Chronic Unpredictable Stress Mice Research Articles

Brain-derived neurotrophic factor in 5-HT neurons regulates susceptibility to depression-related behaviors induced by subchronic unpredictable stress

Chronic stress is a major risk factor for the development of depression. Brain-derived neurotrophic factor (BDNF) plays an important role in neural functions and exhibits antidepressant effects. However, studies on depression-related behavioral response to BDNF have mainly focused on the limbic system, whereas other regions of the brain still require further exploration. Here, we report that exposure to chronic unpredictable stress (CUS) can induce depression-associated behaviors in mice. CUS could decrease total Bdnf mRNA and protein levels in the dorsal raphe nucleus (DRN), which correlated with depression-related behaviors. A corresponding reduction in exon-specific Bdnf mRNA was observed in the DRN of CUS mice. Bdnf was highly expressed in 5- Hydroxytryptamine (5-HT) neurons from the DRN. Selective deletion of Bdnf in 5-HT neurons alone could not induce anhedonia and behavioral despair in male or female mice, as indicated by the unchanged female urine sniffing time and preference for sucrose/saccharin. However, it could increase the latency to food in female mice, but not in male mice as shown by novelty-suppressed food test. Nevertheless, enhanced stress-induced susceptibility is observed in these male mice as suggested by the decrease in female urine sniffing time, and for female mice by the reduced sucrose preference and increased immobility in forced swim test. Furtherly, total Bdnf mRNA levels in DRN were correlated with depression-related behaviors of female, but not male 5-HT neurons specific Bdnf knockout mice. Our results indicate that BDNF might act on 5-HT neurons to regulate depression-related behaviors and stress vulnerability in a sex-dependent manner.

Antidepressive properties of microglial stimulation in a mouse model of depression induced by chronic unpredictable stress

The decrease of microglia in the hippocampus is a novel mechanism for depression onset. Reversal of this decrease can ameliorate stress-induced depression-like behaviors in rodents. However, the property of this therapeutic strategy remains unclear. We addressed this issue by designing a series of behavioral experiments. Results showed that a single lipopolysaccharide (LPS) injection at the dose of 75 and 100 μg/kg, but not at 30 or 50 μg/kg, produced obvious antidepressant effects in chronic unpredictable stress (CUS) mice at 5 h after the drug administration. In the time-dependent experiment, a single LPS injection (100 μg/kg) ameliorated the CUS-induced depression-like behaviors in mice at 5 and 8 h, but not at 3 h, after the drug administration. The antidepressant effect of a single LPS injection persisted at least 10 days and disappeared at 14 days after the drug administration. 14 days after the first injection, a second LPS injection (100 μg/kg) still produced antidepressant effects in chronically-stressed mice who re-displayed depression-like behaviors at 5 h after the drug administration. The antidepressant effect of LPS appears to be dependent on microglia, as at 5 h after LPS administration (100 μg/kg), the CUS-induced decrease in microglial numbers and Iba-1 mRNA levels in the hippocampus was reversed markedly, and inhibition of microglia by minocycline (40 mg/kg) or PLX33297 (290 mg/kg) prevented the antidepressant effect of LPS in CUS mice. These results indicate that a single LPS injection displays rapid and sustained antidepressant effects in chronically stressed mice likely through stimulating hippocampal microglia.

Chronic unpredictable stress promotes cell-specific plasticity in prefrontal cortex D1 and D2 pyramidal neurons

Exposure to unpredictable environmental stress is widely recognized as a major determinant for risk and severity in neuropsychiatric disorders such as major depressive disorder, anxiety, schizophrenia, and PTSD. The ability of ostensibly unrelated disorders to give rise to seemingly similar psychiatric phenotypes highlights a need to identify circuit-level concepts that could unify diverse factors under a common pathophysiology. Although difficult to disentangle a causative effect of stress from other factors on medial prefrontal cortex (PFC) dysfunction, a wealth of data from humans and rodents demonstrates that the PFC is a key target of stress. The present study sought to identify a model of chronic unpredictable stress (CUS) which induces affective behaviors in C57BL6J mice and once established, measure stress-related alterations in intrinsic excitability and synaptic regulation of mPFC layer 5/6 pyramidal neurons. Adult male mice received 2 weeks of ‘less intense’ stress or 2 or 4 weeks of ‘more intense’ CUS followed by sucrose preference for assessment of anhedonia, elevated plus maze for assessment of anxiety and forced swim test for assessment of depressive-like behaviors. Our findings indicate that more intense CUS exposure results in increased anhedonia, anxiety, and depressive behaviors, while the less intense stress results in no measured behavioral phenotypes. Once a behavioral model was established, mice were euthanized approximately 21 days post-stress for whole-cell patch clamp recordings from layer 5/6 pyramidal neurons in the prelimbic (PrL) and infralimbic (IL) cortices. No significant differences were initially observed in intrinsic cell excitability in either region. However, post-hoc analysis and subsequent confirmation using transgenic mice expressing tdtomato or eGFP under control of dopamine D1-or D2-type receptor showed that D1-expressing pyramidal neurons (D1-PYR) in the PrL exhibit reduced thresholds to fire an action potential (increased excitability) but impaired firing capacity at more depolarized potentials, whereas D2-expressing pyramidal neurons (D2-PYR) showed an overall reduction in excitability and spike firing frequency. Examination of synaptic transmission showed that D1-and D2-PYR exhibit differences in basal excitatory and inhibitory signaling under naïve conditions. In CUS mice, D1-PYR showed increased frequency of both miniature excitatory and inhibitory postsynaptic currents, whereas D2-PYR only showed a reduction in excitatory currents. These findings demonstrate that D1-and D2-PYR subpopulations differentially undergo stress-induced intrinsic and synaptic plasticity that may have functional implications for stress-related pathology, and that these adaptations may reflect unique differences in basal properties regulating output of these cells.

Chronic histamine 3 receptor antagonism alleviates depression like conditions in mice via modulation of brain-derived neurotrophic factor and hypothalamus-pituitary adrenal axis

The last two decades of research has established histamine (HA) as a neurotransmitter. Since H3R antagonists are known to modulate several neurotransmitters besides HA, H3R antagonists have shown potential for the treatment of different central nervous system disorders, including depression. However, molecular mechanisms underlying the beneficial effects of H3R antagonism in depression are not clear, yet. In the present study, we investigated the antidepressant potential of ciproxifan, a selective H3R antagonist, in chronic unpredictable stress (CUS) model of depression in C57BL/6 J mice. We observed that chronic treatment of CUS mice with ciproxifan (3 mg/kg i.p.; for three weeks) alleviates depression-like symptoms such as helplessness measured by forced swim and tail suspension test (FST and TST), anhedonia measured by sucrose preference test (SPT) and social deficit measured in social behavior test. Chronic ciproxifan treatment restored CUS induced BDNF expression in the prefrontal cortex (PFC) and hippocampus. We also observed that ciproxifan modulates CUS induced NUCB2/nesfatin-1 and CRH expression in the hypothalamus and plasma corticosterone. We also determined the direct effect of HA on BDNF expression in neurons by western blotting and immunocytochemistry, and found that HA significantly induced BDNF expression, which was blocked by the H4R selective antagonist, but not by other HA receptor selective antagonists. Furthermore, ciproxifan significantly modulated NMDA glutamate receptor subunits NR2B and NR2A. Thus, these results suggest that increased HA signaling in the brain produces antidepressant-like effects in mice and modulates BDNF expression and HPA-axis.

Chronic stress leads to epigenetic dysregulation in the neuropeptide-Y and cannabinoid CB1 receptor genes in the mouse cingulate cortex

Persistent stress triggers a variety of mechanisms, which may ultimately lead to the occurrence of anxiety- and depression-related disorders. Epigenetic modifications represent a mechanism by which chronic stress mediates long-term effects. Here, we analyzed brain tissue from mice exposed to chronic unpredictable stress (CUS), which induced impaired emotional and nociceptive behaviors. As endocannabinoid (eCB) and neuropeptide-Y (Npy) systems modulate emotional processes, we hypothesized that CUS may affect these systems through epigenetic mechanisms. We found reduced Npy expression and Npy type 1 receptor (Npy1r) signaling, and decreased expression of the cannabinoid type 1 receptor (CB1) in the cingulate cortex of CUS mice specifically in low CB1-expressing neurons. Epigenetic investigations revealed reduced levels of histone H3K9 acetylation (H3K9ac) associated to Npy and CB1 genes, which may represent a factor determining the dysregulation occurring at expression and signaling level. CUS mice also showed increased nuclear protein levels and activity of the histone deacetylase type 2 (HDAC2) in the cingulate cortex as compared to controls. Chronic administration of URB597, an inhibitor of anandamide degradation, which is known to induce anxiolysis in CUS mice, reversed the epigenetic changes found in the Npy gene, but was ineffective in alleviating the dysregulation of Npy at transcriptional and signaling level. Our findings suggest that epigenetic alterations in the Npy and CB1 genes represent one of the potential mechanisms contributing to the emotional imbalance induced by CUS in mice, and that the Npy and eCB systems may represent therapeutic targets for the treatment of psychopathologies associated with or triggered by chronic stress states.

Antidepressant activity of fingolimod in mice.

Recent findings indicate that fingolimod, the first oral drug approved for the treatment of multiple sclerosis (MS), acts as a direct inhibitor of histone deacetylases (HDACs) and enhances the production of brain-derived neurotrophic factor (BDNF) in the CNS. Both mechanisms are relevant to the pathophysiology and treatment of major depression. We examined the antidepressant activity of fingolimod in mice subjected to chronic unpredictable stress (CUS), a model of reactive depression endowed with face and pharmacological validity. Chronic treatment with fingolimod (3 mg kg−1, i.p., once a day for 4 weeks) reduced the immobility time in the forced swim test (FST) in a large proportion of CUS mice. This treatment also caused anxiogenic-like effects in the social interaction test without affecting anxiety-like behavior in the elevated plus maze or spatial learning in the water maze. CUS mice showed reduced BDNF levels and enhanced HDAC2 levels in the hippocampus. These changes were reversed by fingolimod exclusively in mice that showed a behavioral response to the drug in the FST. Fingolimod treatment also enhanced H3 histone K14-acetylation and adult neurogenesis in the hippocampus of CUS mice. Fingolimod did not affect most of the parameters we have tested in unstressed control mice. The antidepressant-like activity of fingolimod was confirmed in mice chronically treated with corticosterone. These findings show for the first time that fingolimod exerts antidepressant-like effect acting in a “disease-dependent” manner, and raise the interesting possibility that the drug could relieve depressive symptoms in MS patients independently of its disease-modifying effect on MS.

5HT3 receptor antagonist (ondansetron) reverses depressive behavior evoked by chronic unpredictable stress in mice: Modulation of hypothalamic–pituitary–adrenocortical and brain serotonergic system

Chronic stress is one of the major causes of depression, associated with behavioral and biochemical impairments. 5HT3 receptor antagonists (such as ondansetron) have shown alleviation of depressive symptomology in preclinical and in few clinical studies. However, their effects in chronic stress-induced depressive behavior and the underlying mechanism(s) are yet to be known. In the present study, the effects of a 5HT3 receptor antagonist, ondansetron were evaluated in chronic unpredictable stress (CUS)-evoked depressive behavior. In addition, the possible mechanism was determined by measuring plasma corticosterone (CORT) as a marker of hypothalamic–pituitary–adrenocortical (HPA)-axis activity and serotonin levels in the discrete brain regions. Mice were subjected to a battery of unpredictable stressors for 28days. Ondansetron (0.05, 0.1 and 1mg/kg, p.o.) and fluoxetine (10mg/kg, p.o.) were administered during the last 14days (day 15–28th) of CUS testing paradigm. The results showed that the 4-week CUS produced significant depressive behavior in mice, which included increased despair effects in forced swim test (FST) and reward-related deficits in sucrose preference test. Biochemical assays demonstrated a significant increase in percentage of plasma CORT and decrease in percentage of serotonin levels in the discrete brain regions of CUS mice. Chronic ondansetron treatment, similar to that of positive control fluoxetine, significantly reversed despair effects in FST and reward-related deficits in sucrose preference test. In addition, ondansetron and fluoxetine treatments significantly increased percentage of serotonin levels in the measured brain regions and attenuated HPA-axis hyperactivity, as evidenced by low percentage of plasma CORT levels in CUS mice. These findings indicate the potential role of ondansetron (a 5HT3 receptor antagonist) in reversing CUS-induced depressive behavior, which is possibly mediated by its modulating effects on the HPA-axis and serotonergic system. Further, the study represents that 5HT3 receptor antagonists can be a potential therapeutic candidate for stress-related depressive disorders.

Psychological stress in adolescent and adult mice increases neuroinflammation and attenuates the response to LPS challenge

BackgroundThere is ample evidence that psychological stress adversely affects many diseases. Recent evidence has shown that intense stressors can increase inflammation within the brain, a known mediator of many diseases. However, long-term outcomes of chronic psychological stressors that elicit a neuroinflammatory response remain unknown.MethodsTo address this, we have modified previously described models of rat/mouse predatory stress (PS) to increase the intensity of the interaction. We postulated that these modifications would enhance the predator-prey experience and increase neuroinflammation and behavioral dysfunction in prey animals. In addition, another group of mice were subjected to a modified version of chronic unpredictable stress (CUS), an often-used model of chronic stress that utilizes a combination of stressors that include physical, psychological, chemical, and other. The CUS model has been shown to exacerbate a number of inflammatory-related diseases via an unknown mechanism. Using these two models we sought to determine: 1) whether chronic PS or CUS modulated the inflammatory response as a proposed mechanism by which behavioral deficits might be mediated, and 2) whether chronic exposure to a pure psychological stressor (PS) leads to deficits similar to those produced by a CUS model containing psychological and physical stressors. Finally, to determine whether acute PS has neuroinflammatory consequences, adult mice were examined at various time-points after PS for changes in inflammation.ResultsAdolescent mice subjected to chronic PS had increased basal expression of inflammation within the midbrain. CUS and chronic PS mice also had an impaired inflammatory response to a subsequent lipopolysaccharide challenge and PS mice displayed increased anxiety- and depressive-like behaviors following chronic stress. Finally, adult mice subjected to acute predatory stress had increased gene expression of inflammatory factors.ConclusionOur results demonstrate that predatory stress, an ethologically relevant stressor, can elicit changes in neuroinflammation and behavior. The predatory stress model may be useful in elucidating mechanisms by which psychological stress modulates diseases with an inflammatory component.

Author index

Brain-derived neurotrophic factor (BDNF) is a biomarker of depression. Recent studies have found adenosine deaminase acting on RNA1 (ADAR1) is a novel target being sensitive to stress at epigenetic level. The epigenetic regulation mechanism of stress-related depression is still unclear so far. To explore the potential regulating mechanism of ADAR1 on BDNF, over and low expression of ADAR1 in PC12 and SH-SY5Y cell lines are prepared. In the meanwhile, chronic unpredictable stress (CUS) mice are treated with ADAR1 inducer (interferon-γ, IFN-γ). ADAR1 regulates BDNF expression, which is proven by that over and low expressions of ADAR1 increase and decrease BDNF mRNA and protein respectively in vitro. Additionally, ADAR1 inducer alleviates the depressive-like behavior of CUS mice by recovering the decreased BDNF protein in brain and serum. Moreover, over and low expressions of ADAR1 reduce and enhance microRNA-432 (miR-432) expression respectively in vitro. Furtherly, over and low miR-432 expressions lead to decreased and increased BDNF and ADAR1 mRNA, protein and immunoreactivity respectively in vitro. The above results demonstrate that ADAR1 is involved in antidepressant action by regulating BDNF via miR-432. Those novel findings can provide a new idea for the study of epigenetic regulation mechanism, early diagnosis, and effective treatment of stress-related depression.