Days Of Chronic Unpredictable Stress Research Articles

L-type calcium channel regulation of depression, anxiety and anhedonia-related behavioral phenotypes following chronic stress exposure

Exposure to chronic and unpredictable stressors can precipitate mood-related disorders in humans, particularly in individuals with pre-existing mental health challenges. L-type calcium channels (LTCCs) have been implicated in numerous neuropsychiatric disorders, as LTCC encoding genes have been identified as candidate risk factors for neuropsychiatric illnesses. In these sets of experiments, we sought to examine the ability of LTCC blockade to alter depression, anxiety, and anhedonic-related behavioral responses to chronic unpredictable stress (CUS) exposure in female and male rats. Rats first underwent either 21 days of CUS or no exposure to chronic stressors, serving as home cage controls (HCC). Then rats were examined for anhedonia-related behavior, anxiety and depression-like behavioral responses as measured by the sucrose preference test (SPT), elevated plus maze (EPM), and forced swim test (FST). CUS exposed females and males showed anhedonic and anxiogenic-like behavioral responses on the SPT and EPM, respectively, when compared to HCCs. In female and male rats, systemic administration of the LTCC blocker isradipine (0.4 mg/kg and 1.2 mg/kg, I.P.) attenuated the CUS-induced decrease in sucrose preference and reversed the CUS-induced decrease in open arm time. In the FST, systemic isradipine decreased immobility time across all groups, consistent with an antidepressant-like response. However, there were no significant differences in forced swim test immobility time between HCC and CUS exposed animals. Taken together, these data point to a role of LTCCs in the regulation of mood disorder-related behavioral phenotype responses to chronic stress exposure.

Chronic Unpredictable Stress Induces Mitochondrial Dysfunction in Hypothalamic Pituitary Adrenal Axis Regions

Background: Stress elicits physiological and behavioral responses via adaptive responses like the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic nervous system (SNS) activation. Under chronic stress, however, these responses become dysfunctional leading to pathological changes in behavior and health. Mitochondria are dynamic organelles that provide cellular energy, activate immune responses, and synthesize glucocorticoids in the adrenal glands in response to stress. Thus, the mitochondrion is a key candidate linking the effects of chronic stress and health, however, the effects of chronic stress on mitochondrial function in the HPA axis, particularly with respect to sex differences, are largely unexplored. Hypothesis: Chronic unpredictable stress (CUS) decreases mitochondrial function and dynamics in key regions within the HPA axis. Methods: Adult male and female C57Bl6/J mice were exposed to 28 consecutive days of CUS or handling (non-CUS) with weekly weighing. Mice were euthanized 24h after the last stress exposure and the brain and adrenal glands dissected and weighed. Mitochondrial function was assessed using high resolution respirometry in hypothalamus and adrenal tissues. Protein expression was assessed by immunoblot. Results: CUS decreased mitochondrial respiration within key stress centers, with stressed males exhibiting decreased respiration in the amygdala (20%, p=0.05), and stressed females exhibiting decreased respiration in the hypothalamus (20%, p=0.004) and adrenal glands (17%, p=0.06). CUS increased mitochondrial complex protein in male adrenal gland (47%, p=0.01) and mitochondrial mass in hypothalamus (p=0.007). Finally, cleaved Gasdermin D expression was increased nearly 2-fold in male hypothalamus (p=0.03). Conclusions: Our data show sex- and region-dependent decreases in HPA axis mitochondrial function concurrent with upregulated protein expression and pore formation. These observations point to sex-specific effects of CUS to induce mitochondrial dysfunction. This work was supported by P20GM109091 (F.H.), R01HL130972-01A1, R01HL5949, BX000168-10A1, BX005320 (F.G.S.), R01DK132948 (C.W. & F.P.) R01 MH129798 (SKW); VA VISN7 RDA (F.H.), 1U54HL169191-01 & BX002604 (M.J.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the abstract. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

(250) NLRP3 Inflammasome Might Contribute to Erectile Dysfunction Induced by Chronic Unpredictable Stress in Male Mice

Abstract Introduction Introduction: The association of stress with erectile dysfunction (ED) is well known, however, it is not clear whether psychological stressors affect the vasculature and the erectile tissue in a manner that could contribute to a vasculogenic erectile dysfunction. Besides hormonal changes, cellular damage induced by stress could lead to the activation of the NLRP3, to increase production of cytokines associated with ED. In this study, we aimed to evaluate the reactivity of the corpus cavernosum and pudendal artery of male mice undergoing chronic unpredictable stress (CUS). Objective Objective: We hypothesize that CUS leads to mitochondrial damage and activates the NLRP3 inflammasome leading to impaired reactivity of the pudendal artery and corpus cavernosum which might cause erectile dysfunction. Methods Methods: Male C57Bl/6 mice were submitted to 28 days of chronic unpredictable stress (CUS). The pudendal artery and corpus cavernosum were removed and mounted in a myograph to evaluate reactivity. Tissues were kept in physiological salt solution at 37°C, constantly aerated with 95%O2/5%CO2 and concentration-response curves to acetylcholine (ACh; 1 nM – 30 uM) and phenylephrine (PE; 1 nM – 30 uM) were obtained. Using non-linear regression, we obtained the maximal response (Emax) and potency (pEC50) of ACh and PE. Expression of NLRP3, pro-caspase1 and caspase1 were measured in arteries by Western blot. Data are expressed as mean ± S.E.M. P<0.05 was considered statistically different. Results Results: CUS led to a significant decrease in the maximal relaxation induced by acetylcholine in the pudendal artery (Non-CUS: 96 ± 3% vs CUS: 84 ± 7%, n=6 and 7, respectively) and in the corpus cavernosum (Non-CUS: 88 ± 4% vs CUS: 75 ± 4%, n=7 each group) as well as an impaired contraction to PE in the corpus cavernosum (non-CUS: 1.72 ± 0.17% vs CUS: 1.07 ± 0.15%, n= 7 each group). In arteries, there were no differences in the expression of NLRP3, however, the ratio of caspase1/pro-caspase-1 was increased in CUS, suggesting that CUS increases the NLRP3 inflammasome pathway. Taken together, our data suggest that CUS led to an activation of the NLRP3 inflammasome pathway, and impaired the relaxation of the pudendal artery and corpus cavernosum by a mechanism that might be associated with decreased nitric oxide bioavailability (endothelium-dependent relaxation), whereas impaired contraction might be associated with desensitization of alpha-adrenergic receptors in response to stress. Conclusions Conclusion: Our findings demonstrate that stress impairs the reactivity of the corpus cavernosum and pudendal artery likely associated with the activation of NLRP3 inflammasome, which might play a role in the development of erectile dysfunction in stressed males. Disclosure No.

Abstract 027: Chronic Unpredictable Stress Affects Mitochondrial Function In The Hypothalamic-Pituitary-Adrenal Axis And Impairs Vascular Reactivity In Male And Female Mice

Chronic stress is associated with hypertension but the underlying mechanisms linking the interplay between the changes in the hypothalamic-pituitary-adrenal axis and vascular dysfunction remain unclear. We hypothesize that chronic unpredictable stress (CUS) induces mitochondrial dysfunction in the HPA axis and in the vasculature, associated with GasderminD pore formation in the mitochondrial membrane. Adult male and female C57Bl6/J mice were exposed to 28 consecutive days of CUS or handling (CTL). Anxiety-like behavior, coping behavior, and systolic blood pressure (SBP) were assessed. Mitochondrial function was assessed using high-resolution respirometry in the hypothalamus, amygdala, and adrenal tissues. The vascular reactivity of mesenteric resistance arteries (MRA), pudendal arteries (PA), and aorta were assessed in response to phenylephrine or acetylcholine. GasderminD protein expression was measured in the MRA. Data are presented as mean ± S.E.M and significance was set at p<0.05. In both sexes, CUS increased SBP by 12.1±4.9%. CUS decreased body weight gain by 53% (p=0.04) and increased anxiety-like behavior (p=0.01) in males (vs CTL) but did not affect females. CUS decreased latency to immobility in the forced swim test by 46% (p=0.0001) in both sexes. Sex-dependent decreases in mitochondrial respiration were observed as follows: in the amygdala of males (20%, p=0.05) and the hypothalamus (20%, p=0.004) and adrenal glands (17%, p=0.06) of females. In the vasculature, CUS led to endothelial dysfunction observed by reduced potency of ACh in the MRA (pEC 50 CUS 6.17±0.09 vs CTL 6.81±0.11; p=0.003) and PA (pEC 50 CUS 6.31 ±0.07 vs CTL 6.65±0.09; p=0.008) of male mice, as well as in the MRA of females (pEC 50 CUS 5.94±0.30 vs CTL 6.98±0.15; p=0.041). Contractile responses to PE were significantly reduced in the aorta of CUS males only (E MAX CTL: 4.70±0.48 mN vs CUS 3.01± 0.23 mN; p=0.007). The expression of gasderminD was increased in the MRA of female mice only. CUS increased SBP in males and females, induced sex-specific changes in the mitochondrial function in key areas of the brain, and caused vascular dysfunction, suggesting that behavioral, brain, and vascular adaptations to CUS are sex-dependent.

(230) Chronic Unpredictable Stress Impairs Corpus Cavernosum Reactivity in Mice

Abstract Introduction Stress is associated with erectile dysfunction (ED), however, it is not clear whether psychological stressors affect the vasculature and the erectile tissue in a manner that could contribute to a vasculogenic erectile dysfunction. A positive correlation between hyperprolactinemia, stress and erectile dysfunction has been demonstrated, and interestingly, prolactin was shown to decrease endothelium nitric oxide and endothelium- dependent vasorelaxation in different vascular beds. In this study, we aimed to evaluate the relaxation of the corpus cavernosum of male and pudendal artery of male and female mice undergoing chronic unpredictable stress (CUS). Objective We aimed to test the hypothesis that vascular impairment caused by CUS leads to impaired relaxation of the pudendal artery and corpus cavernosum which may cause erectile dysfunction. Methods Male and female C57Bl/6 mice were submitted to 28 days of chronic unpredictable stress (CUS). The pudendal artery and corpus cavernosum were removed and mounted in a myograph to evaluate reactivity. Tissues were kept in physiological salt solution at 37°C, constantly aerated with 95%O2/5%CO2 and concentration-response curves to acetylcholine (ACh; 1 nM – 30 uM) and phenylephrine (PE; 1 nM – 30 uM) were obtained. Using non-linear regression, we obtained the maximal response (Emax) and potency (pEC50) of ACh and PE. Data are expressed as mean ± S.E.M. P<0.05 was considered statistically different. Results In the pudendal artery, neither the potency nor the maximal response to ACh was changed by the stress. Contraction to PE was higher in males, compared to females, and there was a trend to be further increased in stressed males. On the other hand, in the corpus cavernosum of stressed males there was a significant decrease in the maximal relaxation induced by acetylcholine (control: 88 ± 4% vs stress: 75 ± 4%, n=7 each group) as well as impaired contraction to PE (control: 1.72 ± 0.17% vs stress: 1.07 ± 0.15%, n= 7 each group). The impaired relaxation might be associated with impaired nitric oxide availability, whereas impaired contraction can be associated with a desensitization of alpha-adrenergic receptors in response to stress. Conclusions Our findings demonstrate that stress impairs the reactivity of the corpus cavernosum, which may contribute to the development of erectile dysfunction in stressed males. Disclosure No

Chronic Unpredictable Stress Impairs Vascular Reactivity in Male and Female Mice

Background: Chronic stress is known to induce vascular inflammation causing endothelial and vascular smooth muscle dysfunction. Changes in the hypothalamic-pituitary-adrenal axis lead to increased sympathetic and decreased parasympathetic drive, causing the release of inflammatory cytokines in the vasculature, by mechanisms that are unclear. Hypothesis: We hypothesize that chronic unpredictable stress (CUS) increases reactive oxygen species and induces gasdermin pore formation and voltage-dependent anion channel (VDAC) oligomerization in the membrane of the mitochondria, which releases mitochondrial DNA (mtDNA) to the cytosol and induces TNF-α, IL-1β and IL-18 production through activation of the NLRP-3 inflammasome pathway. Methods: Adult male and female C57Bl6/J mice were exposed to 28 consecutive days of chronic unpredictable stress (CUS) or handling (CTL). Systolic blood pressure (SBP) was measured at the end of the stress period by tail cuff plethysmography. Vascular function using myographs was assessed on isolated mesenteric resistance arteries (MRAs), pudendal artery (PA) and aorta from male and female CUS and CTL mice. The arterial rings were kept in physiological salt solution at 37°C, constantly aerated with 95%O2/5%CO2. Concentration-response curves to phenylephrine (PE; 1nM-30μM) and acetylcholine (ACh; 1nM-30μM) were performed. Expression of gasderminD was measured in the MRA by western blot. Concentration-response curves were analyzed using non-linear regression analysis. Data are presented as mean ± S.E.M. Statistical significance set at p<0.05. Data were analyzed with GraphPad Prism 9.2.0. Results: SBP was increased by 12.1±4.9% in CUS mice. In CUS mice, compared to the CTL, the potency of ACh-induced relaxation was decreased in the MRA (pEC50 CUS 6.17±0.09 vs CTL 6.81±0.11; p=0.003) and PA (pEC50 CUS 6.65±0.07 vs CTL 6.31±0.09; p=0.008) but not in the aorta of male mice. In females, the potency to ACh was decreased only in the MRA (pEC50 CUS 5.94±0.30 vs CTL 6.98±0.15; p=0.041). There were no differences in the contractile responses to PE between controls and mice exposed to CUS in the MRA and PA of both males and females, while in the aorta of CUS males, there was a significant reduction in maximal response (EMAX CTL: 4.70±0.48 mN vs CUS 3.01± 0.23mN; p=0.007). The expression of gasderminD was increased in MRA of female mice only. Conclusion: Our results show that, in both sexes, CUS increased SBP and induced vascular dysfunction observed by impaired reactivity to ACh and PE, with increased expression of gasderminD in MRA of female mice only. These results suggest that the vascular adaptations to CUS are sex-dependent. This work was supported by NIH grants P20GM109091 (F.H.), R01HL130972-01A1 (F.G.S.), R01HL5949 (F.G.S.), RO1DK132948 (C.W. & F.P.) and R01 MH129798 (SKW); VA grants: VISN7 RDA (F.H.), Merit awards: BX000168-10A1 (F.G.S.), BX005320 (F.G.S.), and BX002604 (M.J.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the abstract. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Impact of ketamine administration on chronic unpredictable stress-induced rat model of depression during extremely low-frequency electromagnetic field exposure: Behavioral, histological and molecular study.

In the study, we examined the effects of ketamine and extremely low-frequency electromagnetic fields (ELF-EMF) on depression-like behavior, learning and memory, expression of GFAP, caspase-3, p53, BDNF, and NMDA receptor in animals subjected to chronic unpredictable stress (CUS). After applying 21 days of chronic unpredictable stress, male rats received intraperitoneal (IP) of ketamine (5mg/kg) and then were exposed to ELF-EMF (10-Hz, 10-mT exposure conditions) for 3 days (3h per day) and behavioral assessments were performed 24h after the treatments. Instantly after the last behavioral test, the brain was extracted for Nissl staining, immunohistochemistry, and real-time PCR analyses. Immunohistochemistry (IHC) was conducted to assess the effect of ketamine and ELF-EMF on the expression of astrocyte marker (glial fibrillary acidic protein, GFAP) in the CA1 area of the hippocampus and medial prefrontal cortex (mPFC). Also, real-time PCR analyses were used to investigate the impacts of the combination of ketamine and ELF-EMF on the expression of caspase3, p53, BDNF, and NMDA receptors in the hippocampus in rats submitted to the CUS procedure. Results were considered statistically significant when p<.05. Our results revealed that the combination of ketamine and ELF-EMF increased depression-like behavior,increased degenerated neurons and decreased the number of GFAP (+) cells in the CA1 area and mPFC, incremented the expression of caspase-3, and reduced the expression of BDNF in the hippocampus but showed no effect on the expression of p53 and NMDA-R. These results reveal that combining ketamine and ELF-EMF has adverse effects on animals under chronic unpredictable stress (CUS).

Depletion of microglial BDNF increases susceptibility to the behavioral and synaptic effects of chronic unpredictable stress

In the medial prefrontal cortex (PFC), chronic stress reduces synaptic expression of glutamate receptors, leading to decreased excitatory signaling from layer V pyramidal neurons and working memory deficits. One key element driving these changes is a reduction in brain-derived neurotrophic factor (BDNF) signaling. BDNF is a potent mediator of synaptic growth and deficient BDNF signaling has been linked to stress susceptibility. Prior studies indicated that neurons are the primary source of BDNF, but more recent work suggests that microglia are also an important source of BDNF. Adding to this, our work showed that 14 days of chronic unpredictable stress (CUS) reduced Bdnf transcript in PFC microglia, evincing its relevance in the effects of stress. To explore this further, we utilized transgenic mice with microglia-specific depletion of BDNF (Cx3cr1Cre/+:Bdnffl/fl) and genotype controls (Cx3cr1Cre/+:Bdnf+/+). In the following experiments, mice were exposed to a shortened CUS paradigm (7 days) to determine if microglial Bdnf depletion promotes stress susceptibility. Analyses of PFC microglia revealed that Cx3cr1Cre/+:Bdnffl/fl mice had shifts in phenotypic markers and gene expression. In a separate cohort, synaptoneurosomes were collected from the PFC and western blotting was performed for synaptic markers. These experiments showed that Cx3cr1Cre/+:Bdnffl/fl mice had baseline deficits in GluN2B, and that 7 days of CUS additionally reduced GluN2A levels in Cx3cr1Cre/+:Bdnffl/fl mice, but not genotype controls. Behavioral and cognitive testing showed that this coincided with exacerbated stress effects on temporal object recognition in Cx3cr1Cre/+:Bdnffl/fl mice. These results indicate that microglial BDNF promotes glutamate receptor expression in the PFC. As such, mice with deficient microglial BDNF had increased susceptibility to the behavioral and cognitive consequences of stress.

Effects of liraglutide on depressive behavior in a mouse depression model and cognition in the probe trial of Morris water maze test

BackgroundWe investigated the effects of liraglutide, a glucagon-like peptide-1 (GLP-1) agonist, on a depression-like phenotype in mice exposed to chronic unpredictable stress (CUS). Learning and memory were also assessed using the Morris water maze (MWM) test. MethodsLiraglutide (0.3 mg/kg/day for 21 days) was administered to mice with or without exposure to CUS. After 21 days of CUS, the forced swim test (FST) was performed to assess its antidepressant effect. To evaluate cognitive function, liraglutide was administered to mice under stress-free conditions for 21 days, and then the MWM test was performed on 6 consecutive days. ResultsChronic liraglutide treatment reduced FST immobility in mice with and without CUS. In the probe trial of the Morris water maze test, the search error rate was reduced and the time spent and path length in the target quadrant and the number of platform crossings were increased. LimitationAdditional animal model experiments and molecular level studies are needed to support the results obtained in this study. ConclusionsLiraglutide appears to exert antidepressant effects and could improve cognitive function. Based on these results, GLP-1 agonists could have potential as novel antidepressants.

Gonadal hormone trigger the dynamic microglial alterations through Traf6/TAK1 axis that correlate with depressive behaviors

Gonadal hormone deficiency is associated with the development of depression, but what mediates this association is unclear. To test the possibility that it reflects neuroimmune and neuroinflammatory processes, we analyzed how gonadal hormone deficiency and replacement affect microglial activation and inflammatory response during the development of depressive symptomatology in gonadectomized male mice. Testosterone level and the ratio of testosterone to estradiol in the serum and brain tissue of mice exposed to 3–35 days of chronic unpredictable stress were much lower than in control animals. Gonadal hormone sustained deficiency in gonadectomized mice and subsequent led to acute inflammation at day 7 following castration. Activating microglia in mice exposed to 7 days of castration subsequently suppressed the proliferation of microglia, such that their numbers in hippocampus and cortex were lower than the numbers in sham-operated mice after 30 days of castration. Here, we showed that gonadal hormone deficiency induces Traf6-mediated microglia activation, a type of inflammatory mediator. Microglia treated in this way for long time showed down-regulation of activation markers, abnormal morphology and depressive-like behaviors. Restoration and maintenance of a fixed ratio of testosterone to estradiol significantly suppressed microglial activation, neuronal necroptosis, dramatically inducing hippocampal neurogenesis and reducing depressive behaviors via the suppression of Traf6/TAK1 pathway. These findings suggest that activated or immunoreactive microglia contribute to gonadal hormone deficiency-induced depression, as well as testosterone and estradiol exert synergistic anti-depressant effects via suppressing microglial activaton in gonadectomized male mice, possibly through Traf6 signaling.

Chronic Unpredictable Stress Impairs the Reactivity of the Pudendal Artery and Corpus Cavernosum in Male Mice

Erectile dysfunction (ED) can be associated with psychological disorders as well as vascular disorders. It is known that stress is associated with erectile dysfunction, however, it is not clear if psychological stressors could lead to a vascular and cavernosal dysfunction that could contribute to a vasculogenic erectile dysfunction. A recent study reported a positive correlation between hyperprolactinemia, stress and erectile dysfunction, and interestingly, prolactin was shown to decrease endothelium nitric oxide and endothelium‐ dependent vasorelaxation of aorta and coronary arteries. In this study, we aimed to evaluate the relaxation of the corpus cavernosum and pudendal artery of mice subject to chronic unpredictable stress (CUS). We hypothesize that CUS will lead to an impairment of the relaxation of the pudendal artery and corpus cavernosum leading to erectile dysfunction.MethodsMale C57Bl/6 mice were submitted to 28 days of chronic unpredictable stress (CUS). The pudendal artery and corpus cavernosum were removed and mounted in a myograph to evaluate reactivity. Tissues were kept in physiological salt solution at 37°C, constantly aerated with 95%O2/5%CO2 and concentration‐response curves to acetylcholine (ACh; 1 nM – 30 uM)) were obtained. Using non‐linear regression, we obtained the maximal response (Emax) and potency (pEC50) of ACh. Data are expressed as mean ± S.E.M. P&lt;0.05 was considered statistically different.ResultsNeither the potency nor the maximal response to acetylcholine was changed in the pudendal artery of stressed animals (Fig. 1A) whereas in the corpus cavernosum, there was a significant decrease in the maximal relaxation induced by acetylcholine (control: 88 ± 4% vs stress: 75 ± 4%, n=7 each group; Fig. 1B).ConclusionOur findings demonstrate that CUS for 4 weeks impairs acetylcholine‐induced relaxation of the corpus cavernosum. This may contribute to the development of erectile dysfunction in stressed males.

Repeated episodes of transient reduction of oxygen exposure simulating aircraft cabin conditions enhance resilience to stress in mice.

Pilots and crew of domestic flights are exposed to transient periods of mild reductions of partial pressure of inspired oxygen each day, and this might have functional consequence on their performance in the long range. Here, we exposed mice to mild reductions of oxygen exposure (ROE) four times per day for 21 days by lowering oxygen partial pressure to levels corresponding to an altitude of about 2300 m, which is the quote of pressurization of the air cabin. Four groups of mice were studied: unstressed or stressed mice exposed to ROE or normoxic conditions. Mice were exposed to chronic unpredictable stress (CUS) for 28 days, and ROE was delivered in the last 21 days of CUS. In normoxic mice, CUS caused anhedonia in the sucrose preference test, anxiety-like behaviour in the open field test, learning impairment in the Morris water maze, reduced hippocampal neurogenesis, increased serum corticosterone levels and increased expression of depression-related genes (Pclo, Mthfr and Grm5) in the hippocampus. All these changes were reversed by ROE, which had little or no effect in unstressed mice. These findings suggest that ROE simulating air cabin conditions of domestic flights may enhance resilience to stress improving mood, anxiety and learning ability.

Chronic Unpredictable Stress Alters Brain Tryptophan Metabolism and Impairs Working Memory in Mice without Causing Depression-Like Behaviour.

Chronic stress is a well-known risk factor in major depressive disorder and disrupts the kynurenine and serotonin pathways of tryptophan metabolism. Here, we characterize the temporal central and peripheral changes in tryptophan metabolism and concomitant depressive-like behavioural phenotype induced during the progression of chronic unpredictable stress (CUS). Mice were exposed to 0, 10, 20, or 30 days of CUS followed by a panel of behavioural assays to determine depressive-like phenotypes. Immediately after behavioural testing, plasma and brain tissue were collected for metabolic analysis. While anhedonia-like and anxiety-like behaviours were unaffected by stress, nesting behaviour and cognitive deficits became apparent in response to CUS exposure. While CUS caused a transient reduction in circulating quinolinic acid, no other tryptophan metabolites significantly changed in response to CUS. In the brain, tryptophan, kynurenine, picolinic acid, and 5-hydroxyindoleacetic acid concentrations were significantly elevated in CUS-exposed mice compared with non-stress control animals, while kynurenic acid, xanthurenic acid, and serotonin decreased in CUS-exposed mice. Metabolic turnover of serotonin to the major metabolite 5- hydroxyindoleacetic acid was markedly increased in response to CUS. These results suggest that CUS impairs hippocampal-dependent working memory and enhances nascent nesting behaviour in C57BL/6J male mice, and these behaviours are associated with increased brain kynurenine pathway metabolism leading to accumulation of picolinic acid and a significant reduction in serotonin levels.

Norepinephrine and Glucocorticoids Modulate Chronic Unpredictable Stress-Induced Increase in the Type 2 CRF and Glucocorticoid Receptors in Brain Structures Related to the HPA Axis Activation.

The stress response is multifactorial and enrolls circuitries to build a coordinated reaction, leading to behavioral, endocrine, and autonomic changes. These changes are mainly related to the hypothalamus-pituitary-adrenal (HPA) axis activation and the organism's integrity. However, when self-regulation is ineffective, stress becomes harmful and predisposes the organism to pathologies. The chronic unpredictable stress (CUS) is a widely used experimental model since it induces physiological and behavioral changes and better mimics the stressors variability encountered in daily life. Corticotropin-releasing factor (CRF) and glucocorticoids (GCs) are deeply implicated in the CUS-induced physiological and behavioral changes. Nonetheless, the CUS modulation of CRF receptors and GR and the norepinephrine role in extra-hypothalamic brain areas were not well explored. Here, we show that 14days of CUS induced a long-lasting HPA axis hyperactivity evidenced by plasmatic corticosterone increase and adrenal gland hypertrophy, which was dependent on both GCs and NE release induced by each stress session. CUS also increased CRF2 mRNA expression and GR protein levels in fundamental brain structures related to HPA regulation and behavior, such as the lateral septal nucleus intermedia part (LSI), ventromedial hypothalamic nucleus (VMH), and central nucleus of the amygdala (CeA). We also showed that NE participates in the CUS-induced increase in CRF2 and GR levels in the LSI, reinforcing the locus coeruleus (LC) involvement in the HPA axis modulation. Despite the CUS-induced molecular changes in essential areas related to anxiety-like behavior, this phenotype was not observed in CUS animals 24h after the last stress session.

Synaptic and behavioral effects of chronic stress are linked to dynamic and sex-specific changes in microglia function and astrocyte dystrophy

Emerging evidence indicates that males and females display different neurobiological responses to chronic stress which contribute to varied behavioral adaptations. In particular, pyramidal neurons undergo dendritic atrophy and synapse loss in the prefrontal cortex (PFC) of male, but not female, mice. Our recent work shows that chronic stress also provokes microglia-mediated neuronal remodeling, which contributes to synaptic deficits in the PFC and associated behavioral consequences in males. Separate studies indicate that chronic stress promotes astrocyte dystrophy in the PFC which is associated with behavioral despair. Notably, these prior reports focused primarily on stress effects in males. In the present studies, male and female mice were exposed to 14 or 28 days of chronic unpredictable stress (CUS) to assess molecular and cellular adaptations of microglia, astrocytes, and neurons in the medial PFC. Consistent with our recent work, male, but not female, mice displayed behavioral and cognitive deficits with corresponding perturbations of neuroimmune factors in the PFC after 14 days of CUS. Fluorescence-activated cell sorting and gene expression analyses revealed that CUS increased expression of select markers of phagocytosis in male PFC microglia. Confocal imaging in Thy1-GFP(M) mice showed that CUS reduced dendritic spine density, decreased GFAP immunolabeling, and increased microglia-mediated neuronal remodeling only in male mice. After 28 days of CUS, both male and female mice displayed behavioral and cognitive impairments. Interestingly, there were limited stress effects on neuroimmune factors and measures of microglial phagocytosis in the PFC of both sexes. Despite limited changes in neuroimmune function, reduced GFAP immunolabeling and dendritic spine deficits persisted in male mice. Further, GFAP immunolabeling and dendritic spine density remained unaltered in the PFC of females. These findings indicate that chronic stress causes sex-specific and temporally dynamic changes in microglial function which are associated with different neurobiological and behavioral adaptations. In all, these results suggest that microglia-mediated neuronal remodeling, astrocyte dystrophy, and synapse loss contribute to stress-induced PFC dysfunction and associated behavioral consequences in male mice.

Ursolic acid abrogates depressive-like behavior and hippocampal pro-apoptotic imbalance induced by chronic unpredictable stress.

Emerging evidence has shown that ursolic acid exerts antidepressant-like effects, however, its ability to elicit an antidepressant-like response in rodents subjected to stress model that mimics behavioral and neurochemical alterations found in depression remains to be determined. Thus, this study investigated the possible antidepressant-like effect of ursolic acid in mice subjected to chronic unpredictable stress (CUS) for 14 days, and whether this effect could be associated with the modulation of serum corticosterone levels and hippocampal Bcl-2/Bax mRNA expression. Our results indicated that CUS induced a depressive-like behavior, as demonstrated by an increase in the immobility time and latency to first grooming in the tail suspension test and splash test, respectively. Conversely, the repeated administration of ursolic acid (0.1mg/kg, p.o.) or fluoxetine (10mg/kg, p.o.) in the last 7 days of CUS completely prevented CUS-induced behavioral alterations, suggesting an antidepressant-like effect. Additionally, CUS significantly increased the mRNA expression of Bax (pro-apoptosis marker), but not Bcl-2 (anti-apoptosis marker) in the hippocampus. Moreover, reduced hippocampal mRNA expression of Bcl-2/Bax ratio was detected in CUS-exposed mice. Ursolic acid, but not fluoxetine, prevented CUS-induced increase in the expression of Bax, but both ursolic acid and fluoxetine prevented CUS-induced reduction on Bcl-2/Bax ratio. Furthermore, neither CUS nor treatments with ursolic acid or fluoxetine altered serum corticosterone levels. Our study unveils the ability of ursolic acid to prevent the depressive-like behavior induced by stress and the modulation of Bcl-2/Bax expression could be associated with this response.

Memantine treatment exerts an antidepressant-like effect by preventing hippocampal mitochondrial dysfunction and memory impairment via upregulation of CREB/BDNF signaling in the rat model of chronic unpredictable stress-induced depression

Mitochondrial and cognitive dysfunctions have long been associated with major depressive disorders (MDDs). Studies have shown that Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, possesses an antidepressant-like effect. Hence, the NMDA receptor can be a better therapeutic target for MDD. Therefore, the present study was designed to study the impact of Memantine on mitochondrial functional status and depression-like symptoms in the chronic unpredictable stress (CUS) model of depression. CUS for 28 days resulted in depression-like symptoms (as indicated by increased immobility time in the forced swim test) and a decline in the spatial learning and retention memory in the Morris water maze (MWM) test, which was prevented by Memantine (10 mg/kg/day) treatment. We observed elevated plasma corticosterone (CORT) levels, microdialysates glutamate concentration, and synaptosomal calcium (Ca2+) ion levels after 28 days of CUS. Memantine treatment prevented only increased plasma CORT and synaptosomal Ca2+ ion levels. Memantine treatment also restored CUS induced increase in oxidative stress parameters [increased neuronal nitric oxide synthase (nNOS) expression, nitric oxide (NO) levels, lipid peroxidation (LPO) and superoxide dismutase (SOD) activity], decrease in mitochondrial electron transport chain (ETC) enzymes activity and mitochondrial membrane potential (MMP). CUS also reduced the expression of cell survival genes, cyclic-AMP response element-binding protein (CREB), and brain-derived nerve growth factor (BDNF), which was reversed by treatment with Memantine. CUS, however, caused a non-significant decrease in the hippocampal adenosine triphosphate (ATP) levels and a non-significant increase in the expression of pro-apoptotic genes, Caspase 3, and the number of TUNEL positive cells, indicating that hippocampal mitochondrial dysfunction caused due to CUS was not severe enough to affect overall energy production, mitochondrial integrity, and cellular apoptosis status. Thus, Memantine treatment exerts an antidepressant-like effect by preventing CUS induced excitotoxicity, oxidative stress, and enhancing CUS induced decrease in mitochondrial functioning and expression of cell survival genes via upregulation of stress-responsive CREB/BDNF signaling.

Early life selective knockdown of the TrkB receptor and maternal separation modulates adult stress phenotype

Brain Derived Neurotrophic Factor (BDNF) is a neurotrophin that has been implicated in mood and anxiety disorders and is upregulated by antidepressants. It has marked effects on synaptic and extra-synaptic plasticity through tropomyosin receptor kinase B (TrkB). Importantly, early life stressors that affect BDNF production are known to predispose individuals towards the later development of depression or anxiety disorders. Yet, few studies have actually assessed the long-term impact of selective early life BDNF manipulation. Therefore, we utilized a knock-in transgenic mouse line (TrkBF616A) with a mutation on the full-length TrkB receptor (TrkB.tk+), to reversibly block early postnatal BDNF/TrkB signaling. This was done during exposure to early life stress (maternal separation) followed by exposure to 35 days of chronic unpredictable stress (CUS) in adulthood. The TrkB.tk + mice that received the stressor treatments displayed a blunted anhedonic-like response (sucrose preference), compared to stressed animals that did not have the transient early life TrkB knockdown. But the TrkB.tk + mice actually showed an enhanced anxiety-like response in an open field in response following the CUS. This was paralleled by reductions of BDNF within the PFC and hippocampus of stressed TrkB.tk + mice, but basal elevations of BDNF was evident in the nucleus accumbens. These data are consistent with the contention that early in life, BDNF may program stress responsive circuits but this may differentially map onto depressive and anxiety-like responses in adulthood.

Gonadal Hormone Trigger Traf6-Mediated Dynamic Microglial Alterations that Correlate with Depressive Behaviors

Gonadal hormone deficiency is associated with the development of depression, but what mediates this association is unclear. To test the possibility that it reflects neuroimmune and neuroinflammatory processes, we analyzed how gonadal hormone deficiency and replacement affect microglial activation and inflammatory response during the development of depressive symptomatology in gonadectomized male mice. Testosterone level and the ratio of testosterone to estradiol in the serum and brain tissue of mice exposed to 3-35 days of chronic unpredictable stress were much lower than in control animals. Gonadal hormone sustained deficiency in gonadectomized mice and subsequent led to acute inflammation at day 7 following castration. Activating microglia in mice exposed to 7 days of castration subsequently suppressed the proliferation of microglia, such that their numbers in hippocampus and cortex were lower than the numbers in sham-operated mice after 30 days of castration. Here, we showed that gonadal hormone deficiency induces Traf6-mediated microglia activation, a type of inflammatory mediator. Microglia treated in this way for long time showed down-regulation of activation markers, abnormal morphology and depressive-like behaviors. Restoration and maintenance of a fixed ratio of testosterone to estradiol significantly suppressed microglial activation, neuronal necroptosis, dramatically inducing hippocampal neurogenesis and reducing depressive behaviors via the suppression of Traf6/TAK1 pathway. These findings suggest that activated or immunoreactive microglia contribute to gonadal hormone deficiency-induced depression, as well as testosterone and estradiol exert synergistic anti-depressant effects via suppressing microglial activaton in gonadectomized male mice, possibly through Traf6 signaling. Funding: This study was supported by National Nature Science Foundation of China (No. 81572069, 81501815, 81772265). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: Experiments in ICR male mice were approved by the Animals Ethics and Welfare Committee of Renmin Hospital of Wuhan University. All the experiments were conducted in accordance with the Directives of Wuhan University on animal ethics and welfare.

Early Blood Profile of C57BL/6 Mice Exposed to Chronic Unpredictable Stress.

Physiological responses to psychological stressors are protective in acute fight or flight situations; however, there is increasing evidence suggesting the detrimental impact of chronic psychological stress on disease. Chronic stress has been associated with inflammation, poor prognosis, increased morbidity, and poor outcome in many diseases including atherosclerosis, cancer, and pulmonary disease. Given the systemic impact of stress, and the role of the hematopoietic system as a rapid responder to homeostatic insults, we hypothesized that early blood profile changes and biochemical alterations could be detected in a model of chronic stress. To test this hypothesis, a variation of the chronic unpredictable stress (CUS) model was employed. Following 10 days of CUS, C57BL/6 mice exhibited a chronic-stress-associated corticosterone profile. Complete blood count (CBC) revealed mild normochromic, normocytic anemia, and reduced monocyte and lymphocyte count. Serum analysis demonstrated hypoferremia with unchanged total iron binding capacity and serum ferritin levels. These findings are consistent with clinical diagnostic parameters for anemia of chronic disease and indicate that CUS results in significant changes in blood and serum biochemical profile in C57BL/6 mice. These studies identify early changes in blood parameters in response to CUS and identify hematopoietic and biochemical alterations that are often associated with increased morbidity in patients experiencing chronic-stress-associated mental health disease.