, relatively little is known about the role of glutamatergic system components in chronic psychosocial stress, neither in rodents nor in humans. Recently, drug discovery efforts at the metabotropic receptor subtypes of the glutamatergic system (mGlu1-8 receptors) led to the identification of pharmacological tools with emerging potential in psychiatric conditions. But again, the contribution of individual mGlu subtypes to the manifestation of physiological, molecular, and behavioral consequences of chronic psychosocial stress remains still largely unaddressed. The current review will describe animal models typically used to analyze acute and particularly chronic stress conditions, including models of psychosocial stress, and there we will discuss the emerging roles for mGlu receptor subtypes. Indeed, accumulating evidence indicates relevance and potential therapeutic usefulness of mGlu2/3 ligands and mGlu5 receptor antagonists in chronic stress-related disorders. In addition, a role for further mechanisms, e.g. mGlu7-selective compounds, is beginning to emerge. These mechanisms are important to be analyzed in chronic psychosocial stress paradigms, e.g. in the chronic subordinate colony housing (CSC) model. We summarize the early results and discuss necessary future investigations, especially for mGlu5 and mGlu7 receptor blockers, which might serve to suggest improved therapeutic strategies to treat stress-related disorders.

Immunization with a heat-killed preparation of the environmental bacterium Mycobacterium vaccae promotes stress resilience in mice.

The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Repeated immunization with a heat-killed preparation of Mycobacterium vaccae, an immunoregulatory environmental microorganism, reduced subordinate, flight, and avoiding behavioral responses to a dominant aggressor in a murine model of chronic psychosocial stress when tested 1-2 wk following the final immunization. Furthermore, immunization with M. vaccae prevented stress-induced spontaneous colitis and, in stressed mice, induced anxiolytic or fear-reducing effects as measured on the elevated plus-maze, despite stress-induced gut microbiota changes characteristic of gut infection and colitis. Immunization with M. vaccae also prevented stress-induced aggravation of colitis in a model of inflammatory bowel disease. Depletion of regulatory T cells negated protective effects of immunization with M. vaccae on stress-induced colitis and anxiety-like or fear behaviors. These data provide a framework for developing microbiome- and immunoregulation-based strategies for prevention of stress-related pathologies.

Chronic subordinate colony housing paradigm: a mouse model to characterize the consequences of insufficient glucocorticoid signaling.

Chronic, in particular chronic psychosocial, stress is a burden of modern societies and known to be a risk factor for numerous somatic and affective disorders (in detail referenced below). However, based on the limited existence of appropriate, and clinically relevant, animal models for studying the effects of chronic stress, the detailed behavioral, physiological, neuronal, and immunological mechanisms linking stress and such disorders are insufficiently understood. To date, most chronic stress studies in animals employ intermittent exposure to the same (homotypic) or to different (heterotypic) stressors of varying duration and intensity. Such models are only of limited value, since they do not adequately reflect the chronic and continuous situation that humans typically experience. Furthermore, application of different physical or psychological stimuli renders comparisons to the mainly psychosocial stressors faced by humans, as well as between the different stress studies almost impossible. In contrast, rodent models of chronic psychosocial stress represent situations more akin to those faced by humans and consequently seem to hold more clinical relevance. Our laboratory has developed a model in which mice are exposed to social stress for 19 continuous days, namely the chronic subordinate colony housing (CSC) paradigm, to help bridge this gap. The main aim of the current review article is to provide a detailed summary of the behavioral, physiological, neuronal, and immunological consequences of the CSC paradigm, and wherever possible relate the findings to other stress models and to the human situation.

Blocking metabotropic glutamate receptor subtype 7 (mGlu7) via the Venus flytrap domain (VFTD) inhibits amygdala plasticity, stress, and anxiety-related behavior.

The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7's extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7's Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.

Dose-dependent effects of chronic central infusion of oxytocin on anxiety, oxytocin receptor binding and stress-related parameters in mice.

Chronic psychosocial stress is a recognized risk factor for various affective and somatic disorders. In an established murine model of chronic psychosocial stress, exposure to chronic subordinate colony housing (CSC) results in an alteration of physiological, behavioral, neuroendocrine and immunological parameters, including a long-lasting increase in anxiety, adrenal hypertrophy and thymus atrophy. Based on the stress-protective and anxiolytic properties of oxytocin (OXT) after acute administration in rodents and humans, the major aims of our study were to assess whether chronic administration of OXT dose-dependently affects the behavior and physiology of male mice, as for therapeutic use in humans, mostly chronic treatment approaches will be used. Further, we studied, whether chronic administration during CSC prevents stress-induced consequences. Our results indicate that chronic intracerebroventricular (ICV) infusion of OXT (15 days) at high (10ng/h), but not at low (1ng/h) dose, induces an anxiogenic phenotype with a concomitant reduction of OXT receptor (OXTR) binding within the septum, the basolateral and medial amygdala, as well as the median raphe nucleus. Further, we demonstrate that chronic ICV infusion of OXT (1ng/h) during a 19-day CSC exposure prevents the hyper-anxiety, thymus atrophy, adrenal hypertrophy, and decreased in vitro adrenal ACTH sensitivity. Thus, given both negative, but also beneficial effects seen after chronic OXT treatment, which appear to be dose-dependent, a deeper understanding of long-lasting treatment effects is required before OXT can be considered for long-term therapeutic use for the treatment of psychopathologies such as autism, schizophrenia or anxiety-disorders.

HPA axis changes during the initial phase of psychosocial stressor exposure in male mice.

Chronic subordinate colony (CSC) housing for 19 days results in unaffected basal morning corticosterone (CORT) levels despite a pronounced increase in adrenal mass, likely mediated by an attenuation of adrenal corticotropin (ACTH) responsiveness. Given that the pronounced increase in basal morning plasma CORT levels returns to baseline as early as 48 h after the start of CSC, it is likely that the attenuated ACTH responsiveness develops already during this initial phase. This was tested in the present study. In line with previous findings, basal morning plasma CORT levels were elevated following 10 h, but not 48 h, of CSC exposure. Basal morning plasma ACTH concentrations and relative in vivo adrenal CORT content were increased following 10 h and to a lesser extent following 48 h of CSC exposure, positively correlating. Relative in vitro adrenal CORT secretion in response to ACTH (100 nM) and kidney protein expression of 11ß-hydroxysteroid dehydrogenase type 2 (HSD11B2) were unaffected following both time points. Adrenal mRNA expression of key steroidogenic enzymes was unaffected/decreased following 10 h and unaffected/increased following 48 h of CSC exposure. Together, our findings suggest that basal plasma hypercorticism during the initial CSC phase is mainly prevented by an attenuation of pituitary ACTH release. An increased absolute adrenal weight following 10 h, but not 48 h, of CSC exposure indicates that restoration of normal adrenal mass also adds to a lesser extent to prevent basal hypercorticism. A contributing role of alterations in enzymatic CORT degradation and steroidogenic enzyme availability is likely, but has to be further addressed in future studies.

Chronic psychosocial stress in male mice causes an up-regulation of scavenger receptor class B type 1 protein in the adrenal glands.

Mice exposed to chronic subordinate colony housing (CSC, 19 days) show an exaggerated adrenal corticosterone response to an acute heterotypic stressor (elevated platform (EPF), 5 min) despite no difference from EPF-exposed single-housed control (SHC) mice in corticotropin (ACTH) secretion. In the present study, we asked the question whether this CSC-induced increase in adrenal capability to produce and secrete corticosterone is paralleled by an enhanced adrenal availability and/or mobilization capacity of the corticosterone precursor molecule cholesterol. Employing oil-red staining and western blot analysis we revealed comparable relative density of cortical lipid droplets and relative protein expression of hormone-sensitive lipase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and low-density lipoprotein receptor (LDL-R) between CSC and SHC mice. However, relative protein expression of the scavenger receptor class B type 1 (SR-BI) was increased following CSC exposure. Moreover, analysis of plasma high-density lipoprotein-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) revealed increased LDL-C levels in CSC mice. Together with the pronounced increase in adrenal weight, evidently mediated by hyperplasia of adrenocortical cells, these data strongly indicate an enhanced adrenal availability of and capacity to mobilize cholesterol in chronic psychosocially-stressed mice, contributing to their increased in vivo corticosterone response during acute heterotypic stressor exposure.

Time matters: pathological effects of repeated psychosocial stress during the active, but not inactive, phase of male mice.

Recent findings in rats indicated that the physiological consequences of repeated restraint stress are dependent on the time of day of stressor exposure. To investigate whether this is also true for clinically more relevant psychosocial stressors and whether repeated stressor exposure during the light phase or dark phase is more detrimental for an organism, we exposed male C57BL/6 mice to social defeat (SD) across 19 days either in the light phase between Zeitgeber time (ZT)1 and ZT3 (SDL mice) or in the dark phase between ZT13 and ZT15 (SDD mice). While SDL mice showed a prolonged increase in adrenal weight and an attenuated adrenal responsiveness to ACTH in vitro after stressor termination, SDD mice showed reduced dark phase home-cage activity on observation days 7, 14, and 20, flattening of the diurnal corticosterone rhythm, lack of social preference, and higher in vitro IFNγ secretion from mesenteric lymph node cells on day 20/21. Furthermore, the colitis-aggravating effect of SD was more pronounced in SDD than SDL mice following dextran sulfate sodium treatment. In conclusion, the present findings demonstrate that repeated SD effects on behavior, physiology, and immunology strongly depend on the time of day of stressor exposure. Whereas physiological parameters were more affected by SD during the light/inactive phase of mice, behavioral and immunological parameters were more affected by SD during the dark phase. Our results imply that repeated daily SD exposure has a more negative outcome when applied during the dark/active phase. By contrast, the minor physiological changes seen in SDL mice might represent beneficial adaptations preventing the formation of those maladaptive consequences.

Chronic psychosocial stress results in sensitization of the HPA axis to acute heterotypic stressors despite a reduction of adrenal in vitro ACTH responsiveness.

Although chronic psychosocial stress is often accompanied by changes in basal hypothalamo-pituitary-adrenal (HPA) axis activity, it is vital for a chronically-stressed organism to mount adequate glucocorticoid (GC) responses when exposed to acute challenges. The main aim of the present study was to test whether this is true or not for the chronic subordinate colony housing (CSC, 19 days) paradigm, an established and clinically relevant mouse model of chronic psychosocial stress. As shown previously, CSC mice are characterized by unaffected morning and decreased evening plasma corticosterone (CORT) levels despite enlarged adrenals, suggesting a maladaptive breakdown of adrenal functioning. Plasma CORT levels, determined by repeated blood sampling via jugular vein catheters, as well as relative right adrenal CORT content were increased in CSC compared with single-housed control (SHC) mice in response to acute elevated platform (EPF, 5min) exposure. However, in vitro stimulation of adrenal explants with physiological and pharmacological doses of ACTH revealed an attenuated responsiveness of both the left and right adrenal glands following CSC, despite mRNA and/or protein expression of melanocortin 2 receptor (Mc2r), Mc2r accessory protein (MRAP), and key enzymes of steroidogenesis were not down-regulated. Taken together, we show that chronic psychosocial stressor exposure impairs in vitro ACTH responsiveness of both the left and right adrenal glands, whereas it increases adrenal responsiveness to an acute heterotypic stressor in vivo. This suggests that an additional factor present during acute stressor exposure in vivo rescues left and right adrenal ACTH sensitivity, or itself acts as CORT secretagogue in chronically stressed CSC mice.